硫化氢活化ERK抵抗内质网应激诱导的心肌细胞凋亡

Hydrogen Sulfide Inhibits Endoplasmic Reticulum Stress-induced Myocardial Cell Apoptosis by Activating ERK

目的探讨硫化氢(H2S)对心肌的保护作用是否通过激活细胞外信号调节激酶(ERK)通路来抵抗心肌缺血诱导的内质网应激所致的心肌细胞凋亡。方法 60只雄性SD大鼠随机分为对照组、ISO模型组、NaHS+ISO组及PD98059阻断组,每组各15只。对照组大鼠注射等体积生理盐水;ISO模型组大鼠第1、2天腹腔注射生理盐水,第3、4天注射完生理盐水30min后背部皮下分别注射10mg/kg和5mg/kg的ISO;NaHS+ISO组大鼠腹腔注射NaHS 14μmol/kg,1次/d,连续2d后,改为2次/d,连续2d,并在后2d的第1次注射30min后,于背部皮下分别注射10mg/kg和5mg/kg的ISO,1次/d;PD98059阻断组大鼠在上述NaHS+ISO组大鼠处理基础上,于注射ISO之前经尾静脉注射MEK/ERK抑制剂PD98059(4mg/kg),1次/d,连续2d。每组最后一次注射ISO并禁食12h后,检测心电图、心功能指标;测定血浆中H2S浓度变化;TTC染色测定心肌梗死面积;TUNEL法检测心肌细胞凋亡指数(AI);免疫组织化学方法检测心肌中GRP78、CHOP及ERK磷酸化(p-ERK)蛋白的表达。结果 ISO模型组大鼠血浆的H2S含量明显低于对照组(P<0.01);14μmol/kg NaHS可以显著改善心肌缺血引起的心功能改变,而PD98059阻断组可以逆转NaHS的心肌保护作用;与ISO模型组相比,NaHS+ISO组大鼠心肌组织中GRP78、CHOP的表达明显减少(均P<0.05),pERK表达明显增多(P<0.01),AI、心肌梗死面积明显减小(均P<0.05);与NaHS+ISO组相比,PD98059阻断组大鼠心肌组织中GRP78、CHOP的表达、AI、梗死面积均显著增加(均P<0.05),心功能明显降低(P<0.05),而p-ERK无表达。相关分析显示大鼠心肌细胞CHOP表达、AI与p-ERK的表达呈负相关(P<0.01)。结论内源性H2S浓度的降低及内质网应激可能参与急性缺血心肌损伤的发生与发展,H2S对缺血损伤的心肌保护作用机制可能与其激活ERK进而抑制内质网应激诱导的心肌细胞凋亡有关。

Objective To investigate whether hydrogen sulfide（H2S）protects cardiomyocytes from endoplasmic reticulum stress-induced apoptosis by activating the extracellular signal regulation kinase（ERK）.Methods A total of 60 SD rats were randomly divided into control group,ISO group,NaHS＋ISO group and PD98059group（n=15in each group）.Animals in control group was injected with saline;those in ISO group was intraperitoneally injected with saline at the same volume as control group for 4days,and subcutaneously injected with ISO at 10mg/kg and 5mg/kg,respectively,on the back,30 min after the intraperitoneal injection with saline at the later 2days;those in NaHS＋ISO group was intraperitoneally injected with 14μmol/kg NaHS once a day for 2days,twice a day for 2days,and then subcutaneously injected with ISO 10mg/kg and 5mg/kg,respectively,on the back,30 min after NaHS injection at the later 2days;those in PD98059 group was given the same treatment as NaHS＋ISO group except intravenous injection with 4 mg/kg PD98059（ERK inhibitor）via the caudal vein before ISO injection.Animals were fasted for 12 hafter last injection with ISO.The heart function was monitored with physiologic signal analysis system and the electrocardiogram was recorded.The H2 S concentration in plasma was biochemically determined.The myo-cardial infarction size and the apoptosis index（AI）of cardiomyocytes was measured by TTC staining and TUNEL assay,respectively.And the expression of GRP78,CHOP and p-ERK were detected in the myocardial tissue by using immunohistochemistry.Results The H2 S concentration was much lower in ISO group than in control group（P0.01 for all）.NaHS（14μmol/kg）could significantly improve the impaired heart function caused by myocardial ischemia,which could be reversed by PD98059.The expression of GRP78 and CHOP was significantly decreased,that of p-ERK was significantly increased and the AI and the myocardial infarction size were profoundly reduced in NaHS＋ISO group when compared with ISO group（P0.05）.However,the myocardial infarction size,the AI,the expression of GRP78 and CHOP were obviously increased,while the heart function was significantly attenuated in PD98059 group when compared with NaHS＋ISO group（all P0.05）.There was no statistical difference in p-ERK expression between the two groups.Moreover,the correlation analysis showed that the expression of CHOP and AI had a negative correlation with the expression of p-ERK in myocardial tissues（P0.01）.Conclusion The decrease of endogenous H2 S and the endoplasmic reticulum stress may be involved in the occurrence and development of acute ischemic myocardial injury.The protective effect of H2 S on ischemic myocardia may be associated with the activation of ERK and thereby inhibit the apoptosis induced by the endoplasmic reticulum stress.