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Objectives: To observe the effect of electroacupuncture (EA) stimulation of "Dazhui"(GV14) and "Mingmen" (GV4) of the Governor Vessel (GV) on the apoptosis of neurons in rats with spinal cord injury (SCI) by regulating the expression of glycogen synthase kinase-3β (GSK-3β) protein and its phosphorylation level, so as to reveal partial mechanism for the treatment of SCI with EA of GV., Methods: A total of 45 male SD rats were randomly divided into sham-operation, model and EA groups, 15 rats in each group. The SCI model was established by using the modified Allen's percussion device. EA stimulation (2 Hz, 1 mA) was applied to GV14 and GV4 for 20 min, once daily for 28 days. Basso-Beattie-Bresnahan (BBB) locomotor rating scale was used to assess the rat's hind limb motor function at the 1 st , 7 th , 14 th , 21 th and 28 th day after modeling. H.E. staining was used to observe the histopathological changes of the injured spinal cord tissue. The expressions of GSK-3β and its phosphorylation at Ser9 site in the spinal cord were detected by immunohistochemistry and Western blot, respectively. The TUNEL staining was used to observe the apoptosis of neuron in the spinal cord., Results: In comparison with the control group, the BBB scores from day 1 to day 28, and the expression and immunoactivity of p-GSK-3β were significantly decreased ( P <0.01), while the expression and immunoactivity of GSK-3β and the apoptosis index were significantly increased ( P <0.01) in the model group. In contrast to the model group, the EA group had a remarkable increase in the BBB score from day 7 to day 28, and the expression and immunoactivity of p-GSK-3β ( P <0.05, P <0.01), and an obvious decrease ( P <0.05, P <0.01) in the expression and immunoactivity of GSK-3β and the apoptosis index. H.E. staining showed that in the model group, the spinal cord structure of the SCI region was disorganized, with tissue cavity, blurred gray matter boundary, severe inflammatory infiltration, reduction of normal neurons, loss of cytoplasm, and nuclear contraction, which was relatively milder in the EA group., Conclusions: EA at GV14 and GV4 can improve the symptoms of neurological function injury and reduce the apoptosis of neurons in rats with SCI, which may be achieved by regulating GSK-3β protein and its phosphorylation level.

Objective: To investigate the role and mechanism of COL11A1 in lung adenocarcinoma migration and invasion. Methods: Surgical pathological tissues of 4 patients with lung adenocarcinoma admitted to the Affiliated Hospital of Guizhou Medical University from September to November 2020 were used. Immunohistochemical methods were used to identify lung adenocarcinoma tissues, para-cancerous tissues and parallel transcriptome sequencing. Genetic prognostic analysis was conducted by TCGA and GTEx databases.The expression level of COL11A1 gene in lung adenocarcinoma and adjacent tissues was detected by Western blotting.The primary human lung adenocarcinoma cells cultured. The COL11A1 siRNA was transfected into primary human lung adenocarcinoma cells, then the transcriptome sequencing of differential genes was performed,and KEGG enrichment analysis of differential gene enrichment pathway was conducted. Protein expression and phosphorylation were detected by Western blot method. Cell migration was detected by scratch healing test. Cell proliferation was detected by CCK8 method and invasion ability was detected by Transwell method. Results: Ten differentially expressed genes were screened by transcription sequencing in lung adenocarcinoma. Prognostic analysis of single gene showed that COL11A1 gene expression level was correlated with survival rate ( P <0.001). The expression of COL11A1 in lung adenocarcinoma was higher than that in adjacent tissues by Western blot ( P <0.001). Transcriptome sequencing of COL11A1 siRNA transfection into primary human lung adenocarcinoma cells showed that differential genes were concentrated in PI3K-akt pathway. The expression of tumor suppressor gene PTEN in siRNA transfection group was significantly higher than that in control group and negative transfection group by Western blot. The expression of Aktp-Akt 473 p-Akt 308 p-PTENp-PDK1p-c-Rafp-GSK-3 β was down-regulated (all P <0.05).Compared with the negative control group, the ability of migration, proliferation and invasion of primary human lung adenocarcinoma cells in siRNA transfection group decreased (all P <0.05). COL11A1 regulates PI3K/Akt/GSK-3 β pathway to promote migration and invasion of primary human lung adenocarcinoma cells. Conclusion: COL11A1 regulates PI3K/Akt/GSK-3 β pathway to promote migration and invasion of primary human lung adenocarcinoma cells.

Chronic inflammatory pain is mainly manifested by peripheral sensitization. Baimai Ointment(BMO), a classical Tibetan medicine for external use, has good clinical efficacy in the treatment of chronic inflammatory pain, while its pharmacodynamics and mechanism for relieving peripheral sensitization remain unclear. This study established an animal model of chronic inflammatory pain induced by complete Freund's adjuvant to explore the mechanism of BMO in the treatment of chronic inflammatory pain by behavioral test, side effect assessment, network analysis, and experimental verification. The pharmacodynamics experiment showed that BMO increased the thresholds of mechanical pain sensitivity and thermal radiation pain sensitivity of chronic inflammatory pain mice in a dose-dependent manner, and had inhibitory effect on foot swelling, inflammatory mediator, and the expression of transient receptor potential vanilloid-1(TRPV1) and transient receptor potential A1(TRPA1). The results of body weight monitoring, pain sensitivity threshold detection in normal mice, rotarod performance test, and forced swimming test showed that BMO had no obvious toxic or side effect. The network analysis of 51 candidate active molecules selected according to the efficacy of BMO, content of main components, and ADME parameters showed that the inhibitory effect of BMO on chronic inflammatory pain was associated with the core regulatory elements of tumor necrosis factor(TNF) and T cell receptor signaling pathways. BMO down-regulated the protein levels of mitogen-activated protein kinase 14(MAPK14), MAPK1, and prostaglandin-endoperoxide synthase 2(PTGS2), and up-regulated the phosphorylation le-vel of glycogen synthase kinase 3 beta(GSK3 B) in the plantar tissue of mice. In conclusion, BMO can effectively relieve peripheral sensitization of chronic inflammatory pain without inducing tolerance and obvious toxic and side effects. The relevant mechanism may be related to the regulation of BMO on core regulatory elements of TNF and T cell receptor signaling pathways in surrounding tissues.

