姜黄素对环孢素A诱导激活的大鼠牙龈成纤维细胞TGF-β1/Smad3通路的抑制作用

Effect of curcumin on TGF-β1/Smad3 pathway in rat gingival fibroblast treated with cyclosporine A

目的:体外实验研究姜黄素(curcumin,Cur)对环孢素A(cyclosporine A,CsA)作用大鼠牙龈成纤维细胞TGF-β1/Smad3通路的影响,为进一步探讨Cur抑制CsA所致药物性牙龈增生的发生机制提供理论依据。方法:使用CCK-8法观察0、5、10、20、30μmol/L Cur对大鼠牙龈成纤维细胞增殖的影响,20μmol/L Cur+200 ng/mL CsA共同作用对大鼠牙龈成纤维细胞增殖的影响。实时荧光定量PCR检测20μmol/L Cur+200 ng/mL CsA共同作用下,牙龈成纤维细胞中转化生长因子TGF-β1、Smad3、平滑肌肌动蛋白α-SMA和I型胶原蛋白COL-I的mRNA表达变化;蛋白免疫印迹实验检测TGF-β1、Smad3、p-Smad3、α-SMA和COL-I的蛋白表达变化。细胞划痕实验观察20μmol/L Cur+200 ng/mL CsA对牙龈成纤维细胞迁移能力的影响。采用SPSS 23.0软件包对数据进行统计学分析。结果:大鼠牙龈成纤维细胞在20μmol/L Cur+200 ng/mL CsA共同作用下,细胞增殖和迁移能力明显降低;20μmol/L Cur显著下调了牙龈成纤维细胞中TGF-β1、α-SMA和COL-I的m RNA表达,蛋白免疫印迹实验提示,TGF-β1、p-Smad3、α-SMA和COL-I的表达同样显著下调。结论:Cur可能通过抑制CsA激活的牙龈成纤维细胞TGF-β1/Smad3信号通路,从而降低牙龈成纤维细胞增殖、迁移、平滑肌肌动蛋白和胶原分泌,改善牙龈增生。

PURPOSE:The aim of the present study was to investigate the effect of curcumin(Cur)on TGF-β1/Smad3 pathway of rat gingival fibroblast treated with cyclosporine A(CsA)in vitro,and to provide theoretical basis for the mechanism of curcumin inhibiting drug-induced gingival hyperplasia induced by CsA.METHODS:Healthy Sprague-Dawley rat gingival fibroblasts were cultured with different concentrations of Cur(0,5,10,20,30μmol/L)and Cur(20μmol/L)+CsA(200 ng/mL),cell proliferation was assessed with CCK-8 assay.The mRNA levels of TGF-β1,Smad3,α-SMA and collagen typeⅠin gingival fibroblasts were detected by real-time PCR under Cur(20μmol/L)+CsA(200 ng/mL);the protein level of TGF-β1,Smad3,p-Smad3,α-SMA and collagen typeⅠwere determined through Western blot.The effect of Cur(20μmol/L)+CsA(200 ng/mL)on migration ability of gingival fibroblasts was observed through Scratch woundhealing assay.The data were analyzed with SPSS 23.0 software package.RESULTS:Cell proliferation and migration ability of rats gingival fibroblasts were significantly reduced under Cur(20μmol/L)+CsA(200 ng/mL).20μmol/L Cur significantly decreased mRNA expression of TGF-β1,α-SMA and collagen typeⅠin gingival fibroblasts,and Western blot suggested significantly down-regulated expression of TGF-β1,p-Smad3,α-SMA,and collagen typeⅠ.CONCLUSIONS:Cur may inhibit TGF-β1/Smad3 signaling pathway of gingival fibroblasts activated by CsA,thereby weakening proliferation and migration,reducing secretion of smooth muscle actin and collagen of gingival fibroblasts,and ameliorating gingival hyperplasia.