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AbstractContext Although Tongguan capsule (TGC) is used in the treatment of coronary atherosclerotic disease, the exact mechanism remains unclear.Objective Network pharmacology and experimental validation were applied to examine the mechanism of TGC for treating myocardial ischemia-reperfusion injury (MIRI).Materials and methods The components and candidate targets were searched based on various databases such as TCMSP, TCMID, BATMAN-TCM. The binding ability was determined by molecular docking. The ischemia-reperfusion (I/R) model was constructed by ligating the left anterior descending (LAD) coronary artery. APOE-/- mice were divided into three groups (n = 6): Sham group, I/R group, and TGC group (1 g/kg/d). To further verification, HCAEC cells were subjected to hypoxia-reoxygenation (H/R) to establish in vitro model.Results The compounds, such as quercetin, luteolin, tanshinone IIA, kaempferol and bifendate, were obtained after screening. The affinity values of the components with GSK-3β, mTOR, Beclin-1, and LC3 were all

Bian-Se-Tong mixture (BSTM) is an optimized formulation based on the classical prescription “Zhizhu pill”, which is widely used in the clinical treatment of slow-transit constipation (STC). The potential molecular mechanism of BSTM therapy for STC was investigated by network pharmacology prediction combined with animal experiments. The active components of BSTM were screened via the TCMSP platform. The GeneCards, OMIM and DrugBank databases were used to search for STC targets. With the help of the Biogenet tool, a protein interaction network between drugs and disease targets was constructed, and the intersection network of the two was extracted to obtain the key targets of BSTM in the treatment of STC. GO and KEGG enrichment analyses of key targets were carried out with Metascape. Loperamide hydrochloride was used to establish an STC rat model, and the key targets and related pathways were preliminarily verified. The important signaling pathways included the PI3K-Akt, MAPK, IL-17, cAMP, and cell cycle signaling pathways. The experimental results showed that BSTM treatment increased the body weight of STC rats and increased the fecal particle number, fecal water content and intestinal carbon ink promotion rate within 24 h. Further pathological changes in the colon of the rats were also observed. In-depth mechanistic studies have shown that BSTM can significantly reduce the apoptosis of intestinal Cajal cells, downregulate the expression of Bax and c-Caspase 3, upregulate the expression of Bcl-2 and c-kit, and promote the phosphorylation of AKT. The results showed that BSTM can significantly relieve constipation in STC rats via a mechanism related to activating the PI3K-Akt signaling pathway and improving Cajal cell apoptosis.

Abstract Background Toxoplasma gondii is a neurotropic single-celled parasite that can infect mammals, including humans. Central nervous system infection with T. gondii infection can lead to Toxoplasma encephalitis. Toxoplasma infection can cause endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, which ultimately can lead to apoptosis of host cells. The dense granule protein GRA3 has been identified as one of the secretory proteins that contribute to the virulence of T. gondii; however, the mechanism remains enigmatic. Methods The expression of the GRA3 gene in RH, ME49, Wh3, and Wh6 strains was determined using quantitative real-time polymerase chain reaction (qRT–PCR). pEGFP-GRA3Wh6 was constructed by inserting Chinese 1 Wh6 GRA3 (GRA3Wh6) cDNA into a plasmid encoding the enhanced GFP. Mouse neuro2a (N2a) cells were transfected with either pEGFP or pEGFP-GRA3Wh6 (GRA3Wh6) and incubated for 24–36 h. N2a cell apoptosis and ER stress-associated proteins were determined using flow cytometry and immunoblotting. Furthermore, N2a cells were pretreated with GSK2656157 (a PERK inhibitor) and Z-ATAD-FMK (a caspase-12 inhibitor) before GRA3Wh6 transfection, and the effect of the inhibitors on GRA3Wh6-induced ER stress and apoptosis were investigated. Results GRA3 gene expression was higher in the less virulent strains of type II ME49 and type Chinese 1 Wh6 strains compared with the virulent strains of type I RH strain and type Chinese 1 Wh3 strain. Transfection with GRA3Wh6 plasmid induced neuronal apoptosis and increased the expression of GRP78, p-PERK, cleaved caspase-12, cleaved caspase-3, and CHOP compared with the control vector. Pretreatment with GSK2656157 and Z-ATAD-FMK decreased apoptosis in N2a cells, and similarly, ER stress- and apoptosis-associated protein levels were significantly decreased. Conclusion GRA3 induces neural cell apoptosis via the ER stress signaling pathway, which could play a role in toxoplasmic encephalitis. Graphical Abstract

Traditional Chinese medicine (TCM) has attracted a great deal of attention in the treatment of cerebral ischemia is credited with the remarkable neuroprotective effects. However, the imperfect functional mechanism of TCM is a major obstacle to their application. Many studies have been conducted to illustrate the pathophysiology of post-ischemic cerebral ischemia by elucidating the neuronal cell death pathway. Meanwhile, a new type of cell death, ferroptosis, is gradually being recognized in various diseases and is becoming a new pathway of therapeutic intervention strategy to solve many health problems. Especially since ferroptosis has been found to be closely involved into the pathogenesis of cerebral ischemia, it has been considered as a key target in the treatment of cerebral ischemia. Therefore, this paper reviews the latest research findings about the treatment of cerebral ischemia with TCM focused on ferroptosis as a target. Also, in order to explores the possibility of a new approach to treat cerebral ischemia with TCM, we discusses the correlation between ferroptosis and other cell death pathways such as apoptosis and autophagy, which would provide references for the following researches.

Abstract Background Adjacent segmental intervertebral disk degeneration (ASDD) is a major complication secondary to lumbar fusion. Although ASSD pathogenesis remains unclear, the primary cause of intervertebral disk degeneration (IVDD) development is apoptosis of nucleus pulposus (NP). Raloxifene (RAL) could delay ASDD by inhibiting NP apoptosis. Methods An ASDD rat model was established by ovariectomy (OVX) and posterolateral spinal fusion (PLF) on levels 4–5 of the lumbar vertebrae. Rats in the treatment groups were administered 1 mg/kg/d RAL by gavage for 12 weeks, following which, all animals were euthanized. Lumbar fusion, apoptosis, ASDD, and vertebrae micro-architecture were evaluated. Results RAL maintained intervertebral disk height (DHI), delayed vertebral osteoporosis, reduced histological score, and inhibited apoptosis. The OVX+PLF+RAL group revealed upregulated expression of aggrecan and B-cell lymphoma-2 (bcl2), as well as significantly downregulated expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), metalloproteinase-13 (MMP-13), caspase-3, BCL2-associated X (bax), and transferase dUTP nick end labeling (TUNEL) staining. Micro-computed tomography (Micro-CT) analysis revealed higher bone volume fraction (BV/TV), bone mineral density (BMD), and trabecular number (Tb.N), and lower trabecular separation (Tb.Sp) in OVX+PLF+RAL group than in the OVX+PLF group. Conclusions RAL can postpone ASDD development in OVX rats through inhibiting extracellular matrix metabolic imbalance, NP cell apoptosis, and vertebral osteoporosis. These findings showed RAL as a potential therapeutic target for ASDD.

Oncolytic viruses have been emerging as a promising therapeutic option for cancer patients, including lung cancer. Orf virus (ORFV), a DNA parapoxvirus, can infect its natural ungulate hosts and transmit into humans. Moreover, the ORFV has advantages of low toxicity, high targeted, self-amplification and can induce potent Th1-like immunity. This study explored the therapeutic potential of ORFV infection for human lung cancer therapy and investigated the molecular mechanisms. We used a previously described ORFV NA1/11 strain and tested the oncolysis of ORFV NA1/11 in two lines of lung cancer cells in vitro and in vivo. Treatment of both cell lines with ORFV NA1/11 resulted in a decrease in cell viability by inducing cell cycle arrest in G2/M phase, suppressing cyclin B1 expression and increasing their apoptosis in a caspase-dependent manner. The ORFV NA1/11-infected lung cancer cells were highly immunogenic. Evidently, ORFV NA1/11 infection of lung cancer cells induced oncolysis of tumor cells to release danger-associated molecular patterns, and promoted dendritic cell maturation, and CD8 T cell infiltration in the tumors by enhancing CXCL16 secretion. These findings may help to understand the molecular mechanisms of ORFV oncolysis and aid in the development of novel therapies for lung cancer.

