摘要
目的探讨真武汤通过腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对扩张型心肌病(DCM)小鼠心功能、心肌细胞自噬的影响及其机制。方法将C57BL/6J小鼠10只作为正常组(A),cTnTR141W转基因扩张型心肌病小鼠30只,按照体质量随机分为模型组(B)、真武汤组(C)、卡托普利组(D),每组10只。实验用心脏超声检测各组小鼠心脏左室舒张末期内径(LVEDD)、左室收缩末期内径(LVESD)、左室短轴缩短率(FS)、左室射血分数(EF)的变化;光学显微镜观察心肌细胞形态及排列;实时荧光定量PCR(qPCR)检测重组人自噬相关蛋白3(ATG3)和重组人自噬相关蛋白7(ATG7),蛋白免疫印迹法(Western blot)检测自噬相关蛋白p62、AMPKα、mTOR、unc-51样激酶1(ULK1)、结节性硬化复合物2(TCS2)表达及其磷酸化水平。结果cTnT^(R141W)转基因扩张型心肌病小鼠,显微镜下心肌纤维排列紊乱,胶原纤维含量明显增多,真武汤干预后可见细胞排列渐规整、心肌纤维化减少,超声测量cTnT^(R141W)转基因扩张型心肌病小鼠心室扩大,心室射血分数及短轴缩短率较正常组明显降低,真武汤和卡托普利治疗后心脏超声提示左室舒张末、收缩末内径明显减小,射血分数明显增加。荧光定量PCR 2^(-△△CT)值比较发现,扩心病小鼠ATG3和ATG7表达增加,经过真武汤治疗后进一步上调ATG3和ATG7表达。Western blot结果中,与正常组比较,模型组AMPKα、p-AMPKα、p62、p-ULK1表达上调,mTOR、p-mTOR、ULK1、TCS2、磷酸化结节性硬化复合物2(p-TCS2)表达下调,相较于模型组,真武汤干预后明显上调p-AMPKα蛋白表达,下调AMPKα、p-mTOR、p62、p-ULK1、P-TCS2蛋白表达。结论真武汤通过温阳利水之功效治疗扩张型心肌病小鼠,可明显改善心脏功能,减少心肌纤维化,其机制可能与真武汤调控AMPKα/mTOR通路增加细胞自噬有关,此外真武汤的治疗还上调ATG3、ATG7表达,下调了p62、p-ULK1
Objective To investigate the effect of Zhenwu Decoction(真武汤)on cardiac function and cardiomyocyte autoph-agy in mice with dilated cardiomyopathy(DCM)through AMP-activated protein kinase(AMPK)/mammalian target of rapamy-cin(mTOR)signaling pathway and its mechanism.s Methods Ten C57BL/6J mice were randomly divided into normal group(A)and 30 cTnT^(R141W) transgenic dilated cardiomyopathy mice according to body weight,and divided into model group(B),Zhenwu Decoction group(C)and captopril group(D),with 10 mice in each group.The changes of left ventricular end-diastolic diame-ter(LVEDD),left ventricular end-systolic diameter(LVESD),left ventricular fractional shortening(FS)and left ventricular ejection fraction(EF)were detected by echocardiography.The morphology and arrangement of myocardial cells were observed by light microscopy.The expression and phosphorylation of recombinant human autophagy-related protein 3(ATG3)and recombi-nant human autophagy-related protein 7(ATG7)were detected by real-time PCR(RT-PCR).The expressions and phos-phorylation of autophagy-related protein p62,AMPKα,mTOR,unc-51-like kinase 1(ULK1)and tuberous sclerosis com-plex 2(TCS2)were detected by Western blot.s Results In cTnT^(R141W) transgenic dilated cardiomyopathy mice,myocardial fibers were disorganized and collagen fibers were significantly increased under microscope.After Zhenwu Decoction intervention,cell arrangement was gradually regular and myocardial fibrosis was reduced.Echocardiography measured ventricular enlargement,ventricular ejection fraction and fractional shortening in cTnT^(R141W) transgenic dilated cardiomyopathy mice were significantly re-duced compared with those of the normal group.After Zhenwu Decoction and captopril treatment,cardiac ultrasound revealed that LVEDD and LVESD were significantly reduced and ejection fraction was significantly increased.Quantitative fluorescence PCR showed that the expressions of ATG3 and ATG7 were increased in mice with dilated cardiomyopathy,and the expressions of ATG3 and ATG7 w
作者
隋吉峰
原艺
李铮
李明阳
李文杰
SUI Jifeng;YUAN Yi;LI Zheng;LI Mingyang;LI Wenjie(Liaoning University of Traditional Chinese Medicine,Shenyang 110847,Liaoning,China;The Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110032,Liaoning,China)
出处
《中华中医药学刊》
CAS
北大核心
2023年第10期41-47,I0012,共8页
Chinese Archives of Traditional Chinese Medicine
基金
国家自然科学基金面上项目(81173184)
辽宁省博士科研启动基金计划项目(2020-BS-165)。
