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Objective To explore the molecular mechanism of naringenin in preventing and treating insulin resistance in rats with polycystic ovary syndrome (PCOS) based on the aspect of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway. Methods SD rats were injected with dehydroepiandrosterone subcutaneously on the back daily to establish a PCOS model. The model rats were randomly divided into the model group, the naringenin group, the PI3K inhibitor group, and the naringenin+PI3K inhibitor group, with 15 rats in each group. At the same time, 15 SD rats were subcutaneously injected with oil 2 mL/kg on the neck and back, and they were used as the normal control group. The levels of blood glucose, insulin, blood lipids, and reproductive hormones were detected in each group. The morphology of ovary was observed with HE staining. The positive expression of p-PI3K was detected with immunohistochemical method. The protein expression of PI3K/AKT pathway phosphorylated protein, and pathway related protein-insulin receptor substrate-1 (IRS-1), glycogen synthase-3β (GSK-3β), glucose transporter factor-4 (GLUT4), phosphatase and tensin homologue deleted on chromosome 10 (PTEN) were detected with Western blot assay. Results Compared with the normal control group, the ovarian tissue of the model group showed symptoms of pathological damage such as follicular cystic dilatation, decreased corpus luteum number and granulosa cell layer, and increased atretic follicles, increased levels of blood lipid and blood sugar, increased insulin resistance, and disordered secretion of reproductive hormones, decreased PI3K/AKT pathway phosphorylated proteins and pathway-related proteins IRS-1, GSK-3β phosphorylated protein and GLUT4 protein expression, and increased expression of PTEN protein (P＜0.05). Naringenin could reduce pathological damage such as cystic dilatation of follicles, promote PI3K/AKT pathway phosphorylated proteins and pathway-related proteins IRS-1, GSK-3β phosphorylated protein and GLUT4 protein expression, improve the disorder of reproductive hormone secretion, reduce insulin resistance, blood glucose and blood lipid levels and PTEN protein expression (P＜0.05). PI3K inhibitors could attenuate the above effects of naringenin (P＜0.05). Conclusion Naringenin can promote the activation of PI3K/AKT pathway, reduce blood glucose and blood lipid levels and insulin resistance, and improve reproductive hormone disorders and ovarian polycystic changes in PCOS rats. [ABSTRACT FROM AUTHOR]

: Objective To investigate the effect and mechanism of gastrodin on head twitch behavior in rats with Tourette syndrome (TS). Methods 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induction method was used to replicate TS rat model. Forty Wistar rats were randomly divided into four groups (n=10): the normal group (gastric administration of normal saline), the TS model group (from the first day of modeling, intragastric administration with normal saline at 2 h after DOI injection), the TS+ gastrodin group (from the first day of modeling, intragastric administration with 30 mg/kg gastrodin at 2 h after DOI injection), and the TS+gastrodin+CHIR-99021 group [glycogen synthase kinase-3β (GSK3β) inhibitor] (from the first day of modeling, 2 h after DOI injection, intraperitoneal injection with 2 mg/kg CHIR-99021 firstly, and then intragastric administration with 30 mg/kg gastrodin), continuous administration for 21 d. After the last administration, the rat’s head twitching behavior within 30 min was counted. The levels of striatal 5-hydroxyindole (5-HT), 5-hydroxyindole acetic acid (5-HIAA), and dopamine (DA) were detected by kits. The number of DA neurons in the substantia nigra was detected by immunohistochemical method. The expression of 5-HT transporter (SERT), 5- hydroxyindole 2A receptor (5-HT2AR), 5-hydroxyindole 2C receptor (5-HT2CR) and GSK-3β phosphorylation in the striatum were detected by Western blot assay. Results Compared with the normal group, the number of head twitches, DA level, number of DA neurons, SERT level and p-S9-GSK-3β, p-S9-GSK-3β/t-GSK-3β level increased in the TS model group, and the 5-HT level decreased (P＜0.05). Compared with the TS model group, the number of head twitches, DA level, number of DA neurons, SERT level and p-S9-GSK-3β, p-S9-GSK-3β/t-GSK-3β level decreased in the TS+gastrodin group, and the 5-HT level increased (P＜0.05). Compared with the TS+gastrodin group, the number of head twitches, DA level, number of DA neurons, SERT level and p-S9-GSK-3β, p-S9-GSK-3β/t-GSK-3β level increased, and the 5-HT level decreased in the TS+gastrodin+CHIR-99021 group (P＜0.05). Conclusion Gastrodin can improve the head twitch behavior of TS rats, and its mechanism may be by inhibiting the phosphorylation of GSK-3β, regulating the expression of SERT, affecting the level of 5-HT, and then inhibiting the release of DA in striatum. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect of tripipartite motif 59 (TRIM59) expression interference on the chemosensitivity of daunorubicin (DNR) in chronic myeloid leukemia (CML) K562 cells and the related molecular mechanism., Methods: The expressions of TRIM59 mRNA in bone marrow tissues of patients with CML and K562 cells were detected by RT-qPCR. Liposome-based transfection technology was used to transfect TRIM59-specific siRNA (si-TRIM59) into K562 cells which then were treated with DNR. The proliferation and apoptosis of cells were detected by CCK-8 assay and flow cytometry, respectively, and the expressions of apoptosis-related protein and Wnt/β-catenin signaling pathway-related protein were detected by Western blot., Results: Compared with the bone marrow tissue of CML patients at the time of initial treatment, the expression of TRIM59 mRNA in bone marrow tissue of CML patients at the time of chemotherapy resistance was significantly increased ( P <0.05). Compared with control group, the cell proliferation inhibition rate and apoptosis rate in si- TRIM59 group and DNR group were significantly increased ( P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased ( P <0.05), while the expressions of Bcl-2, Wnt3α, GSK-3β protein and the ratio of p-β-catenin/β-catenin were significantly decreased ( P <0.05). Compared with si- TRIM59 group and DNR group, the proliferation inhibition rate and apoptosis rate of si- TRIM59 +DNR group were significantly increased ( P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased, while the expression of Bcl-2, Wnt3α, GSK-3β protein and the ratio of p-β-catenin/β-catenin were significantly decreased ( P <0.05)., Conclusion: TRIM59 expression interference may enhance the chemosensitivity of K562 cells to DNR, and its mechanism may be related to the regulation of Wnt/β-catenin signaling pathway.

Objectives: To explore the mechanism of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD)., Methods: The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacology methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriching analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblastoid cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT and CCK-8 methods were used to quantify SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS), and immunofluorescence staining was used to detect the generation of Aβ 1-42 in a co-cultured model. Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aβ deposition in the pharynx was detected by Thioflavin S staining., Results: Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were identified. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP) and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aβ 1-42 (both P <0.01), inhibit the expression of β-secretase 1 (BACE1), APP and Aβ 1-42 proteins (all P <0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3β/GSK3β in SKNMC cells (all P <0.05). The in vivo studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aβ deposition., Conclusions: Anemarrhenae Rhizoma may reduce the production of Aβ in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/Akt/GSK-3β pathway.

Objective: To investigate the role of Rho GTPase-activating protein 21 (ARHGAP21) in regulating the migration and metastasis of non-small cell lung cancer (NSCLC) cells., Methods: TCGA, CPTAC database were used to analyze the correlation of ARHGAP21 expression level in NSCLC and the patients' prognosis. The expression of ARHGAP21 in clinical specimens of NSCLC tissues was examined using Western blotting and immunohistochemistry. The effect of ARHGAP21 knockdown on migration ability of lung cancer cell lines was examined using Transwell assay and wound healing assay. A nude mouse model with injection of lung cancer H1299 cells via the tail vein was used to examine the effect of ARHGAP21 knockdown on the metastatic ability of the tumor cells. The possible mechanism of ARHGAP21 was predicted by bioinformatics analysis and verified using Western blotting., Results: A low ARHGAP21 expression was associated with poor prognosis of patients with NSCLC ( P < 0.05). ARHGAP21 expression was significantly downregulated in lung cancer tissues as compared with the adjacent tissues ( P < 0.001). In cultured lung cancer cells, ARHGAP21 knockdown obviously promoted the migration ability of the cells ( P < 0.001). In the nude mouse models, injection of H1299 cells with ARHGAP21 knockdown, as compared with the negative control cells, resulted in a greater number of metastatic lung cancer nodules ( P < 0.05), which expressed higher levels of N-cadherin and vimentin. Bioinformatic analysis showed a close correlation of ARHGAP21 with APC, GSK3β, and Axin ( P < 0.001). Western blotting showed that ARHGAP21 knockdown significantly decreased ubiquitination of β-catenin, upregulated N-cadherin and activated the WNT signaling pathway in the lung cancer cells., Conclusion: ARHGAP21 downregulation can significantly promote the migration and metastatic ability of NSCLC possibly as a result of WNT signaling pathway activation, which reduces the ubiquitination of β-catenin by affecting the expressions of APC, GSK3β, and Axin.

Objective: To observe the effects of Yizhi Tiaoshen (benefiting mental health and regulating the spirit) acupuncture on learning and memory function, and the expression of phosphorylated tubulin-associated unit (tau) protein in the hippocampus of Alzheimer's disease (AD) model rats, and explore the effect mechanism of this therapy on AD., Methods: A blank group and a sham-operation group were randomly selected from 60 male SD rats, 10 rats in each one. AD models were established in the rest 40 rats by the intraperitoneal injection of D-galactose and okadaic acid in the CA1 region of the bilateral hippocampus. Thirty successfully-replicated model rats were randomly divided into a model group, a western medication group and an acupuncture group, 10 rats in each one. In the acupuncture group, acupuncture was applied to "Baihui" (GV 20), "Sishencong" (EX-HN 1), "Neiguan" (PC 6), "Shenmen" (HT 7), "Xuanzhong" (GB 39) and "Sanyinjiao" (SP 6); and the needles were retained for 10 min. Acupuncture was given once daily. One course of treatment was composed of 6 days, with the interval of 1 day; the completion of treatment included 4 courses. In the western medication group, donepezil hydrochloride solution (0.45 mg/kg) was administrated intragastrically, once daily; it took 7 days to accomplish one course of treatment and a completion of intervention was composed of 4 courses. Morris water maze (MWM) and novel object recognition test (NORT) were used to assess the learning and memory function of the rats. Using HE staining and Nissl staining, the morphological structure of the hippocampus was observed. With Western blot adopted, the protein expression of the tau, phosphorylated tau protein at Ser198 (p-tau Ser198), protein phosphatase 2A (PP2A) and glycogen synthase kinase-3β (GSK-3β) in the hippocampus was detected., Results: There were no statistical differences in all of the indexes between the sham-operation group and the blank group. Compared with the sham-operation group, in the model group, the MWM escape latency was prolonged ( P <0.05), the crossing frequency and the quadrant stay time in original platform were shortened ( P <0.05), and the NORT discrimination index (DI) was reduced ( P <0.05); the hippocampal cell numbers were declined and the cells arranged irregularly, the hippocampal neuronal structure was abnormal and the numbers of Nissl bodies decreased; the protein expression of p-tau Ser198 and GSK-3βwas increased ( P <0.05) and that of PP2A decreased ( P <0.05). When compared with the model group, in the western medication group and the acupuncture group, the MWM escape latency was shortened ( P <0.05), the crossing frequency and the quadrant stay time in original platform were increased ( P <0.05), and DI got higher ( P <0.05); the hippocampal cell numbers were elevated and the cells arranged regularly, the damage of hippocampal neuronal structure was attenuated and the numbers of Nissl bodies were increased; the protein expression of p-tau Ser198 and GSK-3β was reduced ( P <0.05) and that of PP2A was increased ( P <0.05). There were no statistically significant differences in the above indexes between the acupuncture group and the western medication group ( P >0.05)., Conclusion: Acupuncture therapy of "benefiting mental health and regulating the spirit" could improve the learning and memory function and alleviate neuronal injure of AD model rats. The effect mechanism of this therapy may be related to the down-regulation of GSK-3β and the up-regulation of PP2A in the hippocampus, and then to inducing the inhibition of tau protein phosphorylation.