Glioblastoma (GBM) is the most lethal primary brain tumor in the central nervous system with limited therapeutic strategies to prolong the survival rate in clinic. TNF-related apoptosis-inducing ligand (TRAIL)-based strategy has been demonstrated to induce cell death in an extensive spectrum of tumor cells, including GBM, while a considerable proportion of malignant cells are resistant to TRAIL-induced apoptosis. MiR-137 is highly expressed in the brain, but significantly decreases with advanced progression of GBM. However, the functional link between miR-137 and TRAIL-induced apoptosis in GBM cells has not been established. Here, GBM cells were transfected with miR-137, and gene expression levels were examined by qRT-PCR and western blot. Apoptotic cells were measured by Annexin-V staining and TUNEL assay. Our data showed that miR-137 sensitizes GBM cells to the TRAIL-mediated apoptosis. Mechanistically, we identified that XIAP is a bona fide target of miR-137, which is essential for miR-137-regulated sensitivity of TRAIL-induced cell death in GBM cells. Finally, in a xenograft model, combined utilization of miR-137 and TRAIL potently suppresses tumor growth in vivo. Collectively, we demonstrate that a miR-137-XIAP axis is required for the sensitivity of TRAIL-induced cell death and shed a light on the avenue for the treatment of GBM.

Aflatoxin B1 (AFB1) is an unavoidable environmental pollutant commonly found in feed and foodstuffs. It is the most toxic one of all the aflatoxins, which can cause severe impairment to testicular development and function. Yet, the underlying mechanisms of reproductive toxicity in rams sheep remain inconclusive. The study was designed to explore the effects of AFB1 on sheep testes through rumen-microbiota, oxidative stress and apoptosis. Six-month-old male Dorper rams (n = 6) were orally administrated with 1.0 mg/kg AFB1 (dissolved in 20 mL 4% ethanol) 24 h before the experiment. At the same time, rams in the control group (n = 6) were intragastrically administrated with 20 mL 4% ethanol. It was observed that acute AFB1 poisoning had significant (p

Background: Oxidative stress-induced endothelial cell death, such as apoptosis and autophagy, plays a critical role in ischemia-reperfusion injury. Protocatechualdehyde (PCA) is a major bioactive component of the traditional Chinese medicine Salvia miltiorrhiza Bunge (Lamiaceae), and it has been proved to be effective in the prevention and treatment of ischemic cardiovascular and cerebrovascular diseases. However, its role in oxidative stress-induced endothelial cell death and its underlying mechanisms remains unclear. This study aims to investigate the effects and mechanisms of PCA on endothelial cell apoptosis and autophagy induced by oxygen-glucose deprivation/reoxygenation (OGD/R) injury.Methods: After OGD/R induction, human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of PCA. Cell viability, apoptosis, and autophagy were detected by Cell Counting Kit-8 assay, flow cytometry, and monodansylcadaverine assay, respectively. Western blot was applied to explore the effects of PCA on the expression levels of relevant protein factors.Results: The results show that PCA significantly promoted cell survival rate and cell proliferation and enhanced the antioxidant activity in OGD/R-induced HUVECs. PCA inhibited HUVECs apoptosis, as evidenced by decreased expression of cleaved-caspase-3, Bcl2-associated X (BAX), and increased expression of Bcl-2. PCA induced autophagy by reducing the expression of P62 while increasing the expression of Beclin-1 and LC3 II/I. Meanwhile, PCA enhanced the expression of Sirtuin 1 (SIRT1) and suppressed the expression of P53. When SIRT1 was inhibited by selisistat or SIRT1 small-interfering RNA, the anti-apoptotic and pro-autophagy abilities of PCA were attenuated.Conclusion: These results demonstrated that PCA rescued HUVECs from OGD/R-induced injury by promoting autophagy and inhibiting apoptosis through SIRT1 and could be developed as a potential therapeutic agent against ischemic diseases.

Abstract Background Ophiorrhiza pumila, belonging to the genus Ophiorrhiza (Rubiaceae), is distributed throughout tropical and subtropical Asia. In this study, we evaluated for the first time the anti-proliferation and anti-migration effects of ethanol extract of O. pumila (OPE) on HepG2 and SMMC-7721 cells, and explored the related mechanism. Methods OPE was prepared by percolation with 95% ethanol and its main compounds were analyzed by HPLC-MS2. The anti-proliferation effect of OPE was evaluated by the CCK-8 assay and colony formation assay. Cell cycle distribution, apoptosis, and reactive oxygen species (ROS) level were detected by flow cytometry. Migration and invasion abilities were detected by Transwell migration/invasion assays. The expression of correlated proteins was determined using western blotting. Results A total of 5 tentative compounds were identified from OPE, including pumiloside, deoxypumiloside, camptothecin, aknadinine, and β-stigmasterol. OPE displayed strong cytostatic effects on HepG2 and SMMC-7721 cells. OPE induced G2/M phase cell cycle arrest, increased apoptosis, and augmented ROS production in these cell lines. In addition, OPE possessed a significant inhibition on cell migration and invasion by reduction of MMP-9 and MMP-2 expression. Moreover, OPE significantly suppressed the phosphorylation of p65. Conclusions Our data showed that OPE suppresses liver cancer cell proliferation and migration, which is possibly involved with the inhibition of the NF-κB pathway.

Aflatoxin B1 (AFB1) is a common mycotoxin in food and in the environment that lead to multi-organ injury in humans and animals. The objective of this study was to evaluate the detoxification properties of dietary total flavonoids of Rhizoma drynariae (TFRD), a Chinese herbal, on aflatoxin B1 (AFB1)-induced hepatic oxidative damage and apoptosis of liver of broiler chickens. A total of 160 healthy specific pathogen free (SPF) 21-day-old broilers were randomly allocated to 4 groups, including the CON group (basal diet), TFRD group (basal diet with 125 mg/kg TFRD), AFB1 group (100 μg/kg body weight), and AFB1 (100 μg/kg body weight) + TFRD (basal diet with 125 mg/kg TFRD) group. The exposure of AFB1 continued for seven days. The results showed that TFRD treatment alleviated the abnormal changes of growth performance and liver morphology, reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Moreover, TFRD promoted the antioxidant capacity of serum, increased the activities of total superoxide dismutase (T-SOD), oxidized glutathione (GSSG) and glutathione (GSH) (p 0.05). Meanwhile, supplementation of TFRD significantly increased the expression of antioxidant-related genes (SOD, CAT, GST, and GPX1) in liver (p

The widespread fungal toxin Aflatoxin B1 (AFB1) is an inevitable pollutant affecting the health of humans, poultry, and livestock. Although studies indicate that AFB1 is hepatotoxic, there are few studies on AFB1-induced hepatotoxicity in sheep. Thus, this study examined how AFB1 affected sheep liver function 24 h after the animals received 1 mg/kg bw of AFB1 orally (dissolved in 20 mL, 4% v/v ethanol). The acute AFB1 poisoning caused histopathological injuries to the liver and increased total bilirubin (TBIL) and alkaline phosphatase (AKP) levels. AFB1 also markedly elevated the levels of the pro-inflammatory cytokines TNF-α and IL-6 while considerably reducing the expression of antioxidation-related genes (SOD-1 and SOD-2) and the anti-inflammatory gene IL-10 in the liver. Additionally, it caused apoptosis by dramatically altering the expression of genes associated with apoptosis including Bax, Caspase-3, and Bcl-2/Bax. Notably, AFB1 exposure altered the gut microbiota composition, mainly manifested by BF311 spp. and Alistipes spp. abundance, which are associated with liver injury. In conclusion, AFB1 can cause liver injury and liver dysfunction in sheep via oxidative stress, inflammation, apoptosis, and gut-microbiota disturbance.

AIM: To discuss the effect and mechanism of melatonin on diabetic retinopathy in rats and its mechanism.METHODS:Totally 40 healthy male SD rats were choose and randomly divided into 4 groups, normal control group, diabetid group, low dose melatonin group and high dose melatonin group. The changes of retinal structure were observed by hematoxylin-eosin staining(HE staining), ultrastructure of retinal ganglion cells were observed by electron microscopy, superoxide dismutase(SOD)and malondialdehyde(MDA)level in the retina tissue were detected by xanthine oxidase method, and protein expression levels of B cell lymphoma/lewkmia-2(Bcl-2), Bcl-2 associated X protein(Bax)and P53 were detected by Western Blot.RESULTS: HE staining results showed that the rat retina tissue structure of the normal control group was clear, the retinal tissue of diabetes group rats were not clear, the nerve fiber layer was edema, the retina cells of low dose melatonin group were probably distinct, the interlayer cells were arranged neatly, and the inner and outer nuclear layers were slightly disordered, rats retinal structure in high dose melatonin group were further improved than that in low dose melatonin group; Electron microscope results showed that compared with diabetes group, retinal ganglion cells form in melatonin group were improved in different extent; Xanthine oxidase method detected that SOD level in retinal tissue of melatonin group rats were higher than that in diabetic group, the MDA level were lower than that in diabetic group, compared with diabetes group the differences both have statistically significant(PPCONCLUSION: Melatonin could improve the morphological changes of retina in diabetic rats and inhibit diabetic retinopathy to some extent.

