摘要
目的 从线粒体自噬角度探讨5-氟尿嘧啶(5-Fu)心肌毒性及黄芪甲苷(AS-Ⅳ)的干预机制。方法 体外培养大鼠心肌细胞,将细胞分为5组:正常组(A组)、5-Fu模型组(B组)、AS-Ⅳ干预组(C组)、线粒体自噬抑制剂组(D组)、线粒体自噬激活组(E组)。采用细胞计数试剂盒-8(CCK-8)法、TUNEL染色检测心肌细胞功能改变,心肌线粒体腺苷三磷酸(ATP)及膜电位水平观察线粒体功能改变,LC3Ⅱ免疫荧光染色检测线粒体自噬活性,采用实时荧光定量聚合酶链反应(RT-PCR)、蛋白质印迹(Western blot)检测PINK1/Parkin通路PINK1、Parkin、Beclin1、LAMP、LC3基因及蛋白表达情况。结果各组细胞存活率比较,A组>D组>C组>E组>B组(F=566.153,P<0.05);各组ATP含量、膜电位比较,A组>D组>C组>B组>E组(F=15.967、13.608,P<0.05);各组细胞凋亡率、LC3Ⅱ表达水平、PINK1/Parkin通路相关mRNA表达及蛋白比较,E组>B组>C组>D组>A组(F=92.799、38.130、15.921、7.287、20.921、13.387、5.012、16.824、8.167、23.291、14.622、6.571,P<0.05)。结论 5-Fu可诱导心肌线粒体损伤,且线粒体可呈现自噬过度状态,AS-Ⅳ可通过抑制线粒体自噬减轻5-Fu心肌毒性。
Objective To investigate the myocardial toxicity of 5-fluorouracil(5-Fu)and the in-tervention mechanism of astragaloside(AS-Ⅳ)from the perspective of mitochondrial autophagy.Methods Rat cardiomyocytes were cultured in vitro,and the cells were divided into five groups:normal group(group A),5-Fu model group(group B),AS-Ⅳintervention group(group C),mitochondrial autophagy inhibitor group(group D),and mitochondrial autophagy activation group(group E).Cell Counting Kit-8(CCK-8)method and TUNEL staining were used to detect functional alterations in cardiomyocytes,mitochondrial ade-nosine triphosphate(ATP)and membrane potential levels in cardiac mitochondria,LC3Ⅱimmunofluores-cence staining was used to detect mitochondrial autophagic activity,and real-time fluorescence quantitative polymerase chain reaction(RT-PCR)and protein blotting(Western blot)were used to detect PINK1/Parkin pathway PINK1,Parkin,Beclin1,LAMP,LC3 gene and protein expression.Results Comparison of cell survival rate in each group,group A>group D>group C>group E>group B(F=566.153,P<0.05).Com-parison of ATP content and membrane potential in each group,group A>group D>group C>group B>group E(F=15.967,13.608,P<0.05).Comparison of apoptosis rate,LC3Ⅱexpression level,PINK1/Parkin pathway related mRNA expression and protein in each group,group E>group B>group C>group D>group A(F=92.799,38.130,15.921,7.287,20.921,13.387,5.012,16.824,8.167,23.291,14.622,6.571,P<0.05).Conclusion 5-Fu can induce myocardial mitochondrial damage,and mitochondria can show excessive autophagy.AS-Ⅳcan reduce 5-Fu myocardial toxicity by inhibiting mitochondrial autophagy.
作者
周湘忠
杨书凤
李玉捷
陈立军
魏刚
ZHOU Xiangzhong;YANG Shufeng;LI Yujie;CHEN Lijun;WEI Gang(Department of Cardiovascular Medicine,Dagang Hospital,Tianjin,China,300450;Department of On-cology,Dagang Hospital,Tianjin,China,300450)
出处
《分子诊断与治疗杂志》
2023年第10期1791-1795,共5页
Journal of Molecular Diagnostics and Therapy
基金
天津市滨海新区卫生健康委员会重点支持科技项目(2019BWKZ004)。
关键词
线粒体
自噬
5-氟尿嘧啶
心肌毒性
黄芪甲苷
Mitochondria
Autophagy
5-Fluorouracil
Myocardial toxicity
AstragalosideⅣ
作者简介
通信作者:周湘忠,E⁃mail:tjbhyl120@163.com。
