miR-21-5p对川崎病内皮细胞焦亡的作用及其机制研究

Effect of miR-21-5p on pyroptosis of endothelial cells in Kawasaki disease mouse model and its mechanism

目的探究miR-21-5p对川崎病(KD)内皮细胞焦亡的作用及其机制。方法采用随机数字表法将16只C57BL/6小鼠分为KD组和PBS组,每组8只。利用干酪乳杆菌细胞壁提取物(LCWE)腹腔注射小鼠,构建KD模型。利用LCWE处理小鼠冠状动脉内皮细胞(MCAECs),构建KD体外细胞模型。在使用LCWE处理的基础上,根据转染miR-21-5p抑制剂(miR-21-5p inhibitor)或inhibitor阴性对照物(inhibitor-NC)、miR-21-5p模拟物(miR-21-5p mimics)或mimics阴性对照物(miR-NC)、用或不用含NLR家族Pyrin域蛋白3(NLRP3)抑制剂MCC950,将MCAECs分组为:(1)LCWE组和对照组;(2)LCWE+inhibitor-NC组和LCWE+miR-21-5p inhibitor组;(3)LCWE组、LCWE+MCC950组、LCWE+MCC950+miR-NC组和LCWE+MCC950+miR-21-5p mimics组。采用HE染色观察小鼠冠状动脉组织病理学变化;采用qRT-PCR法检测miR-21-5p表达变化;采用Western blot检测焦亡相关蛋白NLRP3、凋亡相关斑点样蛋白(ASC)、含半胱氨酸的天冬氨酸蛋白水解酶-1(caspase-1)、caspase-1前体(pro-caspase-1)、消皮素D蛋白(GSDMD)p30、IL-1β、IL-18的蛋白表达;采用细胞计数盒法检测细胞活力;采用乳酸脱氢酶(LDH)试剂盒检测LDH活性。结果与PBS组相比,KD组小鼠冠状动脉组织病变明显,冠状动脉组织中NLRP3介导的细胞焦亡、miR-21-5p表达水平均升高(均P<0.01)。与对照组相比,LCWE组MCAECs细胞活力明显降低并且miR-21-5p表达水平明显升高(均P<0.01)。与LCWE+inhibitor-NC组相比,LCWE+miR-21-5p inhibitor组细胞miR-21-5p表达水平、NLRP3、ASC、caspase-1、GSDMD p30、IL-1β、IL-18的蛋白表达以及LDH活性均明显降低(均P<0.01),而细胞活力明显升高(P<0.05)。与LCWE组相比,LCWE+MCC950组NLRP3、ASC、caspase-1、GSDMD p30、IL-1β、IL-18的蛋白表达以及LDH活性均明显降低(均P<0.01),且细胞活力明显升高(P<0.01),而进一步转染miR-21-5p mimics明显逆转了以上变化(均P<0.01)。结论在KD中,miR-21-5p通过激活NLRP3炎症小体调节血管内皮细胞焦亡,从而加剧血管内皮细胞损伤。提示抑制miR-21-5p可以部分改善KD期间的血管内皮细胞损伤,miR-21-5p可以作为治疗KD的潜在干预靶点。

Objective To explore the effect and mechanism of miR 21-5p on focal death of endothelial cells in Kawasaki disease(KD)mouse model.Methods According to the random number table method,16 C57BL/6 mice were divided into KD group and PBS group,with 8 mice in each group.KD model was induced by intraperitoneal injection of lactobacillus casei cell wall extract(LCWE)in mice.Mouse coronary artery endothelial cells(MCAECs)were treated with LCWE to establish a KD cell model.On the basis of LCWE treatment,MCAECs were transfected with miR-21-5p inhibitor or the negative control of the inhibitor(inhibitor-NC),miR 21-5p mimics or the negative control of the mimics(miR-NC),and inhibi-pretreated with or without NLRP3 inhibitor MCC950.HE staining was used to observe the histopathological changes of coronary artery;qRT-PCR was used to detect the expression of miR 21-5p;Western blot was used to detect the protein expression of recombinant NLR family,Pyrin domain containing protein 3(NLRP3),apoptosis-related speckle-like protein(ASC),cysteinyl aspartate specific proteinase(caspase-1),pro-caspase-1,gasdermin D(GSDMD)p30,IL-1β,and IL-18.Cell vitality was determined with cell counting kit-8 test and lactate dehydrogenase(LDH)activity was determined with LDH test kits.Results Compared with the PBS group,the KD mice had obvious coronary artery tissue lesions,and the level of pyroptosis mediated by NLRP3 inflammasomes and the expression of miR-21-5p were increased in the coronary artery tissue of KD mice(all P<0.01).Compared with the control group,the activity of MCAECs cells in LCWE group was significantly decreased and the expression of miR 21-5p was significantly increased(both P<0.01).Compared with LCWE+inhibitors NC group,the expression of miR-21-5p,the protein expression of NLRP3,ASC,caspase-1,GSDMD p30,IL-1β,IL-18 and LDH release quantity in LCWE+miR 21-5p inhibitor group were significantly lower(all P<0.01),and the cell vitality increased significantly(P<0.05).Compared with LCWE group,LDH release and the protein expression of NLRP3,ASC,caspase-1,GSDMD p30,IL-1βand IL-18 in LCWE+MCC950 group were significantly decreased(all P<0.01),and the cell activity increased significantly(P<0.01).Further transfection of miR-21-5p mimics significantly reversed the above changes(all P<0.01).Conclusion In KD,miR 21-5p regulates vascular endothelial cell pyroptosis by activating NLRP3 inflammasome to aggravate vascular endothelial cell injury.These results suggest that inhibition of miR 21-5p can partially ameliorate vascular endothelial cell injury in KD,and miR 21-5p might be used as a potential intervention target for the treatment of KD.