Fasudil在EAM小鼠中通过抑制NOTCH信号通路下调IL-6表达

Fasudil exerts a protective effect on down-regulation of IL-6 expression by inhibiting the NOTCH signaling pathway in EAM mice

目的探究法舒地尔(Fasudil)在实验性自身免疫性心肌炎(experimental autoimmune myocarditis,EAM)小鼠治疗中发挥的作用及机制,为临床上Fasudil治疗心肌炎提供理论依据。方法以Balb/c雄性小鼠为研究对象,使用MyHC-α614-629多肽构建EAM小鼠模型。单个核细胞分离和培养检测小鼠脾脏中单个核细胞的数量变化,HE染色、Masson染色和免疫组化分别检测小鼠心肌组织的炎症浸润情况、纤维化情况和IL-6的表达,Western blotting检测Notch1和白细胞介素-6(IL-6)蛋白的表达,qRT-PCR检测促炎因子(IL-1α、IL-1β和IL-6)、TLRs和NOTCH信号通路的关键基因表达。结果EAM小鼠心脏明显增大发白、HW增加、BW下降和HW/e-BW增大,心肌组织炎症浸润和纤维化加重。EAM小鼠经过Fasudil治疗后,上述的症状或者病理特征得到改善。进一步分析发现,EAM小鼠心肌组织中促炎因子IL-1α、IL-1β和IL-6明显增加,但是经过Fasudil治疗后只有IL-6的表达下调具有统计学差异,此外,还发现TLRs信号可能也在Fasudil治疗EAM发挥重要作用。IL-6和Notch1的表达具有一致性,经过Fasudil治疗后,NOTCH信号通路的关键基因(Notch1、Hes1和Jag2)显著下调。结论Fasudil在EAM小鼠中通过抑制NOTCH信号通路下调IL-6的表达,从而发挥保护作用。

Objective To explore the effect and mechanism of Fasudil in the treatment of experimental autoimmune myocarditis(EAM)in mice so as to provide a theoretical basis for the clinical use of Fasudil in treating myocarditis.Methods Balb/c male mice were used as the research objects,and the EAM mice model was constructed using MyHC-α614-629 polypeptide.Mononuclear cells were isolated and cultured to detect the number of mononuclear cells in mouse spleen.Inflammation infiltration,fibrosis and IL-6 expression in mouse myocardial tissue were detected by HE staining,Masson staining and immunohistochemistry,respectively.The protein expressions of Notch1 and IL-6 were detected by Western blotting.qRT-PCR was used to detect the expressions of pro-inflammatory factors(IL-1α,IL-1βand IL-6)as well as key genes of TLRs and NOTCH signaling pathway.Results EAM mice showed increased HW,decreased BW,increased HW/e-BW,and increased inflammatory infiltration and fibrosis in myocardial tissue.The above-mentioned symptoms or pathological features were improved in EAM mice treated with Fasudil.The analysis showed that the pro-inflammatory factors IL-1α,IL-1βand IL-6 in the myocardial tissue of EAM mice were significantly increased,but only the expression of IL-6 was statistically different after Fasudil treatment compared with the control group.In addition,TLRs signaling pathway might also play an important role in the EAM mice treated with Fasudil.The expressions of IL-6 and Notch1 were consistent,and the expressions of the key genes of NOTCH signaling pathway(Notch1,Hes1 and Jag2)were down-regulated after Fasudil treatment.Conclusion Fasudil exerts a protective effect on down-regulation of IL-6 expression by inhibiting the NOTCH signaling pathway in EAM mice.