南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (05): 640-646.doi: 10.12122/j.issn.1673-4254.2020.05.05

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二甲双胍抑制大鼠胆管成纤维细胞胶原生成的分子信号机制

卢家美，张晶晶，吕 毅，王 博，贾丽娜，师建华   

  • 出版日期:2020-05-20 发布日期:2020-05-20
  • 基金资助:

Metformin inhibits collagen production in rat biliary fibroblasts: the molecular signaling mechanism

  

  • Online:2020-05-20 Published:2020-05-20

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摘要： 目的 探讨二甲双胍是否通过活化腺苷酸激活蛋白激酶（AMPK）抑制转化生长因子-β1（TGF-β1）刺激的大鼠胆管成纤维细胞胶原生成并揭示其分子信号机制。方法 取雌雄各半的SD大鼠共50只（体质量150~200 g），CO2窒息法处死后，无菌条件下分离并培养大鼠胆管成纤维细胞，倒置显微镜观察所培养的细胞，发现不同体质量及性别的大鼠所获取的细胞形态及活性无差异。采用随机数字表法进行随机分组，分别设置对照组、TGF-β1组、Smad3 siRNA干预组、结缔组织生长因子（CTGF）siRNA干预组、二甲双胍干预组、Compound C干预组，每组3个样本。设置对照组：大鼠胆管成纤维细胞正常培养；TGF-β1组：10 ng/mL TGF-β1孵育细胞；Smad3 siRNA干预组：Smad3 siRNA转染24 h后＋10 ng/mL TGF-β1孵育细胞；CTGF siRNA干预组：CTGF siRNA 转染 24 h 后＋10 ng/mL TGF-β1 孵育细胞；二甲双胍干预组：10 mmol/L 二甲双胍＋10 ng/mL TGF-β1 孵育细胞；Compound C干预组：10 μmol/L Compound C＋10 mmol/L二甲双胍＋10 ng/mL TGF-β1孵育细胞。以TGF-β1刺激大鼠胆管成纤维细胞分泌胶原，以Smad3 siRNA或CTGF siRNA转染细胞以抑制Smad3或CTGF蛋白的表达，以二甲双胍孵育细胞以激活AMPK，以Compound C预处理细胞以抑制AMPK的活性。采用ELISA或Western-blot的方法检测CTGF、胶原（I Col I）蛋白水平；Western-blot的方法检测p-Smad3/t-Smad3、p-AMPK/t-AMPK、CTGF水平。结果 TGF-β1以时间和剂量依赖的方式刺激胆管成纤维细胞胶原I生成，二甲双胍激活的AMPK也剂量依赖性抑制TGF-β1诱导的胶原I生成；AMPK抑制剂Compound C预孵育细胞，可显著逆转二甲双胍激活的AMPK抑制胶原I生成的作用（P<0.01）。进一步发现，二甲双胍激活的AMPK对TGF-β1刺激的Smad3磷酸化无抑制作用，却可抑制其下游的CTGF蛋白表达（P<0.01）及胶原I生成（P<0.01），而AMPK抑制剂可逆转二甲双胍对CTGF及胶原I的上述作用。结论 二甲双胍通过激活AMPK抑制TGF-β1/Smad3信号通路诱导的CTGF蛋白表达，进而抑制胆管成纤维细胞胶原I生成。

Abstract: Objective To clarify the molecular signaling mechanism underlying the inhibitory effect of metformin on transforming growth factor-β1 (TGF-β1)-stimulated collagen I production in rat biliary fibroblasts. Methods Primary biliary fibroblasts were isolated under aseptic condition from 50 Sprague-Dawley rats (half male and half female), and microscopic observation identified no obvious difference in the morphology or viability of the cells from rats with different sexes or body weight. The cells were treated with TGF-β1 (10 ng/mL), Smad3 siRNA+TGF-β1, CTGF siRNA+TGF-β1, metformin (10 mmol/L)+ TGF-β1, or Compound C (10 μmol/L)+metformin+TGF-β1. The expressions of CTGF and collagen I in the treated cells were determined using ELISA kit or Western blotting; the phorsphorylated and total Smad3 and AMPK expressions were detected using immunoblotting. Results TGF-β1 time- and dose-dependently induced collagen I production in rat biliary fibroblasts. The activated AMPK by metformin dose-dependently inhibited TGF-β1-induced collagen I production. Pre-incubation of cells with the AMPK inhibitor Compound C restored the inhibitory effect of AMPK on TGF-β1-induced collagen I secretion (P< 0.01). Activation of AMPK by metformin significantly reduced TGF-β1-induced collagen I production by suppressing Smad3-driven CTGF expression (P<0.01), and the application of Compound C reversed such changes in the fibroblasts (P< 0.01). Conclusion Metformin inhibits TGF-β1-stimulated collagen I production by activating AMPK and inhibiting Smad3- driven CTGF expression in rat biliary fibroblasts.