瓜蒌薤白半夏汤对缺血性心肌损伤大鼠的线粒体功能障碍和AMPK/PGC-1α信号通路的影响

Effect of Gualou Xiebai Banxiatang on Mitochondrial Dysfunction and AMPK/PGC-1α Signaling Pathway in Rats with Ischemic Myocardial Injury

目的:观察瓜蒌薤白半夏汤对缺血性心肌损伤大鼠的线粒体功能障碍及腺苷酸活化蛋白激酶(AMPK)/过氧化物酶体增殖物激活受体γ辅激活因子^(-1)α(PGC^(-1)α)信号通路的影响。方法:70只雄性SD大鼠,随机选取6只作为正常组(CON),其余给予高脂饲料结合注射异丙肾上腺素(5 mg·kg^(-1)·d^(-1),7 d),建立以高血脂为基础的缺血性心脏病模型。将造模成功者随机分为模型组(MOD)、瓜蒌薤白半夏汤高剂量(GXBD-H)组、瓜蒌薤白半夏汤中剂量(GXBD-M)组、瓜蒌薤白半夏汤低剂量(GXBD-L)组和美托洛尔(MET)组。其中,GXBD-H组、GXBD-M组和GXBD-L组大鼠分别给予11.2、5.6、2.8 g·kg^(-1)·d^(-1)的瓜蒌薤白半夏汤水煎液,MET组给予美托洛尔2.6 mg·kg^(-1)·d^(-1),CON组和MOD组给予等体积纯净水,连续28 d。心导管法测量大鼠血流动力学;透射电镜分析心肌组织线粒体变化;酶联免疫吸附测定法(ELISA)检测血清脑钠肽(BNP)和心肌肌钙蛋白T(cTnT)水平;线粒体膜电位检测试剂盒(JC^(-1)法)检测线粒体膜电位;实时荧光定量聚合酶链式反应(Real-time PCR)检测线粒体DNA拷贝数的变化;分光光度计法检测心肌组织三磷酸腺苷(ATP)含量。蛋白免疫印迹法(Western blot)检测心肌组织磷酸化AMPK(p-AMPK)、AMPK、PGC^(-1)α、核呼吸因子1(NRF1)和线粒体转录因子A(TFAM)的表达水平。结果:与CON组比较,MOD组的左室收缩末期压(LVESP)和左室舒张末期压(LVEDP)明显升高(P<0.05,P<0.01),以及心室收缩时室内压最大上升速率(+dp/dtmax)和左室舒张时室内压最大下降速率(-dp/dtmax)显著降低(P<0.01);心指数和心肌间质纤维化面积均显著升高(P<0.01)及线粒体破坏损伤;血清BNP、cTnT和丙二醛(MDA)显著升高(P<0.01),以及血清超氧化物歧化酶(SOD)水平显著降低(P<0.01);心肌线粒体膜电位、DNA拷贝数和ATP水平均显著降低(P<0.01);心肌组织的p-AMPK/AMPK、PGC^(-1)α、NRF1和TFAM蛋白水平显著减少(P<0.01)。与MOD组比较,GXBD-H和GXBD-M组的LVESP、LVEDP、+dp/dt_(max)和-dp/dt_(max)明显改善(P<0.05,P<0.01);心指数和心肌间质纤维化面积均明显降低(P<0.05,P<0.01)及线粒体损伤减轻;血清BNP、cTnT和MDA明显降低(P<0.05,P<0.01),及血清SOD水平显著升高(P<0.01);心肌线粒体膜电位、DNA拷贝数和ATP水平均明显增加(P<0.05,P<0.01);心肌组织的p-AMPK/AMPK、PGC^(-1)α、NRF1和TFAM蛋白水平明显升高(P<0.05,P<0.01)。结论:瓜蒌薤白半夏汤可以降低心肌损伤模型大鼠的心功能和心肌病理形态变化,抑制线粒体功能障碍,并上调心肌组织p-AMPK/AMPK、PGC^(-1)α、NRF1和TFAM蛋白表达变化。该研究为深入探索经典复方防治心肌损伤的效应机制,提供了新的实验室证据。

