参附注射液调控HIF-1α/BNIP3通路介导线粒体自噬抗心肌缺血再灌注损伤作用  

Effect of Shenfu Injection of Anti-myocardial Ischemia-Reperfusion Injury via Modulating HIF-1α/BNIP3 Pathway-Mediated Mitophagy

作　　者：谌治安 袁颢 孙晗 刘思彤 张帅 姜爽 宋伍 刘智 CHEN Zhi-an;YUAN Hao;SUN Han;LIU Si-tong;ZHANG Shuai;JIANG Shuang;SONG Wu;LIU Zhi(School of Clinical Medicine,Changchun University of Traditional Chinese Medicine,Changchun 130117)

机构地区：[1]长春中医药大学临床医学院,长春130117

出　　处：《中国中西医结合杂志》2024年第9期1086-1093,共8页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金资助项目(No.81603324);吉林省卫生健康科技能力提升项目(No.2022JC038)。

摘　　要：目的探讨参附注射液调节线粒体自噬减轻大鼠心肌缺血再灌注损伤(MIRI)的作用机制。方法50只大鼠随机分成5组:假手术组,模型组,参附注射液低、中、高剂量[3.34、6.68、13.36 mL/(kg·d)]组,每组10只,造模前给药。干预组大鼠腹腔注射给药,假手术组及模型组腹腔注射生理盐水,每天1次,连续给药7天后造模。末次给药24 h后,将大鼠左冠状动脉前降支结扎30 min,再灌注120 min建立MIRI模型。超声评估心脏结构和心功能;ELISA法检测血清肌酸激酶(CK-MB)、乳酸脱氢酶(LDH)、肌钙蛋白(cTnI)含量;TTC染色观察心肌梗死面积;HE染色评估心肌病理变化;Western Blot、q RT-PCR检测缺氧诱导因子1α(HIF-1α)、Bcl-2相互作用蛋白3(BNIP3)、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、微管相关蛋白1轻链3-Ⅰ(LC3-Ⅰ)蛋白及m RNA表达;TUNEL法检测心肌细胞凋亡率;电镜观察线粒体及自噬小体数量。结果与假手术组比较,模型组射血分数(LVEF)、短轴缩短率(LVFS)、左室舒张末期内径(LVEDd)降低(P<0.05),收缩末期内径(LVESd)升高(P<0.05),梗死面积增加(P<0.01),CK-MB、LDH、cTnI含量升高(P<0.05),心肌细胞坏死和炎症浸润增加;HIF-1α、BNIP3表达及LC3-Ⅱ/LC3-Ⅰ比值升高(P<0.05),HIF-1α、BNIP3、LC3 mRNA表达升高(P<0.05);心肌细胞凋亡率、单位面积内线粒体明损伤及自噬小体数量增多(P<0.01,P<0.05)。与模型组比较,参附注射液组LVEF、LVFS、LVEDd和LVESd升高(P<0.01);除参附注射液高剂量组LC3Ⅱ/LC3Ⅰ值降低,参附注射液组HIF-1α、BNIP3及LC3Ⅱ/LC3Ⅰ表达升高(P<0.01,P<0.05),HIF-1α、BNIP3、LC3 mRNA表达升高(P<0.01);参附注射液组梗死面积减少(P<0.01),CK-MB、LDH、cTnI含量降低(P<0.05),细胞凋亡率、线粒体损伤及自噬小体数量减少(P<0.01,P<0.05)。结论参附注射液具有保护MIRI大鼠心肌的作用,其机制与促进HIF-1α/BNIP3通路介导线粒体自噬有关。Objective To explore the mechanism of Shenfu Injection in regulating mitophagy to alleviate myocardial ischemia-reperfusion injury(MIRI)in rats.Methods Fifty rats were randomly divided into 5 groups,i.e.,the sham group,the model group,the low,medium,and high dose Shenfu Injection group,10 rats in each.The rats were administered before modelling.The rats in the Shenfu Injection groups were administered intraperitoneally(3.34,6.68,and 13.36 mL·kg^(-1)·d-1),the rats in the sham group and the model group were injected intraperitoneally with saline once a day for 7 consecutive days to establish model.Twentyfour hours after the last administration,the anterior descending branch of the left coronary artery was ligated for 30 min,and then reperfused for 120 min to establish the MIRI model.The cardiac structure and cardiac function were assessed by ultrasound.The levels of serum creatine kinase(CK-MB),lactate dehydrogenase(LDH),and troponin(cTnI)were detected by ELISA.The infract size was determined by TTC.The changes of pathological in myocardial tissues were assessed by HE staining.Western Blot and qRT-PCR were used to detect the protein and mRNA expression of hypoxia-inducible factor 1α(HIF-1α),Bcl-2 interacting protein 3(BNIP3),microtubuleassociated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),and microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅰ).The apoptosis rate of cardiomyocytes were detected by TUNEL method.The number of mitochondrion and autophagosome were observed by electron microscopy.Results Compared with the sham group,the left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),and left ventricular end-diastolic diameter(LVEDd)decreased(P<0.05),left ventricular end-systolic diameter(LVESd)increased(P<0.05),infract size increased(P<0.01),the levels of CK-MB,LDH,and cTnI increased(P<0.05),necrosis and inflammatory infiltration in cardiomyocytes increased;the expressions of HIF-1α,BNIP3,and LC3-Ⅱ/LC3-Ⅰratio increased(P<0.05),HIF-1α,BNIP3,LC3 mRNA expressions increased(

关 键 词：参附注射液 心肌缺血再灌注损伤 缺氧诱导因子1Α Bcl-2相互作用蛋白3 线粒体自噬 中成药 

分 类 号：R285.5[医药卫生—中药学]