Objective: To investigate the effects and mechanism of astragalus polysaccharide (APS) on wound healing of deep partial-thickness burns in rats. Methods: The experimental study method was used. Fifty 7-week-old male Sprague-Dawley rats were divided into normal group, simple burn group, APS group, inhibitor group, and inhibitor+APS group according to the random number table, with 10 rats in each group. Except for normal group, rats in the other 4 groups were inflicted with a deep partial-thickness burn wound on the back. Rats in normal group and simple burn group were intraperitoneally injected with normal saline, and rats in the other three groups were injected with APS and/or integrin-linked kinase (ILK) inhibitor, respectively. After 7 days of injection, the wound healing rate of rats with burns in the four groups was calculated, and the serum content of interferon-γ, interleukin-2 (IL-2), and tumor necrosis factor α (TNF-α) in rats in 5 groups was determined by enzyme-linked immunosorbent assay (ELISA). The normal skin tissue of rats in normal group and wound tissue of rats with burns in the four groups were taken, the water content was determined and the water ratio was calculated, the content of interferon-γ, IL-2, and TNF-α was detected by ELISA, the mRNA expressions of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and ILK were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction, and the protein expressions of ILK, protein kinase B (Akt), phosphorylated Akt (p-Akt), glycogen synthetic kinase-3β (GSK-3β), and phosphorylated GSK-3β (p-GSK-3β) were detected by Western blotting. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: After 7 days of injection, the wound healing rate of rats in APS group was (67±5)%, which was significantly higher than (52±4)% in simple burn group and (59±5)% in inhibitor+APS group (with all the P values <0.05). The wound healing rate of rats in inhibitor+APS group was significantly higher than (48±4)% in inhibitor group ( P <0.05). After 7 days of injection, compared with those in serum or normal skin tissue of rats in normal group, the serum content of interferon-γ, TNF-α, IL-2 and the water ratio of wound tissue of rats in simple burn group were significantly increased ( P <0.05); compared with those in APS group, the serum content of interferon-γ, TNF-α, IL-2 and the water ratio of wound tissue of rats in simple burn group and inhibitor+APS group were significantly increased ( P <0.05); compared with those in inhibitor group, the serum content of interferon-γ, TNF-α, IL-2 and the water ratio of wound tissue of rats in inhibitor+APS group were significantly decreased ( P <0.05). After 7 days of injection, compared with that in normal skin tissue of rats in normal group, the content of interferon-γ, TNF-α, and IL-2 in wound tissue of rats in simple burn group was significantly increased ( P <0.05); compared with that in APS group, the content of interferon-γ, TNF-α, and IL-2 in wound tissue of rats in simple burn group and inhibitor+APS group was significantly increased ( P <0.05); compared with that in inhibitor group, the content of interferon-γ, TNF-α, and IL-2 in wound tissue of rats in inhibitor+APS group was significantly decreased ( P <0.05). After 7 days of injection, compared with those in normal skin tissue of rats in normal group, the mRNA expressions of EGF, bFGF, ILK and protein expressions of ILK, p-Akt, p-GSK-3β in wound tissue of rats in simple burn group were significantly increased ( P <0.05); compared with those in APS group, the mRNA expressions of EGF, bFGF, ILK and protein expressions of ILK, p-Akt, p-GSK-3β in wound tissue of rats in simple burn group and inhibitor+APS group were significantly decreased ( P <0.05); compared with those in inhibitor group, the mRNA expressions of EGF, bFGF, ILK and protein expressions of ILK, p-Akt, p-GSK-3β in wound tissue of rats in inhibitor+APS group were significantly increased ( P <0.05). There were no statistically significant differences in the protein expressions of Akt and GSK-3β in normal skin tissue of rats in normal group and wound tissue of rats with burns in the four groups ( P >0.05). Conclusions: APS can alleviate systemic and local inflammation, alleviate tissue edema, and promote the expressions of healing factors in rats with deep partial-thickness burns, thus to promote the wound healing, possibly by activating ILK/Akt/GSK-3β signaling pathway.

Background: Acquired and primary resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is still the bottleneck of clinical treatment of advanced non-small cell lung cancer (NSCLC). STE029 is a novel anticancer drug which consists of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) inhibitor and novel cancer cell membrane targeting molecular. This study aimed to investigate the reversal mechanism of EGFR-TKI resistance by STE029 in lung adenocarcinoma., Methods: CCK8 test was used to test the cell viability and survival rate of EGFR mutated PC9 cell (Gefitinib sensitive), PC9/BB4 cell (acquired Gefitinib resistant), and EGFR wild type A549 cell after treatment of STE029, Gefitinib or combination of both. EdU test was applied to detect changes in cell cycle and Hoechst 33258 was applied to detect apoptosis rate in overcoming the EGFR-TKI resistance. The activity of EGFR/PI3K/Akt, cell cycle and apoptosis signal pathways were examined. In vivo, nude mice were exposed to STE029, Gefitinib and STE029+Gefitinib for 5 wk. And the the tumor volume was measured and tumor weight was obtained on the last day., Results: (1) PC9 cells was highly sensitive to Gefitinib, while PC9/BB4 and A549 cell showed significant resistance to Gefitinib treatment; (2) STE029+Gefitinib treatment could significantly decrease the 50% inhibitory concentrarion (IC₅₀) of Gefitinib in PC9, PC9/BB4 and A549 cells (P<0.05, respectively); (3) In PC9 and PC9/BB4 cells, STE029+Gefitinib can block cell cycle and inhibit cell proliferation (P<0.001), while there was no significant difference in apoptosis rate among three drug intervention groups (P>0.05); However, apoptosis rate was increased in STE029+Gefitinib group in A549 cell (P<0.01), while no significance detected in cell proliferation (P>0.05). (4) In PC9 and PC9/BB4 cells, the combination of STE029 and Gefitinib could downregulate p-EGFR, p-Akt, p-Cyclin D1 and Cyclin D1 (P<0.001), and upregulate the expression of GSK-3β (P<0.001), and the expression of cleaved caspase-8, caspase-8 cleaved caspase-9, caspase-9 showed no difference among groups (P>0.05). In A549 cells, the combination of STE029 and Gefitinib could downregulate p-Akt (P<0.001) and upregulate cleaved caspase-8 and cleaved caspase-9 (P<0.001); (5)In vivo, the combination of STE029 and Gefitinib effectively inhibited tumor development and progression compared to STE029 alone or Gefitinib alone, with significant difference (P<0.05) in PC9 and PC9/BB4 xenografted tumor., Conclusions: STE029 could sensitize Gefitinib by inhibiting EGFR/PI3K/Akt pathway, blocking the tumor cell cycle and proliferation and inducing apoptosis through caspase-8 and caspase-9 dependent pathway. STE029 deserves further investigations in overcoming EGFR-TKI resistance in lung cancer.

Objective: To investigate the effects of the peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the proliferation of primary rat proliferation of pulmonary artery smooth muscle cells ( PASMCs ) induced by hypoxia, in order to discover new drugs for the treatment and prevention of pulmonary vascular remodeling. Methods: The PASMCs in the control group were cultured with 21% oxygen, while the PASMCs in the hypoxia group were cultured with 3% oxygen to induce cell proliferation. PASMCs were incubated with GW501516 at the concentrations of 10, 30 and 100 nmol/L under hypoxic conditions for different time points (12, 24, and 48 h) to find out the appropriate concentrations of GW501516 for inhibition the proliferation. PASMCs were incubated with 100 nmol/L GW501516 and ( or ) protein kinase B (AKT) agonist SC79 for 24 h to explore related mechanisms of GW501516 in regulating the proliferation. The proliferation and DNA synthesis were determined by CCK-8 and BrdU kit. The cell cycle progression was analyzed by flow cytometry. The mRNA expressions of Cyclin D1 and the cyclin kinase inhibitor p27(p27) were measured by quantitative real-time PCR (RT-PCR). The expressions of PPARδ, total and phosphorylated forms AKT and glycogen synthase kinase 3β (GSK3β) were detected by Western blot. Results: Compared with the hypoxia group, PASMCs incubated with different concentrations of GW501516 (10, 30, 100 nmol/L) for 12, 24, 48 h under hypoxic conditions could inhibit the proliferation and DNA synthesis, and the greatest level of suppression of proliferation was induced by GW501516 at the concentration of 100 nmol/L( P ＜0.05 or P ＜0.01). Compared with the control group, the expression of PPARδ was upregulated markedly in PASMCs incubated with 100 nmol/L GW501516 for 24 h,while hypoxia could downregulate the expression of PPARδ significantly( P ＜0.01). Compared with the hypoxia group, 100 nmol/L GW501516 blocked the proliferation and DNA synthesis of PASMCs significantly( P ＜0.01), increased the proportion of PASMCs in G0 /G1 phase while decreased the proportion of PASMCs in S phase and G2 /M phase( P ＜0.05 or P ＜0.01), markedly downregulated the mRNA expression of cyclin D1 and upregulated the mRNA expression of p27( P ＜0.01), significantly inhibited the protein expressions of phosphorylated AKT and GSK3β( P ＜0.01). Compared with the 100 nmol/L GW501516 hypoxia group, AKT agonist SC79 reversed all the above effects of 100 nmol/L GW501516 on hypoxia stimulated PASMCs( P ＜0.05 or P ＜0.01). Conclusion: GW501516 inhibits hypoxia induced proliferation in PASMCs via inactivating AKT/GSK3β signaling pathway.

Objective: To investigate the effects of glycogen synthase kinase-3β (GSK3β)/eukaryotic extension factor kinase 2 (eEF2K) signaling pathway on the process of pulmonary fibrosis through in vivo experiments, and find new ideas for clinical treatment of pulmonary fibrosis. Methods: The pulmonary fibrosis model of C57BL/6 male mice was induced by bleomycin with intratracheal injection at the dose of 2 mg/kg. After 14 days of modeling, animals were divided into model group, negative inhibition group and inhibition group ( n =5 for each group), and control group was not processed. The inhibition group was treated with TDZD-8 (4 mg/kg) after modeling, the negative inhibition group was given DMSO solution after modeling, and the samples were collected after 28 days. Hematoxylin-eosin staining method was used to detect lung fibrosis in mice and scored according to Ashcroft scale. Expression levels of GSK3β, p-GSK3β, eEF2K, p-eEF2K (Ser70, Ser392, Ser470), precursor protein of matrix metalloproteinase-2 (pro-MMP-2), matrix metalloproteinase-2 (MMP-2), collagen I (Col I), collagen Ⅲ (Col Ⅲ) and α-smooth muscle actin (α-SMA) were detected by Western blot. Results: Compared with control group, the fibrosis score was up-regulated, the expression levels of GSK3β, p-GSK3β, p-eEF2K (Ser70, Ser392, Ser470), pro-MMP-2, MMP-2, Col I, Col Ⅲ and α-SMA were increased, while that of eEF2K was decreased in model group ( P ＜0.05). Compared with model group, the fibrosis score, expression levels of GSK3β, p-GSK3β, p-eEF2K (Ser70, Ser392, Ser470), pro-MMP-2, MMP-2, Col I, Col Ⅲ and α-SMA were decreased, but the expression level of eEF2K was increased in inhibition group ( P ＜0.05). Conclusion: GSK3β can activate eEF2K by phosphorylation at the sites of Ser70, Ser392 and Ser470, increase the contents of fibrosis indicators, promote the formation of pulmonary fibrosis, and aggravate lung tissue lesions.

Objective: To explore the influence of long non-coding (lnc) RNA Gm15645 on the podocyte injury in mice with diabetic nephropathy., Methods: Male db/db mice (with Type 2 diabetes) with a genetic background of C57BLKs/J and db/m mice (healthy) born in littermates were randomly divided into three groups. db/db group was injected with lncRNAGm15645 shRNA lentivirus with a podocyte-specific marker NPHS2; db/db blank group was injected with saline, and db/db control group was injected withnon-sense lentivirus. The results of PAS staining, pathological changes of renal tissue, relative expression of GSK-3beta, and podocin expression were compared., Results: lncRNAGm15 645 was overexpressed and podocin was down-regulated in the lentivirus overexpressed group. Mesangial cell proliferation, mesangial matrix hyperplasia, thickened basement membrane, widely fused foot process, and podocyte injury were observed by PAS staining. The expression of Gm15645 in the db/db group was significantly lower than that of the db/db blank group and db/db control group (P< 0.05), while the expression of podocin was higher (P< 0.05). Gm15645 was co-stained with podocin in renal tissue, and the target gene was GSK-3beta., Conclusion: lncRNAGm15645 may provide an early biomarker for the occurrence of podocyte injury in diabetic nephropathy. The mechanism may be related to the feedback regulation of GSK-3beta gene.