The therapeutic approach of liver fibrosis is still an unsolved clinical problem worldwide. Notably, the accumulation of extracellular matrix (ECM) in the liver is mediated by the production of cytokines and growth factors, such as transforming growth factor-β1 (TGF-β1) in hepatic stellate cells (HSCs). Ring finger protein 2 (RNF2) was identified as the catalytic subunit of polycomb repressive complex 1 (PRC1), mediating the monoubiquitination of histone H2A. In recent years, a growing amount of evidence suggests that RNF2 may play an important role in multiple pathological processes involved in cancer. Here, we explored the role of RNF2 in liver fibrogenesis and its potential mechanisms. The results showed that RNF2 was up-regulated in human fibrotic liver tissue. Knockdown of RNF2 led to a decreasing expression of collagen1 and α-smooth muscle actin (α-SMA) in LX-2 cells, which was upregulated by RNF2 overexpression. Moreover, RNF2 overexpression significantly promoted TGF-β1-induced LX-2 cell proliferation but decreased apoptosis. Furthermore, knockdown of RNF2 inhibited the activation of ERK/p38 signaling pathways induced by TGF-β1. These data suggested that RNF2 is an effective pro-fibrogenic factor for HSC activation via ERK/p38 signaling pathway. RNF2 inhibition might be a promising therapeutic target for liver fibrosis.

Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene, which is involved in the RAS/MAPK cell signaling transduction process. SHP2 has been shown to contribute to the progression of various cancers and is emerging as an important target for anti-tumor drug research. However, past efforts to develop SHP2 inhibitors into drugs have been unsuccessful owing to the positively charged nature of the active site pocket tending to bind negatively charged groups that are usually non-drug-like. Here, a series of uncharged pyrazoline derivatives were designed and developed as new SHP2 inhibitors using a structure-based strategy. Compound 4o, which exhibited the strongest SHP2 inhibitory activity, bound directly to the catalytic domain of SHP2 in a competitive manner through multiple hydrogen bonds. Compound 4o affected the RAS/MAPK signaling pathway by inhibiting SHP2, and subsequently induced apoptosis and growth inhibition of HCT116 cells in vitro and in vivo. Notably, the oral administration of compound 4o in large doses showed no obvious toxicity. In summary, our findings provide a basis for the further development of compound 4o as a safe, effective and anti-tumor SHP2 inhibitor.

Abstract Background Our previous study showed that during in vitro experiments changes in calcium concentration were associated with apoptosis. We presumed that the calcium ion might play a role as intermediate messenger for apoptosis-related genes. No such evidence has been reported in the literature. Here, we investigate the effect of calcium ionophore A23187 on the apoptosis of rat hepatic stellate cells (HSCs) stimulated by transforming growth factor-β1 (TGF-β1) to explore the mechanism of apoptosis through the endoplasmic reticulum stress pathway. Methods The apoptotic rate was determined using flow cytometry. The changes in Ca2+ level in HSCs were examined with laser confocal microscopy. The expressions of caspase-12 GRP78 and caspase-9 were assayed via western blot. Results The respective apoptosis rates for the blank group, the TGF-β1 group and the TGF-β1 + low, medium and high dose calcium ionophore A23187 groups were 3.40 ± 0.10%, 1.76 ± 0.12%, 5.86 ± 0.31%, 11.20 ± 0.48% and 15.08 ± 0.75%, with significant differences between the groups (p

The main aim of this study was to investigate the potent anti-apoptosis and anti-pyroptosis effects of apple polyphenols extract (APE) on dextran sulfate sodium model group (DSS)-induced acute ulcerative colitis (UC) and the protective effect of APE against acute UC-related neuroinflammation and synapse damage. Forty-three C57BL/6 male mice were randomly divided into a control group (CON), a 3% DSS model group (DSS), a 500 mg/(kg·bw·d) APE group (HAP), and a 125 (LD) or 500 (HD) mg/(kg·bw·d) APE treatment concomitantly with DSS treatment group. The results showed that APE significantly ameliorated DSS-induced acute UC through inhibiting intestinal epithelial cell (IEC) apoptosis and the Caspase-1/Caspase-11-dependent pyroptosis pathway, with increased BCL-2 protein expression and decreased protein levels of NLRP3, ASC, Caspase-1/11, and GSDND. Furthermore, APE significantly reduced acute UC-related neuroinflammation and synapse damage, supported by decreased mRNA levels of hypothalamus Cox-2 and hippocampus Gfap and also increased the mRNA levels of hypothalamus Psd-95. The increased protein expression of ZO-1 and Occludin improved the intestinal barrier integrity and improved the function of goblet cells by upregulating the protein level of MUC-2 and TTF3 accounted for the beneficial effects of APE on UC-associated neuroinflammation. Therefore, APE might be a safe and effective agent for the management of acute UC.

Abstract Background Mesenchymal stem cells (MSCs) are emerging as an extremely promising therapeutic agent for tissue repair. However, limitations exist such as the low numbers of MSCs obtained from donors, and the poor survival and function of donor cells. Omentin-1, a new fat depot-specific secretory adipokine, exerts proproliferation, prosurvival, and proangiogenic functions in certain cells via an Akt-dependent mechanism; however, little is known about the influence of omentin-1 on MSCs. Methods MSCs were isolated from 60–80 g donor rats. Cell proliferation was assessed with CCK-8 and EdU assay. Cell cycle, apoptosis ratio, reactive oxygen species concentration, and mitochondrial membrane potential were detected by flow cytometry. Hoechst 33342 dye was used to assess morphological changes of apoptosis. Expression levels of Akt, FoxO3a, GSK-3β, and apoptosis- and cell cycle-associated proteins were detected by Western blotting. Tube formation assay was used to test the angiogenesis role of conditioned medium from MSCs in vitro. The cytokine secretion was assessed by ELISA. Results After treatment with omentin-1 (100–800 ng/ml), MSCs displayed a higher proliferative capacity with an increasing number of cells in the S and G2 phase of the cell cycle. Moreover, omentin-1 preconditioning for 1 h could protect MSCs against H2O2-induced apoptosis in a concentration-dependent manner. Furthermore, omentin-1 pretreatment reduced the excessive reactive oxygen species. Western blots revealed that increased Bcl-2 and decreased Bax appeared in MSCs after omentin-1 incubation, which inhibited the mitochondrial apoptosis pathways with evidence showing inhibition of caspase-3 cleavage and preservation of mitochondrial membrane potential. Omentin-1 could enhance angiogenic growth factor secretion and elevate the ability of MSCs to stimulate tube formation by human umbilical vein endothelial cells (HUVECs). Furthermore, omentin-1 enhanced Akt phosphorylation; however, blockade of the PI3K/Akt pathway with an inhibitor, LY294002 (20 μM), suppressed the above beneficial effects of omentin-1. Conclusion Omentin-1 can exert beneficial effects on MSCs by promoting proliferation, inhibiting apoptosis, increasing secretion of angiogenic cytokines, and enhancing the ability for stimulating tube formation by HUVECs via the PI3K/Akt signaling pathway. Thus, omentin-1 may be considered a candidate for optimizing MSC-based cell therapy.

Background: Cyclosporine A (CsA) is a commonly used clinical immunosuppressant. However, CsA exposure in rabbits during the gestation period was shown to cause a postnatal decrease in the number of nephrons, with the effects remaining unknown. In this study, we aimed to explore the effects of CsA on metanephros development in the pregnant BALB/c mice. Methods: Pregnant mice were randomly divided into two groups, and CsA (10 mg·kg−1·d−1) was subcutaneously injected from gestation day 10.5 to day 16.5 in the CsA group, whereas a comparable volume of normal saline was given to the control group. All of the mice were sacrificed on gestation day 17.5 and serum CsA concentration was measured. The fetuses were removed and weighed, and their kidneys were prepared for histological assessment and polymerase chain reaction assay. In an in vitro experiment, embryo kidneys of fetal mice on gestation day 12.5 were used, and CsA (10 μmol/L) was added in the culture of the CsA group. The growth pattern of the ureteric bud and nephrons was assessed by lectin staining. Results: No significant differences in the weight of embryo (4.54 ± 1.22 vs. 3.26 ± 1.09 mg) were observed between the CsA and control groups, the thickness of the cortical (510.0 ± 30.3 vs. 350.0 ± 29.7 μm, P < 0.05) and nephrogenic zone (272.5 ± 17.2 vs. 173.3 ± 24.0 μm, P < 0.05), and the number of glomeruli (36.5 ± 0.7 vs. 27.5 ± 2.1, P < 0.05) were reduced in the CsA group when compared to the control group. The cell proliferation of Ki-67 positive index between control and CsA group (307.0 ± 20.0 vs. 219.0 ± 25.0, P < 0.05) in the nephrogenic zone was decreased with the increase of apoptotic cells (17.0 ± 2.0 vs. 159.0 ± 33.0, P < 0.05). The mRNA expression of WT-1, Pax2, and Pax8 was downregulated by CsA treatment. As for the in vitro CsA group, the branch number of the ureteric bud was decreased in the CsA-treated group with the nephrons missing in contrast to control after the incubation for 24 h and 72 h (all P < 0.0001). Conclusion: Treatment of CsA suppressed metanephros development in the pregnant mice; however, the potential action of mechanism needs to be further investigated.

