青蒿琥酯调节PERK/ATF4/CHOP信号通路对OGD/R诱导的心肌细胞铁死亡的影响

Effect of Artesunate on OGD/R-induced Cardiomyocyte Ferroptosis by Modulating PERK/ATF4/CHOP Signaling Pathway

目的:探讨青蒿琥酯调节蛋白激酶R样内质网激酶(PERK)/激活转录因子-4(ATF4)/C/EBP同源蛋白(CHOP)信号通路对氧糖剥夺/复氧(OGD/R)诱导的心肌细胞铁死亡的影响。方法:体外培养大鼠心肌细胞H9c2构建OGD/R模型,通过CCK-8法检测0、1.0、2.5、5、10、20μmol/L青蒿琥酯处理24h后各组细胞活力,筛选合适的青蒿琥酯作用浓度。将体外培养H9c2细胞随机分为对照组、模型组、青蒿琥酯低剂量组、青蒿琥酯高剂量组、MK-28(PERK激活剂)组、青蒿琥酯高剂量+MK-28组,除对照组外的其余各组细胞构建OGD/R模型,然后马上使用青蒿琥酯和MK-28分组处理,采用CCK-8法和流式细胞术分别检测各组H9c2细胞活力和凋亡率;ELISA检测各组H9c2细胞培养基上清中心肌肌钙蛋白I(cTnI)、肌酸激酶同工酶(CK-MB)、肌红蛋白(MB)、炎性因子[前列腺素E2(PGE2)、环氧化酶-2(COX-2)、白细胞介素(IL)-1β]水平;化学荧光法检测活性氧(ROS)水平,微量法检测铁含量、丙二醛(MDA)水平,微板法检测谷胱甘肽(GSH)水平;免疫印迹实验检测各组H9c2细胞PERK/ATF4/CHOP通路相关蛋白表达。结果:与对照组比较,模型组细胞活力、GSH水平降低(P<0.05),凋亡率、细胞培养基上清中cTnI、CK-MB、MB、PGE2、COX-2及IL-1β水平、细胞ROS相对水平、铁含量、MDA水平、p-PERK/PERK及ATF4、CHOP蛋白表达升高(P<0.05)。与模型组比较,青蒿琥酯低剂量组、青蒿琥酯高剂量组细胞活力、GSH水平均升高(P<0.05),凋亡率、细胞培养基上清中cTnI、CK-MB、MB、PGE2、COX-2及IL-1β水平、细胞ROS相对水平、铁含量、MDA水平、p-PERK/PERK及ATF4、CHOP蛋白表达均降低(P<0.05);青蒿琥酯高剂量组细胞活力、GSH水平相较青蒿琥酯低剂量组进一步升高(P<0.05),凋亡率、细胞培养基上清中cTnI、CK-MB、MB、PGE2、COX-2及IL-1β水平、细胞ROS相对水平、铁含量、MDA水平、p-PERK/PERK及ATF4、CHOP蛋白表达较青蒿琥酯低剂量组进一步降低(P<0.05);MK-28组细胞活力、GSH水平降低(P<0.05),凋亡率、细胞培养基上清中cTnI、CK-MB、MB、PGE2、COX-2及IL-1β水平、细胞ROS相对水平、铁含量、MDA水平、p-PERK/PERK及ATF4、CHOP蛋白表达升高(P<0.05)。与青蒿琥酯高剂量组比较,青蒿琥酯高剂量+MK-28组细胞活力、GSH水平降低(P<0.05),凋亡率、细胞培养基上清中cTnI、CK-MB、MB、PGE2、COX-2及IL-1β水平、细胞ROS相对水平、铁含量、MDA水平、p-PERK/PERK及ATF4、CHOP蛋白表达升高(P<0.05)。结论:青蒿琥酯可通过抑制PERK/ATF4/CHOP信号激活而抑制OGD/R诱导的心肌细胞炎症、铁蓄积和脂质过氧化,减轻铁死亡,增强其细胞活力,缓解细胞损伤。

