PI3K/Akt调节线粒体Cx43蛋白表达在H_2S后处理离体大鼠心肌中的保护作用

目的探讨PI3K/Akt信号通路是否通过调节线粒体缝隙连接蛋白Cx43而在硫化氢(H2S)后处理中减轻离体大鼠心脏缺血/再灌注(I/R)损伤。方法 56只♂SpragueDawley(SD)大鼠随机分为4组(n=14):缺血/再灌注组(I/R组),PI3K/Akt信号通路抑制剂LY294002组(LY组),硫化氢后处理组(NP组),硫化氢后处理+PI3K/Akt信号通路抑制剂LY294002组(N+L组)。采用离体心脏Langen-dorff灌注模型,平衡灌注20 min后停灌30 min复灌60 min。记录平衡末及灌注结束时的心率(HR)、左室舒张末期压(LVEDP)、左室发展压(LVDP)、左室内压上升最大速率(+dp/dtmax)、左室内压下降最大速率(-dp/dtmax);灌注结束时,TTC法染色心肌切片并计算心肌梗死面积百分比;TUNEL法检测心肌细胞凋亡,计算凋亡指数(AI);Westernblot半定量线粒体总的Cx43(total connexin 43,tCx43)和磷酸化Cx43(phosphorylated connexin 43,pCx43)表达水平。结果平衡灌注末各组间心功能指标差异无统计学意义。再灌注后,与I/R组比较,NP组心功能的各项指标明显改善(P<0.05);心肌梗死面积减少(26.5±4.2)%vs(44.5±5.3)%(P<0.05);凋亡指数降低(25.9±3.0)%vs(43.1±1.9)%(P<0.05);线粒体tCx43和pCx43蛋白表达水平明显升高。LY294002逆转了H2S后处理产生的心肌保护效应,使N+L组心功能指标及线粒体中tCx43和pCx43的表达水平降低(P<0.05),心肌梗死面积及凋亡指数均增加(P<0.05)。结论 PI3K/Akt信号通路通过上调线粒体缝隙连接蛋白Cx43蛋白的表达而在硫化氢(H2S)后处理中减轻离体大鼠I/R损伤。

Cardioprotective effect of connexin 43 expression regulated by PI3K/Akt on hydrogen sulfide postconditioning in isolated ischemic and reperfused rat hearts

Aim To investigate whether the PI3K/Akt signaling pathway regulates connexin 43 expression to protect isolated rat hearts against ischemia/reperfusion(I/R)injury in hydrogen sulfide postconditioning.Methods Hearts of 56 male SD rats were isolated and linked to the Langendorff apparatus.They were randomly divided into 4 groups(n=14): ischemia reperfusion group(I/R),PI3K/Akt signaling pathway inhibitor LY294002 group(LY),hydrogen sulfide postconditioning group(NP),and hydrogen sulfide with LY294002 group(N+L).The heart rate(HR),the left ventricular diastolic pressure(LVEDP),the left ventricular developed pressure(LVDP),the maximum rate of increase or decrease of left ventricular pressure(±dp/dtmax) were recorded at 20 min of equilibrium at 30 min of reperfusion and at the end of reperfusion,respectively.Myocardial infarct size was measured by triphenyltet tetrazolium chloride(TTC) staining.Myocardial TUNEL staining was determined by in situ cell death detection kit.The ratio of TUNEL positive nuclei to all the nuclei counted was used as apoptotic index(AI).The expression of total Cx43 and phosphorylation Cx43 in mitochondria were determined with Western blot analysis at the end of reperfusion.Results No differences in baseline hemodynamics were observed among the experimental groups(P>0.05).After reperfusion,compared with I/R group,NP group had better hemodynamics;the myocardial infarct size and cardiocyte apoptotic index were much lower(P<0.05);the expression of tCx43(total connexin 43,tCx43) and pCx43(phosphorylated connexin 43,pCx43) in mitochondria increased significantly.However,LY294002 abolished the cardioprotection offered by hydrogen sulfide postconditioning and the increase in tCx43 and pCx43 expression in mitochondria.Conclusion The PI3K/Akt pathway upregulates connexin 43 expression to protect isolated rat hearts against ischemia/reperfusion(I/R)injury in hydrogen sulfide postconditioning.