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 important roles of Wnt signaling in the processes of 0.5Gy X-ray promoting osteoblast differentiation, and make clear the molecular mechanisms involved. Methods: Flow cytometry was employed to detect the apoptosis after osteoblast exposure to 0.5 Gy X-ray radiation.The protein level of osteoblast differentiation markers, such as collagen Iα (Col1α), alkaline phosphatase (ALP), osteocalcin (OCN), were detected by Western-blot and ALP activity staining was performed. Real-time PCR and Western-blot were utilized to evaluate the variations of key factors in Wnt signaling pathways, while specific inhibitor of Wnt/β-Catenin, XAV939 was used to block the Wnt signaling. Results: Low-dose (0.5 Gy) X-ray induced significant decline in MC3T3-E1 osteoblast apoptosis at three days after radiation.The dynamic variations in the expression of osteoblast differentiation markers, including Col1α, ALP, OCN, were observed after 0.5 Gy X-ray irradiation by Western blot analysis.The protein levels of Col1α have a reduction temporarily at 4 days of radiation (34.5%±5.8%, t =9.912, P <0.001), then a significant increase is detected at 10 day after radiation (162.5%±6.5%, t =2.673, P <0.05). OCN levels dropped by 83% ( t =3.968, P <0.01) at 4 day after 0.5 Gy X-ray radiation, and raised at 10 day (39.5%±4.1%, t =3.219, P <0.05) and 14 day (79.4%±7.5%, t =6.708, P <0.001), respectively. ALP levels increased at 7 day (79.7%±22.3%, t =6.257, P <0.001) and 10 day(128.3%±6.1%, t =4.340, P <0.01)after radiation. At the same time, 0.5 Gy X-ray radiation can activate Wnt/GSK-3/β-Catenin signaling.The mRNA levels of Wnt3a、LPR5 and TCF-4 increased by 1.7 fold ( t =6.573, P <0.001), 1.1 fold ( t =5.323, P <0.05) and 1.4 fold ( t =3.054, P <0.05) at 7 day after radiation.In addition, p-GSK-3β level reduced by 42.1% ( t =4.460, P <0.01), and active β-Catenin increased by 1.9 fold ( t =3.528, P <0.05). However, the specific inhibitor of Wnt/β-Catenin, XAV939 completely abrogated Wnt/β-Catenin signaling and the increase in ALP expression and activity induced by 0.5 Gy X-ray radiation. Conclusion: These results demonstrated that low dose X-ray radiation promoted osteoblast survival at early differentiation, and promoted differentiation at middle and late stage, in which Wnt signaling participated the regulation processes.

Objective To observe the regulation of Shuanghuang Shengbai Granule (SHSBG) on regulating Wnt signaling pathway in tumor-bearing mice with chemotherapy induced myelosuppression. Methods Chemotherapy induced myelosuppression model was established in Lewis lung tumor bearing mice by intraperitoneal injection of cyclophosphamide (CTX). And then they were intervened by SHSBG. Routine white blood cell (WBC) count, red blood cell (RBC) count, platelet count, and tumor mass were calculated. Ratios of bone marrow hematopoietic stem cell (Sca, CD34 double positive cells) were detec- ted by flow cytometry. mRNA expression of main genes in Wnt signaling pathway (Wnt, β-catenin, Frizzted, DSH, GSK3) were detected using real time fluorescent quantitative PCR. Results The number of WBC and ratio of hematopoietic stem cells in the treatment group were higher than those in the model group (P<0. 05). Expressions of Wnt, β-catenin, Frizzted, DSH, and GSK3 mRNA in the bone marrow were higher in the treatment group than in the model group (P <0. 05). Expressions of Wnt, β-catenin, Frizzted, and DSH mRNA expression in tumors were lower in the treatment group than in the model group (P <0. 05). There was no statistical difference in counts of RBC and platelet, tumor mass, or GSK3 mR- NA expression among all groups (P >0. 05). Conclusions The mechanism for SHSBG treating myelo-suppression was related to regulating Wnt signaling pathway. Besides, it had dual regulation effect on Wnt signaling pathway, up-regulating expressions of main genes in Wnt signaling pathway while inhibiting ex- pressions of partial genes in tumors.

Glycogen synthase kinase3 (GSK3α and GSK3β) are serine/threonine protein kinases acting on numerous substrates and involved in the regulation of various cellular functions such as their proliferation, survival, glycogen metabolism, and autophagy. Accumulating evidence indicates that the expression of GSK3α is increased mainly in androgendependent while that of GSK3β in androgenindependent prostate cancer, and that GSK3β is also involved in the regulation of the transactivation of the androgen receptor (AR) and growth of prostate cancer. Animal experiments have proved that some GSK3 inhibitors, such as lithium, can significantly suppress tumor growth in different animal models of prostate cancer. The GSK3 inhibitor is promising to be an important agent for the clinical management of prostate cancer.

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 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.

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 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 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 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 study the molecular mechanism of curcumin in human esophageal carcinoma cell line (EC109)., Methods: EC109 cells were cultivated in vitro. When 80%-90% confluence was reached, they were treated with curcumin in different concentrations (15-120 µmol/L). The effects on cell proliferation were examined by CCK-8 colorimetry. The ultrastructure of EC109 cells were detected with transmission electron microscope(TEM). The cells apoptosis was observed with laser confocal microscope(LCM) by AnnexinV-FITC/PI double staining. The proteins level of PTEN, AKT, GSK3β and Caspase 3 were tested by flow cytometry(FCM) ., Results: CCK-8 test showed that curcumin could inhibit the proliferation of EC109 cells in a time- and concentration-dependent manner. TEM and LCM examinations indicated that curcumin could make EC109 cells apoptosis. The data of FCM showed that curcumin could increase the expression of PTEN, GSK3β and Caspase 3, decreased the expression of AKT., Conclusion: The effects of curcumin on inhibiting proliferation and promoting apoptosis of EC109 cells were related with increased expression of PTEN and inhibition of PI3K/AKT signaling pathway.

Objective: To investigate the effect of irradiated human lung fibroblasts (HLFs) on the canonical Wnt/β-catenin signaling pathway in human umbilical cord mesenchymal stem cells (HUMSCs)., Methods: HUMSCs were cultured alone (single group) or co-cultured with HLFs exposed to 5Gy X-rays (co-culture group). The protein levels of GSK-3β, p-GSK-3β, FRAT1 and β-catenin in HUMSCs were examined by Western blotting 3 days after culture or co-culture. WISP-1 protein levels in conditioned medium were examined by ELISA., Results: The levels of p-GSK3β/GSK3β (0.15 ± 0.05), FRAT1 (0.48 ± 0.07) and β-catenin (0.50 ± 0.07) in co-cultured HUMSCs significantly decreased compared to those in single group (0.55 ± 0.05, 1.16 ± 0.13 and 2.39 ± 0.15, all P<0.05). The supernatant level of WISP-1 in co-culture group was significantly decreased [(602.23 ± 161.47) ng/mL], compared to the single group [(977.77 ± 110.56) ng/mL, P<0.05]., Conclusion: Irradiated HLFs attenuate the activation of canonical Wnt/β-catenin signaling pathway in HUMSCs in vitro.

Objective: To analyze the role of glycogen synthase kinase 3β (GSK3β) in hepatocyte apoptosis induced by oxidative stress., Methods: Human HL-7702 hepatoma cells were induced by H₂O₂/antimycin A to establish oxidative stress-induced cell apoptosis models. SB216763, a specific inhibitor of GSK3β, was given to the cells two hours before H₂O₂/antimycin A induction. Cell survival was observed using calcein acetoxymethyl ester/propidium iodide (PI) double staining, and cell apoptosis was detected using annexin V-FITC/PI staining combined with flow cytometry. In the meanwhile, the cell culture supernatant was subjected to lactate dehydrogenase (LDH) assay to evaluate the extent of cell death. The expressions of p-GSK3β, GSK3β, caspase-3, cleaved caspase-3, c-Jun N-terminal kinase (JNK) and cytochrome C (CytC) proteins were examined using Western blotting., Results: Oxidative stress triggered by H₂O₂/antimycin A promoted GSK3β activity; inhibition of GSK3β activity by SB216763 relieved oxidative stress and reduced cell apoptosis induced by oxidative stress. Compared with the model groups, SB216763 intervened group showed that the cell apoptosis rate and the level of LDH were reduced significantly, and that the expressions of cleaved caspase-3, JNK, CytC proteins decreased., Conclusion: GSK3β is an important signaling molecule in the apoptosis pathway induced by oxidative stress. The inhibition on GSK3β may alleviate the oxidative stress-induced hepatocyte apoptosis.

