当归补血汤通过miR-27a/TGF-β1/Smad3通路抑制HK-2细胞纤维化作用及机制研究

Study on the Effect and Mechanism of Danggui Buxue Decoction Inhibiting HK-2 Cell Fibrosis through miR-27a/TGF-β1/Smad3 Pathway

目的 探讨当归补血汤通过miR-27a调控HK-2细胞纤维化的机制。方法 HK-2细胞分为对照组、高糖组、高糖+空白血清组、高糖+当归补血汤含药血清组。采用MTT法检测各组细胞增殖情况;采用Western blot法检测纤维化相关因子波形蛋白(Vimentin)、Ⅳ型胶原(COLⅣ)、α-平滑肌肌动蛋白(α-SMA)、p-Smad3/Smad3和转化生长因子(TGF)-β1蛋白的表达情况;qPCR法检测miR-27a及纤维化相关因子mRNA的表达情况;通过转染miR-27a抑制剂及Smad3-siRNA并向培养基中加入TGF-β1,观察miR-27a的表达和纤维化相关蛋白表达的情况。结果 MTT结果显示,与对照组相比,高糖组细胞增殖率明显降低(P<0.05,P<0.001),当归补血汤含药血清作用24、48 h后细胞增殖率明显提高(P<0.05,P<0.001)。Western blot结果显示,当归补血汤含药血清明显抑制高糖诱导的HK-2细胞纤维化相关蛋白COLⅣ、α-SMA、Vimentin、TGF-β1、p-Smad3/Smad3的表达(P<0.05,P<0.01)。qPCR结果显示,当归补血汤含药血清明显抑制高糖诱导的HK-2细胞COLⅣ、α-SMA、Vimentin、TGF-β1 mRNA和miR-27a的表达(P<0.05,P<0.01)。TGF-β1处理细胞后,细胞纤维化加重,miR-27a的表达升高(P<0.05,P<0.01),当归补血汤含药血清可明显降低TGF-β1诱导的纤维化细胞中miR-27a的表达升高(P<0.001)。转染miR-27a抑制剂及Smad3-siRNA可明显降低TGF-β1诱导的细胞纤维化蛋白COLⅣ、α-SMA、Vimentin、TGF-β1、p-Smad3/Smad3表达的升高(P<0.05,P<0.01)。结论 当归补血汤含药血清可促进HK-2细胞增殖,并通过miR-27a/TGF-β1/Smad3信号通路调控HK-2细胞纤维化。

OBJECTIVE To investigate the mechanism of Danggui Buxue Decoction regulating HK-2 cell fibrosis through miR-27a.METHODS HK-2 cells were divided into control group,high glucose group,high glucose+blank serum group,and high glucose+Danggui Buxue Decoction-containing serum group.The proliferation of cells in each group was detected by MTT assay;The expression of the fibrosis-related factors Vimentin,COLⅣ,α-SMA,p-Smad3/Smad3 and TGF-β1 protein was measured by Western blot;The qPCR was used to detect the expression of miR-27a and fibrosis-related factors mRNA;The expression of miR-27a and fibrosis-related proteins were observed by transfecting miR-27a inhibitor and Smad3-siRNA,as well as adding TGF-β1 to the culture medium.RESULTS MTT results showed that compared with the control group,the cell proliferation rate in the high glucose group significantly decreased(P<0.05,P<0.001),and Danggui Buxue Decoction significantly promoted cell proliferation(P<0.05,P<0.001).Western blot showed that Danggui Buxue Decoction significantly inhibited the expression of fibrosis-related proteins COLⅣ,α-SMA,Vimentin,TGF-β1 and p-Smad3/Smad3 in HK-2 cells induced by high glucose(P<0.05,P<0.01).The qPCR results were consistent with Western blot results.After TGF-β1 treatment of cells,cell fibrosis aggravated,and the expression of miR-27a increased(P<0.05,P<0.01),Danggui Buxue Decoction significantly reduced the elevation of miR-27a in TGF-β1-induced cellular fibrosis(P<0.001).Transfection of miR-27a inhibitor and Smad3-siRNA could significantly reduce the increase of TGF-β1-induced cellular fibrosis protein expression(P<0.05,P<0.01).CONCLUSION Danggui Buxue Decoction-containing serum can promote the proliferation of HK-2 cells and regulate HK-2 cell fibrosis through miR-27a/TGF-β1/Smad3 signaling pathway.