Objective: To explore the inhibitory effects of ginsenoside compound K (CK) on pulmonary arterial smooth muscle cells (PASMCs) proliferation and phenotypic conversion in vitro and investigate its related mechanisms., Methods: PASMCs cultured in vitro were examined in the study. They were induced with platelet-derived growth factor-BB (PDGF-BB) and then treated with CK. The cells were randomly assigned to the control group (receiving no treatment), the model group (PDGF-BB, 20 ng/mL), and the intervention group (20 ng/mL PDGF-BB+5 μmol/L CK). The cell proliferation was measured by CCK-8 assay (on the basis of the above group assignment, concentrations of CK was set at 1, 3, and 5 μmol/L in the intervention group, and the drug group was added, receiving 1, 3, and 5 μmol/L CK, respectively). Cell cycle and apoptosis were examined by flow cytometry. The levels of mRNA and proteins of α-smooth muscle actin ( α-SMA ) and smooth muscle 22α ( SM 22 α ), markers of phenotypic conversion, were detected by quantitative real-time PCR and Western blot. The levels of protein expression related to Wnt/β-catenin signaling pathway were examined by Western blot., Results: Compared with the model group, CK significantly inhibited PDGF-BB-induced proliferation of PASMCs in a dose-dependent way. The results of 5 μmol/L CK intervention were not significantly different from that of the control group ( P >0.05). Hence, 5 μmol/L CK was chosen for subsequent experiments. Separate treatment of PASMCs with CK at doses of 1, 3, and 5 μmol/L did not reveal any cytotoxicity to PASMCs ( P >0.05). CK also arrested the cell cycle of PASMCs at the G 0 /G 1 phase, promoted the apoptosis of PASMCs, and reversed the mRNA and protein expression of α-SMA and SM 22 α ( P <0.01). In addition, CK down-regulated the expressions of cyclin D1 and β-catenin, while it up-regulated the protein expressions of phosphorylated glycogen synthase kinase-3β (pGSK-3β)/glycogen synthase kinase-3β (GSK-3β) ( P <0.01)., Conclusion: CK was capable of inhibiting the abnormal proliferation of PASMCs and reversing the phenotypic conversion, and its acting mechanism may be related to the Wnt/β-catenin signaling pathway, suggesting the therapeutic potential of CK in controlling pulmonary arterial hypertension., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Objective To explore the effect of dexmedetomidine(Dex)on sevoflurane-induced cognitive impairment in neonatal rats through Wnt signaling pathway. Methods Sixty 7-day-old SD rats were assigned into five groups:control group(without any intervention),Dex group(intraperitoneal injection of 25 μg/kg Dex),sevoflurane group(3% sevoflurane treatment for 4 hours),sevoflurane+Dex group(inhalation of 3% sevoflurane after injection of 25 μg/kg Dex for 4 hours),and sevoflurane+Dex+Wnt inhibitor group(Wnt inhibitor XAV393 and 25 μg/kg Dex were injected and 3% sevoflurane was inhaled for 4 hours).Three weeks later,Morris water maze was used to detect the cognitive function;TdT-mediated dUTP nick end labeling(TUNEL)staining was performed to detect the apoptosis of hippocampal neurons;neuronal nuclei (NeuN) staining was conducted to detect the survival of hippocampal neurons;Western blot was carried out to detect the expression of apoptosis-related proteins.The expression of the factors involved in Wnt/GSK-3β/β-catenin signaling pathway was detected by fluorescence quantitative polymerase chain reaction,and Western blot. Results Compared with the control group,there was no significant difference in the escape latency of Dex group(t=0.304,P=0.768);the escape latency in sevoflurane group(t=5.823,P=0.002),sevoflurane+Dex group(t=3.188,P=0.010),and sevoflurane+Dex+Wnt inhibitor group(t=5.784,P=0.002)was significantly prolonged.Compared with that in the sevoflurane group,the escape latency in sevoflurane+Dex group(t=3.646,P=0.005)was significantly shortened.Compared with that in sevoflurane+Dex group,the escape latency in sevoflurane+Dex+Wnt inhibitor group(t=3.296,P=0.008)was prolonged.Compared with that in the control group,the times of crossing platform in sevoflurane group(t=5.179, P=0.004),sevoflurane+Dex group(t=2.309,P=0.043),and sevoflurane+Dex+Wnt inhibitor group(t=3.871, P=0.003)decreased.Compared with that in sevoflurane group,the times of crossing platform in sevoflurane+Dex group(t=3.296,P=0.008)significantly increased.Compared with that in sevoflurane+Dex group,the times of crossing platform in sevoflurane+Dex+Wnt inhibitor group(t=2.361, P=0.041)reduced.Compared with the control group,there was no significant difference in the number of apoptotic cells in Dex group(t=1.920,P=0.127),and the number of apoptotic cells in sevoflurane group,sevoflurane+Dex group,and sevoflurane+Dex+Wnt inhibitor group increased by 16%(t=13.436,P=0.002),5%(t=7.752, P=0.001),and 11.5%(t=12.612,P=0.002),respectively.Compared with that in the sevoflurane group,the number of apoptotic cells in sevoflurane+Dex group and sevoflurane+Dex+Wnt inhibitor group decreased by 11%(t=8.521,P=0.002)and 5.5%(t=3.123,P=0.036),respectively.Compared with that in the sevoflurane+Dex group,the number of apoptotic cells in sevoflurane+Dex+Wnt inhibitor group increased by 6.5%(t=6.250,P=0.003).Compared with that in the control group,the number of positive cells in 0.15 mm 2 area did not show significant difference in Dex group(t=0.898,P=0.136)and sevoflurane+Dex group(t=0.203,P=1.519),and that in sevoflurane group and sevoflurane+Dex+Wnt inhibitor group decreased by 31(t=4.702,P=0.009)and 26(t=3.948,P=0.014),respectively.Compared with that in sevoflurane group,the number of positive cells in sevoflurane+Dex group and sevoflurane+Dex+Wnt inhibitor group increased by 17(t=3.415,P=0.018)and 5(P=0.001),respectively.Compared with that in sevoflurane+Dex group,the number of positive cells in sevoflurane+Dex+Wnt inhibitor group decreased by 12(t=3.010,P=0.039).Western blot was used to detect the gray values of caspase-3,Bax,and Bcl-2 in the hippocampus,which showed no significant difference between Dex group and control group(t=0.612,P=0.573; t=1.225,P=0.288;t=0.961,P=0.391).Compared with that in the control group,the expression of Bax(t=13.440,P=0.002;t=8.520,P=0.001; t=13.320,P=0.002)and caspase-3(t=9.860,P=0.001;t=6.120,P=0.004;t=11.620,P=0.003)were up-regulated and that of Bcl-2(t=7.671,P=0.002;t=2.880,P=0.045;t=6.280,P=0.003)was down-regulated in sevoflurane group,sevoflurane+DEX group,and sevoflurane+Dex+Wnt inhibitor group.Compared with that in sevoflurane group,the expression of Bax(t=8.130,P=0.001)and caspase-3(t=7.120,P=0.002)was down-regulated and that of Bcl-2(t=6.201,P=0.003)was up-regulated in sevoflurane+Dex group.Compared with that in sevoflurane+Dex group,the expression of Bax(t=7.310,P=0.002)and caspase-3(t=7.750,P=0.002)were up-regulated and that of Bcl-2(t=4.206,P=0.013)was down-regulated in sevoflurane+Dex+Wnt inhibitor group.The mRNA expression levels of Wnt3a,GSK-3β,and β-catenin in hippocampus were detected by fluorescence quantitative polymerase chain reaction.The mRNA expression levels of Wnt3a,GSK-3β,and β-catenin in the control group did not differ significantly from those in Dex group(t=1.230,P=0.290;t=0.901,P=0.418;t=1.837,P=0.140);the mRNA expression levels of Wnt3a and β-catenin in the control group did not differ significantly from those in sevoflurane+Dex group(t=1.102,P=0.332;t=1.030,P=0.361).Compared with that in the control group,the protein expression of Wnt3a and β-catenin in sevoflurane group(t=5.790,P=0.004;t=7.130,P=0.002)and sevoflurane+Dex+Wnt inhibitor group(t=7.130,P=0.002; t=5.500,P=0.005)was down-regulated,and the GSK-3β expression was up-regulated in sevoflurane group(t=4.800,P=0.009),sevoflurane+Dex group(t=2.940,P=0.045),and sevoflurane+Dex+Wnt inhibitor group(t=3.100,P=0.042).Compared with that in sevoflurane group,the mRNA expression of Wnt3a(t=4.460,P=0.011)and β-catenin(t=6.390,P=0.003)was up-regulated while that of GSK-3β(t=4.160,P=0.004)was down-regulated in sevoflurane+Dex group.Compared with that in sevoflurane+Dex group,the mRNA expression of Wnt3a(t=5.730,P=0.005)and β-catenin(t=4.640,P=0.010)was down-regulated while that of GSK-3β(t=3.240,P=0.117)was up-regulated in sevoflurane+Dex+Wnt inhibitor group.Compared with that in the control group,the protein expression of Wnt3a and β-catenin in Dex group(t=0.735,P=0.503;t=0.245,P=0.819)and sevoflurane+Dex group(t=1.623,P=0.180;t=1.159,P=0.311)did not differ significantly,while that in sevoflurane group(t=7.280,P=0.002; t=5.640,P=0.005)and sevoflurane+Dex+Wnt inhibitor group(t=7.240,P=0.002;t=4.970,P=0.008)was down-regulated. Compared with that in sevoflurane group,the protein levels of Wnt3a(t=6.410,P=0.003)and β-catenin(t=4.640,P=0.015)were up-regulated in sevoflurane+Dex group.Compared with that in sevoflurane+Dex group,the protein expression of Wnt3a(t=6.360,P=0.003)and β-catenin(t=4.640,P=0.016)was down-regulated in sevoflurane+Dex+Wnt inhibitor group.Compared with that in the control group,the expression(gray value)of P(ser9)-GSK-3β/GSK-3β in sevoflurane group(t=11.280,P=0.002),sevoflurane+Dex group(t=7.080,P=0.002),and sevoflurane+Dex+Wnt inhibitor group(t=9.970,P=0.001)were down-regulated.Compared with that in the sevoflurane group,the expression of P(ser9)-GSK-3β/GSK-3β were up-regulated in sevoflurane+Dex group(t=8.310,P <0.001).Compared with that in sevoflurane+Dex group,the expression of P(ser9)-GSK-3β/GSK-3β in sevoflurane+Dex+Wnt inhibitor group was down-regulated(t=5.510,P=0.005). Conclusion Dex can mediate Wnt/GSK-3β/β-catenin signaling pathway to inhibit sevoflurane-induced cognitive impairment in neonatal rats.

The apoptosis of nucleus pulposus cells (NPCs) is the main cellular process of intervertebral disc degeneration (IVDD). Our previous studies showed that 17β-estradiol (E 2 ) protects rat NPCs from interleukin-1β (IL-1β)-induced apoptosis via the PI3K/Akt signaling pathway. This study was aimed to investigate whether downstream proteins of PI3K/Akt pathway were involved in inhibition of E 2 on NPCs' apoptosis. Primary culture of rat NPCs was isolated by trypsin digestion. Being pretreated with E 2 and different inhibitors of downstream proteins of PI3K/Akt pathway, the NPCs were treated with IL-1β. Cellular apoptosis was detected by Annexin V/PI staining. Cell viability was detected by CCK-8. Cell adhesion was evaluated by cell-collagen binding assay. Phosphorylation levels of mammalian target of Rapamycin (mTOR), glycogen synthase kinase-3β (GSK-3β) and nuclear factor κB (NF-κB) were detected by Western blot. The results showed that E 2 significantly inhibited the IL-1β-induced apoptosis of NPCs, reversed the decrease of cell viability and adhesion induced by IL-1β, and inhibited the down-regulation of mTOR phosphorylation level induced by IL-1β. Rapamycin could block these protective effects of E 2 . These results suggest that E 2 may inhibit IL-1β-induced NPCs' apoptosis through the PI3K/Akt/mTOR signaling pathway.

Objective: To investigate the antileukemia activity of phosphatidylinositol-3 kinase (PI3K) inhibitor ZSTK474 on human leukemia cell line U937., Methods: MTT, soft agar assay, flow cytometric analysis and western blot were used to detect the effect of ZSTK474 on U937 cell proliferation, tumorigenicity, cell cycle, cell apoptosis and phosphorylation levels of the key factor of PI3K/AKT pathway. Chou-Talalay method was used to evaluate the combination of ZSTK474 with Cytarabine or Homoharringtonine., Results: PI3K inhibitor ZSTK474 could inhibit the proliferation and tumorigenicity of U937 cell, induce G 1 cell cycle arrest and promote cell apoptosis, and enhance intracellular ROS production and decrease MMP, downregulate Cyclin D1, p-Rb, BCL-2 and upregulate p27, caspase-9, caspase-3, PARP and BAX. Furthermore, the phosphorylation of PDK1, GSK-3β, AKT and mTOR could be downregulated by ZSTK474. The combination of ZSTK474 with Homoharringtonine was synergistic., Conclusion: ZSTK474 can inhibit the pathway of PI3K/AKT, ZSTK474 alone or in combination with Homoharringtonine shows potential antileukemia activity on U937 cells.

Gastrodiae Rhizoma-Uncariae Ramulus cum Uncis is the most frequently used herbal pair in the treatment of Parkinson's disease(PD). Gastrodin and isorhynchophylline are important components of Gastrodiae Rhizoma-Uncariae Ramulus cum Uncis herb pair with anti-Parkinson mechanism. This study aimed to investigate the effect of gastrodin combined with isorhynchophylline on 1-methyl-4-phenylpyridinium(MPP~+)-induced apoptosis of PC12 cells and their antioxidant mechanism. The leakage of lactate dehydrogenase(LDH) from cells to media was analyzed by spectrophotometry. Apoptotic cells were labeled with Annexin V-fluorescein isothiocyanate(FITC) and propidium iodide(PI) and analyzed by flow cytometry. The cell cycle was analyzed using propidium iodide(PI) staining. Lipid peroxidation(LPO) level was analyzed by spectrophotometry. The mRNA expression of caspase-3 was examined by Real-time RT-PCR. The protein expressions of heme oxygenase 1(HO-1) and NADPH: quinoneoxidore-ductase 1(NQO-1) were determined by Western blot. Gastrodin combined with isorhynchophylline reduced the percentage of Annexin V-positive cells and cell cycle arrest in MPP~+-induced PC12 cells. Gastrodin combined with isorhynchophylline down-regulated the mRNA expression of caspase-3, up-regulated the protein expressions of HO-1 and NQO-1, and reduced LPO content in MPP~+-induced PC12 cells. PD98059, LY294002 or LiCl could partially reverse these changes pretreated with gastrodin combined with isorhynchophylline, suggesting that gastrodin combined with isorhynchophylline inhibited MPP~+-induced apoptosis of PC12 cells and oxidative stress through ERK1/2 and PI3 K/GSK-3β signal pathways. Our experiments showed that gastrodin combined with isorhynchophylline could down-re-gulate the mRNA expression of caspase-3 and up-regulate the protein expressions of HO-1 and NQO-1, so as to reduce oxidative stress and inhibit apoptosis.