Objective: To investigate the effect of Wnt/β-catenin pathway activation by glycogen synthase kinase-3β inhibitor 4,6-disubstituted pyrrolopyrimidine (TWS119) on proliferation and phenotypic characteristics of human nature killer (NK) cells., Methods: The peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and added to the complete medium containing recombinant human interleukin-2 (rhIL-2) and human AB serum to isolate NK cells from PBMCs. After co-cultured with 0-8.0 μmol/L TWS119 for 72 hours, growth curve and Wnt/β-catenin activation of NK cells in each group were determined by CCK-8 and Western blotting. The CD107a and CD62L (L-selectin) expressions in the NK cells were detected using flow cytometry., Results: NK cells were amplified to (61.76 ± 3.74)% after human PBMCs were cultured for 10 days. The 0-2.0 μmol/L TWS119 could promote the growth of NK cells in a dose-dependent manner, and the proliferation rate gradually dropped when TWS119 was more than 2.0 μmol/L. 0-8.0 μmol/L TWS119 could activate Wnt/β-catenin pathway and up-regulate the expression of CD62L in a dose-dependent manner, but it decreased the expression of CD107a., Conclusion: Human NK cells isolated from peripheral blood treated with TWS119 gave rise to early mature CD62L⁺ NK cells.

Objective: To investigate the effect of risperidone, an antipsychotic drug, on the Akt-GSK3β pathway and the role of PI3K in dopamine D2 receptor (DRD2) expression and Akt-GSK3β signal pathway., Methods: Human glioma cells (U251) were cultured in vitro. Cells without any treatment as control, Western blotting was used for measuring the expression of Akt (Thr308 and Ser473) and GSK3β (Ser9) protein phosphorylation by risperidone and LY294002 in U251 cell, and real-time PCR was used for detecting the expression of DRD2 mRNA., Results: Risperidone has significantly enhanced the expression of phosphorylated Akt and phosphorylated GSK3β (P< 0.05), but did not alter the mRNA expression of DRD2. LY294002 could reduce the phosphorylation of Akt and GSK3β (P< 0.01, P< 0.05), and also decrease the DRD2 mRNA (P<0 .05)., Conclusion: Risperidone can activate the Akt-GSK3β signaling pathway in the U251 cells, and PI3K is a common regulatory site in Akt-GSK3β signaling and D2 receptor gene expression.

Objective: To observe the effects of electroacupuncture (EA) at "Dazhui" (GV 14) on Wnt-β-catenin signal pathway in annulus fibrosus cells in intervertebral disc in rats with cervical spondylosis., Methods: Forty SD rats were randomized into a control group, a model group, an EA group and a medication group, 10 rats in each one. Rats in the control group were treated with sham operation, only incision on local skin; rats in the remaining groups were made into cervical spondylosis models. After model establishment, rats in the control group and model group received fixed treatment under identical condition; rats in the EA group were treated with EA at "Dazhui" (GV 14), 30 min per treatment; rats in the medication group were treated with intragastric administration of meloxicam tablets. Treatments were both given once a day, and 14 days were taken as one session; there was an interval of 2 days between two sessions, and totally two sessions were given. After the treatments, immunohistochemistry was applied to measure the expression of Wnt, glycogen synthase kinase-3β (GSK-3β) and Axin in annulus fibrosus cells; western blot was used to test the expression of P-β-catenin., Results: In the control group, there were more positive cells of Wnt, GSK-3β and Axin, which were intensively distributed, deeply colored, and strongly positive; In the model group, there were less positive cells of Wnt, GSK-3β and Axin, which were sparsely distributed and weakly positive. The expression of Wnt, GSK-3β, Axin and P-β-catenin in the model group was less than that in the control group (all P < 0.05); expression of Wnt, GSK-3β, Axin and P-β-catenin in the EA group and medication group was higher than that in the model group (all P < 0.05); expression of Wnt, GSK-3β, Axin and P-β-catenin was not significantly different between EA group and medication group (all P > 0.05)., Conclusion: EA could delay the degeneration of intervertebral disc, which may be related to EA inhibiting signal pathway of Wnt-β-catenin.

Objective: To investigate the effect of Biejiajian Pills on the expressions of the signal molecules and target genes of Wnt signal pathway in HepG2 cells and explore the mechanisms by which Biejiajian pills suppress the invasiveness of hepatocellular carcinoma., Methods: HepG2 cells were cultured for 48 h in the presence of serum collected from rats fed with Biejiajian Pills. The expressions of β-catenin, GSK-3β and P-GSK-3β in the cultured cells were assessed by Western blotting and the expressions of CD44v6 and VEGF were detected using immunohistochemistry., Results: HepG2 cells cultured with the serum of rats fed with Biejiajian Pills showed lowered expressions of β-catenin protein both in the cytoplasm and the nuclei with also inhibition of phosphorylation of GSK-3β and reduced expression of CD44v6 and VEGF., Conclusion: Biejiajian Pills can significantly reduce the expression of β-catenin by decreasing the phosphorylation of GSK-3β and blocking the Wnt/β-catenin signaling pathway to cause down-regulation of the target genes CD44v6 and VEGF, which may be one of the molecular mechanisms by which Biejiajian Pills suppress the proliferation and invasiveness of hepatocellular carcinoma.

Objective: To discuss the changes in Wnt pathway inhibiting factors in esophageal precancerosis lesions induced by methyl benzyl nitrosamine (MBNA) and the effect of Gexia Zhuyu decoction., Method: Wistar rats were subcutaneously injected with MBNA (3.5 mg x kg(-1) for twice per week to establish the model. Since the 1st day after the model establishment, they were orally administered with Gexia Zhuyu decoction (16, 8 mg x kg(-1)). At the 10th week, esophageal tissues were collected to observe the pathological changes of esophageal mucosa, detect SFRP1, sFRP4, Axin1, Axin2 and GSK-3β mRNA levels.by fluorescent quantitation PCR analysis and β-catenin protein level by Western blotting., Result: Being induced by MBNA, rats in the model group showed slight atypical hyperplasia in the histopathological examination. Compared with the normal group, Gexia Zhuyu decoction dose high and low groups showed no significant pathomorphological and histological changes. The model group showed lower gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and higher β-catenin protein expression level (P < 0.01) than the normal control group. The Gexia Zhuyu decoction low dose group showed higher gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and lower β-catenin protein expression level (P < 0.01) than the normal control group., Conclusion: Up-regulated β-catenin protein level and down-regulated Wnt pathway could enhance Wnt pathway activity of MBNA-induced esophageal precancerous lesions. Gexia Zhuyu decoction could down-regulate the β-catenin protein level and up-regulate the transcription level of Wnt pathway inhibiting factors, but could not block MBNA-induced esophageal precancerosis lesions.

