鹿芪方调控PAM信号通路抑制HIF-1α介导的心肌细胞自噬改善病理性心肌肥大的机制研究

Mechanism research of Luqi Formula regulating PAM signaling pathway to suppress HIF-1α-mediated autophagy in cardiomyocytes to ameliorate pathological cardiac hypertrophy

目的:探究鹿芪方治疗压力超负荷诱导的病理性心肌肥大(CH)的作用机制。方法:将60只C57/BL6J小鼠随机分为空白组、模型组、鹿芪方组、抑制剂组[3-甲基腺嘌呤(3-MA)抑制剂]。除空白组外均采用主动脉弓缩窄术诱导构建CH模型,28 d取材。明场成像观察心脏整体形态,WGA和α-actinin染色观察心肌细胞表面积,免疫荧光明确自噬标志物LC3B表达水平,Western Blot检测磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶Bα(Akt1)、磷酸化蛋白激酶Bα(p-Akt1)、哺乳动物雷帕霉素靶蛋白(mTOR)、缺氧诱导因子-1α亚基(HIF-1α)、微管相关蛋白1轻链3B(LC3B)、Beclin-1蛋白表达。RT-qPCR检测心房利钠肽前体A(Nppa)、心房利钠肽前体B(Nppb)mRNA的表达。结果:体内实验结果显示,鹿芪方可改善小鼠心肌肥大,缩小心肌细胞表面积;抑制自噬相关蛋白LC3B、Beclin-1的表达(P<0.01,P<0.05);多组学测序结果和实验验证显示鹿芪方显著抑制PI3K/Akt/mTOR通路(PAM信号通路)和HIF-1α蛋白表达(P<0.05)。体外实验结果显示,与模型组比较,鹿芪方组心肌细胞表面积缩小;Nppa、Nppb mRNA表达水平降低(P<0.05);LC3B、Beclin-1蛋白表达水平降低(P<0.05)。与模型组比较,鹿芪方组PAM信号通路和HIF-1α蛋白表达降低(P<0.01,P<0.05)。结论:鹿芪方通过抑制PAM信号通路介导的HIF-1α蛋白表达,抑制心肌细胞自噬,改善CH。

Objective:To investigate the mechanism of Luqi Formula(LQF)in treating pressure overload-induced pathologic cardiac hypertrophy(CH).Methods:Sixty C57/BL6J mice were randomly divided into blank group,model group,LQF group,and inhibitor group[3-methyladenine(3-MA)inhibitor].The CH model was constructed by aortic arch constriction induction in all except the blank group,and was sacrificed at 28 days.Bright-field microscopy was taken to observe the overall morphology of the heart,WGA andα-actinin staining was used to observe the surface area of cardiomyocytes,immunofluorescence was used to clarify the expression level of the autophagy marker LC3B,and Western Blot was used to detect the protein expression of PI3K,Akt1,p-Akt1,mTOR,HIF-1α,LC3B,and Beclin-1.RT-qPCR was used to detect Nppa,Nppb mRNA expression.Results:The results obtained for the in vivo experiments demonstrated that the LQF group ameliorated myocardial hypertrophy,reduced cardiomyocytes surface,and inhibit the expression of autophagy-related proteins LC3B and Beclin-1(P<0.01,P<0.05);the results of multi-omics sequencing and experimental validation demonstrated that the LQF significantly inhibited the expression of the PI3K/Akt/mTOR pathway(PAM signaling pathway)and HIF-1αproteins(P<0.05).The in vitro results revealed that compared with the model group,the cardiomyocytes surfuce in the LQF group was reduced;the expression levels of Nppa and Nppb mRNA were decreased(P<0.05);and the expression levels of LC3B and Beclin-1 proteins were decreased(P<0.05).Compared with the model group,PAM signaling pathway and HIF-1αprotein expression were reduced in the LQF group(P<0.01,P<0.05).Conclusion:LQF inhibited cardiomyocyte autophagy and ameliorated CH by inhibiting HIF 1αprotein expression mediated by PAM signaling axis.