当归芍药散对AD大鼠线粒体稳态及AMPK/SIRT1/PGC-1α信号通路的影响

Effect of Danggui Shaoyaosan on Mitochondrial Homeostasis and AMPK/SIRT1/PGC-1α Signaling Pathway in Rats Model of Alzheimer’s Disease

目的:探讨当归芍药散对阿尔茨海默病(AD)大鼠线粒体形态和功能的影响及作用机制。方法:采用双侧侧脑室注射链脲佐菌素(STZ)构建AD模型大鼠。40只大鼠随机分为假手术组、模型组、当归芍药散低、中、高剂量(12、24、36 g·kg^(-1))组,连续灌胃14 d后透射电镜观察大鼠海马区线粒体形态变化,原位末端标记法(TUNEL)检测细胞凋亡情况,免疫荧光检测活性氧(ROS)、过氧化物酶体增殖物激活受体γ辅激活因子-1α(PGC-1α)的表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测线粒体动力相关蛋白1(Drp1)、线粒体融合蛋白2(MFN2)、细胞色素C氧化酶亚基Ⅳ(COXⅣ)、PGC-1α mRNA的表达,蛋白免疫印迹法(Western blot)检测腺苷酸活化蛋白激酶(AMPK)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、沉默信息调节因子1(SIRT1)、PGC-1α蛋白的表达。结果:与假手术组比较,模型组大鼠海马区线粒体损伤明显,ROS产生和细胞凋亡率显著增高(P<0.01),MFN2、COXⅣ、PGC-1α mRNA表达显著下调,Drp1 mRNA表达显著上调,p-AMPK/AMPK、SIRT1、PGC-1α蛋白明显下调(P<0.05,P<0.01);与模型组比较,当归芍药散中、高剂量组明显改善线粒体损伤,ROS产生减少,细胞凋亡率下降(P<0.01),MFN2、COXⅣ、PGC-1α mRNA表达显著上调,Drp1 mRNA表达显著下调,p-AMPK/AMPK、SIRT1、PGC-1α蛋白显著上调(P<0.01)。结论:当归芍药散可以显著改善AD模型大鼠线粒体稳态失衡。

Objective:To investigate the effect of Danggui Shaoyaosan(DSS)on the morphology and function of mitochondria in rats model of Alzheimer’s disease(AD)and its possible mechanism. Method: Rats model of AD was established by injection of streptozocin(STZ)into bilateral ventricles of SD rats. The 40 rats were randomly divided into sham group,model group,low,medium and high dosages of Danggui Shaoyaosan(12,24,36 g·kg^(-1)·d^(-1))groups,observed the morphological changes of mitochondria in hippocampus of rats by electron microscopy after 14 days of continuous gavage. In situ end labeling(TUNEL) staining used to detect apoptosis and immunofluorescencereactive used to observe the expression of reactive oxygen species(ROS)and peroxisome proliferators-activated receptor γ coactivator lalpha(PGC-1α),quantitative Real-time polymerase chain reaction(Real-time PCR)detected the mRNA expression of dynamin-related protein 1(Drp1),mitochondrial fusion protein 2(MFN2),cytochrome C oxidase subunit Ⅳ(COX Ⅳ)and PGC-1α. Western blot detected the protein expression of adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK),phosphorylation(p)-AMPK,recombinant Sirtuin 1(SIRT1)and PGC-1α. Result: Compared with the sham group,the results of model group showed that the damage of mitochondria in hippocampus was more obvious,accelerated the ROS production and apoptosis rate(P<0.01),decreased the mRNA level of MFN2,COX Ⅳ,PGC-1α,increased the mRNA level of Drp1,and descended the protein of p-AMPK/AMPK,SIRT1,PGC-1α(P<0.05,P<0.01). Compared with the model group,the medium and high dose of DSS group notably improved the damage of mitochondria,reduced the production of ROS and apoptosis rate(P<0.01),promoted the mRNA expression of MFN2,COX Ⅳ,PGC-1α,inhibited the mRNA expression of Drp1,and up-regulated the protein of p-AMPK/AMPK,SIRT1,PGC-1α(P<0.01). Result: DSS can significantly ameliorate the mitochondrial homeostasis imbalance in AD rats.