Background: Resolvin D1 (RvD1) is a newly found anti-inflammatory bioactive compound derived from polyunsaturated fatty acids. The current study aimed to explore the protective effect of RvD1 on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) and its possible mechanism. Methods: Both in vivo and in vitro studies were conducted. Male BALB/c mice were randomly divided into control group (saline), LPS group (LPS 5 mg/kg), RvD1 group (RvD1 5 μg/kg + LPS 5 mg/kg), and blockage group (Boc-MLP 5 μg/kg + RvD1 5 μg/kg + LPS 5 mg/kg). Boc-MLP is a RvD1 receptor blocker. The mice were intraperitoneally injected with these drugs and recorded for general condition for 48 h, while the blood and kidneys were harvested at 2, 6, 12, 24, and 48 h time points, respectively (n = 6 in each group at each time point). Human proximal tubule epithelial cells (HK-2) were randomly divided into control group (medium only), LPS group (LPS 5 μg/ml), RvD1 group (RvD1 10 ng/ml + LPS 5 μg/ml), and blockage group (Boc-MLP 10 ng/ml + RvD1 10 ng/ml + LPS 5 μg/ml). The cells were harvested for RNA at 2, 4, 6, 12, and 24 h time points, respectively (n = 6 in each group at each time point). Blood creatinine was tested by using an Abbott i-STAT portable blood gas analyzer. Tumor necrosis factor-α (TNF-α) level was detected by ELISA. Kidney pathology was observed under hematoxylin and eosin (HE) staining and transmission electron microscope (TEM). We hired immune-histological staining, Western blotting, and fluorescence quantitative polymerase chain reaction to detect the expression of RvD1 receptor ALX, nuclear factor-kappa B (NF-κB) signaling pathway as well as caspase-3. Kidney apoptosis was evaluated by TUNEL staining. Results: RvD1 receptor ALX was detected on renal tubular epithelials. Kaplan–Meier analysis indicated that RvD1 improved 48 h animal survival (80%) compared with LPS group (40%) and RvD1 blockage group (60%), while RvD1 also ameliorated kidney pathological injury in HE staining and TEM scan. After LPS stimulation, the mRNA expression of toll-like receptor 4, myeloid differentiation factor 88, and TNF-α in both mice kidneys and HK-2 cells were all up-regulated, while RvD1 substantially inhibited the up-regulation of these genes. Western blotting showed that the phosphorylated-IκB/IκB ratio in LPS group was significantly higher than that in the control group, which was inhibited in the RvD1 group. RvD1 could inhibit the up-regulation of cleaved-caspase-3 protein stimulated by LPS, which was prohibited in RvD1 blockage group. RvD1 group also had a lower proportion of apoptotic nuclei in mice kidney by TUNEL staining compared with LPS group. Conclusion: In LPS-induced AKI, RvD1 could decrease TNF-α level, ameliorate kidney pathological injury, protect kidney function, and improve animal survival by down-regulating NF-κB inflammatory signal as well as inhibiting renal cell apoptosis.

Ischemic stroke (IS) is the second leading cause of death and disability in the world. Pyroptosis, a form of programmed cell death initiated by caspases, participates in the occurrence and development of IS. Because it can increase cell membrane permeability, mediate the release of inflammatory factors, and aggravate inflammation, inhibiting this process can significantly reduce the pathological injury of IS. The nucleotide binding oligomerization domain-like receptor family pyrin domain protein 3 (NLRP3) is a multiprotein complex whose activation is the core link of pyroptosis. In recent years, studies have reported that traditional Chinese medicine (TCM) could regulate pyroptosis mediated by NLRP3 inflammasome through multi-channel and multi-target networks and thus exert the effect against IS. This article reviews 107 papers published in recent years in PubMed, Chinese National Knowledge Infrastructure (CNKI), and WanFang Data in recent years. It has found that the activation factors of NLRP3 inflammasome include ROS, mitochondrial dysfunction, K+, Ca2+, lysosome rupture, and trans-Golgi breakdown. TLR4/ NF-κB/NLRP3, ROS/TXNIP/NLRP3, AMPK/Nrf2/NLRP3, DRP1/NLRP3, TAK1/ JNK/NLRP3 signaling pathways regulate the initiation and assembly of the NLRP3 inflammasome, subsequently induce pyroptosis, affecting the occurrence and development of IS. TCM can affect the above signaling pathways and regulate the pyroptosis mediated by NLRP3 inflammasome, so as to play a protective role against IS, which provides a new entry point for discussing the pathological mechanism of IS and a theoretical basis for developing TCM treasure house. [ABSTRACT FROM AUTHOR]

Cantharidin (CTD) is a bioactive ingredient isolated from Cantharis vesicatoria (blister beetles), which has potential therapeutic value as an anticancer agent. Magnesium Demethylcantharidate (MDC) is a recently developed derivative of Cantharidin (CTD), and previous studies have illustrated its excellent anticancer activity on HCC cells. However, the effect and mechanism of MDC remains unclear and need to be further studied. In particular, whether MDC can cause ER stress in HCC is still unknown. In this study, we demonstrated that endoplasmic reticulum stress (ERS)-related proteins were changed in SMMC-7721 and Bel-7402 cells after being exposed to MDC. Moreover, we found that MDC could significantly inhibit the growth of xenograft tumor in nude mice. In summary, we confirmed that MDC could induce ERS in HCC cells and thus induce apoptosis. [ABSTRACT FROM AUTHOR]

Neuroinflammation contributes to secondary brain injury following intracerebral hemorrhage (ICH). Triggering receptor expressed on myeloid cells 2 (TREM2) confers strong neuroprotective effect by suppressing neuroinflammatory response in experimental ischemic stroke. This study aimed to clarify the neuroprotective role of TREM2 and potential underlying mechanism in a mouse model of ICH and in vitro. Adeno-associated virus (AAV) and green fluorescent protein-lentivirus (GFP-LV) strategies were employed to enhance TREM2 expression in the C57/BL6 mice and BV2 cells, respectively. The adult male C57/BL6 mice were subjected to ICH by administration of collagenase-IV in 1 month after the AAV particles injection. An in vitro ICH model was performed with oxygen hemoglobin in BV2 cells. Toll-like receptor 4 (TLR4) antagonist TAK242 was applied at 6 h following ICH. Neurological function, TREM2, pro-inflammatory cytokines, brain water content and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were evaluated at 24 h following ICH. TLR4, NF-κB and mitogenactivated protein kinases (MAPK) signaling pathways were also determined by Western blot analysis at the same time point. The levels of TREM2 were increased at 12 h, peaked at 24 h and recovered on 7d following ICH. TREM2 overexpression ameliorated ICH induced neurological dysfunction, inhibited neuroinflammation, and attenuated apoptosis and brain edema. Further mechanistic study revealed that TREM2 overexpression inhibited TLR4 activation and NF-κB and MAPK signaling pathways. ICH increased the percentage of TUNEL-positive cells, which was markedly decreased by TREM2 overexpression. A similar improvement was also observed by the administration of TAK242 following ICH. TREM2 improves neurological dysfunction and attenuates neuroinflammation and neuronal apoptosis in the acute phase of ICH, which is, at least in part, mediated by negatively regulating TLR4 signaling pathway. These findings highlight TREM2 as a potential target for early brain injury following ICH. [ABSTRACT FROM AUTHOR]

