Effect of Wenxin granule on Bcl-2/Bax/Caspase apoptosis pathway gene expression in rats with myocardial infarction

Objective:To explore the cardioprotective mechanism of Wenxin Granules regulating the expression of apoptosis-related genes in cardiomyocytes.Methods:A rat model of myocardial infarction was established and randomly divided into model group,Wenxin granule low-dose group,Wenxin granule high-dose group,metoprolol group and sham operation group.the left ventricular end systole anterior wall thickness(LVAWs),end systole inner diameter(LVIDs),end systole posterior wall thickness(LVPWs),end-diastolic anterior wall thickness(LVAWd),end-diastolic inner diameter(LVIDd),end-diastolic posterior wall thickness(LVPWd)and left ventricular ejection fraction(LVEF)were detected by echocardiography in each group after 2 weeks of treatment.Hematoxylin eosin(HE)staining was used to observe the changes in the cardiac structure of rats in each group.Real-time PCR(Real-time PCR)was used to detect the relative expression of mammalian B-cell lymphoma-2(BCL-2),BCL-2 related X protein(BAX),Caspase-9(Caspase-9),and Caspase-3(Caspase-3)mRNA.TUNEL staining was used to detect changes in the apoptotic rate of rat cardiomyocytes in each group.Results:Compared with the sham operation group,the LVAWs,LVPWs,LVPWd and LVEF of the model group were significantly reduced(P<0.05,P<0.01),and LVIDs and LVIDd were significantly increased(P<0.05,P<0.01).Severe pathological ischemia injury of heart tissue.The relative expression of BCL-2 mRNA and the ratio of BCL-2/BAX in the model group were significantly reduced(P<0.01),while the relative expression of BAX,Caspase-9 and Caspase-3 mRNA was significantly increased(P<0.01).The apoptosis rate was significantly increased(P<0.01).In the low-dose and high-dose groups of Wenxin Granules and the Metoprolol group,LVAWs,LVPWs,LVPW d,and LVEF of rats in each administration group increased significantly(P<0.05,P<0.01),LVIDs,LVIDd was significantly reduced(P<0.05,P<0.01),the pathological damage of the heart tissue was improved,the expression of BCL-2 mRNA and the ratio of BCL-2/BAX were significantly increased(P<0.05,P<0.01),BAX,The expression of Caspase-9 and Caspase-3 mRNA was significantly reduced(P<0.05,P<0.01),and the apoptotic rate of myocardial cells was significantly reduced(P<0.01).Conclusion:Wenxin granule can play a cardioprotective role by regulating the gene expression of BCL-2/BAX/Caspase apoptosis pathway.

Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells

AIM： Thioacetamide （TAA） has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. METHODS： The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAA- induced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAA- induced clone 9 cells were measured by Western blot. RESULTS： The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died （MTT assay） within 24 h after 100 rag/1 TAA was applied. Apoptotic cell percentage （TUNE1 assay） and caspase 3 activities were highest after 100 rag/1 TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspasedependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment. CONCLUSION： These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents, perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.

Total Triterpenoids from Ganoderma Lucidum Suppresses Prostate Cancer Cell Growth by Inducing Growth Arrest and Apoptosis

In this study,one immortalized human normal prostatic epithelial cell line（BPH） and four human prostate cancer cell lines（LNCa P,22Rv1,PC-3,and DU-145） were treated with Ganoderma Lucidum triterpenoids（GLT） at different doses and for different time periods. Cell viability,apoptosis,and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and-3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest（mainly G1 arrest） was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4（CDK4） and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis,which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

A Novel Schiff Base Zinc Coordination Compound Inhibits Proliferation and Induces Apoptosis of Human Osteosarcoma Cells

Various kinds of schiff base metal complexes have been proven to induce apoptosis of tumor cells. However,it remains largely unknown whether schiff base zinc complexes induce apoptosis in human cancer cells. Here,we synthesized a novel schiff base zinc coordination compound（SBZCC） and investigated its effects on the growth,proliferation and apoptosis of human osteosarcoma MG-63 cells. A novel SBZCC was synthesized by chemical processes and used to treat MG-63 cells. The cell viability was determined by CCK-8 assay. The cell cycle progression,mitochondrial membrane potential and apoptotic cells were analyzed by flow cytometry. The apoptosis-related proteins levels were determined by immunoblotting. Treatment of MG-63 cells with SBZCC resulted in inhibition of cell proliferation and cell cycle arrest at G1 phase. Moreover,SBZCC significantly reduced the mitochondrial membrane potential and induced apoptosis,accompanied with increased Bax/Bcl-2 and Flas L/Fas expression as well as caspase-3/8/9 cleavage. Our results demonstrated that the synthesized novel SBZCC could inhibit the proliferation and induce apoptosis of MG-63 cells via activating both the mitochondrial and cell death receptor apoptosis pathways,suggesting that SBZCC is a promising agent for the development as anticancer drugs.