Objective: To study the effect of ginsenoside Rb1 on GSKbeta/IDE signal transduction pathway and Abeta protein secretion in hippocampal neurons of high glucose-treated rats., Method: Hippocampal neurons of 24 h-old newly born SD rats were primarily cultured, inoculated in culture medium under different conditions, and then divided into the normal group, the high glucose group, the LiCl group and the Rb1 group. After being cultured for 72 h, the expressions of their phosphorylated GSK3beta, total GSK3beta and IDE protein were detected by Western blotting analysis. The mRNA expressions of GSK3beta and IDE were determined by RT-PCR. The ELISA assay was used to detect the secretion of Abeta protein in cell supernatant., Result: Compared with the normal group, the high glucose group showed increase in the p/tGSK3beta protein ratio and the secretion of Abeta protein and decrease in IDE protein and mRNA (P < 0.05). Compared with the high glucose group, both Rb1 and LiCl groups showed decrease in the p/tGSK3beta protein ratio and the expression of Abeta protein and increase in IDE protein and mRNA expression (P < 0.05). Compared with the LiCl group, the Rb1 group showed no significant difference in the expressions of p/tGSK3beta protein, IDE protein, mRNA and Abeta protein expression. In addition, the GSK3beta mRNA expression of the four groups had no significant difference., Conclusion: Ginsenoside Rb1 may reduce the secretion of Abeta protein in hippocampal neurons by reducing the phosphorylation of GSK3beta, down-regulating the ratio of pGSK3beta/GSK3beta and upregulating the expression of IDE.

Objective: To investigate the changes in the functional activity of glycogen synthase kinase-3 (GSK-3) in the hepatic tissue after endotoxin (lipopolysaccharide, LPS) tolerance and explore the effects of LPS-induced GSK-3 inhibition on glycogen metabolism in the liver., Methods: Male SD rats were randomly divided into normal control, endotoxin pretreatment and GSK-3 inhibitor (lithium chloride) groups with corresponding pretreatments prior to a large dose of LPS challenge (10 mg/kg) to induce liver injury. Glycogen deposition and content in the hepatic tissue was detected using periodic acid-Schiff (PAS) staining and a glycogen quantification kit, respectively. Western blotting was performed for semi-quantitative analysis of protein level and inhibitory phosphorylation of GSK-3, and a Coomassie brilliant blue G-250-based colorimetric assay was used to detect calpain activity in the liver., Results: Glycogen content in the liver decreased significantly after LPS challenge in all the 3 groups (P<0.05) but showed no significant difference among the groups (P>0.05). Both LPS and lithium chloride pretreatments caused a significant increase of liver glycogen content (P<0.05). LPS pretreatment induced inhibitory phosphorylation of GSK-3β (P<0.05) and partial cleavage of GSK-3α but did not affect the expression of GSK-3 protein (P>0.05). Large-dose LPS challenge significantly increased the activity of calpain in the liver tissue (P<0.05) to a comparable level in the 3 groups (P>0.05)., Conclusion: Endotoxin pretreatment induces inhibitory phosphorylation of GSK-3β and partial cleavage of GSK-3α and promotes the deposition of liver glycogen but does not affect the activity of calpain, which may contribute to an increased glycogen reserve for energy supply in the event of large-dose LPS challenge.

Objective: To explore the regulation of insulin sensitivity in liver cells by androgen signaling., Methods: Eleven adult female C57BL/6 mice were injected daily with testosterone (group T) for 24 weeks. And 10 control mice received sesame oil only (group Con). HepG2 liver cells were initially pretreated with different doses of testosterone (10(-9)-10(-5) mol/L ) for 0-36 h or with 10(-7) mol/L testosterone for 0-96 h followed by a stimulation of 100 nmol/L insulin for 15 min. Later HepG2 cells were pretreated with 10(-7) mol/L testosterone for 36 h followed by a stimulation of 100 nmol/L insulin for 15 min and then a restimulation of 100 nmol/L insulin for 15 min at 4 h and 6 h interval respectively. Phosphorylation and protein expression of Akt and GSK3β in C57BL/6 mice liver tissues and HepG2 cells were analyzed by Western blot., Results: The 24-week treatment of testosterone decreased the phosphorylation of Akt and GSK3β in C57BL/6 adipose and liver tissues (43.1% ± 3.2% vs 77.1% ± 6.7%, 14.7% ± 6.7% vs 82.3% ± 2.0% respectively, P < 0.05). Pretreatment with 10(-8)-10(-6) mol/L testosterone within 36 h obviously increased the phosphorylation of Akt and GSK3β (P < 0.05). However pretreatment with 10(-5) mol/L within 36 h or with 10(-7) mol/L for 96 h had no effect on the phosphorylation of Akt and GSK3β compared with control group (P > 0.05). Pretreatment with 10(-7) mol/L testosterone for 36 h followed by insulin stimulation and restimulation after 6 h interval obviously decreased the phosphorylation of Akt and GSK3β (P < 0.05)., Conclusion: Androgen signaling may contribute to insulin resistance in liver cells.

Objective: To investigate the effects of coenzyme Q10 pretreatment on the expressions of Bcl-2, Bax and glycogen synthase kinase-3β (GSK-3β) in rats suffering from ischemia/reperfusion injury., Methods: Thirty-six adult male SD rats were randomly assigned into 3 groups: sham-operated group (sham), ischemia/reperfusion group (I/R) and coenzyme Q10 preconditioning group (Q10). Focal cerebral ischemia/reperfusion models were established in experimental rats by blocking middle cerebral artery with suture. Histological changes of hippocampal neurons were observed by HE staining. The expressions of Bcl-2, Bax and GSK-3β were detected by immunohistochemistry and Western blotting., Results: Immunohistochemistry showed that the percentage of Bcl-2 positive cells increased in the hippocampus, while the percentages of Bax and GSK-3β positive cells decreased in Q10 group compared with I/R group. Western blotting revealed that the expression level of Bcl-2 was higher and the expression levels of Bax and GSK-3β were lower in Q10 group than in I/R group. There were significant differences between the two groups (P<0.05)., Conclusion: Coenzyme Q10 promoted the expression of Bcl-2 and suppressed the expressions of Bax and GSK-3β in the hippocampus of rats exposed to cerebral ischemia/reperfusion.

LPS stimulation of macrophages production of IFN-beta plays a key role in innate immunity defending the microbial invasion. In this study, the effect of S632A3 promoting LPS-induced IFN-beta production and the underlying mechanism were investigated, mRNA level was measured by real-time PCR, cytokine production was determined by ELISA, GSK-3beta activity was investigated by kinase assay, protein phosphorylation and expression were evaluated by Western blotting. The results revealed that S632A3 significantly augmented IFN-beta production by LPS-stimulated macrophages. S632A3 inhibition of the activation of GSK-3beta, reduced the threonine 239 phosphorylation of transcription factor c-Jun but increased the total level of c-Jun in LPS-stimulated macrophages. Moreover, small interfering RNA-mediated knockdown of c-Jun level abrogated the ability of S632A3 to augment IFN-beta. The study thus demonstrates S632A3 being a new anti-inflammation lead compound and provides a molecular mechanism by which S632A3 promoted LPS-induced IFN-beta production in macrophages through inhibiting the activation of GSK-3beta.