Objective: To study the significance of mir-141 expression in COPD (chronic obstructive pulmonary diseases) mononuclear cells, and to demonstrate its effect on proliferation and apoptosis of fibroblasts Possible to ZEB1(zinc finger E-box binding homeobox 1). Methods: 40 cases acute phase of COPD patients, 80 cases of stable period COPD patients and 110 normal controls in our hospital were collected. RT-PCR was used to detect mir-141 and statistical analysis. The mir-141 mimic was constructed and transfected into lung fibroblasts. The expression of mir-141 was analyzed by RT-PCR. Cell proliferation was analyzed by CCK8 and cell apoptosis was detected by flow cytometry. Targetscan was used to predict the binding site of ZEB1 and mir-141. The target relationship between ZEB1 and mir-141 was identified by RT-PCR and western blot. Results: The expression of mir-141 in mononuclear cells of acute phase of COPD and stable period of COPD was significantly higher than that of normal controls. Mir-141 levels in acute phase of COPD were significantly higher than that of stable period of COPD. Mir-141 mimic transfection significantly up-regulated the expression of mir- 141, promoted cell activity and decreased cell apoptosis rate compared with the empty control group. ZEB1 and mir-141 had binding sites predicted by Targetscan database and verified by dual luciferase assays. After mir-141 mimic transfection hat, ZEB1 mRNA and protein expression were down-regulated. Conclusions: High expression of mir-141 in COPD may promote the proliferation of lung fibroblasts and inhibit apoptosis by targeting ZEB1. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common primary bone tumor that occurs in children and adolescents. Osteosarcoma has a poor prognosis and is often unresponsive to chemotherapy. Therefore, it remains a challenge to identify a novel strategy to effectively treat osteosarcoma. The present study demonstrated a novel opportunity in osteosarcoma treatment using the natural compound plumbagin. Plumbagin reduced cell viability in osteosarcoma cells but not normal bone cells, as determined by MTT assay and colony formation assay. Plumbagin induced cell apoptosis by mitochondrial dysfunction, which in turn promoted Ca2+ release and endoplasmic reticulum (ER)‑stress, as determined by DAPI staining assay, DNA fragmentation assay, flow cytometry and western blotting analysis. In addition, plumbagin improved reactive oxygen species (ROS) generation, as determined by flow cytometry. Finally, these apoptotic cascades activated caspase‑3 and caspase‑9 to elicit apoptosis response. Our results demonstrated the anticancer effect of plumbagin by inducing cell apoptosis in osteosarcoma cells. In conclusion, plumbagin activated the apoptosis signaling pathway through eliciting ROS, ER stress, mitochondria dysfunction, and finally causing caspase activation. These results indicated that plumbagin may serve as potential antitumor drug by its multifunctional effects in osteosarcoma. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to evaluate the frequency of RASSF1A hypermethylation in patients with acute myeloid leukemia (AML), in an attempt to modify the current molecular model for disease prognosis. Materials and methods: Aberrant RASSF1A promoter methylation levels were assessed in 226 newly diagnosed non-M3 AML patients and 30 apparently healthy controls, by quantitative methylation-specific polymerase chain reaction. Meanwhile, RASSF1A mRNA levels were detected by real-time quantitative polymerase chain reaction. Furthermore, hematological characteristics, cytogenetic abnormalities, and genetic aberrations were assessed. Finally, associations of RASSF1A hypermethylation with clinical outcomes were evaluated. Results: RASSF1A hypermethylation was observed in 23.0% of patients with non-M3 AML (52/226), but not in controls. Meanwhile, hypermethylation of the RASSF1A promoter was significantly associated with ASXL1 mutation. Furthermore, the log-rank test revealed that RASSF1A hypermethylation indicated decreased relapse-free survival (RFS) and overall survival (OS) in patients with non-M3 AML (P=0.012 and P=0.014, respectively). In multivariate analysis, RASSF1A hypermethylation was an independent prognostic factor for RFS (P=0.040), but not for OS (P=0.060). Conclusion: Hypermethylation of the RASSF1A promoter is associated with ASXL1 mutation in non-M3 AML patients, likely indicating poor outcome. These findings provide a molecular basis for stratified diagnosis and prognostic evaluation. [ABSTRACT FROM AUTHOR]

The aim of the present study was to explore the effect of silencing wild‑type p53‑induced phosphatase 1 (Wip1) on apoptosis of human ovarian cancer SKOV3 cells. SKOV3 cells cultured in vitro were divided into three groups: untreated cells, cells transfected with control small interfering RNA (siRNA) and cells transfected with siRNA targeting Wip1. Flow cytometry analysis was used to detect cell apoptosis. Western blot analysis was performed to determine expression of tumor protein 53 (p53), cleaved caspase‑3, caspase‑3, BCL2 associated X (Bax), BCL2 apoptosis regulator (Bcl‑2), p38 mitogen‑activated protein kinase (p38 MAPK) and phosphorylated (p)‑p38 MAPK. Reverse transcription‑quantitative polymerase chain reaction was used to detect expression of p53, Bax, Bcl‑2 and caspase‑3 mRNAs. Compared with control, apoptosis of SKOV3 cell was significantly increased following Wip1 siRNA silencing. Wip1 silencing also resulted in a significant increase of p53 and p‑p38 MAPK expression, as well as increased cleaved caspase‑3/caspase‑3 and Bax/Bcl‑2 protein ratios. No significant differences were observed in apoptosis and apoptosis‑related protein expression in the control siRNA transfected cells. The present study demonstrated that Wip1 silencing promotes apoptosis of human ovarian cancer SKOV3 cells by activation of the p38 MAPK signaling pathways and through subsequent upregulation of p53, and cleaved caspase‑3/caspase‑3 and Bax/Bcl‑2 protein ratios. Overall, the findings of the present study suggest that targeting Wip1 may be a potential therapeutic avenue for the treatment of human ovarian cancer in the future. [ABSTRACT FROM AUTHOR]

• GLP attenuates CCl 4 -induced liver injury, inflammation, and fibrosis in mice. • GLP inhibits HSCs activation and collagen formation in vivo and in vitro. • GLP elicits dual effects on apoptosis in vivo (inhibit) and in vitro (promote). • GLP inhibits ECM-receptor interaction pathway including integrin expressions. • GLP alleviates liver fibrosis via suppressing the TGF-β/Smad signaling pathway. Ganoderma lucidum polysaccharide (GLP) has many biological properties, however, the anti-fibrosis effect of GLP is unknown at present. This study aimed to examine the anti-fibrogenic effect of GLP and its underlying molecular mechanisms in vivo and in vitro. Both CCl 4 -induced mouse and TGF-β1-induced HSC-T6 cellular models of fibrosis were established to examine the anti-fibrogenic effect of a water-soluble GLP (25 kDa) extracted from the sporoderm-removed spores of G. lucidum.. Serum markers of liver injury, histology and fibrosis of liver tissues, and collagen formation were examined using an automatic biochemical analyzer, H&E staining, Sirius red staining, immunohistochemistry, immunofluorescence, ELISA, Western blotting, and qRT-PCR. RNA-sequencing, enrichment pathway analysis, Western blotting, qRT-PCR, and flow cytometry were employed to identify the potential molecular targets and signaling pathways that are responsible for the anti-fibrotic effect of GLP. We showed that GLP (150 and 300 mg/kg) significantly inhibited hepatic fibrogenesis and inflammation in CCl 4 -treated mice as mediated by the TLR4/NF-κB/MyD88 signaling pathway. We further demonstrated that GLP significantly inhibited hepatic stellate cell (HSCs) activation in mice and in TGF-β1-induced HSC-T6 cells as manifested by reduced collagen I and a-SMA expressions. RNA-sequencing uncovered inflammation, apoptosis, cell cycle, ECM-receptor interaction, TLR4/NF-κB, and TGF-β/Smad signalings as major pathways suppressed by GLP administration. Further studies demonstrated that GLP elicits anti-fibrotic actions that are associated with a novel dual effect on apoptosis in vivo (inhibit) or in vitro (promote), suppression of cell cycle in vivo , induction of S phase arrest in vitro , and attenuation of ECM-receptor interaction-associated molecule expressions including integrins ITGA6 and ITGA8. Furthermore, GLP significantly inhibited the TGF-β/Smad signaling in mice, and reduced TGF-β1 or its agonist SRI-011381-induced Smad2 and Smad3 phosphorylations, but increased Samd7 expression in HSC-T6 cells. This study provides the first evidence that GLP could be a promising dietary strategy for treating liver fibrosis, which protects against liver fibrosis and HSC activation through targeting inflammation, apoptosis, cell cycle, and ECM-receptor interactions that are mediated by TGF-β/Smad signaling. [Display omitted] [ABSTRACT FROM AUTHOR]

