NLRP3炎症小体参与HSV-1诱导病毒性心肌炎的实验研究

NLRP3-inflammasome participated in HSV-1 induced viral myocarditis

目的:观察NOD样受体热蛋白结构域相关蛋白3(Nod-like receptor pyrin domain-containing protein 3,NLRP3)炎症小体是否参与单纯疱疹病毒1型(herpes simplex virus-1,HSV-1)诱导的病毒性心肌炎(viral myocarditis,VMC)的病理过程。方法:培养乳鼠心室肌细胞(neonatal rat ventricular myocytes,NRVM),分别以0.01 PFU和0.1 PFU HSV-1感染NRVM,24 h后通过光学显微镜观察细胞形态学改变,实时定量PCR(quantitative real-time PCR,qRT-PCR)测定NLRP3炎症小体及其下游通路的mRNA表达水平,免疫荧光(immunofluorescence,IF)显示半胱天冬酶(cysteinyl aspartate-specific proteases,Caspase)-1的表达,全自动生化仪测定细胞上清肌酸激酶同工酶MB(creatine kinase-MB,CK-MB)的含量以及酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定细胞上清白细胞介素(interleukin,IL)-18的浓度。结果:HSV-1感染心肌细胞模型组出现细胞病变现象(cytopathic effect,CPE),培养细胞上清细胞损伤标志物CK-MB明显增高(P<0.05),qRT-PCR测定模型组NLRP3、Caspase-1、IL-1β和IL-18 mRNA相对表达量较对照组高5倍以上,IF显示Caspase-1在HSV-1干预后的NRVM胞质内表达明显增高,培养细胞上清IL-18浓度较对照组增高(P<0.05)。结论:NLRP3炎症小体及下游通路在HSV-1诱导的VMC细胞模型中被激活,参与其病理过程,为治疗病毒性心肌炎提供可能的新靶点。

Objective：To observe whether the Nod-like receptor pyrin domain-containing protein 3（NLRP3）-inflammasome participates in the pathologic process of herpes simplex virus-1（HSV-1） induced viral myocarditis（VMC）.Methods：Cultured neonatal rat ventricular cardiomyocytes（NRVM） of neonatal rats were infected with 0.01 and 0.1 PFU HSV-1 for 24 hours,respectively.Morphologic changes of NRVM were observed under light microscope.The gene expression of NLRP3-inflammasome and its downstream pathways were measured by quantitative real-time PCR（qRT-PCR）.The expression and location of cysteinyl aspartatespecific proteases-1（Caspase-1） were evaluated by immunofluorescent（IF） method.Moreover,creatine kinase-MB（CK-MB） content was detected by automatic biochemical analyzer and supernatant concentration of interleukin-18（IL-18） was measured by enzymelinked immunosorbent assay（ELISA）.Results：Cytopathic effect（CPE） was observed in NRVM infected with HSV-1.The supernatant concentration of CK-MB,one of the myocardial injury biomarkers,was significantly increased（P〈 0.05）.Compared with the control group,the mRNA levels of NLRP3,Caspase-1,IL-1β and IL-18 were up-regulated over 5 times in HSV-1 infected NRVM.IF showed that the expression of Caspase-1 was significantly increased.The concentration of supernatant IL-18 was increased compared with that of the control group（P〈 0.05）.Conclusion：NLRP3-inflammasome and its downstream pathways were activated in cell model of HSV-1 infected VMC.NLRP3-inflammasome may participate in pathologic process of VMC,and became a potential target for VMC therapy.