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

    摘要
  • 摘要:
      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(COL Ⅳ)、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COL Ⅳ、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COL Ⅳ、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COL Ⅳ、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。

     

    Abstract:
      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, COL Ⅳ, 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 COL Ⅳ, 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.

     

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