Background/Aims: Extensive studies have demonstrated that Bleomycin (BLM) is a glycopeptide antibiotic that has been used as an anticancer chemotherapeutic reagent. It can induce both single- and double-strand DNA damage, inhibit synthesis of DNA, suppress proliferation, and induce apoptosis in cancer cells. Smad signaling transducers are considered as important molecules in tumor development and progression, and may closely be related to the biological behaviors of some malignant carcinomas, including gastric cancer. Methods: The effects of different concentrations of BLM on the proliferation, cell cycle, apoptosis, migration, and invasion on gastric cancer cell lines MKN45 and AGS were assayed by using CCK-8 assay, Annexin V/PI double staining, PI staining, and transwell assay. Western blot and Immunohistochemistry were applied to analyze the potential mechanism(s). Results: BLM treatment resulted in a low proliferation, high apoptosis, low migration and invasion in MKN45 and AGS cells. Furthermore, the possible mechanisms underlying that Smad3 activity could be changed after binding with BLM, and subsequently the Smad signaling pathway had a cascade response. Conclusion: These results highlight BLM as an exciting theme for gastric cancer treatment, which may represent an effective clinical therapeutic reagent for gastric cancer patients. [ABSTRACT FROM AUTHOR]

Chondrosarcoma is a highly malignant cartilage-forming bone tumor that has the capacity to invade locally and cause distant metastasis. Moreover, chondrosarcoma is intrinsically resistant to conventional chemotherapy or radiotherapy. The novel benzofuran derivative, BL-038 (2-amino-3-(2,6-dichlorophenyl)-6-(4-methoxyphenyl)benzofuran-4-yl acetate), has been evaluated for its anticancer effects in human chondrosarcoma cells. BL-038 caused cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353, but not in primary chondrocytes. Treatment of chondrosarcoma with BL-038 also induced reactive oxygen species (ROS) production. Furthermore, BL-038 decreased mitochondrial membrane potential (MMP) and changed mitochondrial-related apoptosis, by downregulating the anti-apoptotic activity members (Bcl-2, Bcl-xL) and upregulating pro-apoptotic members (Bax, Bak) of the B-cell lymphoma 2 (Bcl-2) family of proteins, key regulators of the apoptotic machinery in cells. These results demonstrate that in human chondrosarcoma cells, the apoptotic and cytotoxic effects of BL-038 are mediated by the intrinsic mitochondria-mediated apoptotic pathway, which in turn causes the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), to elicit apoptosis response. Our results show that the benzofuran derivative BL-038 induces apoptosis in chondrosarcoma cells. [ABSTRACT FROM AUTHOR]

Background: Sodium selenite and ginsenoside Rh2 (G-Rh2) are well known for their anticancer properties and have been exploited as a new therapeutic approach. In this study, we are interested to evaluate if sodium selenite and G-Rh2 combination results in a synergistic anticancer effect that could contribute to lower systemic toxicity. Methods: We observed the synergistic antitumor effect by combination of sodium selenite and G-Rh2 on HCT-116 human colorectal carcinoma cells in vitro. Cell growth, viability, cell cycle progression and cell apoptosis, Bax/Bcl2 ratio, caspase-3 expression, reactive oxygen species (ROS) production and autophagy were evaluated. Results: The results showed that sodium selenite and G-Rh2 combination have a synergistic effect on cell growth inhibition (57%) compared with sodium selenite (25%) and G-Rh2 alone (28%) after 24h of treatment. This combination also induced G1 and S phase arrest simultaneously and increased apoptosis rate. The results also indicated that Bax/Bcl2 ratio and caspase-3 expression, known as proapoptotic factors, were increased in the presence of sodium selenite and G-Rh2 alone. However, combined drug treatment results in a more significant increase in Bax/Bcl2 ratio and caspase-3 expression (P < 0.05). In addition, this combination significantly induces a depletion of ROS production and autophagy, compared to control, sodium selenite and G-Rh2 alone (P < 0.05). Conclusion: Sodium selenite and ginsenoside Rh2 combination may be a more effective treatment for human colorectal carcinoma and is a promising chemotherapeutic approach for malignant tumors. [ABSTRACT FROM AUTHOR]

Background Post-stroke cognitive impairment seriously affects the quality of life and functional rehabilitation of patients with stroke. Objective To examine the effects of electroacupuncture (EA) at GV20 and GV24 on cognitive impairment and apoptosis including expression of apoptosis-related genes Bcl-2 and Bax in a rat model of cerebral ischaemia-reperfusion (IR) induced by middle cerebral artery occlusion (MCAO). Methods Thirty-five Sprague-Dawley rats were allocated to a sham operation control group (SC group, n=10) or underwent surgery and MCAO (n=25). Postoperatively the latter group was randomly subdivided into EA or untreated (IR) groups. Cognitive impairment was assessed using the Morris water maze (MWM). Apoptosis was examined by detection of Bcl-2 and Bax expression in the cerebral cortex. Results The EA group had significantly decreased neurological deficit scores compared to the IR group (p<0.05). In the MWM test, significant differences in escape latency and route were observed between the EA and IR groups (p<0.05). Rats in the EA group performed better in the probe trial than those in the IR group (p<0.05). EA treatment markedly reduced the number of TUNEL-positive cells compared to the IR group (20.13 ±4.30% vs 38.40±3.38%; p<0.001). Reverse transcription-polymerase chain reaction (RT-PCR) results showed the Bcl-2/Bax ratio was significantly increased in the EA group compared to the IR group (1.61 ±0.19 vs 0.50 ±0.05, p<0.01). Conclusions These findings suggest that EA ameliorates cognitive impairment of rats with IR injury by modulating Bcl-2 and Bax expression. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is a primary malignant tumor of bone and is most prevalent in children and adolescents. OS is frequently associated with pulmonary metastasis, which is the main cause of OS-related mortality. OS has a poor prognosis and is often unresponsive to conventional chemotherapy. In this study, we determined that Nimbolide, a novel anti-cancer therapy, acts by modulating multiple mechanisms in osteosarcoma cells. Nimbolide induces apoptosis by increasing endoplasmic reticulum (ER) stress, mitochondrial dysfunction, accumulation of reactive oxygen species (ROS), and finally, caspase activation. We also determined that Nimbolide inhibits cell migration, which is crucial for metastasis, by reducing the expression of integrin αvβ5. In addition, our results demonstrate that integrin αvβ5 expression is modulated by the PI3K/Akt and NF-κB signaling cascade. Nimbolide has potential as an anti-tumor drug given its multifunctional effects in OS. Collectively, these results help us to understand the mechanisms of action of Nimbolide and will aid in the development of effective therapies for OS. [ABSTRACT FROM AUTHOR]

The toxicities of conventional chemotherapeutic agents to normal cells restrict their dosage and clinical efficacy in acute leukemia; therefore, it is important to develop novel chemotherapeutics, including natural products, which selectively target cancer-specific pathways. The present study aimed to explore the effect of the chemopreventive agent asiatic acid (AA) on the proliferation and apoptotic rate of the leukemia cell line HL-60 and investigated the mechanisms underlying its anti-tumor activity. The effect of AA on the proliferation of HL-60 cells was evaluated using the MTT assay. Annexin V-fluorescein isothiocyanate/propidium iodide double staining followed by flow cytometric analysis as well as Hoechst 33258 staining were used to analyze the apoptotic rate of the cells. Furthermore, changes of survivin, B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 expressions were detected by western blot analysis. AA blocked the growth of HL-60 cells in a dose- and time-dependent manner. The IC50-value of AA on HL-60 cells was 46.67±5.08 µmol/l for 24 h. AA induced apoptosis in a dose-dependent manner, which was inhibited in the presence of Z-DEVD-FMK, a specific inhibitor of caspase. The anti-apoptotic proteins Bcl-2, Mcl-1 and survivin were downregulated by AA in a dose-dependent manner. Concurrently, AA inhibited ERK and p38 phosphorylation in a dose-dependent manner, while JNK phosphorylation was not affected. In conclusion, the present study indicated that the p38 and ERK pathways, as well as modulation of Bcl-2 family and survivin proteins were key regulators of apoptosis induced in HL-60 cells in response to AA. [ABSTRACT FROM AUTHOR]