Objective: To investigate the effect and possible mechanism of up-regulation of p-Akt by doxycycline (DOX) on myeloma cell line H929., Methods: Multiple myeloma cell line H929 was treated with DOX at different concentrations for different times, and cell proliferation rate was measured by CCK-8 assay. The protein expression level of p-Akt, PTEN, p-PDK1, p-mTOR, p-GSK-3β, and p-BAD was analyzed by Western blot. The mRNA levels of mTOR, BCL-2, and NF-κB was analyzed by RT-PCR. PI3K inhibitor Wortmannin was used to antagonize the up-regulation of p-Akt, and the cell proliferation and p-Akt protein expression level were analyzed by CCK-8 assay and Western blot respectively., Results: DOX could inhibit the proliferation of H929 cells and up-regulate the expression of p-Akt at the same time. The protein levels of both p-PDK1 and PTEN in H929 cells did not alter significantly during DOX treatment. The expressions of p-BAD and p-GSK-3β were up-regulated in H929 cells after treated with DOX, but the expression of p-mTOR was not altered. The mRNA levels of mTOR, BCL-2, and NF-κB in H929 were all down-regulated in H929 cells during DOX treatment. The effect up-regulating p-Akt level by DOX was suppressed when DOX combined with PI3K inhibitor Wortmannin and Wortmannin could enhance the inhibitory effect of DOX in H929 cells., Conclusion: DOX can activate PI3K/Akt signaling pathway in H929 cells, and antagonizing this effect of DOX may enhance its cytotoxicity to myeloma cells.

Objective: To study the effect and related signaling pathways of ginsenoside Rb1 in the treatment of coronary artery lesion (CAL) in a mouse model of Kawasaki disease (KD)., Methods: BALB/c mice were randomly divided into a control group, a model group, an aspirin group, a low-dose ginsenoside Rb1 group (50 mg/kg), and a high-dose ginsenoside Rb1 group (100 mg/kg), with 12 mice in each group. All mice except those in the control group were given intermittent intraperitoneal injection of 10% bovine serum albumin to establish a mouse model of KD. The mice in the aspirin group, the low-dose ginsenoside Rb1 group, and the high-dose ginsenoside Rb1 group were given the corresponding drug by gavage for 20 days after modeling. Hematoxylin and eosin staining was used to observe the pathological changes of coronary artery tissue. ELISA was used to measure the levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in serum and coronary artery tissue. Western blot was used to measure the relative expression levels of proteins involved in the regulation of the AMPK/mTOR autophagy signaling pathway and the PI3K/Akt oxidative stress signaling pathway in coronary artery tissue., Results: The observation of pathological sections showed that compared with the model group, the high-dose ginsenoside Rb1 group had significant improvement in the symptoms of vascular wall thickening, intimal edema, fiber rupture, and inflammatory infiltration of endothelial cells. Compared with the control group, the model and low-dose ginsenoside Rb1 groups had significant increases in the levels of TNF-α, IL-6, and IL-1β in serum and coronary artery tissue (P<0.05); the model group had significant increases in the expression levels of P-AMPK/AMPK, P-mTOR/mTOR, and P-P70S6/P70S6 in coronary artery tissue (P<0.05) and significant reductions in the expression levels of P-PI3K/PI3K, P-AKT/AKT, and P-GSK-3β/GSK-3β in coronary artery tissue (P<0.05). Compared with the model group, the aspirin group and the high-dose ginsenoside Rb1 group had significant reductions in the levels of TNF-α, IL-6, and IL-1β (P<0.05); the low- and high-dose ginsenoside Rb1 groups had significant reductions in the expression levels of P-AMPK/AMPK, P-mTOR/mTOR, and P-P70S6/P70S6 (P<0.05) in a dose-dependent manner between the two groups (P<0.05); the low-dose ginsenoside Rb1 group had no significant change in the expression level of P-PI3K/PI3K (P>0.05) and had significant increases in the expression levels of P-AKT/AKT and P-GSK-3β/GSK-3β (P<0.05), while the high-dose ginsenoside Rb1 group had significant increases in the relative protein expression levels of the above three proteins (P<0.05). Compared with the low-dose ginsenoside Rb1 group, the aspirin group and the high-dose ginsenoside Rb1 group had significant reductions in the levels of TNF-α, IL-6, and IL-1β (P<0.05); the high-dose ginsenoside Rb1 group had significant increases in the expression levels of P-PI3K/PI3K and P-AKT/AKT (P<0.05)., Conclusions: Ginsenoside Rb1 can effectively alleviate CAL in a mouse model of KD in a dose-dependent manner, possibly by regulating the AMPK/mTOR/P70S6 autophagy signaling pathway to inhibit CAL inflammation and regulating the PI3K/AKT/GSK-3β oxidative stress signaling pathway to exert a biological activity of protection against coronary artery endothelial cell injury.

Objective: To study the effects of Youguiwan on the osteoglycin (OGN), osteonectin (ON), fibrinogen 2 (FBN2) of articular cartilage tissue in the model of knee osteoarthritis (KOA). Methods: Sixty SD rats were randomly divided into six groups: sham control group, model group, glucosamine sulfate group, Youguiwan (high-dose, middle-dose and low-dose )group. The modified Hulth method was used to establish KOA models for 6 weeks. The sham control group and the model group were treated with normal saline. The rats in Youguiwan high-dose, middle-dose, low-dose groups were treated with Youguiwan at the doses of 4.8, 2.4, 1.2 g/kg by gavage respectively, and the glucosamine sulfate group was treated with glucosamine sulfate 0.17 g/kg. The rats were administrated for 8 weeks according to the dose. After intervening, articular cartilage of rats were obtained, the pathological changes were observed by using HE staining method, and Mankin score was evaluated. The expressions of OGN, ON and FBN2 in articular cartilage were detected by immunohistochemistry. The expression of GSK-3β in articular cartilage was detected by Western blot. Results: Compared with the sham control group, the Mankin score was obviously increased in the model group, the protein expression of FBN2 was increased significantly, yet the protein expressions of OGN, ON and GSK-3β were decreased significantly ( P ＜0.01), articular cartilage was seriously damaged, and chondrocytes were arranged in disorder. Compared with the model group, the Mankin score was declined obviously in the high-dose Youguiwan group, the protein expression of FBN2 was significantly decreased, but the protein expression of GSK-3β was significantly increased, the protein expressions of OGN and ON were significantly increased in the middle-dose and high-dose Youguiwan group ( P ＜0.05 or P ＜0.01), cartilage structure was tended to be normal, the chondrocytes distribution was uneven, and articular cartilage surface was not smooth. Conclusion: Youguiwan can significantly improve the articular cartilage degeneration of KOA rats, its mechanism maybe raise OGN and ON protein expression level to promote the ossification and reconstruction of articular cartilage.

Objective: To investigate the effect of miR-148b-3p on invasion and migration of glioma cells and the possible molecular mechanism. Methods: Human glioma U251 cells were cultures in vitro. MiR-148b-3p mimic or negative control was transfected into U251 cells by Lipofectamine 2000 transfection reagent, which were recorded as miR-148b-3p group and NC group, respectively. A blank control (Ctr group) was set. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the transfection effect. Transwell assay was used to detect the invasive ability of U251 cells in each group. The scratch test was used to detect the migration ability of U251 cells in each group. The concentrations of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in the supernatant of the cells were determined by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the expressions of Wnt signaling pathway-related proteins in cells. Results: The qRT-PCR experiment showed that the expression level of miR-148b-3p in U251 cells of miR-148b-3p group (2.45±0.25) was significantly higher than that of NC group (0.97±0.10) and Ctr group (1.00±0.11) ( P <0.05). Transwell and scratch experiments showed that the number of invasive cells (50.62±5.36) in miR-148b-3p group was significantly lower than those in NC group (108.84±10.14) and Ctr group (113.40±10.06) ( P <0.05). The results of the scratch experiment showed that the cell migration rate of miR-148b-3p group (23.19±2.50)% was significantly lower than those of NC group (51.81±5.25)% and Ctr group (52.06±5.33)% ( P <0.05). Overexpression of miR-148b-3p inhibited the expressions of MMP-2 and MMP-9, downregulated the expressions of Wnt1 and GSK-3β protein. Conclusion: miR-148b-3p can inhibit the invasion and migration of human glioma U251 cells by inhibiting the activation of Wnt signaling pathway.

Objective: To investigate the effects of PM&#95;(2. 5) exposure on the development of synaptic plasticity and Wnt/β-catenin pathway in hippocampus of offspring rats., Methods: Healthy 7-week-old SPF SD rats(n=36) mated with a male to female ratio of 2∶1. Pregnant rats were randomly divided into three groups, including control group, low PM&#95;(2. 5) group, and high PM&#95;(2. 5) group, with eight rats in each group. The low and high PM&#95;(2. 5) concentrations in dynamic exposure cabinet were approximately two times and four times higher than the annual average PM&#95;(2. 5) concentration in Tangshan city respectively. The exposure started from pregnant day 0, until postnatal day 21(PND21) of offspring rats. After weaning, the offspring rats continued to be exposed to PM&#95;(2. 5) until PND42. PND21 and PND42 pups were subjected to Morris water maze and new object recognition experiments. Western blot was used to detect post synaptic density-95(PSD-95), synaptophysin(SYN), growth associated protein(GAP-43), glycogen synthase kinase 3β(GSK-3β), β-catenin protein levels and phosphorylation levels of GSK-3β and β-catenin in the hippocampus of offspring rats., Results: Compared with the control group, the learning and memory abilities of the pups of each PM&#95;(2. 5) group were significantly decreased with a dose dependent manner. Compared with the control group, the protein level of SYN, GAP-43 and PSD-95 in hippocampus of PND0 rats of each PM&#95;(2. 5)groups were decreased(P&lt;0. 05), and the protein level of SYN of each PM&#95;(2. 5)group and PSD-95 of high PM&#95;(2. 5) group in PND21 and PND42 were decreased(P&lt;0. 05), and the level of GAP-43 of low PM&#95;(2. 5) group in PND42 were decreased(P&lt;0. 05). Compared with the low PM&#95;(2. 5) group, the level of PSD-95 of high PM&#95;(2. 5) group in PND0 and PND21, the level of PSD-95 of high PM&#95;(2. 5) group in PND0 and PND42 were decreased(P&lt;0. 05). Compared with the control group, the level of p-GSK-3β in hippocampus of each PM&#95;(2. 5)group in PND0, PND21 and PND42 was decreased(P&lt;0. 05), and with the increase of PM&#95;(2. 5) exposure dose, the trend is more obvious. The protein level of p-β-catenin in hippocampus of high PM&#95;(2. 5) group in PND0 and PND42 was significantly increased(P&lt;0. 05). The level of p-β-catenin in high-dose PND21 pups compared with the control group was significantly reduced(P&lt;0. 05)., Conclusion: Exposure to PM&#95;(2. 5) in early life can damage the synaptic plasticity and decrease the protein levels of β-catenin and p-GSK-3β in the Wnt/β-catenin pathway of hippocampus in offspring rats.

