从肝治心组方通过Nrf2/HO-1通路对缺氧/复氧损伤H9c2心肌细胞铁死亡的影响

Conggan Zhixin(从肝治心)Decoction Regulates Ferroptosis in H9c2 Cardiomyocytes Exposed to Hypoxia/Reoxygenation Injury via Nrf2/HO-1 Pathway

目的:基于核因子E2相关因子2(Nrf2)/血红素氧合酶1(HO-1)信号通路探讨从肝治心组方对大鼠H9c2心肌细胞铁死亡的影响。方法:本研究采用H9c2心肌细胞缺氧/复氧(H/R)方法模拟心肌缺血再灌注损伤(MIRI)细胞模型,细胞分为:空白对照组、模型对照组、15%正常对照血清组、26.6 g/kg从肝治心组方含药血清15%组、NK25220μmol/L组、ML3855μmol/L组、26.6 g/kg从肝治心组方含药血清15%+ML3855μmol/L组,通过CCK-8法检测细胞活力;DCFH-DA荧光探针检测细胞内活性氧(ROS)水平;TMRE荧光探针检测细胞线粒体膜电位水平;FerroOrange荧光探针检测细胞内Fe^(2+)水平;RT-qPCR、Werstern blot法检测核因子E2相关因子2(Nrf2)、血红素氧合酶1(Ho1)、膜铁转运辅助蛋白(Heph)、二价金属离子转运体(Dmt1)、酰基辅酶A合成酶长链家族成员4(Acsl4)mRNA和蛋白表达。结果:与正常对照组比较,模型对照组线粒体膜电位水平降低,ROS及Fe^(2+)水平升高,Nrf2、Ho1、Heph mRNA及蛋白表达下调,Dmt1、Acsl4 mRNA及蛋白表达上调(P<0.05或P<0.01);与模型对照组比较,从肝治心组方含药血清组和NK252组线粒体膜电位水平升高,ROS及Fe^(2+)水平降低,Nrf2、Ho1、Heph mRNA及蛋白表达上调,Dmt1、Acsl4 mRNA及蛋白表达下调(P<0.05或P<0.01);与ML3855μmol/L组比较,从肝治心组方+ML3855μmol/L组线粒体膜电位水平升高,ROS及Fe^(2+)水平降低,Nrf2、Ho1、Heph mRNA及蛋白表达上调,Dmt1、Acsl4 mRNA及蛋白表达下调(P<0.05或P<0.01)。结论:从肝治心组方可能通过调控Nrf2/HO-1信号通路,减轻缺氧/复氧损伤的H9c2心肌细胞铁死亡。

Objective:To investigate the effects of Conggan Zhixin(从肝治心)Decoction on ferroptosis of H9c2 cardiomyocytes via the nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway.Methods:H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation(H/R)to establish a cell model of myocardial ischemia-reperfusion injury(MIRI).Cells were classified into normal control,model control,15%blank serum control group,26.6 g/kg Conggan Zhixin Decoction-containing serum(15%),NK252(20μmol/L),ML385(5μmol/L),and 26.6 g/kg Conggan Zhixin Decoction-containing serum(15%)+ML385(5μmol/L)groups.The Cell Counting Kit-8(CCK-8)was used to examine cell viability.DCFH-DA,TMRE,and FerroOrange probes were used to measure the levels of reactive oxygen species(ROS),mitochondrial membrane potential,and intracellular Fe^(2+).western blotting and Real-time reverse transcription polymerase chain reaction(RT-qPCR)were employed to determine the protein levels and mRNA of Nrf2,HO-1,hephaestin(Heph),divalent metal transporter 1(DMT1),and acyl-CoA synthetase long-chain family member 4(ACSL4).Results:Compared with the normal control group,the model control group showed decreased mitochondrial membrane potential,elevated ROS and Fe^(2+)levels,down-regulated expression of Nrf2,HO-1,and Heph,and up-regulated expression of DMT1 and ACSL4(P<0.05,P<0.01).Compared with the model control group,26.6 g/kg Conggan Zhixin Decoction-containing serum(15%)and NK252(20μmol/L)groups showed increased mitochondrial membrane potential,lowered ROS and Fe^(2+)levels,up-regulated expression of Nrf2,HO-1,and Heph,and down-regulated expression of DMT1 and ACSL4(P<0.05,P<0.01).Compared with the ML385(5μmol/L)group,the 26.6 g/kg Conggan Zhixin Decoction-containing serum(15%)+ML385(5μmol/L)group showed increased mitochondrial membrane potential,lowered ROS and Fe^(2+)levels,up-regulated expression of Nrf2,HO-1,and Heph,and down-regulated expression of DMT1 and ACSL4(P<0.05,P<0.01).Conclusion:Conggan Zhixin Decoction may attenuate ferroptosis in H9c2 cardiomyocytes exposed to H/R injury by modulating the Nrf2/HO-1 signaling pathway.