Objective:To observe the effect of Gualou Xiebai Banxiatang on mitochondrial dysfunction and adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)/peroxlsome proliferator-activated receptor-γcoactlvator^(-1)α(PGC^(-1)α)signaling pathway in rats with ischemic myocardial injury.Method:Seventy male SD rats were used in this experiment.Six rats from them were randomly selected as the control(CON)group,and the others were given high fat diet combined with isoproterenol injection(5 mg·kg^(-1)·d^(-1),7 d)to induce the rat model of ischemic heart disease based on hyperlipidemia.Successfully modeled rats were then randomly divided into model(MOD)group,high-dose Gualou Xiebai Banxiatang(GXBD-H)group,medium-dose Gualou Xiebai Banxiatang(GXBD-M)group,low-dose Gualou Xiebai Banxiatang(GXBD-L)group,and metoprolol(MET)group.Rats in the GXBD-H,GXBD-M,and GXBD-L groups were given 11.2,5.6,2.8 g·kg^(-1)·d^(-1)Gualou Xiebai Banxiatang,those in the MET group were given 2.6 mg·kg^(-1)·d^(-1)metoprolol,and those in the CON and MOD groups were given equal volume of pure water for 28 d.Hemodynamics were measured in rats by cardiac catheterization.Transmission electron microscopy was used to analyze myocardial mitochondria.Serum brain natriuretic peptide(BNP)and cardiac troponin T(cTnT)levels were detected by enzyme-linked immunosorbent assay(ELISA).Mitochondrial membrane potential assay kit(JC^(-1) method)was applied for detecting mitochondrial membrane potential.The changes in the mitochondrial DNA copy number were measured by real-time quantitative polymerase chain reaction(Real-time PCR).The content of adenosine triphosphate(ATP)in myocardial tissues was determined by spectrophotometer.The expression levels of p-AMPK,AMPK,PGC^(-1)α,nuclear respiratory factor 1(NRF1),and mitochondrial transcription factor A(TFAM)in myocardium was detected by Western blot.Result:As compared with the CON group,left ventricular end-systolic pressure(LVESP)and left ventricular end-diastole pressure(LVEDP)in the MOD group were significantly increased(P<0.05,P<0.01),and+dp/dt_(max) and-dp/dt_(max) were significantly decreased(P<0.01).In the MOD group,cardiac index and myocardial interstitial fibrosis area were significantly increased(P<0.01),accompanied by mitochondrial damage,serum BNP,cTnT,and malondialdehyde(MDA)were significantly increased(P<0.01),and serum superoxide dismutase(SOD)level was significantly decreased(P<0.01).The myocardial mitochondrial membrane potential,DNA copy number,and ATP level were significantly decreased(P<0.01),and the protein expression levels of p-AMPK/AMPK,PGC^(-1)α,NRF1,and TFAM in myocardial tissues were significantly decreased in the MOD group(P<0.01).Compared with the MOD group,the GXBD-H and GXBD-M groups significantly improved LVESP,LVEDP,+dp/dt_(max),and-dp/dt_(max)(P<0.05,P<0.01),significantly decreased heart index and myocardial interstitial fibrosis area(P<0.05,P<0.01),and alleviated mitochondrial damage.In the GXBD-H and GXBD-M groups,serum BNP,cTnT,and MDA were decreased significantly(P<0.05,P<0.01),serum SOD level was increased significantly(P<0.05),and myocardial mitochondrial membrane potential,DNA copy number,and ATP level were significantly increased(P<0.05,P<0.01).The protein levels of p-AMPK/AMPK,PGC^(-1)α,NRF1,and TFAM in myocardial tissues were significantly increased in the GXBD-H and GXBD-M groups(P<0.05,P<0.01).Conclusion:Gualou Xiebai Banxiatang has the effects of reducing the changes in cardiac function and myocardial pathology of rats with myocardial injury,inhibiting mitochondrial dysfunction,and up-regulating the protein expression levels of p-AMPK/AMPK,PGC^(-1)α,NRF1,and TFAM in myocardial tissues.This study provides new laboratory evidence for in-depth exploration of the mechanism of this classical compound in preventing and treating myocardial injury.