To investigate the mechanisms of a compound (FF16), compatibility of Rhodiola crenulata, Cordyceps militaris, and Rheum palmatum, on glucose metabolic disorders, the IRF mice charactered with insulin resistance and glucose metabolic disorders induced by high-fat diet in C57BL/6J mice were randomly divided into 3 groups; IRF, rosiglitazone (Rosi) and FF16. The glucose metabolism was evaluated by fasting blood glucose (FBG) levels and intraperitoneal glucose tolerance test (IPGTT). The insulin sensitivity was estimated by insulin tolerance test (ITT), fasting serum insulin levels and the index of HOMA-IR. The expressions of Akt and its phosphorylation levels, GSK3beta and its phosphorylation levels in liver were detected by Western Blot. The results showed that FF16 significantly improved the glucose metabolic disorders through reducing FBG by 15.1%, decreasing AUC values in glucose tolerance tests by 22.3%. FF16 significantly improved the insulin sensitivity through decreasing AUC values in insulin tolerance tests by 22.1%, reducing the levels of serum insulin by 42.9% and of HOMA-IR by 49.5%, comparing with model control, respectively. After the treatment with FF16, the levels of p-Akt and p-GSK3beta were increased by 116.4% and 24.9%, respectively, in the liver of IRF mice. In conclusion, compound FF16 could improve glucose metabolic disorders in IRF mice through enhancing the glyconeogenesis.

Objective: To study the inhibitory effect of Akt inhibitor deguelin on PC-3 human prostate cancer cell lines and its possible mechanism., Methods: PC-3 human prostate cancer cells were cultured in deguelin at the concentrations of 10, 100, 500 and 1 000 nmol/L for 24, 48 and 72 hours, respectively. Then the inhibitory effect of deguelin on the proliferation of the PC-3 cells was determined by MTT assay and that on the cell cycle was detected by flow cytometry. The expression levels of MDM2 and GSK3beta mRNA were measured by RT-PCR and those of MDM2 and GSK3beta proteins by Western blot., Results: At 24, 48 and 72 hours, the inhibition rates of deguelin on the proliferation of the PC-3 prostate cancer cells were (91.10 +/- 3.75), (86.39 +/- 1.16) and (79.51 +/- 2.63)% at 10 nmol/L, (82.46 +/- 3.65), (76.84 +/- 0.97) and (69.69 +/- 2.30) % at 100 nmol/L, (81.46 +/- 0.41), (75.56 +/- 1.12) and (54.07 +/- 3.21)% at 500 nmol/L, and (66.77 +/- 2.82), (58.22 +/- 0.35) and (39.34 +/- 2.40)% at 1000 nmol/L, all with statistically significant differences from the control group (P < 0.01). Deguelin at 10, 100, 500 and 1 000 nmol/L increased the cell cycles blocked in the G0/G1 phase ([62.4 +/- 2.2], [63.6 +/- 1.1 ], [65.0 +/- 0.3] and [66.5 +/- 1.9]%, P < 0.01) and reduced the percentage of the S-phase cells ([14.7 +/- 2.4], [11.1 +/- 5.2], [5.8 +/- 1.1] and [7.0 +/- 0.6]%, P < 0.01). RT-PCR and Western blot showed markedly up-regulated expressions of GSK3 P3 a3beta down-regulated expressions of MDM2 mRNA and proteins in the PC-3 cells treated with deguelin., Conclusion: Akt inhibitor deguelin can inhibit the proliferation of PC-3 human prostate cancer cells by affecting the down-stream signal molecules GSK3P3 and betaDM2 in the Akt pathway.

Objective: To observe the effects of leptin on activity of GSK-3β and explore its mechanism., Methods: C2C12 myoblasts differentiated for 3 days into myotubes in differentiation medium. Myotubes were stimulated by leptin (100 nmol/L) for 0, 5, 15 or 30 min respectively. Western blot was used to detect the expression levels of GSK-3β and phospho-GSK-3β (ser-9). Co-immunoprecipitation (CO-IP) was performed to determine the relationship among APPL1, leptin receptor and GSK-3β in the presence or absence of leptin. The expression level of GSK-3β at phospho-GSK-3β (ser-9) was detected in APPL1-suppressed C2C12 myotube while that of APPL1 at phospho-APPL1 (ser-401) determined in GSK-3β overexpressed/inhibited C2C12 cell., Results: Leptin time-dependently increased the phosphorylation level of GSK-3β at ser-9 in C2C12 cell, and the pGSK-3β level in cells incubated by leptin for 30 min was as 4.08 times as which in control cells (P < 0.01). The triple complex of APPL1, leptin receptor and GSK-3β, in the presence of leptin, the binding capacity between APPL1 and GSK-3β was stronger. The level of phospho-GSK-3β was significantly lower in APPL1-suppressed C2C12 cell compared with that in control cells. And the phosphorylation of APPL1 at ser-401 could be induced by GSK-3β., Conclusion: Leptin promotes muscle glycogen synthesis by inducing phosphorylation of GSK-3β in C2C12 cell. Such a function may be mediated by the triple complex of APPL1, leptin receptor and GSK-3β. Meanwhile, GSK-3β can also increase the phosphorylation of APPL1 at ser-401.

Objective: To investigate the expressions of the active form of glycogen synthase kinase-3(GSK-3)-pGSK-3α/β (Tyr279/216) and its downstream moleculor X-linked inhibitor of apoptosis protein (XIAP) in cholangiocarcinoma and to analyze their correlation with clinicopathological and survival significance., Methods: Immunohistoehemistry was used to detect the expressions of the active form of GSK-3- pGSK-3α/β (Tyr279/216) and its downstream moleculor XIAP proteins in 50 cholangiocarcinoma tissues and 20 normal bile duct tissues., Results: The positive rates of pGSK-3α/β (Tyr279/216) and XIAP were 62.0% and 68.0% in cholangiocarcinoma, and 10.0% and 25.0% in normal bile duct tissues, respectively. The intensity of pGSK-3α/β (Tyr279/216) and XIAP expressions in cholangiocarcinoma were significantly higher than that in the normal bile duct tissues (P < 0.001), and there was a significant correlation between pGSK-3α/β (Tyr279/216) and XIAP expressions (r = 0.544, P < 0.001). The expression of pGSK-3α/β(Tyr279/216) protein in cholangiocarcinoma was associated with TNM stage (P = 0.042), histological grade (P = 0.031), whereas the expression of XIAP protein in cholangiocarcinoma was correlated with CEA level (P = 0.006). Patients with positive expression of pGSK-3α/β (Tyr279/216) and XIAP demonstrate a significantly worse prognosis than that of patients with negative expression of pGSK-3α/β (Tyr279/216) and XIAP for overall survival (P = 0.002, P = 0.018). Multivariate survival analysis revealed that positive pGSK-3α/β (Tyr279/216) expression provided significant independent prognostic value for overall survival (P = 0.002)., Conclusions: The expressions of pGSK-3α/β(Tyr279/216) and XIAP proteins were significantly associated with the development and progression of cholangiocarcinoma. pGSK-3α/β(Tyr279/216) may be an important prognostic factor for survival of patients with cholangiocarcinoma.

