二甲双胍对增生性瘢痕成纤维细胞增殖及胶原合成的影响及机制

Effects and Mechanism of Metformin on Proliferation and Collagen Synthesis of Hypertrophic Scar Fibroblasts

目的探讨二甲双胍对增生性瘢痕成纤维细胞增殖、胶原合成及TGFβ1/Smad信号通路磷酸化水平的影响。方法取增生性瘢痕组织,收集第3~6代成纤维细胞(Fb),分为空白对照组(PBS处理)和5、10、20、30 mmol·L^(-1)的二甲双胍处理的实验组,每组3孔,观察各组给药刺激24 h后细胞形态变化,给药刺激24 h后提取各组细胞总RNA,检测各组增生性瘢痕成纤维细胞中CollagenⅠ、CollagenⅢ和α-平滑肌肌动蛋白(α-SMA)的mRNA相对表达水平。给药刺激48 h后提取细胞总蛋白,Western blot技术检测TGFβ1、Smad3磷酸化水平、CollagenⅠ、CollagenⅢ和α-SMA的蛋白相对表达水平,采用CCK-8法检测各组细胞增殖活力。结果5、10、20、30 mmol·L^(-1)的二甲双胍处理成纤维细胞后,与空白对照组相比,实验组细胞数目较少,排列较稀疏,且呈剂量依赖性,细胞体积较小,呈长梭形,核小,轮廓较清楚。成纤维细胞的抑制率随二甲双胍剂量的增大而增加,呈剂量依赖性,均高于空白对照组(P均<0.05);经不同浓度二甲双胍处理的成纤维细胞中CollagenⅠ、CollagenⅢ、α-SMA mRNA和蛋白相对表达水平及TGFβ1、Smad3磷酸化水平随二甲双胍剂量的增加而降低,且呈剂量依赖性,均低于空白对照组(P均<0.05)。结论二甲双胍可抑制增生性瘢痕成纤维细胞CollagenⅠ、CollagenⅢ、α-SMA的表达,可能与TGFβ1/Smad信号通路调控有关。

Objective To investigate the effect of metformin on the proliferation and collagen synthesis as well as TGFβ1/Smad signaling pathway phosphorylation in hypertrophic scar fibroblasts.Methods Hypertrophic scar tissue was collected,fibroblasts(Fb)of passages 3 to 6 were collected.The cells were divided into the control group(PBS group)and the experimental group treated with different concentrations of 5,10,20 and 30 mmol·L^(-1) metformin,with 3 wells in each group.The morphological changes of cells in each group were observed 24 hours after drug administration.After 24 hours of drug stimulation,the total RNA of cells in each group was extracted,the relative expression levels of CollagenⅠ,CollagenⅢ,andα-SMA(α-smooth muscle actin)mRNA in hypertrophic scar fibroblasts in each group were detected,and the total cell protein was extracted after 48 hours of drug stimulation.The Western blot technique was used to detect TGFβ1 and the phosphorylation level of Smad 3,and the relative expression levels of CollagenⅠ,CollagenⅢ,andα-SMA.Results After fibroblasts were treated with different concentrations of 5,10,20,and 30 mmol·L^(-1) metformin,compared with the control group,the number of cells in the experimental group decreased,and the arrangement was sparse,and in a dose-dependent manner.The inhibition rate of fibroblasts increased with the dose of metformin in a dose-dependent manner,which was higher than that of the blank control group(P all<0.05).The relative expression levels of CollagenⅠ,CollagenⅢ,α-SMA mRNA and TGFβ1 and the phosphorylation level of Smad 3 proteins in fibroblasts treated with different concentrations of metformin decreased with the increase of metformin in a dose-dependent manner,which were lower than those in the control group(P all<0.05).Conclusion Metformin can inhibit the expression of CollagenⅠ,CollagenⅢandα-SMA in hypertrophic scar fibroblasts,which may be related to the regulation of TGFβ1/Smad signaling pathway.