低分子量透明质酸通过CD44调控S100A4核转移促进心肌纤维化

Low molecular weight hyaluronic acid regulates the nuclear translocation of S100A4 to promote myocardial fibrosis via CD44

目的:研究低分子量透明质酸(low molecular weight hyaluronic acid,LMW-HA)对小鼠心脏成纤维细胞(cardiac fibroblast,CF)表型转化的促进作用,探讨CD44和S100A4在此过程中的作用。方法:从ICR乳小鼠的心脏中分离提取CF并培养,使用LMW-HA进行刺激,采用CCK-8和EdU染色测细胞增殖程度,免疫印迹(Western blot,WB)、实时定量PCR、免疫荧光确定纤维化程度,对CD44和S100A4蛋白质核分离并通过WB和免疫荧光明确核转移。CD44抑制剂BRIC-235处理后再次检测上述指标。结果:CCK-8增殖与EdU染色实验确定LMW-HA的最佳刺激浓度为0.8 mg/mL。WB、PCR和免疫荧光表明LMW-HA的刺激使心脏纤维化标志物(α-SMA和Collagen 3)的水平显著增加,CD44保持不变,S100A4随时间逐渐增加,同时CD44和S100A4蛋白转移到细胞核内。CD44抑制剂BRIC-235能抑制这些改变。结论:LMW-HA的刺激能够促进小鼠CF的表型转化从而促进心肌纤维化,该过程是通过与CD44结合并促进S100A4转移入细胞核内激活下游通路实现的。

Objective:This study aimed to investigate the promotion effect of low molecular weight hyaluronic acid(LMW-HA)on phenotypic transformation of mice cardiac fibroblasts and the role of CD44 and S100 A4 in this process. Methods:Cardiac fibroblasts(CFs)were isolated and cultured from neonatal ICR miceandthen stimulated by LMW-HA. The proliferation of CFs was measured by CCK-8 and EdU. Western blot(WB),quantitative RT-PCR and immunofluorescence were used to determine myocardial fibrosis.Nuclear and cytoplasmic proteins of CD44 and S100 A4 were extracted to specify the nuclear translocation by WB and immunofluorescence. Above indicators were measured again after CFs treated with the CD44 inhibitor BRIC-235. Results:CCK-8 and EdU determined that the optimal concentration of LMW-HA stimulation was 0.8 mg/mL. WB,qRT-PCR and immunofluorescence showed that LMW-HA stimulation significantly increased the level of myocardial fibrosis markers(α-SMA and Collagen 3),but CD44 remained unchanged,and S100 A4 gradually increased with time.The proteins of CD44 and S100 A4 were transferred into the nucleus at the same time. BRIC-235 can inhibit these changes. Conclusion:LMW-HA can promote the differentiation of micecardiac fibroblasts,which is regulated by the nuclear translocation of CD44 and S100 A4 and the activation of downstream signaling pathway.