田蓟苷调控p53/NOX4信号通路抗缺氧/复氧诱导的心肌细胞损伤的机制  

Mechanism of tilianin regulating p53/NOX4signal pathway against hypoxia-reoxygenation-induced cardiomyocyte injury

作　　者：袁勇[1] 李雅琪 黄川生 赵云丽 曹文疆 王新春[1] YUAN Yong;LI Yaqi;HUANG Chuansheng;ZHAO Yunli;CAO Wenjiang;WANG Xinchun(Department of Pharmacy,The First Affiliated Hospital of Shihezi University,Xinjiang Shihezi 832008,China;Department of Pharmacy,Jingzhou Central Hospital,Hubei Jingzhou 434020,China)

机构地区：[1]石河子大学第一附属医院药学部,新疆石河子832008 [2]荆州市中心医院药学部,湖北荆州434020

出　　处：《中国医院药学杂志》2023年第24期2736-2742,共7页Chinese Journal of Hospital Pharmacy

基　　金：国家自然科学基金项目(编号:81960766);兵团重点领域科技攻关计划(编号:2022AB020)。

摘　　要：目的:探究田蓟苷(tilianin,Til)预处理对缺氧/复氧(H/R)诱导的H9c2心肌细胞的保护作用。方法:体外培养H9c2心肌细胞,将其分为正常(Normal)组、模型(H/R)组、田蓟苷(Til)组、p53激动剂(Nutlin-3a)组、田蓟苷+p53激动剂(Til+Nutlin-3a)组,缺氧12 h再复氧6 h,建立H/R模型。CCK-8法测H9c2细胞活力,化学荧光法测ROS水平,试剂盒测LDH、MDA、SOD水平,JC-1法测线粒体膜电位(ΔΨm),Annexin-FITC/PI和Fluo-3 AM分别结合流式细胞仪检测细胞凋亡率和Ca^(2+)荧光强度,RT-PCR测定p53、NOX4、Bax、Bcl-2 mRNA表达,免疫蛋白印迹法检测p53、NOX4、Bax、Bcl-2、Nrf2、HO-1蛋白表达,免疫荧光法测定p53、NOX4蛋白表达。结果:相较于H/R组,Til组H9c2细胞活力增强(P<0.01),ROS、LDH、MDA水平下降(P<0.01),SOD水平上升(P<0.01),ΔΨm增强(P<0.01),早期和晚期凋亡细胞降低(P<0.01),Ca^(2+)荧光强度减弱(P<0.01),p53、NOX4、Bax的mRNA和蛋白表达下降(P<0.05,P<0.01),Bcl-2的mRNA和蛋白表达上升(P<0.01),Nrf2、HO-1的蛋白表达进一步上升(P<0.01),p53、NOX4阳性细胞表达减少;单独应用Nutlin-3a后上述效应被削弱,而当加入Til后又不同程度逆转Nutlin-3a的效应。结论:Til对缺氧/复氧诱导的H9c2心肌细胞损伤具有显著的保护作用,该作用可能是通过调控p53/NOX4通路,抑制氧化应激和凋亡实现的。OBJECTIVE To investigate the protective effect of tilianin(Til)pretreatment on hypoxia/reoxygenation(H/R)-induced H9c2 cardiomyocytes.METHODS H9c2 cardiomyocytes were cultured in vitro and divided into normal(Normal)group,model(H/R)group,Tilianin(Til)group,p53 agonist(Nutlin-3a)group,and Tilianin+p53 agonist(Til+Nutlin-3a)group,and the H/R model was established by hypoxia for 12 h reoxygenation for 6 h.Cell viability of each group was measured by CCK-8 method,and chemical fluorescence method was used to determine the level of ROS,the level of LDH,MDA and SOD was determined by kit,JC-1 method was used to detect the mitochondrial membrane potential(ΔΨm),detection of apoptosis rate and Ca^(2+)fluorescence intensity by Annexin-FITC/PI and Fluo-3 AM in combination with flow cytometry respectively,the expression of p53,NOX4,Bax,Bcl-2 mRNA was determined by RT-PCR,the expression of p53,NOX4,Bax,Bcl-2,Nrf2,HO-1 protein was detected by WB,immunofluorescence assay was used to determine the p53,NOX4 protein localization and expression in H9c2 cells.RESULTS Compared with the H/R group,H9c2 cell viability increased(P<0.01),ROS,LDH and MDA levels decreased(P<0.01),SOD levels increased(P<0.01),ΔΨmenhanced(P<0.01),early and late apoptotic cells decreased(P<0.01),Ca^(2+)fluorescence intensity diminished(P<0.01),p53,NOX4 and Bax mRNA and protein expression decreased(P<0.05,P<0.01),mRNA and protein expression of Bcl-2 increased(P<0.01),protein expression of Nrf2 and HO-1 further increased(P<0.01),and a decrease in p53 and NOX4 positive cells could be observed under fluorescence microscopy;These effects were attenuated by the application of Nutlin-3a alone and reversed to varying degrees when Til was added.CONCLUSION Til has a significant protective effect on hypoxia/reoxygenation-stimulated H9c2 cardiomyocyte injury,which may be achieved by regulating the p53/NOX4 pathway and inhibiting oxidative stress and apoptosis.

关 键 词：田蓟苷 H9C2心肌细胞 缺氧/复氧 p53/NOX4通路 氧化应激 凋亡 

分 类 号：R966[医药卫生—药理学]