Objective: To explore the mechanism of electroacupuncture (EA) underlying improvement of cerebral infarction (CI) by investigating its influence on expression of cerebral Wnt7a, lymphoid enhancer factor-1 (LEF1), glycogen synthase kinase 3β(GSK-3β) and Dickkopf-1(DKK1) mRNA and proteins in CI rats., Methods: A total of 280 male Wistar rats were randomly divided into blank control ( n ＝10), sham-operation, model and EA groups，and 90 rats of the last 3 groups were further divided into 1, 3, 6, 9, 12 and 24 h, and 3, 7 and 12 d subgroups with 10 rats in each subgroup. The CI model was established by occlusion of the middle cerebral artery (MCAO). The sham-operation group received the same surgical operation but without thread embolus insertion. EA (2 Hz/15 Hz, 2 mA) was applied to "Shuigou" (GV26) for 20 min, once a day for 1, 3, 7 and 12 d, respectively. The neurological deficit was evaluated by using Neurological Severity Scores (NSS). The expression levels of Wnt7a，LEF1, GSK-3β and DKK1 mRNAs and proteins in the right ischemic brain tissues were detected by Quantative real-time PCR and Western blot, respectively., Results: After MCAO, the NSS score was significantly increased in the model and EA groups relevant to the blank control and sham-operation groups ( P <0.01) and gradually decreased with the prolongation of ischemia time. After EA, the NSS scores were notably decreased on day 3, 7 and 12 in the EA group compared with the model group ( P <0.05, P <0.01). After modeling, the expression levels of Wnt7a and LEF1 mRNAs from 3 h to 12 d, Wnt7a and LEF1 proteins from 6 h to 12 d were considerably increased ( P <0.01, P <0.05), while those of GSK-3β mRNA at 9, 12 and 24 h, GSK-3β protein at 24 h and 3 d, and DKK1 mRNA at 24 h and 3 d and DKK1 protein at 3 d were obviously decreased in the model group relevant to the sham-operation group ( P <0.05, P <0.01). After the intervention, the expression levels of Wnt7a mRNA at 12 h to 3 d, Wnt7α protein from 24 h to 12 d, LEF1 mRNA from 24 h to 12 d, and LEF1 protein from 3 d to 12 d were further apparently up-regulated ( P <0.01, P <0.05), while those of GSK-3β mRNA at 9 h, 3，7 and 12 d, and GSK-3β protein at 12 h, 7 d and 12 d, and DKK1 mRNA at 12 h, 24 h and 3 d, and DKK1 protein at 24 h to 12 d were obviously down-regulated in the EA group relevant to the model group ( P <0.01, P <0.05). No significant difference was found between the blank and sham-operation groups in the NSS scores and expression levels of Wnt7a, LEF1, GSK-3β and DKK1 mRNAs and proteins at all the time points ( P >0.05)., Conclusion: EA of GV26 can significantly improve the neurological deficit symptoms in MCAO rats, which may be associated with its effects in up-regulating the expression of Wnt7a and LEF1 mRNAs and proteins, and in down-regulating the expression of GSK-3β and DKK1 mRNAs and proteins.

Objective: To investigate the differential expression of serum-and-glucocorticoid-inducible-kinase-2 (SGK2) in hepatocellular carcinoma (HCC) and normal liver tissues and the related mechanism mediating signal transduction of GSK-3 β / β catenin in HCC cells. Methods: Twenty pairs of matched HCC and normal tissues were collected and the situation of expression of SGK2 mRNA was detected by real-time fluorescence quantitative PCR. Western blot was used to detect the levels of SGK2 protein in human HCC cell lines (Huh-7, SMMC-7721) and normal human liver cell line (L02). SGK2 siRNA was used to transfect human HCC cell lines (SMMC-7721 and Huh-7), and then the protein expression levels of GSK-3 β/ β - catenin was successfully detected with the above-mentioned transfected cell line by western blot. Measurement data were expressed as mean ± standard deviation ( x ±s), and the Student t -test was used as the statistical method. Results: SGK2 mRNA expression was up-regulated in all 20 HCC samples than that of the expression of matched normal liver tissues. SGK2 protein levels were significantly higher in Huh-7 and SMMC-7721 than normal human liver cell lines ( P < 0.01). The downregulation of SGK2 expression in human HCC cell lines (SMMC-7721 and Huh-7) had inhibited the expression of unphosphorylated GSK-3 β. In addition, the downregulation of SGK2 expression in HCC cell lines had decreased the dephosphorylation of β - catenin to prevent degradation of the β - catenin proteasome. Conclusion: SGK2 is overexpressed in HCC and mediates GSK-3β/β- catenin signaling in HCC cells.

Objective: To observe the effects of diammonium glycyrrhizinate (DG) on nerve regeneration repair in rats with severe traumatic brain injury (STBI) from the perspective of Wnt/β-catenin signaling pathway., Methods: Seventy-two Sprague-Dawle (SD) male rats were randomly divided into normal group, STBI model group, ganglioside (GA) treatment group and DG treatment group. The STBI animal model was reproduced referring to modified Feeney free fall impact model. No injury was made in normal group. Six hours after modeling, monosialotetrahexosylganglioside sodium injection and DG injection were injected via tail vein of rats in GA treatment group and DG treatment group respectively, once a day for 7 days. Normal group and STBI model group were given the same amount of normal saline. Six rats in each group were sacrificed on the 1st, 3rd and 7th day after the challenge for neurological severity score (NSS), and then the blood of abdominal aorta was drawn and brain tissue was harvested. The contents of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in serum were detected by enzyme linked immunosorbent assay (ELISA). The pathological changes of sub-granular zone (SGZ) were observed under light microscope after hematoxylin eosin (HE) staining. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of Wnt3a, β-catenin, glycogen synthetase kinase-3β (GSK-3β) and Axin., Results: (1) There was no neurological deficit in the normal group and NSS was 0. NSS score of rats increased significantly on the first day after modeling, and then decreased gradually over time. NSS of the rats treated with GA and DG were significantly lower than that of the STBI model rats (score: 7.33±2.07, 6.17±2.23 vs. 9.33±1.63, both P < 0.01). Though NSS gradually decreased over time, the differences were still statistically significant on the 7th day (score: 2.67±0.82, 1.00±0.00 vs. 6.17±2.23, both P < 0.01), and NSS of DG treatment group was significantly lower than that of GA treatment group. (2) In SGZ of rats, cells were arranged in a compact and orderly way in the normal group, but neurons and tissues were damaged and destroyed at different time points in the STBI model group. After either GA or DG treatment, the damage of nerve tissue was improved gradually over time, and the effect of DG was more obvious. (3) In the normal group, the mRNA expressions of Wnt3a and β-catenin were almost not expressed, the mRNA expressions of GSK-3β and Axin were higher, and the contents of BDNF and NGF in serum were less. On the 1st day after STBI, the mRNA expressions of Wnt3a and β-catenin in hippocampus, the contents of BDNF and NGF in serum were significantly increased, and the mRNA expressions of GSK-3β and Axin were significantly decreased. The mRNA expressions of Wnt3a and β-catenin in the hippocampus and the contents of BDNF and NGF in serum were significantly higher than those in the model group 1 day after GA or DG was added, the mRNA expressions of GSK-3β and Axin were significantly decreased, and the effect of DG was more significant than that of GA [Wnt3a mRNA (2 -ΔΔCt ): 3.51±0.14 vs. 2.93±0.05, β-catenin mRNA (2 -ΔΔCt ): 1.90±0.08 vs. 1.75±0.04, BDNF (ng/L): 4.06±0.55 vs. 3.16±0.64, NGF (ng/L): 9.53±1.08 vs. 7.26±0.43, GSK-3β mRNA (2 -ΔΔCt ): 0.75±0.01 vs. 0.79±0.01, Axin mRNA (2 -ΔΔCt ): 0.74±0.02 vs. 0.76±0.02, all P < 0.05]. It was gradually increasing or decreasing over time and the difference was still statistically significant up to the 7th day., Conclusions: DG can promote the recovery of nerve function in rats with STBI, and its mechanism may be related to the regeneration of nerve cells proliferation and differentiation by Wnt/β-catenin signaling pathway and the reconstruction of nerve tissue in SGZ of hippocampus.

Objective: To investigate the role of the protein-serine-threonine kinase (AKT)/glycogen synthase kinase 3β (GSK3β) signaling pathway in nicotinic acetylcholine receptors (nAChRs) aggregation disorder on skeletal muscle cell membranes induced by sepsis., Methods: Mouse C2C12 myoblasts were differentiated into myotubes by horse serum, and then C2C12 myotubes were randomly divided into four groups: the Sham group treated with serum from sham-operated mice, the Sepsis group treated with serum from septic mice, the Sepsis+D group treated with serum from septic mice and dimethyl sulfoxide (DMSO), the Sepsis+SB group treated with serum from septic mice and GSK3β inhibitor SB216763. Agrin was added into the cell culture to induce nAChRs aggregation before the treatment. After serum treatment for 5.5 h, the myotubes were examined for nAChRs clusters using Alexa Fluor 594-conjugated α-bungarotoxin (α- BTX). The expression levels of AKT, GSK3β and CLIP- associated protein 2 (CLASP2) and the phosphorylation of AKT, GSK3β were examined with Western blotting. The phosphorylation of CLASP2 and the interaction between CLASP2 and α-tubulin were detected with co-immunoprecipitation (Co-IP) assay., Results: Compared with the serum from sham-operated mice, the serum from septic mice caused significant reduction in the area and density of nAChRs clusters on C2C12 myotubes, lowered the levels of phosphorylated AKT (p-AKT) and phosphorylated GSK3β (p-GSK3β), increased the expression of phosphorylated CLASP2 (p-CLASP2), and obviously reduced the binding between CLASP2 and α-tubulin. Compared with DMSO, SB216763 significantly increased the area and density of nAChRs clusters on C2C12 myotubes treated with serum from septic mice, decreased the expression of p-CLASP2, and enhanced the interaction between CLASP2 and α-tubulin., Conclusions: Septic mouse serum impairs nAChRs aggregation on C2C12 myotubes possibly by suppressing AKT/GSK3β phosphorylation to cause reduced interaction between CLASP2 and α-tubulin.

Objective: To study the effect of glycogen synthase kinase 3β(GSK3β) in BPA-induced adipose tissue inflammation in high fat diet(HFD) fed mice., Methods: Four-week-old male C57 BL/6 mice were randomly divided into normal diet(ND) group, HFD group, HFD + GSK3β inhibitor group, HFD + 1000 nmol/L BPA group, HFD+1000 nmol/L BPA+GSK3β inhibitor group. The mice were exposed to BPA via drinking water. From the 14 th week of BPA exposure to the end of 16 weeks, the GSK3β inhibitor group was intraperitoneally injected with 21. 5 mg/kg lithium chloride(Li Cl) every two days for a total of 10 times. At the end of 16 weeks, the mice were sacrificed after anesthesia, and the epididymal fat tissue was taken aseptically. The pathological changes were observed by H&E staining. The expressions of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by immunohistochemistry(IHC). The expression of GSK3β protein and its S9 serine(GSK3β-S9) phosphorylation were detected by western blot., Results: Compared with the ND group, the body weight [(34. 97±1. 91) g]and epididymal fat pad coefficient [(3. 25±0. 39) %]of HFD group was significantly up-regulated(P < 0. 05), the adipose tissue inflammatory cell infiltration was increased, the expression of TNF-α(F = 73. 157, P < 0. 05) and IL-1β(F = 42. 788, P < 0. 05) was significantly enhanced, and the phosphorylation degree of GSK3β-S9(F = 57. 991, P < 0. 05) was decreased. The inflammatory cell infiltration of adipose tissue in the HFD+1000 nmol/L BPA group was significantly increased, the body weight [(38. 49±1. 34) g]and epididymal fat pad coefficient [(4. 41±0. 33) %] of the mice were significantly increased, the phosphorylation of GSK3β-S9(F = 57. 991, P <0. 05) was significantly down-regulated, and the expression of TNF-α(F = 73. 157, P <0. 05) and IL-1β(F = 42. 788, P <0. 05) was significantly enhanced compared with that in the HFD group. Compared with the HFD + 1000 nmol/L BPA group, the HFD + 1000 nmol/L BPA+GSK3 inhibitor group was decreased inflammatory cell infiltration in adipose tissue, significantly decreased body weight [(32. 61 ± 3. 34) g] and epididymal fat pad coefficient [(3. 33±0. 66) %], significantly increased GSK3-S9(F = 57. 991, P < 0. 05)phosphorylation, and significantly decreased TNF-α(F = 73. 157, P < 0. 05) and IL-1β(F = 42. 788, P<0. 05) expression., Conclusion: GSK3β inhibitor can down-regulate BPA-induced adipose tissue inflammation, inflammatory cytokine expression and upregulate GSK3β-S9 phosphorylation in HFD-fed mice, suggesting that BPA exposure may regulate the expression of inflammatory cytokines mediating adipose tissue inflammation by affecting the degree of phosphorylation of GSK3β-S9.

Objective: To investigate the effect of Quercetin (Qu) on cell proliferation, apoptosis and cell cycle, as well as the expression changes of Wnt/β-catenin signaling pathway, apoptosis and cell cycle regulators and BCR-ABL in CML susceptible cells K562 and imatinib-resistant cells (IM) K562R., Methods: The trypan blue staining was used to detect the all proliferation. The cell cycle and apoptosis were detected by flow cytometry. The fluorescence quantitative PCR and Western blot were used to detect the expression of mRNA and protein respectively., Results: After administration with 5, 10, 20, 40, 80, 160, 320 μmol/L Qu, the inhibition ratio in K562 cells was 5.07%, 5.98%, 11.09%, 31.88%, 56.89%, 70.44%, 86.63%; and that in K562R cells were 4.99%, 9.75%, 10.54%, 8.93%, 25.13%, 46.89%, 68.60%; IC 50 of K562 and K562R was 76.4 μmol/L and 230.2 μmol/L, respectively. Flow cytometry showed that Qu (50, 100 and 200 μmol/L) could induce cell apoptosis and cell cycle arrest in a dose-dependent manner (r=0.9914, r=0.9871 respectively). After treatment with Qu (100 μmol/L)，the expressions of mRNA (P＜0.05) and protein(except Caspase-9) expression of Caspase-3, 8 and 9, p21 and p27 increased in K562 cells as compared with control, but the protein expression of p27 and Caspase-3 not changed in K562R. Qu (100 μmol/L) could decrease the mRNA(P＜0.05) and protein levels of Wnt/β-catenin signaling pathway members GSK-3β, β-catenin, Lef-1 and the downstream targets PPAR-δ and Cyclin D1 compared with control. The PCR results showed that Qu could reduce the BCR-ABL mRNA expression in CML cells, but the protein expression of BCR-ABL and p-BCR-ABL not obviouly changed., Conclusion: Qu can inhibit the proliferation K562 and K562R cells, and decrease the drug resistance and increase the sensitivity, that relate with inhibiting Wnt/β-catenin signaling pathway, activating apoptosis pathway and cyclins.

