基于PI3K/Akt/GSK3β信号通路研究心宝丸对慢性心力衰竭大鼠心肌肥厚的影响

Effect of Xinbao Pill on Myocardial Hypertrophy in Chronic Hearth Failure Rats by Regulating the PI3K/Akt/GSK3βPathway

目的探讨心宝丸是否能够通过调控PI3K/Akt/GSK3β信号通路抑制慢性心力衰竭(CHF)大鼠模型心肌肥厚。方法将60只雄性SD大鼠按体重随机分为假手术组、模型组、心宝丸低、中、高剂量组和贝那普利组。除假手术组外所有大鼠采用腹主动脉缩窄法复制CHF模型,假手术组只分离腹主动脉,不结扎,术后4周心宝丸药物干预组[40、80、120 mg/(kg·d)]和贝那普利组[1.05 mg/(kg·d))]开始灌胃给药8周,假手术组和模型组用等体积生理盐水灌胃8周。术后12周用超声心动图检测大鼠心脏功能,全部大鼠禁食24 h后处死,HE染色法观察心肌组织形态,小麦胚凝集素染色(WGA)观察心肌细胞大小,ELISA法检测血清NT-proBNP值,Real-time PCR法检测肥厚相关基因ANP、Myh7 mRNA表达水平,Western Blot法检测心肌组织PI3K、p-PI3K、Akt、p-Akt、GSK3β、p-GSK3β蛋白表达水平。结果与假手术组比较,模型组大鼠术后第12周左心室射血分数(LVEF)、短轴缩短率(FS)降低(P<0.01),左心室收缩末期内径(LVIDs)升高(P<0.05),心肌细胞横截面积(CSA)增大(P<0.01),血清NT-proBNP水平升高(P<0.01),ANP、Myh7 mRNA及p-PI3K、p-AKT、p-GSK3β蛋白表达水平上调(P<0.01,P<0.05),GSK3β表达水平下调(P<0.01);与模型组比较,心宝丸低、中、高剂量组和贝那普利组LVEF升高(P<0.05,P<0.01),心肌细胞大小减小(P<0.01),Myh7 mRNA表达水平降低(P<0.05,P<0.01),血清中NT-proBNP水平降低(P<0.01),p-PI3K、p-AKT、p-GSK3β表达水平下调(P<0.01),GSK3β表达水平上调(P<0.01),心宝丸中、高剂量组FS升高(P<0.05),心宝丸中、高剂量组和贝那普利组LVIDs减小(P<0.05),心宝丸高剂量组和贝那普利组ANPmRNA表达水平降低(P<0.01);HE染色结果显示:心宝丸可以改善心肌肥厚,心肌细胞排列紊乱,间隙增宽的病理变化。结论心宝丸可以抗心肌肥厚,提升心脏功能,其机制可能与抑制PI3K/Akt信号的磷酸化激活,抑制GSK3β的磷酸化有关。

Objective To investigate whether Xinbao Pill(XBP)attenuate myocardial hypertrophy in chronic heart failure(CHF)rats by regulating the PI3K/Akt/GSK3βpathway.Methods Totally 60 male Sprague-Dawley rats were randomly divided into sham-operated group,model group,as well as low,medium and high dose XBP groups as well as Benazepril group according to their weight.All the rats except the sham-operated group underwent abdominal aortic banding to establish the CHF model.The sham-operated group were only separated the abdominal aorta without ligation.Four weeks after operation,the XBP treatment groups(40,80,120 mg·kg^-1·d^-1)and the Benazepril group(1.05 mg·kg^-1·d^-1)were intragastrically administered for 8 weeks.The sham-operated group and the model group were intragastrically administered with an equal volume of normal saline for 8 weeks.Cardiac function was measured by echocardiography 12 weeks after operation.All rats were sacrificed after fasting for 24 hours.Myocardial tissue sections were taken for HE staining to observe myocardial morphology.Wheat germ agglutinin staining was used to observe the mycote cross-sectional area(CSA).The serum separated from abdominal aorta blood was used to detected the level of NT-proBNP by ELISA.RT-PCR was used to detect the expressions of the mRNA of ANP and Myh7.The expression levels of PI3K,p-PI3K,Akt,p-Akt,GSK3βand p-GSK3βwere detected by Western Blot.Results Compared with the sham group,the left ventricular eiection function(LVEF)and frac-tional shortening(FS)decreased in the 12 th week after surgery(P<0.01),the LVIDs increased(P<0.05),the CSA increased(P<0.01),the level of NT-proBNP increased(P<0.01),the contents of ANP,Myh7 mRNA in-creased(P<0.01),the protein expression levels of p-PI3K,p-AKT,p-GSK3βupregulated(P<0.01,P<0.05),the level of GSK3βdownregulated(P<0.01),significantly in the model group.Compared with the model group,the LVEF improved(P<0.05,P<0.01),the CSA decreased(P<0.01),the level of Myh7 mRNA decreased(P<0.05,P<0.01),the level of NT-proBNP in serum declined(P<0.01),the protein expression levels of p-PI3K,p-AKT,p-GSK3βdownregulated(P<0.01),the level of GSK3βupregulated(P<0.01),significantly in the XBP for low,medium and high dose groups and Benazepril group,the FS improved significantly in XBP for medium and high dose groups(P<0.05),the LVIDs decreased significantly in XBP for medium and high dose groups and Benazepril group(P<0.05),the level of ANP mRNA decreased significantly in XBP for high dose group and Benazepril group(P<0.01).The results of HE staining exhibited that XBP could improve the pathological changes of cardiac hyper-trophy and myocardial cell arrangement disorder.Conclusion XBP can retard the progression of myocardial hyper-trophy and improve cardiac functions,probably via inhibiting the phosphorylation of GSK3βby inhibiting the phosphorylation of PI3K/Akt pathway.