鹿茸多肽预处理通过miR-133a调控TGF-β/Smad信号通路对TBHP诱导心肌H9c2细胞损伤的保护作用  

Protective effect of Velvet antler polypeptide pretreatment on myocardial H9c2 cell injury induced by TBHP through regulating TGF-β/Smad signaling pathway with miR-133a

作　　者：周高峰 肖静 周佳[1] 刘俊秀 律广富 王雨辰[1] 林贺 黄晓巍[1] ZHOU Gaofeng;XIAO Jing;ZHOU Jia;LIU Junxiu;LYU Guangfu;WANG Yuchen;LIN He;HUANG Xiaowei(Department of Clinical Pharmacy and Pharmacology of Chinese Medicine,School of Pharmacy,Changchun University of Chinese Medicine,Changchun 130117,China;Institute of Medicinal Plant Chinese Academy of Medical Sciences,Beijing 100094,China;Department of Pharmacology of Traditional Chinese Medicine,Jilin Ginseng Academy,Changchun University of Chinese Medicine,Changchun 130117,China)

机构地区：[1]长春中医药大学药学院临床药学与中药药理教研室,吉林长春130117 [2]中国医学科学院药用植物研究所,北京100094 [3]长春中医药大学吉林省人参研究科学院中药药理组,吉林长春130117

出　　处：《吉林大学学报（医学版）》2023年第2期369-376,共8页Journal of Jilin University：Medicine Edition

基　　金：吉林省卫健委技术创新项目(2020J068);吉林省发改委创新能力建设项目(2021C011)。

摘　　要：目的:探讨鹿茸多肽(VAP)预处理对叔丁基过氧化氢(TBHP)诱导大鼠心肌H9c2细胞损伤的保护作用,阐明VAP对miR-133a/转化生长因子β(TGF-β)/Smad轴的作用及其机制。方法:将H9c2心肌细胞分为空白对照组、空白血清组、TBHP组、TBHP+低剂量(100 mg·kg^(-1))VAP组、TBHP+高剂量(400 mg·kg^(-1))VAP组和miR-133抑制剂(miR-133 inhibitor)组。空白对照组不做任何处理,其余各组细胞经VAP处理24 h后,给予200μmol·L^(-1)TBHP处理;miR-133inhibitor组细胞转染miR-133 inhibitor 24 h,给予400 mg·kg^(-1)VAP处理24 h,并给予200μmol·L^(-1)TBHP处理。MTT法检测各组H9c2细胞存活率,酶联免疫吸附试验(ELISA)法检测各组细胞培养上清液中肌钙蛋白T(cTnT)、肌钙蛋白I(cTnI)和肌酸激酶同工酶(CK-MB)水平,实时荧光定量PCR(RT-qPCR)法检测各组H9c2细胞中miR-133表达水平,Western blotting法检测各组H9c2细胞中转化生长因子β1(TGF-β1)、磷酸化Smad2/3(p-Smad2/3)和Smad4蛋白表达水平。结果:MTT法检测,与TBHP组比较,TBHP+低剂量VAP组和TBHP+高剂量VAP组H9c2细胞存活率升高(P<0.05),miR-133inhibitor组H9c2细胞存活率差异无统计学意义(P>0.05)。ELISA法检测,与空白对照组比较,TBHP组H9c2细胞中cTnT、cTnI和CK-MB水平升高(P<0.05);与TBHP组比较,TBHP+低剂量VAP组和TBHP+高剂量VAP组H9c2细胞中cTnT、cTnI和CK-MB水平降低(P<0.05)。RT-qPCR法检测,与空白对照组比较,TBHP组H9c2细胞中miR-133a表达水平降低(P<0.05);与TBHP组比较,TBHP+低剂量VAP组和TBHP+高剂量VAP组H9c2细胞中miR-133a表达水平升高(P<0.05或P<0.01),miR-133 inhibitor组H9c2细胞中miR-133a表达水平明显降低(P<0.01)。Western blotting法检测,与空白对照组比较,TBHP组H9c2细胞中TGF-β1、p-Smad2/3和Smad4蛋白表达水平升高(P<0.05);与TBHP组比较,TBHP+低剂量VAP组和TBHP+高剂量VAP组H9c2细胞中TGF-β1、p-Smad2/3和Smad4蛋白表达水平降低(P<0.05)。结论:VAP预处理通过miR-133a调控TGF-β/Smad信号通�Objective:To explore the protective effect of velvet antler peptide(VAP)pretreatment on the myocardial H9c2 cell injury of the rats induced by tert-butyl hydroperoxide(TBHP),and to clarify the effect of VAP on miR-133a/transforming growth factor-β(TGF-β)/Smad axis and its mechanism.Methods:The H9c2 cells were divided into blank control group,blank serum group,TBHP group,TBHP+low dose(100 mg·kg^(-1))of VAP group,TBHP+high dose(400 mg·kg^(-1))of VAP group,and miR-133 inhibitor(miR-133 inhibitor)group.The cells in blank control group were given no treatment,and the cells in the other groups were treated with VAP for 24 h,then were treated with 200μmol·L^(-1) TBHP;the cells in miR-133 inhibitor group were transfected with miR-133 inhibitor for 24 h,treated with 100 mg·kg^(-1) VAP for 24 h+200μmol·L^(-1) TBHP.MTT assay was used to detect the survival rates of the H9c2 cells in various groups;the levels of troponin T(cTnT),and cardial troponin T(cTnI)in culture supernatant of the cells in various groups were detected by enzyme-linked immunosorbent assay(ELISA)method and creatine kinase-MB(CK-MB);the expression levels of miR-133 in the H9c2 cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method;the expression levels of transforming growth factor-β1(TGF-β1),phosphorylated Smad2/3(p-Smad2/3),and Smad4 proteins the in H9c2 cells in various groups were detected by Western blotting method.Results:The MTT results showed that compared with TBHP group,the survival rates of H9c2 cells in TBHP+low dose of VAP group and TBHP+high dose of VAP group were increased(P<0.05),but the survival rate of the H9c2 cells in miR-133 inhibitor group had no significant difference(P>0.05).The ELISA assay results showed that compared with blank control group the levels of cTnT,cTnI,and CK-MB in the H9c2 cells in TBHP group were increased(P<0.05);compared with TBHP group,the levels of cTnT,cTnI,CK-MB in the H9c2 cells in TBHP+low dose of VAP group and TBHP+high dose of VAP group were decreased(P<0

关 键 词：鹿茸多肽 心肌损伤 微小RNA-133a 转化生长因子β 血清药理学 

分 类 号：R285.5[医药卫生—中药学]