Objective: To explore the role of Long noncoding RNA UFC1 (lincRNA-UFC1) in modulating the metastasis and invasion of hepatocellular carcinoma (HCC) cells and the underlying mechanism., Methods: Human HCC cell line Huh7 was infected with the lentiviral vector carrying lincRNA-UFC1 to obtain a cell line with lincRNA-UFC1 overexpression. A short hairpin RNA (shRNA) targeting lincRNA-UFC1 was delivered in human HCC BEL-7402 cells via a lentiviral vector to obtain a cell line with lincRNA-UFC1 knockdown. Expression levels of lincRNA-UFC1 in the two HCC cell lines were detected using real-time PCR, and the changes in the cell invasion and migration in response to lincRNA-UFC1 overexpression or knockdown were analyzed using Transwell and wound-healing assays. The expressions of GSK-3β/β-catenin-related proteins in the cells were detected with Western blotting. XAV-939, a GSK-3β/β-catenin inhibitor, was used for assessing the impact of lincRNAUFC1 overexpression on the invasion and migration of the HCC cells through Transwell and wound-healing assays., Results: Overexpression of lincRNA-UFC1 significantly promoted the invasion and migration of Huh7 cells as compared with the control cells ( P &lt; 0.001), while lincRNA-UFC1 knockdown obviously suppressed the invasion and migration of BEL-7402 cells ( P &lt; 0.001). The results of Western blotting showed that the expressions of proteins associated with the cell invasion and migration, namely β-catenin and P-GSK-3β, were significantly upregulated in response to lincRNA-UFC1 overexpression, and were obviously lowered after lincRNA-UFC1 knockdown. Treatment of the cells with XAV-939 significantly reversed the effect of lincRNA-UFC1 overexpression on the cell invasion and migration ( P &lt; 0.001)., Conclusions: lincRNA-UFC1 overexpresison promotes cell invasion and migration through the GSK-3β/β-catenin axis in HCC cells in vitro .

Objective: To investigate the effects of gamma aminobutyric acid(GABA) fortified rice diet intervention on oxidative stress and pancreatic injury in type 2 diabetes mellitus(T2 DM) mice., Methods: Of the 70 male ICR mice, 10 were randomly selected as blank control group and they were always fed with the normal white rice feed. The remaining 60 mice were fed with high-fat white rice for 9 weeks. They were fasted for 12 h and injected intraperitoneally with streptozocin(STZ) at a dose of 50 mg/kg body weigh for two consecutive days. The control group was injected with the corresponding volume of normal saline. Subsequently, 50 T2 DM mice with successful modeling were randomly divided into 5 groups according to blood glucose, 10 in each group: T2 DM model control group, germinated brown rice positive control group(GABA content is 0. 2 g/kg feed), GABA-fortified rice low, medium and high dose group(GABA content was 0. 02, 0. 1 and 0. 2 g/kg feed respectively) and each target diet was fed for 6 weeks. Oral glucose tolerance test was performed one week before the end of the experiment to observe the hypoglycemic effect of different doses of GABA fortified rice. After the end of the experiment, HE staining was used to observe the morphology of pancreas. At the same time, the redox indicators from plasma and pancreas of reactive oxygen species(ROS), malondialdehyde(MDA), total antioxidant capacity(T-AOC), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD) were examined in each group; The mRNA expressions of oxidative stress-related genes including glycogen synthase kinase-3β(GSK-3β), nuclear transcription factor 2(Nrf2), heme oxygenase 1(HO-1) and NAD(P)H: quinone oxidoreductase1(NQO1), insulin secretion related genes including pancreatic and duodenal homeobox 1(PDX-1), mus musculus v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A(MafA), glucokinase(GCK), glucose transporter 2(GLUT2) and the apoptosis associated genes including b-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax) and caspase-3 in pancreas were assayed by real-time fluorescent quantitative PCR., Results: The intervention of GABA fortified rice could alleviate the improvement of the blood glucose level and the lack of insulin secretion in T2 DM mice and relieve plasma and pancreatic oxidative stress. besides, The intervention of GABA fortified rice could up-regulate the expression of insulin secretion-related genes PDX-1, GCK, GLUT2, inhibit the expression of pro-apoptotic gene caspase-3 and promote the expression of anti-apoptosis gene Bcl-2. There was a dose-response relationship between the above result and the 0. 2 g/kg dose group was the most significant, which achieved similar result to germinated brown rice., Conclusion: GABA-fortified rice can significantly improve the plasma and pancreatic redox status of STZ-induced T2 DM mice, regulate the expression levels of oxidative stress-related genes and apoptosis-related genes, thereby protect pancreatic tissue morphology, improve pancreatic insulin secretion and thereby alleviate glucose metabolism.

Objective: To explore the pathogenic role of changes of Wnt/β-catenin signaling pathway in the hippocampus in depression- and anxiety-like behaviors caused by prenatal stress (PS) in offspring rats., Methods: Twelve female SpragueDawley rats weighing 240-260 g were randomly divided into control and restraint stress groups. The rats in the control group received no interventions, and those in restraint stress group were subjected to restraint stress (three times a day, 45 min each time) at the gestational age of 14-20 days. The 1-month-old offspring rats underwent open field test and forced swimming test to assess the anxiety- and depression-like behaviors, and the expressions of Wnt1, Gsk-3β and β-catenin in the hippocampus were detected using Western blotting., Results: In open field test, the offspring rats with PS showed significantly decreased crossings of the center ( P &lt; 0.01) with reduced time spent in the center ( P &lt; 0.05) compared with control offspring rats. In forced swimming test, the offspring rats in PS group exhibited a significantly longer immobility time than in the control rats, and showed obvious depression- and anxiety-like behaviors. Compared with those in the control offspring rats, Gsk-3β expression increased significantly while the expressions of β-catenin and Wnt1 were significantly lowered in the hippocampus of the offspring rats in PS group ( P &lt; 0.01)., Conclusions: PS causes changes in Wnt/β-catenin signaling pathway in the hippocampus to contribute to the occurrence of depression-and anxiety-like behaviors in rats.

The aim of this study was to investigate the mechanisms of mono-functional alkylating agent MNNG to damage human gastric epithelial GES-1 cells and roles of Wnt/β-catenin signaling pathway in the process. The GES-1 cells were treated with MNNG (2 × 10 -5 mol/L) for 24 h. The morphological changes of the GES-1 cells were observed under inverted microscope 2 d after treatment. The cell viability was measured by MTT assay. The apoptosis and cell cycle distribution of the GES-1 cells were analyzed by flow cytometry. The mRNA expressions of β-catenin, GSK-3β, c-Met and MMP7 in the GES-1 cells were detected by qPCR. The protein expressions of β-catenin, GSK-3β, p-GSK-3β and c-Met were determined by Western blot. The results showed that MNNG induced the injury of GES-1 cells and changed the normal cell morphology to irregular long spindle shape. MNNG induced the apoptosis of GES-1 cells and blocked the cell cycle progression obviously. MNNG up-regulated the mRNA expressions of β-catenin, GSK-3β, c-Met and MMP7, and increased the protein expressions of β-catenin, GSK-3β and p-GSK-3β. These results suggest that the damage of GES-1 cells induced by MNNG may be related to the activation of Wnt/β-catenin signaling pathway, which will provide the basis for the study of cell model of gastric mucosal cell injury.

Objective To investigate the effects of long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on cell cycle, proliferation and apoptosis in SH-SY5Y model cells of Alzheimer's disease (AD). Methods The AD model cells (SH-SY5Y human neuroblastoma cells) were constructed using Aβ 25-35 . The AD cells were randomly divided into five groups: control group, over-expressed empty victor (pCDH-EV) group, MALAT1 over-expression (pCDH-MALAT1) group, MALAT1 expression inhibition of empty victor (pSICOR-EV) group, and MALAT1 expression inhibition (pSICOR-shMALAT1) group. The cell proliferation activity of all the groups was detected by MTT assay. The cell cycle and apoptosis rate were detected by flow cytometry after 48 hours of transfection. Western blot analysis was used to test the expressions of BAX, Bcl-2, caspase-3 and PI3K/mTOR/GSK3β proteins in the five groups. Results The AD model cells, over-expression and inhibition expression of MALAT1 vectors were successfully constructed. Inhibited expression of MALAT1 significantly inhibited the proliferation of AD SH-SY5Y cells, promoted apoptosis and arrested cell cycle in G1 phase. Western blotting showed that the inhibited expression of MALAT1 up-regulated the expressions of BAX, caspase-3 and down-regulated the expression of Bcl-2. At the same time, the inhibited expression of MALAT1 significantly inhibited the expression of p-PI3K/p-mTOR/p-GSK3β. In addition, over-expression of MALAT1 reversed the above effects. Conclusion Konck-down of MALAT1 can inhibit the proliferation and promote the apoptosis of AD model cells via down-regulating the activity of PI3K/mTOR/GSK3β.

Purpose: To investigate the expression of Kruppel-like factor 5 (KLF5) in human periodontal ligament cells (hPDLCs) under cyclic tensile stress (CTS) and its effect on proliferation and osteogenic differentiation of hPDLCs., Methods: hPDLCs were primarily cultured in vitro by tissue explant and enzyme digestion method. RT-PCR and Western blot were used to detect mRNA and protein expression of KLF5 in hPDLCs stimulated by 10% CTS at a frequency of 0.5 Hz for 1, 4, 8, 12 and 24 h, respectively．KLF5 was then knocked-down in hPDLCs using siRNA (si-KLF5) and its mRNA expression was determined by RT-PCR. Meanwhile, basic fibroblast growth factor (FGF2) was overexpressed in KLF-5 silenced hPDLCs by using pcDNA3.1-FGF2. After stimulation with CTS for 8 h, the proliferation of hPDLCs was assayed by CCK-8 kit, alkaline phosphatase (ALP) activity was assayed by ALP kit, the mRNA expression of Runx2 and Osterix was examined by RT-PCR, and the protein expression of Runx2, Osterix, FGF2, GSK-3β, P-GSK-3β (ser 9), β-catenin was determined by Western blot. The data were analyzed using SPSS 22.0 software package., Results: Stimulation with 10% CTS time-dependently induced mRNA and protein expression of KLF5 in hPDLCs. Transfection of si-KLF5 significantly reduced the proliferation of hPDLCs, decreased ALP activity and expression of Runx2 and Osterix in hPDLCs stimulated by 10% CTS. Simultaneously, CTS-induced activation of FGF2-GSK-3β/β-catenin signaling pathway was also inhibited by si-KLF5 transfection. However, overexpression of FGF2 can partly neutralized the inhibitiory effect of si-KLF5 on proliferation and osteogenic differentiation of hPDLCs subjected to 10% CTS., Conclusions: KLF5 promotes proliferation and osteogenesis differentiation of hPDLCs subjected to cyclic tensile stress through FGF2-GSK-3β/β-catenin signaling pathway.

Objective: To investigate the potential signaling pathway that regulates the proliferation of human CD34 + cells stimulated by prostaglandin E2 receptor 4 agonist (EP4A) in vitro., Methods: Twenty samples of peripheral blood containing stem cells were collected from the G-CSF mobilized healthy donors in our department of hematology. Human CD34 + cells were isolated by magnetic activated cell sorting (MACS) microbeads kit. The Cell Counting Kit-8 (CCK8) assay was used to determine the optimal concentration and time of EP4A to promote human CD34 + cell proliferation in vitro. Under the optimal condition, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect mRNA level of β-catenin, and Western blot was used to assay protein expression of β-catenin and P-GSK-3β in human CD34 + cells treated with EP4A., Results: Culturing with 10 µmol/L EP4A for 72 h, it was found that EP4A promoted human CD34 + cell proliferation significantly, and the proliferation rate of human CD34 + cells was 1.36 times higher than that of the control(P=0.002). Under the optimal condition, it was also found that EP4A enhanced the β-catenin expression at both mRNA and protein levels, and up-regulated phosphorylation of GSK-3β in human CD34 + cells, but these effects could be inhibited by the EP4A antagonist EP4AA., Conclusion: EP4A can enhance human CD34 + cell proliferation in vitro by activating Wnt/β-catenin signaling pathway.