Knockdown of Bmi1 Inhibits Bladder Cancer Cell Growth Both In Vitro and In Vivo by Blocking Cell Cycle at G_1 Phase and Inducing Apoptosis

Bmi1 is a member of the polycomb group family of proteins,and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. However,its role in the initiation and progression of bladder cancer is not clearly known. The present study aimed to investigate the function of Bmi1 in the development of bladder cancer. Bmi1 expression was detected in human bladder cancer tissues and their adjacent normal tissues（n=10） by immunohistochemistry,q RT-PCR and Western blotting,respectively. Bmi1 small interference RNA（si RNA） was synthesized and transfected into human bladder carcinoma cells（EJ） by lipofectamine 2000. The Bmil expression at m RNA and protein levels was measured in EJ cells transfected with Bmil si RNA（0,80,160 nmol/L） by q RT-PCR and Western blotting,respectively. Cell viability and Ki67 expression（a marker of cell proliferation） were determined in Bmi1 si RNA-transfected cells by CCK-8 assay and q RT-PCR,respectively. Cell cycle of transfected cells was flow-cytometrically determined. Immunofluorescence and Western blotting were used to detect the expression levels of cell cycle-associated proteins cyclin D1 and cyclin E in the cells. Pro-apoptotic proteins Bax and caspase 3 and anti-apoptotic protein Bcl-2 were detected by Western blotting as well. Additionally,xenograft tumor models were established by inoculation of EJ cells（infected with Bmil sh RNA/p LKO.1 lentivirus or not） into nude mice. The tumor volumes were measured every other day for 14 days. The results showed that the Bmil expression was significantly increased in bladder tumor tissues when compared with that in normal tissues（P〈0.05）. Perturbation of Bmi1 expression by using si RNA could significantly inhibit the proliferation of EJ cells（P〈0.05）. Bmi1 si RNA-trasnfected EJ cells were accumulated in G1 phase and the expression levels of cyclin D1 and cyclin E were down-regulated. Bax and caspase-3 expression levels were significantly increased and Bcl-2 levels decreased after Bmi1 knockdown. Tumor volume was conspicuously reduced in mice injected with EJ cells with Bmi1 knockdown. Our findings indicate that Bmi1 is a potential driver oncogene of bladder cancer and it may become a potential treatment target for human bladder cancer.

Advances in the knowledge on the role of apoptosis repressor with caspase recruitment domain in hemorrhagic stroke

The apoptosis repressor with caspase recruitment domain(ARC)plays a critical role in extrinsic apoptosis initiation via death receptor ligands,physiological stress,infection response in a tissue-dependent manner,endoplasmic reticulum(ER)stress,genotoxic drugs,ionizing radiation,oxidative stress,and hypoxia.Recent studies have suggested that regulating apoptosis-related pathways can improve outcomes for patients with neurological diseases,such as hemorrhagic stroke.ARC expression is significantly correlated with acute cerebral hemorrhage.However,the mechanism by which it mediates the anti-apoptosis pathway remains poorly known.Here,we discuss the function of ARC in hemorrhagic stroke and argue that it could serve as an effective target for the treatment of hemorrhagic stroke.

Upregulation and activation of caspase-3 or caspase-8 and elevation of intracellular free calcium mediated apoptosis of indomethacin-induced K562 cells

Background A nonsteroidal anti-inflammatory drug, indomethacin, has been shown to have anti-leukemic activity and induce leukemic cell opoptosis. This study was to elucidate the mechanism of indomethacin-induced K562 cell apoptosis.Methods K562 cells were grown in RPMI 1640 medium and treated with different doses of indomethacin (0 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L, 800 μmol/L) for 72 hours. The cells were harvested, and cell viability or apoptosis was analyzed using MTT assay and AO/EB stain, combining laser scanning confocal microscopy (LSCM) technique separately. For the localization and distribution of intracellular caspase-3 or caspase-8 protein, immunofluorescence assay was carried out. To reveal the activation of caspase-3 or caspase-8 in indomethacin-treated cells, Western blot detection was used. The change in intracellular free calcium was determined by Fluo-3/ Am probe labeling combined with LSCM. Results Indomethacin could lead to K562 cell apoptosis and inhibit cell viability in a concentration-dependent manner. An increased expression of intracellular caspase-3 or caspase-8 was observed at higher doses of indomethacin (400-800 μmol/L). Western blot results showed upregrulation and activation in both caspase-3 and caspase-8 protein. Under indomethacin intervention, the levels of intracellular free calcium showed a significant increase. Blocking the activity of cyclooxygenase did not abolish the effects of indomethacin on K562 cell apoptosis.Conclusions Activation and upregulation of caspase-3 or caspase-8 protein were responsible for Indomethacin-induced K562 cell apoptosis. Variation of intracellular free calcium might switch on the apoptotic pathway and the proapoptotic effect of indomethacin might be cyclooxygenase-independent.