The pathogenesis of atherosclerosis is multi factorial. The proliferation and migration of vascular smooth muscle cells (VSMCs) are significant in the genesis and development of atherosclerosis plaques, and the degradation of VSMCs plays a crucial role in the process. Mimecan is a member of the Keratan sulfate family of proteoglycans, which are leucine-rich proteoglycans. It has been demonstrated that mimecan is associated with arteriogenesis and atherosclerosis. In the present study, the effect of mimecan on the characteristics of cultured human aortic smooth muscle cells (HASMCs) was investigated. In vitro, human mimecan was stably overexpressed in HASMCs using a lentiviral system. It was observed that the proliferation rate of HASMCs transduced with mimecan was lower compared with that of control cells; overexpression of mimecan induced HASMC apoptosis. To determine the effect of mimecan on HASMC migration, a Transwell cell culture chamber and sterile cloning cylinder assays were used, and it was noted that mimecan enhanced the migration of HASMCs horizontally and vertically. These data indicated that mimecan may be involved in the pathogenesis of atherosclerosis by regulating the proliferation, apoptosis and migration of VSMCs. [ABSTRACT FROM AUTHOR]

Objective. The aim of this study was to investigate the expression of smoothened protein (Smo), a sonic hedgehog (Shh) signalling component, in synovium of RA and its role in the survival and apoptosis of endothelial cells. Methods. The expression of Smo pxrotein in RA synovial tissue was examined by immunohistochemistry. Real-time PCR and western blotting techniques were employed to measure the expression of Shh signalling components in EA.hy926 endothelial cells exposed to TNF-α in the presence or absence of cyclopamine (a Smo-specific antagonist). Lastly, the effect of cyclopamine and Smo small interfering RNA on apoptosis induced by TNF-α and actinomycin D (ActD) was determined. Results. We found that Smo was highly expressed in synovial tissues of RA, especially in endothelial cells, compared with the trauma group. TNF-α significantly increased the expression of Shh signalling components in EA.hy926 endothelial cells, while cyclopamine decreased the expression of Shh signalling components. EA.hy926 endothelial cells treated with various concentrations of cyclopamine (2–8 μmol/l) showed a significant decrease in cell viability and cell survival rate, and an increase in the rate of cell apoptosis compared with endothelial cells treated with TNF-α and ActD ( P < 0.05). EA.hy926 endothelial cells transfected with Smo-siRNA also showed a lower cell survival rate and higher apoptotic rate, compared with cells in the control group ( P < 0.05). Conclusion. The Shh signalling pathway plays a role in regulating endothelial cell apoptosis in a Smo-dependent manner. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effects of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway on the Wnt/β-catenin signaling pathway in rats with focal cerebral ischemia-reperfusion injury. A total of 96 rat focal cerebral ischemia-reperfusion models, established according to a modified version of Longa's method, were randomly divided into four groups: Sham-operated (S), cerebral ischemia-reperfusion injury (I), cerebral ischemia-reperfusion + basic fibroblast growth factor (bFGF) post-processing and, finally, cerebral ischemia-reperfusion + bFGF post-processing + PI3K inhibitor LY294002 (LY). Each group consisted of 24 rats and each group was divided into four subgroups according to the indicated reperfusion times of 12, 24, 48 and 72 h. The morphological changes of the cortical tissue and the cellular apoptosis were determined using hematoxylin and eosin staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling method, respectively. The expression levels of phosphorylated (p-)Akt, glycogen synthase kinase-3β (GSK-3β) mRNA and β-catenin in the cortical tissue were detected at different time-points. The number of apoptotic cells and the expression levels of p-Akt, GSK-3β mRNA and β-catenin in the I and LY groups were significantly higher compared with those in the S group (P<0.05). In the bFGF group, the number of apoptotic cells and the mRNA expression levels of GSK-3β were significantly decreased, whereas the expression levels of p-Akt and β-catenin were significantly increased compared with those in the I and LY groups (P<0.05). In cerebral ischemia-reperfusion injury, the PI3K/Akt signaling pathway regulated β-catenin, the main member of the Wnt signaling pathway, via GSK-3β, providing information to assist in further investigation of the mechanism of β-catenin in ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Puerarin, a predominant isoflavonoid compound extracted from the Chinese medicinal herb Radix Puerariae, is considered to exhibit an antitumor effect. In the present study, the effects of puerarin on SMMC-7721 human hepatocellular carcinoma cells were investigated. Cell viability was assessed by MTT assay. Apoptosis was detected by flow cytometry with Annexin V-fluorescein isothiocyante staining and morphological observation of nuclear changes by Hoechst staining. The mitochondrial membrane potential (MMP) was monitored using rhodamine 123. The generation of reactive oxygen species (ROS) was quantified using dichloro-dihydro-fluorescein diacetate. Polymerase chain reaction and western blot analysis were used to detect the expression levels of apoptosis-associated genes. The results revealed that high concentrations of puerarin (500, 1,000 and 1,500 μg/ml) significantly inhibited the proliferation of SMMC-7721 cells in a time- and dose-dependent manner. Simultaneously, apoptotic rates were increased and cell morphology was changed following puerarin treatment. Furthermore, puerarin-induced apoptosis of SMMC-7721 cells was associated with loss of MMP and generation of ROS. Puerarin treatment increased caspase-3,8,9 and apoptosis-inducing factor (AIF) mRNA expression levels in SMMC-7721 cells, while the phosphorylation levels of P38, extracellular signal-regulated kinase (ERK1) and c-Jun N-terminal kinase were also increased. Furthermore, caspase-9 and AIF protein expression was upregulated. In conclusion, puerarin inhibited proliferation and induced apoptosis in SMMC-7721 cells via the mitochondria-dependent pathway; however, the specific mechanisms require further investigation. [ABSTRACT FROM AUTHOR]

The use of usnic acid and usnic acid-containing products is associated with acute liver failure; however, mechanistic studies of hepatotoxicity caused by usnic acid are limited. In this study, we investigated and characterized the possible mechanisms, especially the role of autophagy in usnic acid's toxicity in human HepG2 cells. Usnic acid caused apoptosis as demonstrated by an increased caspase-3/7 activity and an increased subdiploid nucleus formation. Usnic acid-induced autophagy as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, and an increased number of puncta. Inhibition of autophagy by treating cells with autophagy inhibitors (3-methyladenine or chloroquine) or by small interfering RNA against Atg7 aggravated usnic acid-induced apoptosis and decreased cell viability, indicating that autophagy plays a protective role against usnic acid-induced toxicity. Moreover, usnic acid activated the MAPK signaling pathway. Usnic acid-elicited apoptosis was enhanced and autophagy was decreased when JNK was suppressed by a specific inhibitor. Additionally, inhibition of autophagy decreased the activity of JNK. Taken together, our results suggest that usnic acid perturbs various interrelated signaling pathways and that autophagy induction is a defensive mechanism against usnic acid-induced cytotoxicity. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is a relatively rare form of cancer, but OS is the most commonly diagnosed bone cancer in children and adolescents. Chemotherapy has side effects and induces drug resistance in OS. Since an effective adjuvant therapy was insufficient for treating OS, researching novel and adequate remedies is critical. Hyperthermia can induce cell death in various cancer cells, and thus, in this study, we investigated the anticancer method of hyperthermia in human OS (U-2 OS) cells. Treatment at 43 °C for 60 min induced apoptosis in human OS cell lines, but not in primary bone cells. Furthermore, hyperthermia was associated with increases of intracellular reactive oxygen species (ROS) and caspase-3 activation in U-2 OS cells. Mitochondrial dysfunction was followed by the release of cytochrome c from the mitochondria, and was accompanied by decreased anti-apoptotic Bcl-2 and Bcl-xL, and increased pro-apoptotic proteins Bak and Bax. Hyperthermia triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels, as well as increased calpain expression and activity. In addition, cells treated with calcium chelator (BAPTA-AM) blocked hyperthermia-induced cell apoptosis in U-2 OS cells. In conclusion, hyperthermia induced cell apoptosis substantially via the ROS, ER stress, mitochondria, and caspase pathways. Thus, hyperthermia may be a novel anticancer method for treating OS. [ABSTRACT FROM AUTHOR]

Endothelial barrier disruption is a hallmark of multiple organ injury (MOI). However, mechanisms governing the restoration of endothelial barrier function are poorly understood. Here, we uncovered an NF-κB-to-AP-1 switch that regulates the transition from barrier injury to repair following endotoxemic MOI. Endothelial NF-κB mediates barrier repair by inhibiting endothelial cell (EC) apoptosis. Blockade of endothelial NF-κB pathway activated the activator protein (AP)-1 pathway (NF-κB-to-AP-1 switch), which compensated for the anti-apoptotic and barrier-repair functions of NF-κB. The NF-κB-to-AP-1 switch occurred at 24 hours (injury to repair transition phase), but not at 48 hours (repair phase) post-LPS, and required an inflammatory signal within the endothelium. In the absence of an inflammatory signal, the NF-κB-to-AP-1 switch failed, resulting in enhanced EC apoptosis, augmented endothelial permeability, and impeded transition from barrier injury to recovery. The NF-κB-to-AP-1 switch is a protective mechanism to ensure timely transition from endothelial barrier injury to repair, accelerating barrier restoration following MOI. [ABSTRACT FROM AUTHOR]

