Yu Ying, Shi Hui, Yu Yong, Liu Ming, Li Minghui, Liu Xiaoxiao, Wang Yucheng, Chen Ruizhen
Department of General Practice, Zhongshan Hospital, Shanghai Medical College of Fudan University Shanghai 200032, China.
Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Shanghai Medical College of Fudan University Shanghai 200032, China.
Am J Transl Res. 2020 May 15;12(5):1954-1964. eCollection 2020.
This study aimed to verify the effects of calpain on coxsackievirus B3 (CVB3)-induced myocarditis and to further explore the underlying mechanisms. Transgenic mice overexpressing calpastatin, the endogenous calpain inhibitor, were introduced in the present study. The murine model of viral myocarditis (VMC) was established by intraperitoneal injection of CVB3 into transgenic and wild-type mice. Myocardial injury was measured by H&E staining and ELISA for cTnI. CVB3 replication was assessed via capsid protein VP1 detection and virus titration. The fibrotic factors collagen and TGF-β1 were evaluated by Masson staining and real-time PCR analysis, respectively. Moreover, the levels of NLRP3, AIM2, ASC, cleaved caspase-1, cleaved caspase-11 and the pyroptosis indicators GSDMD p30, IL-1β and HMGB1 were determined by real-time PCR, western blot or immunohistochemical analysis. In addition, peripheral IL-1β and HMGB1 were evaluated by ELISA. We observed that CVB3-infected transgenic mice had lower pathological scores, peripheral cTnI levels, viral loads and expression levels of collagen and TGF-β1 in the heart than CVB3-infected wild-type mice. Furthermore, we found decreased levels of NLRP3, ASC, cleaved caspase-1 and cleaved caspase-11 in the hearts of CVB3-infected transgenic mice. However, after CVB3 infection, the levels of AIM2 in transgenic mice and wild-type mice did not differ significantly. Additionally, calpastatin overexpression significantly reduced the levels of GSDMD p30, IL-1β and HMGB1 in the myocardium as well as peripheral IL-1β and HMGB1. Taken together, these findings indicate that calpain inhibition attenuates CVB3-induced myocarditis by suppressing the canonical NLRP3 inflammasome/caspase-1-mediated and noncanonical caspase-11-mediated pyroptosis pathways.
本研究旨在验证钙蛋白酶对柯萨奇病毒B3（CVB3）诱导的心肌炎的影响，并进一步探究其潜在机制。本研究引入了过表达内源性钙蛋白酶抑制剂钙蛋白酶抑制蛋白的转基因小鼠。通过向转基因小鼠和野生型小鼠腹腔注射CVB3建立病毒性心肌炎（VMC）小鼠模型。通过苏木精-伊红（H&E）染色和检测心肌肌钙蛋白I（cTnI）的酶联免疫吸附测定（ELISA）来测量心肌损伤。通过衣壳蛋白VP1检测和病毒滴定评估CVB3复制情况。分别通过Masson染色和实时聚合酶链反应（PCR）分析评估纤维化因子胶原蛋白和转化生长因子-β1（TGF-β1）。此外，通过实时PCR、蛋白质免疫印迹法或免疫组织化学分析测定NLRP3、AIM2、凋亡相关斑点样蛋白（ASC）、裂解的半胱天冬酶-1、裂解的半胱天冬酶-11以及细胞焦亡指标Gasdermin D p30、白细胞介素-1β（IL-1β）和高迁移率族蛋白B1（HMGB1）的水平。另外，通过ELISA评估外周血IL-1β和HMGB1。我们观察到，与感染CVB3的野生型小鼠相比，感染CVB3的转基因小鼠的病理评分、外周血cTnI水平、病毒载量以及心脏中胶原蛋白和TGF-β1的表达水平更低。此外，我们发现感染CVB3的转基因小鼠心脏中NLRP3、ASC、裂解的半胱天冬酶-1和裂解的半胱天冬酶-11的水平降低。然而，CVB3感染后，转基因小鼠和野生型小鼠中AIM2的水平没有显著差异。此外，钙蛋白酶抑制蛋白的过表达显著降低了心肌中Gasdermin D p30、IL-1β和HMGB1的水平以及外周血IL-1β和HMGB1的水平。综上所述，这些发现表明钙蛋白酶抑制通过抑制经典的NLRP3炎性小体/半胱天冬酶-1介导的和非经典的半胱天冬酶-11介导的细胞焦亡途径减轻CVB3诱导的心肌炎。