The aim of this study was to investigate the effects of whole-body vibration on Wnt/β-catenin signaling pathway in bone marrow cells of ovariectomized osteoporosis rats. Thirty-six healthy 3-month old female Sprague Dawley (SD) rats were randomly divided into the following three groups by body weight: sham-operation (Sham), ovariectomized (OVX), and OVX whole-body vibration (WBV) groups. Ten weeks after ovariectomization, the rats of WBV group received vibration treatment (90 Hz, 15 min) twice per day. At the end of 8-week vibration, the whole-body bone mineral density (BMD) and body composition were detected by dual energy X-ray absorptiometry (DEXA) in vivo. The protein expressions of β-catenin and p-GSK3β in both bone marrow cells and bone marrow stromal cells were detected by Western blot. The results showed that, compared with OVX group, WBV group showed decreased fat mass and fat mass content, as well as increased lean body mass content. The BMD of the proximal tibia in WBV group was significantly higher than that in OVX group, however, there was no difference of BMD in whole-body and other positions between the two groups. The β-catenin expression in bone marrow stromal cells showed no difference between OVX and WBV groups. The p-GSK3β expression of bone marrow cells was increased in WBV group compared with that in OVX group, whereas bone marrow stromal cells from two groups did not exhibit the difference of the p-GSK3β expression. These results suggest that whole body vibration can stimulate the protein expression of p-GSK3β in bone marrow cells of ovariectomized osteoporosis rats, which could improve the bone loss induced by ovariectomization.

Objective: To investigate the activity of protein kinase B (Akt) and its downstream protein, glycogen synthase kinase-3beta (GSK-3beta) under hypoxia-ischemia (HI), and the possible regulation for axonal density., Methods: Postnatal day 10 SD rats were suffered the right common carotid artery ligation and 8% mixture of oxygen and nitrogen hypoxia 2.5 h to produce HI model. The expression of total and phosphorylated Akt and GSK-3beta was detected by western blot after HI. After pretreatment of Akt inhibitor, wortmannin or LY294002, Western blot detect the expression of total and phosphorylated of Akt, GSK-3beta at 4 h and 24 h after HI. After pretreatment of wortmannin, axonal density was determined by Bielschowsky silver impregnation, and histological injury was evaluated by hematoxylin and eosin (HE) staining., Results: The expression of total Akt and GSK-3beta remained unchanged after HI. p-Akt protein significantly decreased at 0.5 h, increased at 2 h and reached the highest at 4 h, returned to baseline at 8 h, declined at 24 and 48 h after HI, and finally returned to baseline again at 72 h compared with that of sham controls, p-GSK-3beta protein decreased at 0. 5 h, increased at 2 h, reached the highest at 4 h, returned to baseline at 8 and decreased at 24 h, reached the lowest at 48 h, and returned to baseline at 72 h. Wortmannin or LY294002 intervention didn't change the expression of total Akt and GSK-3beta, while decrease the p-Akt and p-GSK-3beta expression. HI cause decreased axonal density, and the histological injury of brain. Wortmannin pretreatment could aggravate the histological injury and decrease axonal density after HI., Conclusion: The Akt pathway is involved in axonal density and histological brain injury after HI in neonatal rat.

Objective: To observe the effect of Sam68 gene silencing on proliferation of nasopharyngeal carcinoma (NPC) cell line 5-8F and explore its possible molecular mechanism., Methods: The NPC cell line 5-8F was transfected with a small interfering RNA (siRNA) targeting Sam68 and the cell proliferation changes were observed. Quantitative RT-PCR and Western blotting were used to examine the changes in the expressions of Sam68, cell cycle-related proteins, and some up-stream proteins in the transfected cells., Results: Transfection of 5-8F cells with Sam68-specific siRNA significantly lowered the mRNA and proteins levels of Sam68, suppressed cell proliferation, decreased the expression of Cyclin D1, and increased the expression of p27. The transfected cells showed obviously decreased expressions of p-FOXO3a, p-Akt and p-GSK-3β, but the expressions of FOXO3a, Akt and GSK-3β were not obviously affected., Conclusions: Sam68 modulates the proliferation of NPC cells probably by activating Akt/FOXO3a pathway and regulating the cell proliferation-related molecules.

Objective: To explore the relationship among the expression of GSK3β, PI3K/Akt signaling transduction pathway,and cytokines IL6 in Chronic Rhinosinusitis., Methods: We assayed mRNA and protein for GSK3β, PI3K, Akt by using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), and measured the cytokines IL6 mRNA by using reverse transcription polymerase chain reaction (RT-PCR) in the nasal tissue from the patients with Chronic Rhinosinusitis with Nasal Polyps (CRSwNP), Chronic Rhinosinusitis without Nasal Polyps (CRSsNP) and control subjects., Results: The relative expression levels of GSK3β, PI3K, Akt and IL-6 in CRSwNP were 0.6254 ± 0.0584, 0.8239 ± 0.7186, 0.9369 ± 0.0823 and 0.8973 ± 0.0680. But the relative expression levels of GSK3β, PI3K, Akt and IL-6 in control subjects were 0.2684 ± 0.0726, 0.3578 ± 0.0994, 0.6721 ± 0.0590, 0.5898 ± 0.0891. There were significant differences between the groups of CRSwNP and control subjects (t values were 2.358, 3.071, 2.764, and 2.239, respectively, all P < 0.05). There was no significant difference of GSK3β, PI3K, Akt and GSK3β between the groups of CRSwNP and CRSsNP (t values were 1.597, 1.842, 1.468 and 0.926, respectively, all P < 0.05). GSK3β, PI3K, Akt were not only expressed in the cytoplasm of the epithelium and gland cells, but also in the cytoplasm of the inflammatory cell. GSK3β, PI3K, Akt protein in CRS were detected at a higher rate than the normal nasal tissue (values were 16.42, 16.25 and 15.57,respectively, all P < 0.01). However there was no significant difference of GSK3β, PI3K, Akt protein between the groups of CRSwNP and CRSsNP (values were 3.27, 2.85 and 2.46, respectively, all P > 0.05). There was a positive correlation trend among the expression of GSK3β, PI3K and Akt, and IL-6 in CRS (r values were 0.645, 0.617 and 0.583,respectively, all P < 0.01)., Conclusions: The abnormal expression of IL-6, PI3K, Akt and GSK3β in the nasal mucosa of CRS may play a pro-inflammatory role in the occurrence and development of CRS.

