TGF-β/Smads信号通路在姜黄素改善糖尿病大鼠心肌纤维化中的作用

Curcumin ameliorates myocardial fibrosis via TGF-β/Smads signaling pathway in diabetic rats

目的探讨TGF-β/Smads信号通路在姜黄素改善糖尿病心肌纤维化中的作用。方法高糖高脂饮食联合腹腔注射小剂量链脲佐菌素(STZ)35 mg·kg^(-1)建立2型糖尿病大鼠模型,自由饮用300 mg·kg·d^(-1)姜黄素进行干预;天狼猩红染色检测各组大鼠心肌内胶原表达情况;免疫荧光检测各组大鼠心肌内CollagenⅠ和CollagenⅢ的表达情况;葡萄糖(5.5、20、25、30、35、50 mmol·L^(-1))刺激大鼠乳鼠心肌成纤维细胞(CFs)24 h确定最佳高糖造模浓度;30 mmol·L^(-1)高糖加姜黄素(10、25、50、100、200μmol·L^(-1))刺激CFs 24h确定最佳姜黄素给药浓度;Western blot法检测细胞内CollagenⅠ、CollagenⅢ、TGF-β1、p-Smad2、Smad2、p-Smad3、Smad3、TβR-Ⅲ的蛋白表达水平。结果相比于正常组,糖尿病组大鼠心肌组织胶原出现明显沉积,CollagenⅠ和CollagenⅢ表达明显增多,给予姜黄素治疗后,糖尿病大鼠心肌组织胶原沉积明显减少,CollagenⅠ和CollagenⅢ表达明显减少;30 mmol·L^(-1)高糖刺激CFs 24 h可以明显促进细胞增殖(P<0.05);而10μmol·L^(-1)姜黄素可以明显抑制高糖诱导的CFs增殖(P<0.05);30 mmol·L^(-1)高糖诱导CFs24 h以后,CollagenⅠ、CollagenⅢ、TGF-β1、p-Smad2、pSmad3、TβR-Ⅲ的蛋白表达明显增多(P<0.05),给予25μmol·L^(-1)姜黄素以后,上述各蛋白表达水平明显下降(P<0.05)。结论姜黄素可通过TGF-β/Smads信号通路改善糖尿病大鼠心肌纤维化,对糖尿病大鼠心肌具有保护作用。

Aim To investigate the role of TGF-β/Smads signaling pathway in the improvement of myocardial fibrosis in diabetes mellitus by curcumin.Methods A model of type 2 diabetes mellitus was induced by intraperitoneal injection of small dose of streptozotocin(STZ)35 mg·kg-1 with a high glucose and high fat diet,and then intervened by drinking of 300 mg·kg·d-1 curcumin.The expression of myocardial collagen in rats was detected by Sirius red staining.The expressions of Collange I and Collagen III in myocardium of rats were detected by immunofluorescence.Cardiac fibroblasts(CFs)in neonatal rats were stimulated by different concentrations of glucose(5.5,20,25,30,35,50 mmol·L-1)for 24 h to determine the optimum concentration of high glucose model,and rat CFs were stimulated for 24 h by 30 mmol·L-1 high glucose plus different concentrations of curcumin(10,25,50,100,200μmol·L-1)to determine the optimal concentration of curcumin.The expressions of typeⅠandⅢcollagen,TGF-β1,p-Smad2,Smad2,p-Smad3,Smad3 and TβR-Ⅲin CFs were detected by Western blot.Results Compared with the control rats,the collagen deposition in the myocardium of the diabetic rats was more obvious and the expression of CollagenⅠand CollagenⅢsignificantly increased.After treatment of curcumin,the collagen deposition in the myocardium and the expression of Collagen I and CollagenⅢof diabetic rats remarkably decreased.The CFs under the condition of 30 mmol·L-1 high glucose and 24 h had the highest survival rate(P<0.05);10μmol·L-1 curcumin could obviously inhibit the proliferation of myocardial fibroblasts induced by high glucose(P<0.05).After induced by 30 mmol·L-1 high glucose for 24 h,the expression of CollagenⅠand CollagenⅢ,TGF-β1,p-Smad2,Smad2,p-Smad3,Smad3 and TβR-Ⅲproteins in CFs markedly increased(P<0.05),and the expression levels of these proteins were obviously reduced when treated with 25μmol·L-1 curcumin.Conclusion Curcumin could ameliorate myocardial fibrosis in diabetic rats through TGF-β/Smads signaling pathway,exerting the protective effect on myocardium in diabetic rats.