牛磺熊去氧胆酸通过调节内质网应激抑制转化生长因子β1所诱导的心肌成纤维细胞纤维化

Tauroursodeoxycholic acid through modulating endoplasmic reticulum stress on inhibition of transforming growth factorβ1-induced fibrosis in cardiac fibroblasts

目的探讨牛磺熊去氧胆酸(TUDCA)对转化生长因子β1(TGF-β1)所诱导的心肌成纤维细胞(CFs)活化的作用及相关机制,为TUDCA治疗糖尿病心肌纤维化提供新的治疗目标。方法提取原代SD乳鼠CFs和心肌细胞,通过免疫荧光法鉴定CFs的纯度。分别将高糖培养下的CFs用不同时间的TGF-β1干预,用Western blot检测内质网应激(ERS)通路相关蛋白和纤维化指标的表达,探讨TGF-β1对CFs活化及ERS相关通路的影响。用Western blot检测CFs和心肌细胞内同型半胱氨酸内质网应激泛素样结构域1(Herpud1)的表达情况。通过沉默Herpud1的表达及用Transwell和Western blot检测沉默Herpud1后CFs纤维化指标的表达,探讨Herpud1在TGF-β1诱导的CFs活化中的作用。用CCK-8检测不同浓度的TUDCA处理下CFs的活力。用免疫荧光和Western blot检测TUDCA对TGF-β1诱导的CFs中Herpud1、葡萄糖调节蛋白78(GRP78)、转录激活因子6(ATF6)、α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)和Ⅰ型胶原蛋白(CollagenⅠ)表达的影响。为进一步明确Herpud1在TUDCA抑制CFs活化中的作用,用Western blot检测过表达Herpud1后相关蛋白的表达情况。结果在成功构建CFs纤维化模型的基础上,TGF-β1能够诱导CFs活化及ERS通路相关蛋白的表达;Herpud1在CFs中的表达高于心肌细胞;敲除Herpud1可抑制TGF-β1所诱导的CFs活化;TUDCA能显著降低TGF-β1诱导的CFs中GRP78、ATF6、α-SMA、Vimentin和CollagenⅠ的表达水平;此外,过表达Herpud1还可逆转TUDCA对TGF-β1诱导的CFs活化的抑制。结论下调Herpud1基因的表达是TUDCA抑制TGF-β1诱导的CFs纤维化的机制之一。

Objective To investigate the effect of taurodeoxycholic acid(TUDCA)on the activation of cardiac fibroblasts(CFs)induced by transforming growth factorβ1(TGF-β1)and its related mechanisms,and to identify a new therapeutic target for TUDCA in the treatment of diabetic myocardial fibrosis.Methods The CFs and cardiomyocytes of primary neonatal SD rats were extracted,and the purity of CFs was identified by immunofluorescence.CFs cultured with high glucose were treated with TGF-β1 for different duration.The expression of endoplasmic reticulum stress(ERS)pathway related proteins and fibrosis markers were detected by Western blot to explore the effect of TGF-β1 on CFs activation and ERS related pathway.Western blot was used to detect the expression of homocysteine-inducible,ERSinducible,ubiquitin-like domain member 1(Herpud1)in CFs and cardiomyocytes.The role of Herpud1 in TGF-β1-induced CFs activation was explored by silencing the expression of Herpud1 and detecting the expression of CFs fibrosis markers after silencing Herpud1 by Transwell and Western blot.CCK-8 was used to detect the activity of CFs treated with different concentrations of TUDCA.Immunofluorescence and Western blot were used to detect the effects of TUDCA on the expression of Herpud1,glucose regulated protein 78(GRP78),transcriptional activator 6(ATF6),α-smooth muscle actin(α-SMA),vimentin(Vimentin)and typeⅠcollagen(CollagenⅠ)in CFs induced by TGF-β1.In order to further clarify the role of Herpud1 in the inhibition of CFs activation by TUDCA,the expression of related proteins after reaching Herpud1 was detected by Western blot.Results Based on the successful construction of a fibrosis model of CFs,TGF-β1 was proved to induce CFs activation as well as the expression of ERS pathway-related proteins.Expression of Herpud1 was higher in CFs than in cardiomyocytes.Knockdown of Herpud1 showed inhibition of CFs activation induced by TGF-β1.TUDCA significantly reduced TGF-β1-induced activation of CFs in GRP78,ATF6,α-SMA,Vimentin and CollagenⅠexpression levels.In addition,overexpression of Herpud1 reversed the inhibition of TGF-β1-induced activation of CFs by TUDCA.Conclusions Down-regulation of Herpud1 gene expression is one of the mechanisms by which TUDCA inhibits TGF-β1-induced cellular fibrosis in CFs.