Relaxin-3通过抑制HMGB1介导的NLRP3炎性小体活化抑制AngⅡ诱导的心肌成纤维细胞转分化

Relaxin-3 inhibits AngⅡ-induced cardiac fibroblast transdifferentiation by inhibiting HMGB1-mediated activation of NLRP3 inflammasome

目的:探究人松弛素3(relaxin-3)对血管紧张素Ⅱ(AngⅡ)处理心肌成纤维细胞的作用及其可能的作用机制。方法:AngⅡ处理心肌成纤维细胞,建立体外心肌纤维化模型,CCK-8检测细胞活力;EdU试剂盒检测细胞增殖能力;Western blot检测细胞中抗α平滑肌肌动蛋白(α-SMA)、波形蛋白(vimentin)、I型胶原(collagenⅠ)、Ⅲ型胶原(collagenⅢ)、NOD样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、裂解的含半胱氨酸的天冬氨酸蛋白水解酶-1(cleaved caspase-1)和高迁移率族蛋白B1(HMGB1)表达;间接免疫荧光检测细胞中NLRP3的表达。结果:与空白对照组相比,AngⅡ组细胞活力、EdU阳性细胞数明显升高(P<0.05),细胞中α-SMA、vimentin、collagenⅠ和collagenⅢ蛋白表达明显升高(P<0.05),NLRP3荧光强度和NLRP3、ASC、cleaved caspase-1、HMGB1蛋白表达均明显升高(P<0.05);与AngⅡ组相比,relaxin-3组细胞活力、EdU阳性细胞数明显降低(P<0.05),细胞中α-SMA、vimentin、collagenⅠ和collagenⅢ蛋白表达明显降低(P<0.05),NLRP3荧光强度和NLRP3、ASC、cleaved caspase-1、HMGB1蛋白表达均明显降低(P<0.05);relaxin-3组和relaxin-3+oe-NC组两组间的各项指标比较,差异无统计学意义(P>0.05);与relaxin-3+oe-NC组相比,relaxin-3+oe-HMGB1组胞活力、EdU阳性细胞数明显升高(P<0.05),细胞中α-SMA、vimentin、collagenⅠ和collagenⅢ蛋白表达明显升高(P<0.05),NLRP3荧光强度和NLRP3、ASC、cleaved caspase-1、HMGB1蛋白表达均明显升高(P<0.05)。结论:relaxin-3通过下调HMGB1的表达抑制NLRP3炎性小体活化,从而抑制AngⅡ诱导的心肌成纤维细胞转分化。

Objective:To explore the effect of Relaxin-3 on cardiac fibroblasts treated with angiotensinⅡ(AngⅡ)and its possible mechanism.Methods:AngⅡwas used to process myoc ardial fibroblasts to establish an vitro myocardial fibrosis model.CCK-8 was used to detect cell viability,EdU detection kit was used to detect the cell proliferation ability,Western blot was used to detect the expression ofα-SMA,vimentin,collagenⅠ,collagenⅢ,NLRP3,ASC,Cleaved caspase-1 and HMGB1 protein in cells.Indirect immunofluorescence was used to detect the expression of NLRP3 in cells.Results:Compared with the blank control group,the cell viability and the number of EdU positive cells in the AngⅡgroup were significantly increased(P<0.05),and the expression ofα-SMA,vimentin,collagenⅠand collagenⅢprotein in the cells was significantly increased(P<0.05),the intensity fluorescence of NLRP3 and expression of NLRP3,ASC,Cleaved caspase-1 and HMGB1 protein were significantly increased(P<0.05).Compared with the AngⅡgroup,the cell viability and the number of EdU-positive cells in the Relaxin-3 group were significantly reduced(P<0.05),the expression ofα-SMA,vimentin,collagen I and collagenⅢprotein in the cells was significantly reduced(P<0.05),and the fluorescence intensity of NLRP3 and the expression of NLRP3,ASC,Cleaved caspase-1,HMGB1 protein were significantly reduced(P<0.05).There was no significant difference in indicators between the Relaxin-3 group and the Relaxin-3+oe-NC group(P>0.05).Compared with the Relaxin-3+oe-NC group,the cell viability and the number of EdU-positive cells in the Relaxin-3+oe-HMGB1 group were significantly increased(P<0.05),and the expression ofα-SMA,vimentin,collagen I and collagenⅢprotein in the cells was significantly increased(P<0.05),and the expressions of NLRP3,ASC,Cleaved caspase-1 and HMGB1 protein were significantly increased(P<0.05).Conclusion:Relaxin-3 inhibits the activation of NLRP3 inflammasomes by down-regulating the expression of HMGB1,thereby inhibiting the transdifferentiation of cardiac fibroblasts induced by AngⅡ.