PI3K/Akt/Sirt1信号通路介导硫化氢后处理对大鼠缺血心肌的保护作用

PI3K/Akt/Sirt1 signaling pathway mediated hydrogen sulfide postconditioning-induced protection against I/R injury

目的探讨PI3K/Akt/Sirt1信号通路是否参与硫化氢(H_2S)抗心肌缺血/再灌注(I/R)损伤的作用。方法采用Langendorff灌流装置建立大鼠离体心脏I/R损伤模型,平衡灌注20 min后,全心停灌30 min,复灌60 min。60只♂SD大鼠,随机分为5组(n=12);空白组(Control组)、缺血/再灌注组(I/R组)、H_2S后处理组(H_2S组)、抑制剂LY294002组(LY组)、H_2S后处理+抑制剂组(H_2S+LY组)。统计平衡末及再灌注末的左室舒张末期压(LVEDP)、左室发展压(LVDP)、左室内压上升最大速率(+dp/dt_(max))和左室内压下降最大速率(-dp/dt_(max));TTC法测定心肌梗死面积;实时荧光定量PCR法检测Sirt1和PGC-1α的mRNA含量;通过Western blot法检测总的Sirt1和PGC-1α的蛋白表达水平;免疫组化检测Sirt1的细胞分布情况。结果各组间的心功能指标在平衡末差异无统计学意义(P>0.05)。再灌注60min,H_2S组与I/R组相比,心功能的各项指标明显改善(P<0.05),心肌梗死面积减少(26.9±4.9)%vs(48.9±5.6)%(P<0.05);Sirt1和PGC-1α表达水平明显升高(P<0.05);Sirt1的细胞核阳性表达指数增加(P<0.05)。LY294002逆转了H_2S后处理产生的心肌保护效应,使H_2S后处理+抑制剂组心功能指标、Sirt1和PGC-1α的表达及Sirt1的细胞核阳性表达指数降低,心肌梗死面积增加。结论PI3K/Akt/Sirt1信号通路参与了H_2S后处理对大鼠缺血心肌的保护作用。

Aim To explore the role of PI3K/Akt/Sirt1 pathway in cardioprotection of hydrogen sulfide(H_2S) postconditioning against ischemia/reperfusion(I/R) injury.Methods Langendorff perfusion apparatus was used to build an isolated rat myocardial I/R model.Isolated rat hearts were subjected to 30 min global ischemia followed by 60 min reperfusion after 20 min of equilibrium.60 male SD rats were randomly divided into 5 groups(n = 12):control group(Control),ischemia/reperfusion group(I/R),H_2S postconditioning group(H_2S),inhibitor LY294002 group(LY) and H_2S with inhibitor group(H_2S±LY).The left ventricular diastolic pressure(LVEDP),the left ventricular developed pressure(LVDP),the maximum rate of increase or decrease of left ventricular pressure(±dp/dt_(max)) were registered at the end of 20 min equilibrium,30 and 60 min of reperfusion separately.Triphenyl tetrazolium chloride(TTC) staining was used to determine the myocardial infarct size.The levels of Sirtl and PGC-1 mRNA were tested using real-time PCR.The expressions of Sirt1 and PGC-1α were detected with Western blot analysis.Immunohistochemical method was used to determine the location of Sirt1.Results There were no differences in equilibrium hemodynamics observed between the experimental groups(P >0.05).At the end of reperfusion,compared with I/R group,H_2S group had obviously ameliorated functional recovery and significantly decreased the myocardial infarct size(26.9±4.9)%vs(48.9±5.6)%(P < 0.05).Meanwhile,the expression of Sirtl and PGC-la increased significantly.However,LY294002 reversed the cardioprotective effects provided by hydrogen sulfide postconditioning and reduced the level of Sirt1 and PGC-1α,the percentage of Sirt1-positive nuclei.Conclusion PI3K/Akt/Sirt1 signaling pathway mediates the hydrogen sulfide postconditioning-induced protection against I/R injury.