PI3K/Akt/FoxO3a/Bim信号通路介导硫化氢后处理对缺氧H9c2心肌细胞的保护作用

PI3K/Akt/FoxO3a/Bim signaling pathway mediated hydrogen sulfide postconditioning-induced protection against H/R injury

目的探讨硫氢化钠(Na HS)后处理是否通过PI3K/Akt/Fox O3a/Bim信号通路调节细胞凋亡发挥减轻心肌细胞缺氧/复氧(H/R)损伤的作用。方法 H9c2大鼠心肌细胞缺氧3 h/复氧6 h,建立缺氧/复氧损伤模型。将细胞随机分为5组:空白组(Control组)、缺氧/复氧组(H/R组)、硫氢化钠后处理组(H/R+Na HS组)、抑制剂LY294002组(H/R+LY组)、硫氢化钠后处理+LY294002组(H/R+Na HS+LY组)。分别在缺氧前、复氧末检测H9c2心肌细胞的存活率以及LDH释放;流式细胞术检测各组的细胞凋亡率;应用Western blot检测Akt、p-Akt、Fox O3a、p-Fox O3a、Bim蛋白的表达水平;免疫荧光检测Fox O3a的分布情况。结果缺氧前各组心肌细胞存活率、LDH释放量差异无统计学意义(P>0.05)。复氧末,Na HS组与H/R组相比,心肌细胞存活率明显提高(P<0.05),LDH释放量与细胞凋亡率明显降低(P<0.05);p-Akt、p-Fox O3a蛋白表达水平升高,Bim表达降低,同时Fox O3a在细胞质的表达升高。LY294002逆转了Na HS后处理产生的心肌细胞保护作用,使得H/R+Na HS+LY组的心肌细胞存活率降低(P<0.05)、LDH释放和细胞凋亡率升高(P<0.05),p-Akt、p-Fox O3a蛋白表达降低(P<0.05),Bim表达升高(P<0.05),FoxO 3a在细胞质的表达水平降低。结论硫氢化钠(NaH S)后处理通过PI3K/Akt/FoxO 3a/Bim信号通路调控细胞凋亡,减轻心肌细胞缺氧/复氧(H/R)损伤。

Aim To explore the role of PI3K/Akt/FoxO3a/Bim pathway in H9c2 cardiomyocytes cardioprotection of hydrogen sulfide（H2S） postconditioning against hypoxia/reoxygenation（H/R） injury.Methods H9c2 cells were subjected to 3h hypoxia followed by6 h reoxygenation.Cells were divided into 5 groups：control group（Control）,hypoxia/Reoxygenation group（H/R）,Na HS postconditioning group（H/R ＋Na HS）,Na HS with inhibitor LY294002 group（H/R ＋Na HS ＋ LY） and LY294002 group（H/R ＋ LY）.The survival percentage of H9c2 cells and the release of LDH were detected at pre-hypoxia and reoxygenation 6h.The flow cytometry was used to determine cell apoptosis at the end of reoxygenation.The expressions of Akt,p-Akt,Fox O3 a,p-Fox O3 a,Bim were detected by Western blot.Immunofluorescence staining was used to determine the expression of Fox O3 a.Results Therewere no differences between each group before hypoxia（P〈0.05）.At the end of reoxygenation,cell viability in H/R ＋ Na HS group was significantly improved（P〈0.05）,and the release of LDH and cell apoptosis rates were significantly decreased（P〈0.05）.Meanwhile,the expression of p-Akt,p-Fox O3 a and cytoplasm Fox O3 a was significantly improved,and Bim was significantly decreased.But LY294002 abolished the myocardial protection effects of Na HS,and significantly decreased p-Akt,p-Fox O3 a and cytoplasm Fox O3 a level,and increased Bim level.Conclusion PI3K/Akt/Fox O3a/Bim signaling pathway mediates hydrogen sulfide postconditioning-induced protection against H/R injury.