Objective:To explore the effect of artesunate regulating protein kinase R-like endoplasmic reticulum kinase(PERK)/activating transcription factor-4(ATF4)/C/EBP homologous protein(CHOP)signaling pathway on oxygen and glucose deprivation/reoxygenation(OGD/R)-induced cardiomyocyte ferroptosis.Method:Rat cardiomyocytes H9c2 were cultured in vitro to establish an OGD/R model,and the cell viability of each treatment group was detected by CCK-8 method after 0,1.0,2.5,5,10,20 mol/L artesunate treatment for 24 h to screen the appropriate concentration of artesunate.In vitro cultured H9c2 cells were randomly divided into control group,model group,low-dose artesunate group,high-dose artesunate group,MK-28(PERK activator)group,and high-dose artesunate+MK-28 group,except for the control group,the rest of the cells in the other groups constructed the OGD/R model,then artesunate and MK-28 were used to treatment group immediately,and the CCK-8 method and flow cytometry were applied to detect the viability and apoptosis rate of H9c2 cells in each group;Enzyme-linked immunosorbent assay(ELISA)were applied to detect the levels of cardiac troponin I(cTnI),creatine kinase isoenzyme(CK-MB),myoglobin(MB),inflammatory factors[prostaglandin E2(PGE2),cyclooxygenase-2(COX-2),interleukin(IL)-1β]in the supernatant of H9c2 cells in each group.The level of reactive oxygen species(ROS)was detected by chemical fluorescence method,iron content and the level of malondialdehyde(MDA)were detected by micromethod,and the level of glutathione(GSH)was detected by microplate method;the expression of PERK/ATF4/CHOP pathway-related proteins in H9c2 cells in each group was detected by western blotting.Results:Compared with the control group,the cell viability and GSH level in the model group were decreased(P<0.05),the apoptosis rate,cTnI,CK-MB,MB,PGE2,COX-2 and IL-1βlevels in cell culture supernatant,cellular ROS relative level,iron content,MDA level,p-PERK/PERK,the ATF4 and CHOP protein expression were increased(P<0.05).Compared with the model group,the cell viability and GSH level in the low-dose artesunate group and the high-dose artesunate group were increased(P<0.05),the apoptosis rate,cTnI,CK-MB,MB,PGE2,COX-2 and IL-1βlevels in cell culture supernatant,cellular ROS relative level,iron content,MDA level,p-PERK/PERK,the ATF4 and CHOP protein expression all were decreased(P<0.05);compared with the low-dose artesunate group,the cell viability and GSH level in the high-dose artesunate group were further increased(P<0.05),the apoptosis rate,cTnI,CK-MB,MB,PGE2,COX-2 and IL-1βlevels in cell culture supernatant,cellular ROS relative level,iron content,MDA level,p-PERK/PERK,the ATF4 and CHOP protein expression were further decreased(P<0.05);cell viability and GSH level in MK-28 group were decreased(P<0.05),the apoptosis rate,cTnI,CK-MB,MB,PGE2,COX-2 and IL-1βlevels in cell culture supernatant,cellular ROS relative level,iron content,MDA level,p-PERK/PERK,the ATF4 and CHOP protein expression were increased(P<0.05).Compared with the high-dose artesunate group,the cell viability and GSH level in the high-dose artesunate+MK-28 group were decreased(P<0.05),the apoptosis rate,cTnI,CK-MB,MB,PGE2,COX-2 and IL-1βlevels in cell culture supernatant,cellular ROS relative level,iron content,MDA level,p-PERK/PERK,the ATF4 and CHOP protein expression were increased(P<0.05).Conclusion:Artesunate can inhibit OGD/R-induced inflammation,iron accumulation and lipid peroxidation in cardiomyocytes,and reduce ferroptosis by inhibiting the activation of PERK/ATF4/CHOP signaling,and ultimately enhance cell viability and alleviate cell damage.