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目的探究抗炎蛋白TNFAIP3(TNF-αinduced protein 3)(A20)在急性病毒性心肌炎发生发展过程中保护作用。方法90只6-8周龄Balb/c小鼠分为6组:Con组,GFP组,A20组,CVB3组,CVB3+GFP组,CVB3+A20组,每组15只。包装rAAV9-GFF与rAAV9-A20病毒,纯化后经尾静脉注射过表达相应基因,2周后腹腔注射100ul含106 PFU CVB3病毒液或PBS。监测各组小鼠体重变化及小鼠存活情况,病毒注射后第10天行心脏超声及心导管检查,取心脏,行HE染色和免疫组化检测(CD68及Ly6G)。RT-PCR检测心脏炎症因子TNF-α,IL-1β,IL-12,IL-17A,IL-I0,IL-13 mRNA水平。TUNEL检测小鼠心脏凋亡。收集小鼠血浆,检测血浆磷酸肌酸激酶CK-MB和肌钙蛋白cTnI。Western blot检测炎症、凋亡相关蛋白。结果CVB3诱导Balb/c小鼠急性病毒性心肌炎形成,CVB3组和CVB3+GFP组小鼠体重增长缓慢,第4天开始出现死亡,第10天达到高峰,累积生存率分别为40%和46.7%。HE染色可见大量炎症细胞浸润,心肌细胞坏死。免疫组化显示巨噬细胞及中性粒细胞浸润显著。TUUNEL显示大量心肌细胞凋亡。与正常Con相比,心脏超声和心导管显示CVB3组EF、FS、dp/dtmax、-dp/dtmin显著降低,心脏收缩功能下降。小鼠促炎因子TNF-α,IL-1β,IL-12,IL-17A mRNA水平显著增高,抑炎因子IL-10,IL-13 mRNA降低。血浆游离CK-MB和肌钙蛋白cTnI,高于正常组。Western blot示炎症、凋亡相关蛋白激活。高表达A20后,炎症细胞浸润减少,心肌细胞凋亡缓解,炎症通路及凋亡均抑制。促炎因子表达水平降低,血浆肌钙蛋白减少。结论A20可缓解CVB3诱导的急性病毒性心肌炎病理生理学改变,从而发挥保护作用。
Objective To investigate the protective effect of anti-inflammatory protein TNFAIP3 (A20) on the development of acute viral myocarditis. Methods Ninety Balb / c mice aged 6-8 weeks were divided into six groups: Con group, GFP group, A20 group, CVB3 group, CVB3 + GFP group and CVB3 + A20 group. The rAAV9-GFF and rAAV9-A20 viruses were packaged and purified. The corresponding genes were overexpressed by tail vein injection. Two weeks later, 100ul of 106 PFU CVB3 virus solution or PBS were injected intraperitoneally. The changes of body weight and survival of mice in each group were monitored. Cardiac echocardiography and cardiac catheterization were performed on the 10th day after virus injection. The hearts were harvested for HE staining and immunohistochemistry (CD68 and Ly6G). The levels of TNF-α, IL-1β, IL-12, IL-17A, IL-10 and IL-13 mRNA in heart were detected by RT-PCR. TUNEL was used to detect cardiac apoptosis in mice. Mouse plasma was collected and plasma creatine kinase CK-MB and troponin cTnI were measured. Western blot detection of inflammation, apoptosis-related proteins. Results CVB3 induced the formation of acute viral myocarditis in Balb / c mice. The body weight of CVB3 and CVB3 + GFP mice increased slowly. The mice died on the fourth day and reached the peak on the tenth day. The cumulative survival rates were 40% and 46.7% . HE staining shows a large number of inflammatory cell infiltration, myocardial necrosis. Immunohistochemistry showed that macrophages and neutrophils infiltrated significantly. TUUNEL shows massive cardiomyocyte apoptosis. Compared with normal Con, echocardiography and cardiac catheterization showed that the EF, FS, dp / dtmax and -dp / dtmin of CVB3 group were significantly decreased, while the cardiac systolic function was decreased. The levels of TNF-α, IL-1β, IL-12 and IL-17A mRNA in mice increased significantly, and the mRNA expressions of IL-10 and IL-13 decreased. Plasma free CK-MB and troponin cTnI, higher than the normal group. Western blot showed inflammation, apoptosis-related protein activation. After high expression of A20, infiltration of inflammatory cells decreased, cardiomyocyte apoptosis alleviated, inflammatory pathways and apoptosis were inhibited. Pro-inflammatory factor expression decreased, decreased plasma troponin. Conclusion A20 can relieve the pathophysiology of CVB3-induced acute viral myocarditis and play a protective role.