Invasion and metastasis are key factors contributing to the high mortality rate of patients with hepatocellular carcinoma (HCC) involving a complex mechanism. In the invasion and metastasis of HCC, miRNAs can serve as either oncogenes or tumor suppressor genes to regulate the differentiation and proliferation of tumor cells being and play important roles in tumorigenesis, angiogenesis, invasion and metastasis. This review summarizes the recent progress in research of the molecular mechanisms by which miRNAs targeting GSK-3β regulate HCC invasion and metastasis and examines the roles of miRNAs in hepatocellular carcinoma cell proliferation, apoptosis, invasion, metastasis, and GSK-3β regulation.

Objective: To investigate the role of integrin-linked kinase (ILK) signaling pathway in the skin lesions and wound healing in diabetic rats., Methods: Thirty-six SD rats were divided into diabetic wound group (D) and non-diabetic wound group (N) according to the random number table, with 18 rats in each group. 10 g/L streptozocin (60 mg/kg) was intraperitoneally injected in rats in group D, while the rats in group N were given same quantity of sodium citrate buffer. Two weeks after successful reproduction of diabetic model of rats in group D, two full-thickness skin of an area of 2 cm × 2 cm was resected on both sides of back of rats in the two groups. Wounds of three rats of each group were photographed and examined on post injury day (PID) 1, 3, 7, 10, 14, and 21, and the wound healing rates were calculated. The non-injured skin and wound tissue (central part) on back of three rats of the rest 15 rats in the two groups were harvested on PID 3, 7, 10, 14, and 21, respectively. Morphology of the non-injured skin tissue was observed with HE staining, and the thickness of full-thickness skin and epidermis were measured. The mRNA expression levels of ILK, protein kinase B (Akt), and glycogen synthase kinase-3β (GSK-3β) in non-injured skin tissue were determined with real-time fluorescent quantitative RT-PCR. The protein expression levels of ILK, Akt, phosphorylated Akt, GSK-3β, and phosphorylated GSK-3β in non-injured skin tissue, and ILK, phosphorylated Akt in wound tissue were assessed with Western blotting. Data were processed with two independent-sample t test, one-way analysis of variance, SNK test and analysis of variance of factorial design., Results: (1) After injury, the wound scabs of rats in group N were dry, and red granulation tissue with no excretion were seen when the scabs fell off, and the wound healed fast. After injury, excretion under the wound scabs of rats in group D was seen, and the scabs easily fell off with exposure of pink granulation tissue with much excretion, and the wounds healed slowly. Except for PID 3, the wound healing rate of rats in group D was significantly lower than that in group N on other PIDs (with t values from 3.858 to 13.738, P<0.05 or P<0.01). (2) On PID 3, the hair follicles and blood vessels in the non-injured skin tissue of rats in group N were rich, and the epidermis was composed of stratified cells in form of basal cells and keratinocyte, and the hair follicles and blood vessels in the non-injured skin tissue of rats in group D were scarce, and the epidermis was nearly composed of one-layer of cells. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N was similar from PID 3 to 21, and the thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group D on PID 3 was respectively (1 074 ± 66) and (15.1 ± 3.8) μm, and they gradually thinned out to (785 ± 122) and (9.7 ± 2.1) μm on PID 21, respectively. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N were significantly thicker than those in group D on each PID (with t values from 4.620 to 23.549, P values below 0.001). (3) From PID 3 to 21, the mRNA expression levels of ILK and Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values respectively 4.779 and 3.440, P values below 0.05), the mRNA expression levels of GSK-3β in non-injured skin tissue of rats were similar in two groups (t=0.363, P>0.05). (4) From PID 3 to 21, the protein expression levels of ILK, Akt and phosphorylated Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values from 2.630 to 6.209, P<0.05 or P<0.01); the protein expression levels of GSK-3β in non-injured skin tissue of rats in two groups were similar (t=0.652, P>0.05); the protein expression level of phosphorylated GSK-3β in non-injured skin tissue of rats in group D was significantly higher than that in group N (t=4.131, P<0.001). The protein expression levels of ILK in wound tissue of rats in two groups were similar on each PID (with t values from 0.381 to 2.440, P values above 0.05). Except for PID 3, the protein expression levels of phosphorylated Akt in wound tissue of rats in group N were significantly higher than that in group D on other PIDs (with t values from 4.091 to 20.555, P<0.05 or P<0.01). From PID 3 to 21, the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group N were similar (F=2.522, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=117.329, P<0.001); the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group D were similar (F=1.337, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=184.120, P<0.001)., Conclusions: The skin lesion of diabetic rats may be related to the declined expression levels of ILK, Akt and phosphorylated Akt in the ILK signaling pathway. The refractory healing of wound in diabetic rats may be related to the declined expression level of phosphorylated Akt.

Objective: To explore whether nuclear translocation of β-catenin affects functional expression of sodium iodide symporter (NIS) in human thyroid cancer cells., Methods: Two kinds of β-catenin nuclear translocation cell models were firstly established with transfection of hypoxia inducible factor 1α (HIF-1α) and β-catenin cDNA in human thyroid cancer cell line FTC133. Then the expression of β-catenin were further interfered by shRNA in above two cell models. After that, the influence of β-catenin nuclear localization on the functional expression of NIS, iodine uptake potency, epithelial-mesenchymal transition (EMT) characteristics, tumor growth curve and treatment effect inducing by radioactive iodine were comparatively analyzed in vitro and in vivo trials. Additionally, the underlying mechanism of NIS functional expression was also assessed via identifying the activation of β-catenin signal pathway., Results: In vitro assay showed prominent EMT phenotype in the above two β-catenin nuclear translocation cell models, and in vitro proliferation and invasion potential of cancer cells markedly increased (all P<0.05); NIS protein expression translocated from cell membrane to cytoplasma. Cell iodide uptake in vitro decreased about 50.0% and 37.5%, respectively in above two β-catenin translocation cell models (both P<0.05). Moreover, β-catenin nuclear translocation was correlated with phosphorylation expressions of β-catenin Ser45, Y654 and GSK-3β Ser9. Most importantly, all above changes associated with β-catenin nuclear translocation could be effectively reversed after blockade of β-catenin expression. In the animal model, tumor growth potential and tumor mass were significantly inhibited by both blockade of β-catenin and radioactive iodide ((131)I) (all P<0.05). Meanwhile, immunohistochemistry demonstrated that β-catenin nuclear translocation remained in subcutaneous tumor cells after transplantation, and showed negatively associated with NIS membrane expression., Conclusion: β-catenin nuclear translocation is a vital regulatory pathway involved in functional expression of NIS in human thyroid cancer cells.

Objective: To investigate the role of glycogen synthase kinase 3β (GSK-3β) in the maturation and function of murine bone marrow-derived dendritic cells (BMDCs)., Methods: Mature DCs (mDCs) induced by LPS were examined for GSK-3β phosphorylation level with Western blotting before and after LPS exposure. To explore the role of GSK-3β in maturation and function of DCs, we added SB216763, a selective inhibitor of GSK-3β, in the cell culture of immature DCs (iDCs), and examined CD40 and CD86 expressions in the cells by flow cytometry and the expression of IL-6, IL-12 and IL-10 mRNA by real-time PCR; the changes of the immunogenicity of the cells was evaluated by mixed lymphocyte reaction. The expression of GSK-3β and RelB was examined by Western blotting in DC2.4 cells transfected with a lentiviral vector over-expressing murine GSK-3β gene., Results: LPS exposure significantly lowered GSK-3β activity in iDCs as demonstrated by increased Ser9 phosphorylation and reduced Tyr216 phosphorylation. GSK-3β inhibition induced DC maturation by increasing the expression of surface costimulatory molecules CD40 and CD86, lowered the expressions of IL-6 and IL-12 while enhanced the expression of IL-10 in iDCs, and impaired mixed lymphocyte reaction of the cells. In DC2.4 cells, lentivirus-mediated over-expression of GSK-3β obviously down-regulated the expression of RelB., Conclusions: GSK-3β is a crucial enzyme involved in the differentiation and maintenance of an immature phenotype of DCs. GSK-3β is constitutively active in iDCs to inhibit their spontaneous maturation. DCs become phenotypically mature after inhibition of GSK-3β, which also executes a proinflammatory task in DC activation. The reduction of RelB protein levels as a result of GSK-3β overexpression supports GSK-3β as a new target for inducing tolerogenic DCs.

Objective: To explore the impact of glycogen synthase kinase-3β (GSK-3β) on Wnt and NF-κB pathways in a rat model of diabetic nephropathy (DN)., Methods: SD rats were randomly divided into normal control group (NC), DN model group (DM) and GSK-3β inhibitor group (DI). Blood glucose and 24-hour urine protein were monitored in three groups. Renal tissue samples were stained by HE. The expression of GSK-3β and NF-κB proteins was studied by immunohistochemistry. GSK-3β and NF-κB mRNAs were detected by RT-qPCR., Results: Ten weeks after STZ injection, the level of blood glucose increased significantly in DM group [(23.2±5.4) mmol/L] and DI group [(25.0±4.0) mmol/L], compared with NC group, and the level of 24-hour urinary protein increased significantly in DM group [(185.2±35.6) g/24 h] and DI group [(179.6±44.7) g/24 h], compared with NC group. Two weeks after LiCl injection, the level of blood glucose and 24-hour urinary protein decreased in DI group (17.6±2.1) mmol/L, (106.9±30.0) g/24 h], compared with DM Group. Compared with NC group, pathological changes of the kidney of DM group aggravated along with increased mRNA and protein expression of GSK-3β and NF-κB. But the pathological changes of the kidney in DI group alleviated along with declined mRNA and protein expression of GSK-3β and NF-κB as compared with DM group (all P<0.05)., Conclusions: NF-κB protein expression positively correlates with the GSK3β expression. Wnt and NF-κB signal pathways play an important role in the development of diabetic nephropathy.

Objective: To explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on LPS-induced abnormal activation of platelets in peripheral blood of healthy human donors and its possible molecular mechanism., Methods: Venous blood samples were collected from a healthy volunteer, and platelet-rich plasma (PRP) from the blood were isolated by differential centrifugation. The PRP was subpackaged into siliconized test tubes and then divided into control group, LPS group, inactive CORM-2 (iCORM-2) group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group according to the random number table, with 3 tubes in each group. The PRP in control group did not receive any treatment. The PRP in LPS group received LPS (20 mL, 10 µg/mL) stimulation, and the PRP in iCORM-2 group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group underwent the same LPS stimulation and treatment of 50 µmol/L iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2, respectively, with the dosage of 20 mL. After being cultured for 30 min, the platelet adhesion rate was determined by glass bottle method, the number of platelet spreading on fibrinogen was determined with immunofluorescent method, and the platelet aggregation rate was measured by turbidimetric method. The platelet poor plasma (PPP) was prepared from PRP, the levels of ATP in PPP and platelets were determined by chemical fluorescein method. The expressions of platelet glycoprotein I bα (GPIbα) and GPVI were analyzed by flow cytometer. The expressions of glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β were determined by Western blotting and immunoprecipitation, respectively. Measurement of the above indices was repeated for 3 times. Data were processed with one-way analysis of variance and SNK test., Results: Compared with those in control group, the platelet adhesion rates, numbers of platelets spreading on fibrinogen, platelet aggregation rates, expressions of GPIbα and GPVI in PRP, levels of ATP in PPP in LPS and iCORM-2 groups were significantly increased, while levels of ATP in platelets were significantly decreased (with P values below 0.05). Compared with those in LPS group, the former 7 indices in iCORM-2 group showed no significant differences (with P values above 0.05), while the levels of ATP in platelets in the 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly increased, and the other 6 indices in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased (with P values below 0.05). The expression levels of GSK-3β of the platelets in PRP in control, LPS, iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2 groups were 0.550 ± 0.060, 1.437 ± 0.214, 1.210 ± 0.108, 0.720 ± 0.010, and 0.670 ± 0.010, respectively, and the expression levels of the phosphorylated GSK-3β of the platelets in PRP in the above 5 groups were 0.950 ± 0.070, 1.607 ± 0.121, 1.420 ± 0.040, 1.167 ± 0.015, and 0.513 ± 0.122, respectively. Compared with those in control group, both the expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in LPS and iCORM-2 groups were significantly increased (with P values below 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP between LPS group and iCORM-2 group were similar (with P values above 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased compared with those in LPS group (with P values below 0.05)., Conclusions: LPS stimulation can abnormally activate the platelets in peripheral blood of healthy human, but the abnormal activation can be inhibited by CORM-2 intervention, and the mechanism of the latter may involve the phosphorylation of GSK-3β mediated by GP.

