基于NLRP3炎症小体研究C1q/肿瘤坏死因子相关蛋白6对病毒性心肌炎细胞损伤的保护作用

doi:10.3969/j.issn.1006-6233.2023.02.07

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摘要 目的: 研究补体C1q/肿瘤坏死因子相关蛋白6(C1q/tumor necrosis factor-related protein 6,CTRP6)对柯萨奇病毒B3(Coxsackie B3 virus,CBV3)诱导的病毒性心肌炎细胞损伤的调控作用,并探讨其可能的作用机制。方法: 通过CBV3感染人心肌细胞系AC-16来建立病毒性心肌炎的体外细胞模型。通过表达质粒转染技术来实现CTRP6在心肌细胞中的过表达。细胞分为4组:正常组、CBV3组、CBV3 +空载体组和CBV3 + CTRP6载体组。采用实时定量PCR(real-time quantitative PCR,RT-qPCR)方法检测mRNA表达水平。采用Western blotting方法检测蛋白表达水平。采用相应的试剂盒测量肌酸激酶同工酶(creatine kinase-MB,CK-MB)和心肌肌钙蛋白I(cardiac troponin-I,cTn-I)的活性水平。利用原位末端脱氧核苷酸转移酶标记法(terminal deoxynucleotidyl transferase-mediated nick end labeling,TUNEL)检测细心肌细胞胞凋亡。利用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测量炎症因子浓度。采用RNA沉默技术来敲除AdipoR1在心肌细胞中的表达。结果: 与正常组相比,CBV3感染组心肌细胞中CTRP6的表达水平显著降低(P<0.01)。CBV3感染心肌细胞显著提高CK-MB和cTn-I的活性水平,并增加凋亡细胞数量和炎症因子的释放(P<0.01)。与CBV3 +空载体相比,CBV3 + CTRP6载体组心肌细胞中的CK-MB和cTn-I的活性水平显著下降,凋亡细胞数量显著减少,炎症因子的释放显著降低(P<0.01)。与CBV3 +空载体组相比,过表达CTRP6可显著抑制CBV3感染的诱导的NLRP3炎症小体的激活(P<0.01)。敲除AdiopR1则显著阻断CTRP6对心肌炎细胞损伤的保护作用(P<0.01)。结论: CTRP6通过作用于AdiopR1受体抑制NLRP3炎症小体的激活来减轻CBV3诱导的病毒性心肌炎细胞损伤。

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关键词 ：肿瘤坏死因子相关蛋白6, 病毒性心肌炎, 柯萨奇病毒B3, NLRP3炎症小体

Abstract：Objective: To investigate the role of C1q/tumor necrosis factor-related protein 6 (CTRP6) in mediating viral myocarditis-associated cardiac cell injury induced by Coxsackie B3 virus (CVB3) and explore the potential underlying mechanism. Methods: Human cardiac cell line AC-16 was applied to establish an in vitro model of viral myocarditis by infecting CVB3. CTRP6 overexpression was achieved by transfecting the CTRP6 expression vector. Cells were divided into four groups including the normal group, CVB3 infection group, CVB3 + empty vector group, and CVB3 + CTRP6 vector group. The mRNA expression was measured by real-time quantitative PCR (RT-qPCR). The protein expression was measured by Western blotting. The levels of creatine kinase-MB (CK-MB) and cardiac troponin-I (cTn-I) were assessed by corresponding kits. Cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The concentrations of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). Knockdown of AdipoR1 was achieved by RNAi silencing. Results: As compared with the normal group, infection of CVB3 significantly decreased the expression of CTRP6 in cardiac cells (P<0.01). Infection of CVB3 markedly increased the levels of CK-MB and cTn-I, and significantly enhanced the apoptosis and inflammatory cytokine release in cardiac cells (P<0.01). As compared with the CVB3 + empty vector group, overexpression of CTRP6 significantly downregulated the levels of CK-MB and cTn-I and attenuated the apoptosis and inflammatory cytokine release in cardiac cells infected with CVB3 (P<0.01). Overexpression of CTRP6 also inhibited the activation of NLRP3 inflammasome in CVB3-infected cardiac cells compared with the CVB3 + empty vector group (P<0.01). Silencing of AdipoR1 markedly abolished CTRP6-mediated protective effects on CBV3-induced cardiac cell injury (P<0.01). Conclusion: CTRP6 ameliorates viral myocarditis-associated cardiac cell injury induced by CBV3 through inhibition of NLRP3 inflammasome activation via the action on the receptor AdipoR1.

Key words： CTRP6 Viral myocarditis Coxsackie B3 NLRP3 inflammasome

基金资助:陕西省卫生健康科研基金项目,(编号:2022D019)

通讯作者: 董夏

引用本文:

阴睿媛, 王蓓, 许亚静, 董夏. 基于NLRP3炎症小体研究C1q/肿瘤坏死因子相关蛋白6对病毒性心肌炎细胞损伤的保护作用[J]. 河北医学, 2023, 29(2): 214-220.
YIN Ruiyuan, et al. Protective Effect of CTRP6 on Viral Myocarditis-Associated Cardiac Cell Injury Based on NLRP3 Inflammasome. HeBei Med, 2023, 29(2): 214-220.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2023.02.07 或 http://www.hbyxzzs.cn/CN/Y2023/V29/I2/214

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