Studies have shown that electroacupuncture (EA) ameliorates learning and memory after ischemic injury. However, there have been few studies elucidating the mechanisms of EA on learning and memory in cerebral hypoperfusion. In this study, we explored the cAMP response element-binding protein (CREB) signaling pathway-mediated antiapoptotic action involved in EA-induced improvement of learning and memory. EA at GV20 and GV14 acupoints was applied in cerebral hypoperfusion rats. A Morris water maze task was performed, and the immunoreactivities of pCREB, Bcl-2, and Bax in the hippocampal CA1 area were evaluated by the Western blotting technique. Our findings indicated that (1) EA ameliorated spatial learning and memory impairment in cerebral hypoperfusion rats; (2) EA increased the immunoreactivities of pCREB and Bcl-2 and decreased the immunoreactivity of Bax; (3) intracerebroventricular administration of H89 (the inhibitor of protein kinase A) blocked EA-induced, pCREB-mediated antiapoptotic action and improved learning and memory. These results suggest that EA can ameliorate learning and memory via activation of the CREB signaling pathway in the hippocampus to attenuate apoptosis after cerebral hypoperfusion. [ABSTRACT FROM AUTHOR]

In this study, we investigated the anticancer effects of a new benzimidazole derivative, 1-benzyl-2-phenyl -benzimidazole (BPB), in human chondrosarcoma cells. BPB-mediated apoptosis was assessed by the MTT assay and flow cytometry analysis. The in vivo efficacy was examined in a JJ012 xenograft model. Here we found that BPB induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353) but not in primary chondrocytes. BPB induced upregulation of Bax, Bad and Bak, downregulation of Bcl-2, Bid and Bcl-XL and dysfunction of mitochondria in chondrosarcoma. In addition, BPB also promoted cytosolic releases AIF and Endo G. Furthermore, it triggered extrinsic death receptor-dependent pathway, which was characterized by activating Fas, FADD and caspase-8. Most importantly, animal studies revealed a dramatic 40% reduction in tumor volume after 21 days of treatment. Thus, BPB may be a novel anticancer agent for the treatment of chondrosarcoma. [ABSTRACT FROM AUTHOR]

Background& objectives: Tuberculosis (TB) bacilli ingested by macrophages evade host immune responses by multiple mechanisms including the inhibition of apoptosis. As the cytochrome-P-450 system (CYP) contributes to apoptosis it has been suggested that genetic variation in CYP may be associated with susceptibility to TB infection. This study was carried out to evaluate cytochrome P-450 polymorphisms in Chinese Han children and to investigate the effect of these polymorphisms in paediatric TB. Methods: Frequencies for the CYP2C19, CYP3A4, CYP3A5 and CYP2E1 mutated alleles and genotypes were compared between 142 Chinese paediatric TB patients and 150 non-infected controls by real time PCR genotyping on peripheral leukocyte DNA. Results: CYP2C19 (636 G>A, rs4986893) A allele and AG genotype were associated with decreased susceptibility to TB (P = 0.006, OR= 0.33, 95% CI: 0.15-0.76; and P = 0.005, OR =0.31, 95% CI: 0.14- 0.72 respectively), as were the CYP3A5 (6986A>G, rs776746) G allele and particularly homozygous GG (recessive mode) genotype (P = 0.004, OR=0.61, 95% CI: 0.43-0.85; and P=0.002, OR=0.47, 95% CI: 0.29-0.76). Interpretation & conclusions: The data suggested that CYP2C19 and CYP3A5 polymorphisms affect susceptibility to paediatric TB. Further studies are indicated to confirm and elucidate these observations. [ABSTRACT FROM AUTHOR]

Purpose: To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms. Method: CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph- Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo. Results: Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 &mgr;M) and primary CML cells (IC50 was 13.4 &mgr;M for CML-CP and 18 &mgr;M for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could upregulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl. [ABSTRACT FROM AUTHOR]

We here report that miR-17-92 cluster is a novel target for p53-mediated transcriptional repression under hypoxia. We found the expression levels of miR-17-92 cluster were reduced in hypoxia-treated cells containing wild-type p53, but were unchanged in hypoxia-treated p53-deficient cells. The repression of miR-17-92 cluster under hypoxia is independent of c-Myc. Luciferase reporter assays mapped the region responding to p53-mediated repression to a p53-binding site in the proximal region of the miR-17-92 promoter. Chromatin immunoprecipitation (ChIP), Re-ChIP and gel retardation assays revealed that the binding sites for p53- and the TATA-binding protein (TBP) overlap within the miR-17-92 promoter; these proteins were found to compete for binding. Finally, we show that pri-miR-17-92 expression correlated well with p53 status in colorectal carcinomas. Over-express miR-17-92 cluster markedly inhibits hypoxia-induced apoptosis, whereas blocked miR-17-5p and miR-20a sensitize the cells to hypoxia-induced apoptosis. These data indicated that p53-mediated repression of miR-17-92 expression likely has an important function in hypoxia-induced apoptosis, and thus further our understanding of the tumour suppressive function of p53. [ABSTRACT FROM AUTHOR]

The derivatives of 5,8-quinolinedione have been shown to exert anticancer activities. A new synthetic compound 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione (designed as PT-262) derived from 6,7-dichloroquinoline-5,8-dione on its anticancer activity was investigated in this study. PT-262 was synthesized as the following: triethylamine (0.56 ml, 5.1 mmol) was added dropwise to a solution of 6,7-dichloroquinoline-5,8-dione (1.00 g, 4.4 mmol) and piperidine (0.50 ml, 5.1 mmol) in 150 ml of benzene with stirring at room temperature for 5 min, and the solvent was removed using rotary evaporator to give a dark brown solid. PT-262 was purified by flash chromatography using 50% ethyl acetate/hexanes to elute that displayed as brown solids. To examine the induction of apoptosis following PT-262 treatment, the lung cancer cells were subjected to apoptotic cell observation, caspase activation, and mitochondrial functional assays. The protein levels of phosphorylated ERK and CDC2 after treatment with PT-262 were analyzed by Western blot. Treatment with 1–20 μM PT-262 for 24 h induced cytotoxicity via a concentration-dependent manner in human lung cancer cells. PT-262 induced the loss of mitochondrial membrane potential and elevated the caspase-3 activation and apoptosis. Interestingly, the phosphorylation of ERK was inhibited by PT-262. The IC50 value of ERK phosphorylation inhibition was approximate around 5 μM. Treatment with a specific MEK1/2 (the upstream of ERK) inhibitor, PD98059, increased the PT-262-induced cytotoxicity in lung cancer cells. Moreover, PT-262 did not alter the protein expression of tumor suppressor p53. PT-262 elicited the cytotoxicity and accumulated the G2/M fractions in both the p53-wild type and p53-null lung cancer cells. The mitosis-regulated protein levels of cyclin B1 and phospho-CDC2 at Thr14, Tyr15, and Thr161 were repressed by PT-262 in these cells. PT-262 suppresses the phosphorylation of ERK and CDC2 associated with proliferation inhibition via a p53-independent pathway in human lung cancer cells. [ABSTRACT FROM AUTHOR]

Both paired homeo box-2 (Pax-2) and N-myc genes play pivotal roles in renal morphogenesis via their effects on cell proliferation and differentiation, but whether and how they interact have not been addressed. In the present study, we investigated such a potential interaction using embryonic renal cells in vitro. Mouse embryonic mesenchymal (MK4) cells stably transfected with Pax-2 cDNA in sense (+) or antisense (−) orientation were used for experiments. Pax-2 promoter activity was monitored by luciferase assay. Reactive oxygen species (ROS) generation, cell proliferation, and cell apoptosis were evaluated. We found that Pax-2 and N-myc gene expression were upregulated and downregulated in Pax-2 (+) and Pax-2 (−) stable transformants, respectively. ROS generation and apoptosis were significantly reduced both in Pax-2 (+) transformants compared with Pax-2 (−) transformants and in naïve MK4 cells cultured in either normal- (5 mM) or high-glucose (25 mM) medium. Transient transfection of N-myc cDNA into Pax-2 (−) stable transformants restored Pax-2 gene expression and prevented ROS generation induced by high glucose. Our data demonstrate that Pax-2 gene overexpression prevents hyperglycemia-induced apoptosis, and N-myc appears to provide a positive autocrine feedback on Pax-2 gene expression in embryonic mesenchymal cells. [ABSTRACT FROM AUTHOR]