Objective: To investigate the effects of curcumin on the apoptosis of ischemia/reperfusion (I/R) induced H9c2 myocardial cells and the expression of glycogen synthase kinase-3 (GSK-3) and its phosphorylation state., Methods: I/R of H9c2 cells in vitro was simulated by an ischemic Tyrode solution. Cells were randomly divided into 3 groups, i.e., the model group (exposed to ischemic solution for 90 min followed by 30 min reperfusion with the normal Tyrode solution), the curcumin group (7.5 micromol/L curcumin added at the onset of reperfusion for 30 min), and the control group (exposed to normal Tyrode solution for 120 min). Then, the cell apoptosis was detected in 3 groups by flow cytometry. The expression levels of GSK-3, phosphotyrosine-GSK-3 (pTyr-GSK-3), and phosphoserine-GSK-3 (pSer-GSK-3) were detected by Western blot., Results: Compared with the control group,the apoptosis rate was obviously enhanced in the model group (t = 10.439, P = 0.000). And the relative expression levels of both pTyr-GSK-3 and pSer-GSK-3 significantly increased in the model group (t = 5.208, P = 0.006; t = 5.854, P = 0.004, respectively). Compared with the model group, the apoptosis rate and the expression of pTyr-GSK-3 significantly decreased in the curcumin group (t = -8.325, P = 0.001; t = -3.607, P = 0.023). Compared with the model group, the rate of viable cells and the expression of pSer-GSK-3 were significantly enhanced in the curcumin group (t = 9.165, P = 0.001; t = 3.747, P = 0.02)., Conclusions: Both pTyr-GSK-3 and pSer-GSK-3 might participate in the IR injured myocardial cells. Curcumin could reduce apoptosis of I/R injured myocardial cells, which might be correlated with GSK-3 inhibition by decreasing tyrosine phosphorylation and increasing serine phosphorylation.

Diabetes is a global threat threatening human health in the world, with an increasing incidence rate in recent years. The disorder of glucose metabolism is one of the major factors. As relevant glucose metabolic enzymes such as alpha-glucosidase, glucose-6-phosphatase (G-6-P), glycogen phosphorylase (GP) and glycogen synthase kinase-3 (GSK-3) get involved in and control the process of glucose metabolism, the regulation of the activity of glucose metabolic enzymes is of significance to the treatment of diabetes. Traditional Chinese medicines (TCMs) have been widely researched because of their low toxicology and high efficiency, and many extracts and components from TCMs have been proven to be regulators of glucose metabolic enzymes. Compared with anti-diabetic western medicines, anti-diabetic TCMs feature safety, reliability and low price. This essay summarizes the anti-diabetic effect of TCMs on regulating glucose metabolic enzymes.

Objective: To research the effects of glycogen synthase kinase (GSK3β) overexpression and GSK3β inhibitor SB-216763 on the proliferation of hepatic oval cells in rats and its regulatory mechanisms by Wnt signaling pathway., Methods: The hepatic oval cells WBF-344 were divided into the blank control group, GSK3β over-expression group, DMSO control group and GSK3β inhibitor groups, while the inhibitor groups set up three concentration gradients, that was 1, 5, 10 µmol/L. Using the GSK3β over-expression lentivirus, which had been identified correctly, and SB-216763 dealt with the cells WBF-344. The cells morphology of each group was observed under the phase contrast inverted microscope, and the expression of fluorescence in the lentivirus-transfected group was observed under the fluorescent microscope. The proliferation of each group cells was tested by CCK8 kits. The cells' apoptosis was detected by AnnexinV-FITC/PI kits. The expression of GSK3β, β-catenin and cyclin D1 were detected by Western blot., Results: The cells of GSK3β over-expression group were fewer and obvious aging. However, in each inhibitor added group, the cells' division and proliferation was vigorous, and the condition was good. Moreover, the cells' proliferation was getting stronger with the concentration of SB-216763 increasing. A large number of green fluorescence was expressed in the lentivirus-transfected cells. The cells' proliferation in GSK3β over-expression group restrained (t = 7.178, P < 0.01, as compared with control), while the cells' proliferation was vigorous in inhibitor groups (F = 45.030, P < 0.01, as compared with control). Flow Cytometry showed that the cells apoptosis was significant in GSK3β over-expression group. Western blot showed that the expression of GSK3β was increased, while the expression of β-catenin and cyclin D1 was decreased in the over-expression group. The expression of GSK3β had no significant difference among the control group and inhibitor groups. However, the expression of β-catenin and cyclin D1 was significantly increased with the concentration of SB-216763 increasing., Conclusions: The overexpression of GSK3β can inhibit the Wnt signaling pathway, thus restrain the cells' proliferation and promotes apoptosis. SB-216763 can activate the Wnt pathway, thus promotes cells' proliferation.

Objective: To investigate the underlying mechanism of the protective effects of resveratrol on oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes., Methods: H9c2 cells, a permanent cell line derived from embryonic rat cardiac tissue, and then randomly divided into control group [PBS, cells exposed to H2O2 (600 µmol/L) for 20 min to induce mitochondrial oxidant damage], resveratrol group (0.01, 0.1, 1, 5, 10 and 20 µmol/L for 20 min at 20 min before exposing to H2O2), resveratrol plus inhibitor group (1 µmol/L KT5823 for 10 min at 10 min before 5 µmol/L resveratrol treatment) and inhibitor group (1 µmol/L KT5823 for 10 min). Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE) and the mitochondrial permeability transition pore (mPTP) opening was evaluated by measuring the decrease of TMRE fluorescence intensity. Immunofluorescence assay was used to observe GSK-3β phosphorylation. The phosphorylation of GSK-3β and VASP were determined by Western blot. To detect intracellular NO, cells were loaded with DAF-FM DA (specific fluorescent dye of NO) and imaged with confocal microscopy., Results: Compared to the control group, resveratrol (0.01-5 µmol/L) attenuated H2O2-induced mitochondrial damage reflected by attenuating the H2O2-induced TMRE fluorescence intensity decrease in a dose-dependent manner and the efficacy of 10 and 20 µmol/L resveratrol was significantly lower than that of 5 µmol/L resveratrol. Resveratrol also significantly upregulated the protein expression of VASP and increased GSK-3β Ser(9) phosphorylation, which could lead the inactivation of GSK-3β. These effects of resveratrol could be significantly abolished by protein kinase G inhibitor KT5823, while KT5823 alone did not affect GSK-3β and VASP phosphorylation. Confocal microscopy showed that DAF-FM (specific NO indicator) was similar between resveratrol and control group, suggesting that resveratrol did not produce NO., Conclusions: Resveratrol could attenuate oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes by inactivating GSK-3β via cGMP/PKG signaling pathway independent of NO-related mechanism.

Objective: To determine the mechanism underlying the therapeutic activities of glycogen synthase kinase 3b (GSK3b) against hepatic ischemia-reperfusion (H-IR) injury by investigating the inhibitive effects of GSK3b on inflammation mediated by Toll-like receptor 4 (TLR4)., Methods: C57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups: the H-IR untreated model (control group), and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide (LPS) endotoxin alone (inflammation group) or with pretreatment of the SB216763 GSK3b-specific inhibitor (intervention group). To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The cells were then divided into the same three groups as the whole mouse system: control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively., Results: The phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-stimulated ERK, JNK and p38 phosphorylation in macrophages. In the mouse model, GSK3b activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 (control: 0.21 ± 0.08, inflammation: 0.83 ± 0.21, intervention: 1.76 ± 0.67; F = 3.16, P = 0.027) but to significantly inhibit the gene expression of pro-inflammatory cytokines IL-12 (control: 0.11 ± 0.05, inflammation: 0.85 ± 0.11, intervention: 0.43 ± 0.10; F = 2.67, P = 0.038), TNF-a, (control: 0.052 ± 0.012, inflammation: 8.11 ± 0.98, intervention: 3.9 ± 0.82; F = 4.13, P = 0.016), IL-6 (control: 0.22 ± 0.08, inflammation: 6.37 ± 0.81, intervention: 2.11 ± 0.63; F = 3.21, P = 0.024), and IL-1b (control: 0.12 ± 0.07, inflammation: 2.51 ± 0.62, and intervention: 1.28 ± 0.33; F = 2.22, P = 0.030)., Conclusion: Inhibition of GSK3b selectively regulates the expression of anti-inflammatory and pro-inflammatory cytokines in liver Kupffer cells (liver macrophages). This process may be one of the mechanisms underlying the ability of GSK3b to ameliorate hepatic ischemia-reperfusion injury, possibly because inhibition of pro-inflammatory cytokines may indirectly mediate liver cell apoptosis.

