摘要
目的从HIF-1α(Hypoxia-Inducible Factor-1α)/BINP3(B lymphocytoma-2 gene-homology 3)/NIX(Nip-like protein X)介导的线粒体自噬通路探讨柴胡三参胶囊保护心肌缺血再灌注损伤(Myocardial ischemia-reperfusion injury,MIRI)大鼠的生物学机制。方法选取50只SD大鼠,随机分为假手术(Sham)组、模型(I/R)组、柴胡三参胶囊(CHSS)组、HIF-1α抑制剂(2ME2,二甲氧基雌二醇)组、HIF-1α抑制剂+柴胡三参胶囊(2ME2+CHSS)组,每组10只。大鼠灌胃给药,每日1次,连续灌胃4周。末次给药24 h后,结扎左前降支冠状动脉30 min,再灌注120 min建立大鼠MIRI模型。采用酶联免疫吸附(ELISA)法检测血清中CK-MB(Creatinekinase-MB)、cTn I(Troponin)及LDH(Lactatedehydrogenase)含量;HE染色(Hematoxylin-eosin staining)观察心肌组织改变;蛋白免疫印迹法(Western blot)检测大鼠心肌组织自噬相关蛋白HIF-1α、BNIP3、NIX、微管相关蛋白1轻链3-Ⅱ(Microtubule-associated protein 1 light chain 3 B-Ⅱ,LC3-Ⅱ)、微管相关蛋白1轻链3-Ⅰ(Microtubule-associated protein 1 light chain 3 B-Ⅰ,LC3-Ⅰ)表达;流式细胞法检测线粒体膜电位水平;电镜观察线粒体内自噬小体数量。结果与Sham组相比,I/R组血清CK-MB、cTn I、LDH含量升高(P<0.05),心肌组织不同程度损伤,HIF-1α、BNIP3、NIX表达及LC3-Ⅱ/LC3-Ⅰ比值升高(P<0.05),线粒体膜电位水平降低(P<0.05),线粒体自噬小体数量增多(P<0.05);与I/R组相比,CHSS组血清CK-MB、cTn I、LDH含量降低(P<0.05),心肌组织损伤程度较轻,HIF-1α、BNIP3、NIX表达及LC3-Ⅱ/LC3-Ⅰ比值升高(P<0.05),线粒体膜电位水平升高(P<0.05),线粒体自噬小体数量增多(P<0.05);与CHSS组相比,CHSS+2ME2组血清CK-MB、cTn I、LDH明显降低(P<0.05),心肌组织损伤程度加重,HIF-1α、BNIP3、NIX表达及LC3-Ⅱ/LC3-Ⅰ比值最低(P<0.05),线粒体膜电位水平降低(P<0.05),线粒体自噬小体数量减少(P<0.05)。结论柴胡三参胶囊对缺血再灌注大鼠心肌具有保护作用,这种机制与其促进HIF-1α/BNIP3/NIX介导的心肌细胞线粒体自噬通路有关。
Objective From HIF-1α/BINP3/NIX mediated mitochondrial autophagy pathway to explore the biological mechanism of Chaihu Sanshen capsule in protecting myocardial ischemia-reperfusion injury(MIRI)rats.Methods Fifty SD rats were randomly divided into sham group,model group,Chaihu Sanshen capsule(CHSS)group and HIF-1αgroup Inhibitor(2ME2)group,HIF-1αinhibitor+Chaihu Sanshen capsule(2ME2+CHSS)group,10 rats in each group.Rats were administered by gavage once a day for 4 weeks.24 hours after the last administration,the left anterior descending coronary artery was ligated for 30 minutes and reperfusion for 120 minutes to establish the rat Miri model.The expressions of creatine kinase isozyme(CK-MB),troponin I(cTn I)and lactate dehydrogenase(LDH)in serum were detected by ELISA;HE staining was used to observe the changes of myocardial tissue;Autophagy related protein HIF-1αin rat myocardium was detected by Western blot、The expression of BNIP3,NIX,LC3-Ⅱand LC3-Ⅰ;The level of mitochondrial membrane potential was detected by flow cytometry;The number of autophagy bodies in mitochondria was observed by electron microscope.Results Compared with sham group,the contents of CK-MB,cTnI and LDH in serum of I/R group increased(P<0.05),the expression of HIF-1α,BNIP3,NIX and LC3-Ⅱ/LC3-Ⅰratio increased(P<0.05),the level of mitochondrial membrane potential decreased(P<0.05),and the number of mitochondrial autophagy bodies increased(P<0.05);Compared with the I/R group,the levels of CK-MB,cTn I and LDH in CHSS group decreased(P<0.05),the degree of myocardial injury was mild,the expression of HIF-1α、BNIP3、NIX and the ratio of LC3-Ⅱ/LC3-Ⅰincreased(P<0.05),the level of mitochondrial membrane potential increased(P<0.05),and the number of mitochondrial autophagy bodies increased(P<0.05);Compared with CHSS group,serum CK-MB,cTnI and LDH in CHSS+2ME2 group decreased significantly(P<0.05).Conclusion Chaihu Sanshen capsule has a protective effect on myocardial ischemia-reperfusion in rats.This mechanism is related to the promotion of HIF-1α/BNIP3/NIX mediated mitochondrial autophagy pathway in cardiomyocytes.
作者
曹蛟
刘建和
张杼惠
何涛
谭彩
Cao Jiao;Liu Jianhe;Zhang Zhuhui;He Tao;Tan Cai(The First Affiliated Hospital of Hunan University of Traditional Chinese Medicine,Changsha 410007,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2023年第3期993-1001,共9页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
湖南省中医药管理局2021年度湖南省中医药科研计划重点项目(2021011):基于ROS与HIF-1通过线粒体自噬实现的crosstalk探讨柴胡三参胶囊对心肌缺血再灌注损伤的干预机制,负责人:刘建和
关键词
柴胡三参胶囊
缺氧诱导因子-1Α
心肌缺血再灌注损伤
线粒体自噬
Chaihu Sanshen capsule
Hypoxia inducible factor-1α
Myocardial ischemia-reperfusion injury
Mitochondrial autophagy
作者简介
通讯作者:刘建和,教授,主任医师,博士生导师,主要研究方向:中医药防治心血管疾病研究
