Study On The Anti-apoptotic Effect Of Survivin On Cardiomyocytes And Its Mechanism

ERK信号通路和PI3-K信号通路是Survivin的上游激活分子信号通路,并且DADLE还通过减少活性氧(ROS)的产生,阻止线粒体跨膜电位△(?)mt的下降,促进Bcl-2基因的转录和表达和抑制Bax蛋白从胞浆到线粒体的转位,发挥了对心肌细胞的保护作用。Activation of opioid receptors has been shown to be cardioprotective. Although activation of opioid receptors has been shown to regulate infarct size in kinds of cardiomyocytes its signal pathway in neonatal cardiomyocytes apoptosis remains elusive. Survivin is known to be essential for cell division and acts as an inhibitor of apoptosis during embryonic development and in adult cancerous tissues. However, the cardiovascular role of survivin is unknown. The present study aimed to investigate the role of survivin in the antiapoptotic effect ofδ-opioid receptor activation in cultured cardiomyocytes from neonatal rats. Cardiomyocyte apoptosis, as assessed by TdT-mediated dUTP nick end labeling, Hoechst 33258 staining and DNA ladder formation, was induced by serum/glucose deprivation (DEPV). mRNA transcripts of survivin and Bcl-2 were measured by quantitative real time polymerase chain reaction. Translocation of Bax from cytosol to mitochondria was determined by detection of the differential distribution of the protein in cytosol and in mitochondria. Protein levels of survivin, Bcl-2 and Bax were assessed by Western blot analysis. The levels of survivin, Bcl-2 and extracellular signal-regulated kinase (ERK) was positively correlated with the antiapoptotic action of a d-opioid receptor agonist, [D-Ala2, D-Leu5] -enkephalin acetate salt (DADLE). Whilst Bax translocation was inversely correlated with the changes of survivin and Bcl-2．Survivin RNA interference (RNAi) increased DEPV-induced cardiomyocyte apoptosis. Moreover, the antiapoptotic effect of DADLE was blunted by survivin RNAi. Inhibition of ERK prevented DADLE-induced decrease in apoptosis and Bax translocation, and increase in survivin and Bcl-2．DADLE-induced increase in survivin was also blunted by phosphoinositol 3-kinase (PI3-K) inhibition. In conclusion, survivin mediates the antiapoptotic effect ofδ-opioid receptor activation in cardiomyocytes. ERK and PI3-K are upstream regulators of survivin. Bcl-2 expression and Bax translocation are associated with this antiapoptotic action. 内源性的硫化氢(H₂S)被誉为是继一氧化氮(NO)和一氧化碳(CO)之后的第三种重要的气体信号分子,它具有重要的生理意义,在心血管系统,它有舒张血管降低血压,抑制血管平滑肌细胞增殖减轻血管重构以及减小缺血再灌注时的心肌梗死面积等多种生物学效应。但其对缺血缺氧引发的心肌细胞的凋亡是否有直接作用至今还未见报道。我们用原代培养的新生大鼠心肌细胞研究H₂S对心肌细胞的直接作用及其分子信号机制,发现生理浓度下的NaHS预处理心肌细胞能够抑制缺氧/复氧引发的心肌细胞能够增加Survivinå’ŒBcl-2表达,抑制Caspase3的关系是怎样是我们下一步要解决的问题。我们首次发现GSK3β的活性在NaHS抗心肌细胞凋亡中发挥重要作用,但是其作用机制如Survivin的表达是否受到GSK3β的活性的影响,GSK3β的上游调控分子是什么,GSK3β通路与MAPK通路间是否有串话等还有待进一步的研究。Endogenous hydrogen sulfide (H2S) is the third gaseous signaling molecule which discovered recently, having significant physiological functions. Injection of NaHS has been shown to regulate infarct size and decreased the duration and severity of ischemia/reperfusion-induced arrhythmias in the isolated heart while increasing cell viability in cardiac myocytes, its signal pathway in neonatal cardiomyocytes apoptosis remains elusive.The present study aimed to investigate the role of survivin in the antiapoptotic effect of H2S preconditioning in cultured cardiomyocytes from neonatal rats. NaHS preconditioning decreased the ischemia/reperfusion-induced apoptosis both in vivo and in vitro. H2S preconditioning increases survivin and Bcl-2 expression and GSK3(?) phosphorylation, while inhibits the activation of Caspase3 precursor. Survivin mediates the antiapoptotic effect of H2S preconditioning in cardiomyocytes. The upstream regulators of GSK3(?) and the crosstalk between survivin, Bcl-2 and Caspase3 precursor need to be further investigation.文中所用英文缩写及中英文对照3βWild type GSK3-βS9A GSK3-βS9A MutantR96A GSK3-βR96A MutantIPC缺血预适应(ischemia preconditioning)ERK细胞外信号调节激酶(extracellular signal-regulated kinase)ROS活性氧(reactive oxygen species)RNAi RNA干扰(RNA interference)PI3-K磷脂酰肌醇-3-激酶(phosphoinositol 3-kinase)TUNEL脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucleotidyl transferase mediated nick end labeling)IS/ RE Ischemia/ Reperfusion缺血/再灌注PBS磷酸盐缓冲液(phosphote buffered saline)AKT PI3-K controlled serine/threonine kinaseGFP绿色荧光蛋白(green fluorescent protein)PCR多聚酶链反应(polymerase chain reaction)...