This study was proposed to investigate the sensitivity and resistence of HEL cells co-cultured with bone marrow stromal HS-5 cells to chemotherapeutic drugs. HEL cells were cultured in direct contact with HS-5 cells for 6, 12, and 24 h. Cell Counting Kit-8 (CCK-8) was used to determine the sensitivity of HEL cell to cytarabine, methotrexate, VP16, and daunomycin. Cell cycle distribution was determined by using flow cytometry. Real-time RT-PCR was performed to detect the transcription levels of p19, p21, p27, MDR1, ABCG2 and bcl-2. Western blot was performed to determine the protein levels of p-Akt(Ser473), p-glycogen synthase kinase 3β (p-GSK3β(Ser9)), p-signal transducer and activator of transcription (p-STAT3(Tyr705)), Bcl-2, cleaved-Notch1(V1754), and Hes1. The results showed that chemo-sensitivity of HEL cells was remarkably reduced when co-cultured with HS-5 cells. HEL cells were arrested in the G(0)/G(1) phase after co-culture for 24 h. Transcription of p21 was significantly up-regulated at 6 h. Transcription of p19 decreased at 12 h and returned to baseline at 24 h. No significant changes in the mRNA expression of other genes were found. The expressions of p-Akt(Ser473), p-GSK3β(Ser9), cleaved-Notch1(V1754) and Bcl-2 proteins were significantly up-regulated in HEL cells, and Hes1 protein was significantly down-regulated. There was no change in p-STAT3(Tyr705) expression. It is concluded that the direct contact with HS-5 cells can reduce the chemo-sensitivity of HEL cells.

Progressive dementia is described as the first and most prominent symptom of Alzheimer's disease (AD), and hyperphosphorylation of microtubule associated Tau protein (MAPT) plays a key role in neurodegeneration and neuronal dysfunction in AD and other neurodegenerative diseases. This paper reviews several protein kinases and phosphatases which can phosphorylate/dephosphorylate Tau protein, and evaluates a therapeutic strategy based on targeted inhibition of Tau kinases and activation of Tau phosphatases.

Objective: To explore the effects of curcumin on paclitaxel resistance reversal of SKOV3-TR30 cell line and its mechanism., Methods: The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay was performed to determine the sensitivity of curcumin-treated SKOV3-TR30 cells to paclitaxel. The cell cycle distribution of SKOV3-TR30 was analyzed by flow cytometry. And the expression level of glycogen synthase kinase-3 (GSK-3) protein was detected by Western blot., Results: IC(50) of paclitaxel in SKOV3-TR30 decreased with a treatment of curcumin. And the reversal times was 3.0. Flow cytometric analysis of curcumin-treated SKOV3-TR30 cells demonstrated a distinct G(2)-M phase block (78.5 ± 6.4)% after a 12-hour treatment of paclitaxel versus SKOV3-TR30 cells without curcumin. There was a lack of G(2)-M phase arrest (only 27.0% ± 2.9%). The expression of GSK-3 protein in SKOV3-TR30 cells decreased with the 12 and 24-hour treatments of curcumin., Conclusion: Curcumin can partially reverse the paclitaxel-resistance of SKOV3-TR30 cells through a down-regulation of GSK-3.

Objective: To investigate the change in glycogen synthase kinase-3β (GSK-3β) in spinal cord neurons in rats with incisional pain (IP)-remifentanil-induced hyperalgesia., Methods: 32 SD male rats (240 - 260 g) were randomly divided into 4 groups (n = 8 each): group R, group I, group R + I and group C. IP was established as Brennan's description. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured 24h before anesthesia and 2, 6, 24, 48 h after anesthesia. The rats were sacrificed after the last threshold measurement. The expressions of GSK-3β mRNA in rats' spinal cord neurons were determined by real-time PCR. The expressions of GSK-3β and pGSK-3β in rats' spinal cord neurons were determined by Western blot., Results: Remifentanil-induced hyperalgesia developed in group R, I and R + I. The expression of GSK-3β mRNA and the expression of GSK-3β in rats' spinal cord neurons were highest in group R + I. In addition, the ratio of pGSK-3β/GSK-3β was smallest in group R + I., Conclusion: These data indicate that the increased GSK-3β activity in rats spinal cord neurons is involved in remifentanil-induced hyperalgesia.

Objective: To investigate the expressions of phosphorylated protein kinase B (p-AKT), phosphorylated glycogen synthase kinase 3β (p-GSK3β) and β-catenin proteins and to evaluate their relationship with the clinical pathological characteristics in epithelial tumors of the ovary., Methods: The expression of p-AKT, p-GSK3β, and β-catenin was detected with immunohistochemical staining (EnVision method) in 10 cases of benign epithelial neoplasia, 10 cases of borderline epithelial neoplasia and 70 cases of ovarian carcinoma. The relationship of the expression of p-AKT, p-GSK3β and β-catenin with the clinical pathological features was analyzed., Results: The positive expression rates of p-AKT, p-GSK3β and β-catenin in epithelial ovarian carcinoma were 67.1% (47/70), 60.0% (42/70) and 71.4% (50/70), respectively. Compared to the results of benign and borderline epithelial neoplasia, the expression of the three proteins in carcinoma of the ovary was significantly different (all P < 0.05).Positive correlation was found between p-AKT and p-GSK3β, p-GSK3β and β-catenin, and p-AKT and β-catenin in epithelial ovarian carcinoma (r = 0.546, 0.581, 0.500, respectively; all P < 0.05). Compared to the results of benign and borderline epithelial neoplasia, the expression of p-AKT protein in epithelial ovarian carcinoma was significantly different (all P < 0.05). The expression of p-AKT was correlated with the differentiation of epithelial ovarian carcinoma (P < 0.05), but no relationship was found between its expression and histological classification and FIGO staging (P > 0.05). The expression of p-GSK3β and β-catenin in epithelial ovarian carcinoma were both higher than that in benign and borderline epithelial neoplasia (P < 0.05), and correlated with tumor differentiation and FIGO staging (P < 0.05), but no relationship were found between their expression with histological classification (P > 0.05)., Conclusions: Positive correlations are found between p-AKT, p-GSK3β and β-catenin in epithelial ovarian carcinoma. The activation of β-catenin is possibly correlated with inactivation of p-GSK3β that binds to p-AKT.