GSK3β在HGF信号通路中对改善心肌缺血再灌注损伤的作用研究

Study on role of GSK3β for improving myocardial ischemia reperfusion injury in HGF signaling pathway

目的探讨糖原合成激酶3β(GSK3β)的活性在肝细胞生长因子(HGF)信号通路中对改善心肌缺血再灌注损伤的作用及机制。方法研究大鼠离体心肌细胞H9c2,用不同类型的GSK3β腺病毒[不表达GSK3β的重组腺病毒Ad-GFP;野生型GSK3β腺病毒GSK3β-Ad-wt;表达GSK3β不可磷酸化的组成型活性突变体,其第9位丝氨酸残基突变(Ser9)为丙氨酸,从而不能再被磷酸化失活而具有固有活性的GSK3β腺病毒GSK3β-Ad-S9A;表达催化失活的GSK3β复制缺陷型腺病毒载体,其第85位赖氨酸残基突变为甲硫氨酸,从而持续失活的GSK3β腺病毒GSK3β-Ad-K85A]感染细胞和HGF激活剂刺激细胞,分为对照组、缺血再灌注(I/R)组、I/R+HGF组、I/R+HGF+Ad-GFP组、I/R+HGF+GSK3β-Ad-wt组、I/R+HGF+GSK3β-Ad-S9A组和I/R+HGF+GSK3β-Ad-K85A组。Western blot检测GSK3β,下游通路蛋白肝激酶B1(LKB1),腺苷单磷酸激活蛋白激酶(AMPK),自噬相关蛋白Beclin-1、LC3Ⅱ及上游通路蛋白激酶B(Akt)的表达水平。结果与对照组和I/R组比较,HGF激活剂能促进pGSK3β,下游通路蛋白LKB1、AMPK,自噬相关蛋白LC3Ⅱ、Beclin-1,上游蛋白pAkt的表达,差异有统计学意义(P<0.05);而在HGF激活剂的作用下,GSK3β的失活(Adwt和Ad-K85A腺病毒感染组)使下游通路蛋白LKB1、AMPK和自噬相关蛋白Beclin-1、LC3Ⅱ的表达量明显升高,而在I/R+HGF+GSK3β-Ad-S9A组则明显降低,差异有统计学意义(P<0.05)。结论 HGF通路通过激活磷脂酰肌醇3激酶(PI3K)/Akt/GSK3β/AMPK信号通路促进细胞自噬并改善心肌细胞缺血再灌注损伤。

Objective To investigate the role and mechanism of GSK3β activation for improving myocardial ischemia reperfusion injury(IRI)in HGF signaling pathway.Methods The rat isolated cardiomyocytes H9c2 were selected as the study objects.The different kinds of GSK3β adenovirus[not expressing GSK3β recombinant adenovirus Ad-GFP;wild type GSK3β adenovirus GSK3β-Ad-wt;expressing GSK3β nonphosphorylation constitutive type active mutant,its serine residue at site 9(Ser9)mutating as alanine,thus which can not be inactivated again by phosphorylation,and possessing active GSK3β adenovirus GSK3β-AdS9A;expressing catalytically inactivated GSK3β for duplicating deletion type adenovirus vector,its lysine residue at the site 85 mutating as methionine,thus persistently inactivated GSK3β adenovirus GSK3β-Ad-K85 A]were used to infect the cells and HG activator was used to stimulate the cells,which divided into the control group,ischemia and reperfusion(I/R)group,I/R+HGF group,I/R+HGF+ Ad-GFP group,I/R+HGF+GSK3β-Ad-wt group,I/R+HGF+GSK3β-Ad-K85A group and I/R+HGF+GSK3β-Ad-S9A group.The expression levels of downstream pathways proteins LKB1 and AMPK,autophagy-related proteins LC3Ⅱand Beclin1,and upstream pathway protein Akt were detected by Western blot.Results Compared with the control group and I/R group,the HGF stimulator could promote the expressions of pGSK3β,downstream pathway protein LKB1 and AMPK,autophagy-related proteins LC3Ⅱ and Beckin1,and upstream protein pAkt,the differences were statistically significant(P<0.05);while under the stimulation of HGF activator,the inactivation of GSK3β(the Ad-wt and Ad-K85A group)significantly increased the expression levels of its downstream proteins LKB1 and AMPK,autophagy-related proteins LC3Ⅱ and Beclin-1,whereas which significantly decreased in the I/R+HGF+GSK3β-Ad-S9A group,the differences were statistically significant(P<0.05).Conclusion The HGF pathway can promote the cellular autophagy and improve the myocardial ischemia and reperfusion injury by activating the PI3K/Akt/GSK3β/AMPK signaling pathway.