Objective: This study explored how interferon alpha (IFN-α) influenced liver cancer SMCC-7721 cell via PI3K/Akt signaling pathway., Methods: The effect of IFN-α on the viability of SMCC-7721 cell was measured by MTT assay. The effect of IFN-α on SMCC-7721 cell cycle was measured by flow cytometry. The activation of PI3K/Akt signaling pathway and downstream target genes expression level of glycogen synthase kinase 3 beta (GSK-3β), and cell cycle protein (Cyclin D1), p21, C-myc, Survivin were assayed by Western blot., Results: MTT assay indicated IFN-α (5, 10, 15 U/L) could inhibit SMCC-7721 cell viability, and the inhibitory rate was the highest at 48 h. IFN-α made SMCC-7721 cell arrested. Western blotting assays presented IFN-α could down-regulate expression of PI3K and suppress the phosphorylation of Akt, and then inhibit phosphorylation of GSK-3β. It could also reduce the expression levels of Cyclin D1, C-myc and Survivin but increase the level of p21., Conclusion: These results suggested IFN-α could depress the growth and proliferation of liver cancer SMCC-7721 cell via blocking PI3K/Akt signaling pathway, and the molecular proteins Cyclin D1, p21, C-myc, Survivin were involved.

Objective: To investigate the inhibition effect of curcumin on the proliferation of the human esophageal carcinoma cell line Ec109 and its impact on PEN/PI3K/Akt signaling pathway., Methods: Esophageal carcinoma Ec109 cells were cultured in vitro conventionally and were treated with curcumin at different concentrations. The cell proliferation level was examined by MIT colorimetry, the ultrastructure of curcumin-treated Ec109 cells were detected with transmission electron microscope (TEM) and cell apoptosis was observed by FCM with AnnexinV-FITC/PI double staining. The protein levels of PTEN, Akt, GSK3P and Caspase 3 of curcumin-treated Ec109 cells were detected by Western blot., Results: MTT test showed that curcumin could inhibit the proliferation of Ec109 cells in a time and concentration-dependent manner. TEM examination indicated that curcumin could induce Ec109 cell apoptosis. FCM detection showed that Ec109 cell apoptotic rate increased significantly with the increase of drug concentration. On the other hand, curcumin could promote the expression of PTEN, GSK3beta and Caspase 3 yet reduce the expression of Akt., Conclusion: Curcumin could obviously up-regulate the expression of PTEN, GSK3beta and Caspase 3, surpress PI3K/Akt signaling pathway and hence inhibit the proliferation of Ec109 cells.

Objective: To observe the effect of Cornus Officinalis total glycosides (COTG) and Cornus polysaccharides (CP) on myocardial mitochondria and expression levels of glycogen synthase kinase-3β (GSK-3β) of acute myocardial infarction (AMI) rats., Methods: The AMI rat model was established by ligating the left anterior descending branch of coronary artery. Rats were divided into 5 groups according to random digit table, i.e., the sham-operation group, the model group, the COTG prevention group, the CP treatment group, the COTG treatment group, 12 in each group. Normal saline was administered to rats in the normal control group and the model group by gastrogavage. Corresponding medication was respectively administered to rats in the rest 3 groups by gastrogavage. The cardiac function was detected by echocardiography and hemodynamics. The infarct size was determined by Masson trichrome staining. The expression of mitochondrial biogenesis genes such as a subunit of peroxisome proliferators-activated receptor-γ coactivator-1 (PGC-1α), PGC-1β, nuclear respiratory factor-1 (NRF-1), and GSK-3P mRNA were detected by Real-time PCR., Results: Compared with the sham-operation group, the myocardial infarction size increased, cardiac function decreased, the expression of PGC-1α, PGC-1β, and NRF-1 mRNA decreased, and the expression of GSK-3β mRNA increased (all P <0. 05). Compared with the model group, myocardial infarction sizes were reduced, cardiac function was improved, the expression of NRF-1 mRNA was elevated in the COTG prevention group, the CP treatment group, the COTG treatment group; the expression of the PGC-1α and PGC-1β mRNA was elevated in the COTG prevention group and the CP treatment group; the expression of GSK-3β mRNA was reduced in the CP treatment group (all P <0. 05). Compared with the CP prevention group, fractional shortening (FS) and aortic systolic blood pressure (SBP) increased in the CP treatment group; ejection fraction (EF) decreased in the CP treatment group; the expression of PGC-1α, PGC-1β, NRF-1 mRNA were reduced in the the CP treatment group and the COTG treatment group; the expression of GSK-3β mRNA decreased in the CP treatment group (all P <0. 05). Compared with the COTG treatment group, FS, EF, left ventricular end systolic pressure (LVESP), SBP, and the expression of GSK-3β mRNA were reduced in the CP treatment group (P <0. 05)., Conclusions: COTG and CP could improve cardiac function, reduce the myocardial infarction area, and promote biogenesis of myocardial mitochondria. Their protective effects on the mitochondria of cadiocytes might be achieved by GSK-3β signalina pathway.

Aortic valve calcification (AVC) is a pathological process correlated with multiple disease causes and actively regulated by cardiac valve cells. In this study, porcine aortic valve myofibroblasts cultured in vitro were treated with 50 μg z L(-1) of pathological factor tumor necrosis factor α (TNF-α). Tanshinone II A (TSN) with the concentration of 50 mg x L(-1) and TNF-α were combined in incubating cells for 72 h (3 d) and 120 h (5 d). The Western blotting and Real-time PCR were adopted to detect the changes in smooth muscle α actin (α-SMA), bone morphogenetic protein 2 ( BMP2), alkaline phosphatase (ALP) in cells, and expressions of key effect proteins GSK-3β and β-catenin on Wnt/β-catenin signal pathway. According to the findings, TNF-α can significantly increase the expression of myofibroblasts α-SMA and add the transformation activity to them, with nearly no expression of BMP2, ALP and mRNA in the control group and the TSN group but significant increase in their expressions in the TNF-α group (P < 0.01), which showed osteoblast-like phenotype. Moreover, TNF-α down-regulated the expression of up-streaming regulator GSK-3β and mRNA expression (P < 0. 01) , notably increased the expression of key effect protein β-catenin, but with no significant difference in mRNA with the control group and the TSN group. The result demonstrated that TSN showed a certain inhibitory effect on TNF-α's pathological impact (P < 0.05) in a time-dependent manner. Inflammatory factor TNF-α may promote the transformation of aortic valvular myofibroblasts to osteoblast-like phenotype by activating Wnt/β-catenin signal pathway in aortic valvular myofibroblasts, so as to cause AVC. Tanshinone II A can have a preventive effect in AVC by activating GSK-3β proteins and regulating signal transduction of Wnt/β-catenin signal pathway.

Objective: To down-regulate the expression of cylindromatosis (CYLD) gene by shRNA and investigate its effect on proliferation, migration and cell signal pathways of human umbilical vein endothelial cells (HUVECs)., Methods: HUVECs were infected by adenoviruses of CYLD shRNA (experiment group) and GFP (control group), respectively. The proliferation of HUVECs was detected by CCK-8 assay; the cell migration was assessed by a wound healing assay; the related cell signal pathways were analyzed by Western blotting., Results: CCK-8 showed that the proliferation of the CYLD shRNA-infected HUVECs were significantly lower than that of the control group at 1, 2, 3, 4, 5 days after infection. The wound healing assay revealed that the migration rate of the CYLD shRNA-infected HUVECs was significantly reduced compared with the control HUVECs at 48 hours. Western blotting indicated that the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3α/β (GSK3α/β) significantly decreased in the CYLD shRNA-infected HUVECs compared with the control HUVECs., Conclusion: Inhibiting the expression of CYLD gene can suppress the proliferation and migration of HUVECs, which may be attributed to the inhibition of the AKT/GSK3α/β pathway.

Objective: To test the effect of sulindac on autistic behaviors in a rat model and explore the possible mechanisms., Methods: Autistic rat models were established by a single intraperitoneal injection of sodium valproate (VPA) at 12.5 days of pregnancy. The pregnant rats were treated with oral sulindac at a daily dose of 80 mg/kg until weaning of the newborn rats (23 days after being born), which were divided into control, VPA treatment, sulindac treatment, and VPA+ sulindac treatment groups. The social interaction and neuroethology of the newborn rats were evaluated at 35 days, and the levels of β-catenin and phosphorylated Gsk3β in the brain tissues were investigated by Western blotting., Results: Compared with the control rats, the rats treated with VPA showed lower social interaction, longer moving time in central area, and reduced standing times. Treatment with sulindac alone resulted in no obvious changes in the social interaction or neuroethology of the newborn rats, but sulindac treatment corrected VPA-induced autistic-like behaviors. Sulindac also attenuated VPA-triggered p-Gsk3β downregulation and β-catenin upregulation in the prefrontal lobe, seahorse and cerebellum., Conclusion: Sulindac can improve the behaviors of autistic rats possibly by suppressing Wnt signaling pathway.

Objective: To study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells., Methods: MTT assay was used to measure the 50% inhibiting concentration (IC₅₀) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis., Results: After combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC₅₀ of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P < 0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0.4 µg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down-regulation of DNA-PKcs/Akt/GSK-3β signaling pathway, and higher inhibition of MDR1(P-gp) and MRP1 than those treated with TRAIL alone (P < 0.01 for all). The mRNA and protein levels of DR4, DR5, c-myc were significantly decreased after silencing c-myc with specific siRNA in the SGC7901/VCR cells (P < 0.01 for all), and were significantly increased after transfection of recombinant plasmid c-myc into the SGC7901/VCR cells (P < 0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0.25 µg/ml DDP + 10 ng/ml TRAIL and 0.5 µg/ml DDP + 10 ng/ml TRAIL, the relative expression level of c-myc protein in the SGC7901/VCR cells was 0.314 ± 0.012, 0.735 ± 0.026, and 0.876 ± 0.028, respectively, and the relative expression of cytochrome C was 0.339 ± 0.036, 0.593 ± 0.020 and 0.735 ± 0.031, respectively, and the relative expression levels of DR4, DR5, active-caspase 3 and active-caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations., Conclusions: The activation of DNA-PKcs/Akt/GSK-3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi-drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up-regulating c-myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

Objective: To explore the effects of hydrogen-rich saline on Akt/GSK3β signaling pathways and cardiac function during myocardial ischemia-reperfusion (I/R) in rats., Methods: A total of 150 healthy male Sprague-Dawley rats were selected and divided randomly into 5 groups of normal control (I), sham operation (II), myocardial ischemia-reperfusion (III), hydrogen-rich saline (IV) and normal saline group (V) (n = 30 each). Group I had no treatment at all. In group II, anterior descending branch was merely exposed but not ligated. Myocardial I/R was induced by 30-min occlusion of anterior descending branch of left coronary artery followed by 12/24 h reperfusion of Bimbaum. Hydrogen-rich saline 1 ml/100 g were injected intraperitoneally 5 min before reperfusion in group IV. Normal saline 1 ml/100 g was injected intraperitoneally 5 min before reperfusion in group V. The values of ± dp/dt max, left ventricular diastolic pressure (LVDP) and left ventricular systolic pressure (LVSP) during the procedure were recorded by BL-420 biological function experimental system at pre-ischemia, ischemia 30 min, 60 min and 120 min of reperfusion. The animals were sacrificed at 60 min, 120 min of reperfusion and hearts harvested. The pathological changes of myocardial tissue were detected by hematoxylin and eosin staining. And the expressions of Akt and GSK3β in myocardial tissue were examined by Western blot., Results: Compared with groups I and II, the values of ± dp/dt max, LVDP and LVSP significantly increased. And the expressions of Akt and GSK3β in myocardial tissue significantly increased in groups III, IV and V. Compared with groups III and V, the values of ± dp/dt max, LVDP and LVSP significantly decreased. And the expressions of Akt and GSK3β in myocardial tissue significantly decreased in group IV. Compared with T0, the values of ± dp/dt max, LVDP and LVSP significantly decreased at T(1-3) in groups III, IV and V. Compared with T(1), the values of ± dp/dt max, LVDP and LVSP significantly increased at T(2-3) in group IV. Compared with T(2), the expressions of Akt and GSK3β in myocardial tissue significantly decreased at T(3) in group IV., Conclusion: Hydrogen-rich saline can reduce myocardial reperfusion injury and improve heart function. The mechanism may be due to associated with the down-regulated expressions of Akt and GSK3β in myocardial tissue.

Objective: This study was purposed to detect the expressions of β-catenin and P-GSK-3 β in Wnt signaling pathway of patients with mantle cell lymphoma(MCL), and investigate its relationship with the pathogenesis of MCL., Methods: The expression levels of β -catenin protein and P-GSK-3 protein in mantle cell lymphoma and hyperplastic lymphadenitis were detected by using anti-β-catenin, P-GSK-3β polyclonal antibody and S-P staining technique., Results: The abnormal expression of β-catenin protein(73.33%) in mantle cell lymphoma group was significantly higher than that (6.7%) in reactive lymph node hyperplasia group (P<0.05); and the positive rate of P-GSK-3 β(66.67%) in mantle cell lymphoma group was significantly higher than that (16.67%) in reactive hyperplasia of lymph node group (P<0.05). Spearman correlation analysis showed that there was obvious positive correlation (R=0.852, P<0.01)., Conclusion: The abnormal high expressions of β-catenin and P-GSK-3 β protein have been confirmed to appeare in mantle cell lymphoma.