IL-22调控EAM小鼠心肌纤维化的初步研究

Preliminary study on IL-22 regulating myocardial fibrosis in EAM mice

目的:探究IL-22在实验性自身免疫性心肌炎(EAM)发展后期心肌纤维化中的作用及其可能的机制。方法:重组质粒pET28a-IL-22转化和鉴定生成包涵体后,通过变性、复性和纯化得到可溶性蛋白。Western blot、免疫荧光、qRT-PCR检测纤维化相关指标表达水平。ELISA检测细胞培养上清中TGF-β的水平;Western blot检测Smad3及其磷酸化的情况。天狼星红染色检测心脏纤维化的程度;qRT-PCR检测心脏组织中CollagenⅠ/Ⅲ的mRNA表达情况。结果:经过变性、复性和纯化后得到IL-22可溶性蛋白。IL-22降低CollagenⅠ和α-SMA的表达,下调CollagenⅠ/ⅢmRNA的水平。IL-22抑制TGF-β水平及Smad3信号分子的活化。IL-22可以缓解EAM小鼠晚期心脏纤维化进程。结论:IL-22通过抑制血管紧张素Ⅱ(ANGⅡ)激活的TGF-β下游Smad3信号途径缓解EAM小鼠心脏纤维化。

Objective:To explore the role and possible mechanism of IL-22 regulating myocardial fibrosis in the late stage of EAM.Methods:After transformation and identification of recombinant plasmid pET28 a-IL-22 to generate inclusion bodies,soluble protein was obtained by denaturation,renaturation and purification. Western blot,immunofluorescence,and qRT-PCR were used to detect the expression levels of fibrosis-related indicators. ELISA was used to detect the expression level of TGF-β in cell culture supernatant;Western blot was used to detect the expression of Smad3 and its phosphorylation level. Sirius red staining was used to detect the degree of cardiac fibrosis;qRT-PCR was used to detect the mRNA expression of Collagen Ⅰ/Ⅲ in cardiac tissue.Results:After denaturation,renaturation and purification,IL-22 soluble protein was obtained. IL-22 reduces the expression of Collagen I and α-SMA,down-regulates Collagen Ⅰ/Ⅲ mRNA level. IL-22 inhibits the level of TGF-β and activation of Smad3 signaling molecules. IL-22 can alleviate the process of advanced cardiac fibrosis in EAM mice.Conclusion:IL-22 alleviates cardiac fibrosis in EAM mice by inhibiting the Smad3 signaling pathway activated by ANGⅡ downstream of TGF-β.