血管紧张素转换酶2通过抑制IL-6/JAK2/STAT3信号通路抵抗传染性支气管炎病毒诱导细胞炎性反应

Angiotensin converting enzyme 2 alleviates infectious bronchitis virus-induced cellular inflammation by suppressing IL-6/JAK2/STAT3 signaling pathway

摘要:

为探讨血管紧张素转换酶2（angiotensin converting enzyme 2，ACE2）对传染性支气管炎病毒（infectious bronchitis virus，IBV）诱导细胞炎性反应的调控作用及其机制，本研究以IBV重组病毒（IBV-3ab-Luc）分别感染Vero和DF-1细胞，并设立对照组、感染组[IBV-3ab-Luc，感染复数（multiplicity of infection，MOI）=1]、ACE2过表达组[IBV-3ab-Luc+pcDNA3.1（+）-ACE2]及ACE2缺失组（IBV-3ab-Luc+siRNA-ACE2）。待感染组出现合胞体等明显细胞病变后，提取各组细胞总蛋白质和RNA，免疫荧光及免疫印迹试验（Western blotting）鉴定ACE2的过表达与缺失；实时荧光定量PCR （real-time quantitative polymerase chain reaction，RT-qPCR）检测细胞中IBV核衣壳（nucleoprotein，N）、糖蛋白130（glycoprotein 130，gp130）、白细胞介素6（interleukin-6，IL-6）等炎性因子的mRNA表达水平；酶联免疫吸附（enzyme linked immunosorbent assay，ELISA）法测定细胞上清中IL-6水平；Western blotting检测细胞中ACE2表达和酪氨酸蛋白激酶2（janus kinase 2，JAK2）、信号转导和转录激活因子3（signal transducer and activator of transcription 3，STAT3）磷酸化水平。结果表明，Vero和DF-1两种细胞均成功实现ACE2的过表达与缺失。IBV感染可导致Vero细胞变圆、脱落和聚堆，有明显合胞体出现；ACE2过表达后细胞病变减轻；缺失后细胞病变增强。与对照组相比，感染组IBV-N、IL-6和gp130 mRNA表达均显著上调（P<0.05），细胞上清中IL-6水平显著或极显著升高（P<0.05或P<0.01）；ACE2蛋白表达显著下调（P<0.05），JAK2和STAT3蛋白磷酸化水平显著上调（P<0.05）。与感染组相比，ACE2过表达组IBV-N基因mRNA表达水平无显著变化（DF-1细胞）（P>0.05），但gp130 mRNA表达，IL-6水平及mRNA表达，JAK2、STAT3蛋白磷酸化水平显著下调（P<0.05）；缺失组IBV-N也无显著变化（P>0.05），但IL-6水平及mRNA表达显著上调（P<0.05），JAK2和STAT3蛋白的磷酸化水平略下调（P>0.05）。结果首次证实ACE2对IBV在DF-1细胞中的复制无明显影响，但在IBV诱导Vero、DF-1细胞炎性反应中通过抑制IL-6/JAK2/STAT3信号通路发挥一定的抵抗作用。

Abstract:

The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested:the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P<0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P<0.05 or P<0.01); the expression of ACE2 decreased significantly (P<0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P<0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P>0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P<0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P<0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P>0.05), but the IL-6 level and expression of mRNA increased significantly (P<0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P>0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.