毛兰素通过抑制NLRP3炎症体介导的细胞焦亡减轻心肌缺血再灌注损伤

Erianin alleviates myocardial ischemia-reperfusion injury by inhibiting pyroptosis mediated by NLRP3 inflammasome

目的探究毛兰素(ERI)防治心肌缺血再灌注损伤(MIRI)的作用和机制。方法将SD大鼠分为假手术组(n=18)、模型组(n=18)、5 mg ERI组(n=17)、10 mg ERI组(n=16)和20 mg ERI组(n=17)。假手术组和模型组大鼠灌胃1%二甲基亚砜,5 mg ERI组、10 mg ERI组和20 mg ERI组大鼠分别灌胃剂量为5,10,20 mg/(kg·d)的毛兰素,给药7 d后模型组和各ERI组大鼠进行MIRI建模。再灌注48 h后检测各组大鼠左心室射血分数(LVEF)、左室舒张末期内径(LVIDd)、左心室收缩末期内径(LVIDs)和左室缩短分数(LVFS)。然后检测大鼠血清心肌损伤标志物[肌酸激酶同工酶MB(CK-MB)、乳酸脱氢酶(LDH)、心肌肌钙蛋白I(cTnI)]水平,并对大鼠心肌组织进行TTC染色和苏木精-伊红(HE)染色。RT-qPCR检测心肌组织中核苷酸结合寡聚化结构域样受体3(NLRP3)、Caspase-1、凋亡相关斑点样蛋白(ASC)、IL-1β和IL-18 mRNA水平。Western blot检测心肌组织中NLRP3、cleaved Caspase-1、ASC、IL-1β和IL-18蛋白表达水平。结果与假手术组比较,模型组大鼠的LVEF和LVFS降低(P<0.05),LVIDd和LVIDs升高(P<0.05),血清CK-MB、LDH和cTnI水平升高(P<0.05),心肌梗死面积升高(P<0.05),心肌明显损伤,心肌组织中NLRP3、Caspase-1、ASC、IL-1β和IL-18的mRNA表达水平升高(P<0.05),心肌组织中NLRP3、cleaved Caspase-1、ASC、IL-1β和IL-18蛋白表达水平升高(P<0.05)。与模型组比较,5 mg ERI组、10 mg ERI组和20 mg ERI组大鼠的LVEF和LVFS升高(P<0.05),LVIDd和LVIDs降低(P<0.05),血清CK-MB、LDH和cTnI水平降低(P<0.05),心肌梗死面积降低(P<0.05),心肌形态明显改善,心肌组织中NLRP3、Caspase-1、ASC、IL-1β和IL-18的mRNA表达水平降低(P<0.05),心肌组织中NLRP3、cleaved Caspase-1、ASC、IL-1β和IL-18蛋白表达水平降低(P<0.05);且毛兰素对上述指标的影响均呈现剂量依赖性(P<0.05)。结论毛兰素预处理可改善MIRI大鼠的心功能并减轻心肌损伤,毛兰素可能通过抑制NLRP3炎症体介导的细胞焦亡防治MIRI。

Objective To explore the role and its mechanism of erianin(ERI)in prevention and treatment of myocardial ischemia-reperfusion injury(MIRI).Methods SD rats were divided into sham group(n=18),model group(n=18),5 mg ERI group(n=17),10 mg ERI group(n=16)and 20 mg ERI group(n=17).Rats in sham group and MIRI group were given 1%dimethyl sulfoxide,and the rats in 5 mg ERI group,10 mg ERI group and 20 mg ERI group were given 5,10,20 mg/(kg·d)of erianin.Then the rats were induced to establish MIRI models seven days after administration in model group,5 mg ERI group,10 mg ERI group and 20 mg ERI group.Left ventricular ejection fraction(LVEF),left ventricular end-diastolic inner diameter(LVIDd),left ventricular end-systolic inner diameter(LVIDs)and left ventricular fractional shortening(LVFS)were detected at 48 h after reperfusion.The levels of serum myocardial injury markers[creatine kinase isoenzyme MB(CK-MB),lactate dehydrogenase(LDH),cardiac troponin I(cTnI)]were detected,and myocardial tissues were stained by TTC staining and hematoxylin and eosin(HE)staining.The mRNA levels of NLR family pyrin domain containing 3(NLRP3),Caspase-1,apoptosis-associated speck-like protein(ASC),interleukin(IL)-1βand IL-18 in myocardial tissue were detected by RT-qPCR.The protein expression levels of NLRP3,cleaved Caspase-1,ASC,IL-1βand IL-18 in myocardial tissue were detected by Western blot.Results Compared with sham group,LVEF and LVFS were decreased in model group(P<0.05),LVIDd and LVIDs were increased(P<0.05),serum CK-MB,LDH and cTnI levels were increased(P<0.05),myocardial infarction area was increased(P<0.05),myocardial injury was found,the mRNA expression levels of NLRP3,Caspase-1,ASC,IL-1βand IL-18 in myocardial tissue were increased(P<0.05),and the protein expression levels of NLRP3,cleaved Caspase-1,ASC,IL-1βand IL-18 in myocardial tissue were increased(P<0.05).Compared with MIRI group,LVEF and LVFS were increased in 5 mg ERI group,10 mg ERI group and 20 mg ERI group(P<0.05),LVIDd and LVIDs were decreased(P<0.05),serum levels of CK-MB,LDH and cTnI were decreased(P<0.05),the myocardial infarction area was decreased(P<0.05),the myocardial morphology was significantly improved,the mRNA expression levels of NLRP3,Caspase-1,ASC,IL-1βand IL-18 in myocardial tissue were decreased(P<0.05),and the protein expression levels of NLRP3,cleaved Caspase-1,ASC,IL-1βand IL-18 in myocardial tissue were decreased(P<0.05),and the above indexes showed a dose-dependent change by erianin(P<0.05).Conclusion Erianin pretreatment can improve the cardiac function and reduce the myocardial injury in rats with MIRI,and erianin may prevent and treat MIRI by inhibiting NLRP3 inflammasome-mediated pyroptosis.