丹酚酸B调节RhoA/ROCK1信号通路抑制糖尿病大鼠心肌纤维化的作用研究

The study of salvianolic acid B on the improvement of myocardial fibrosisin diabetic rats by RhoA/ROCK1 pathway

目的基于RhoA/ROCK1信号通路,研究丹酚酸B(salvianolic acid B,SalB)对糖尿病大鼠心肌纤维化的作用及其作用机制。方法健康♂SD大鼠尾静脉一次性注射链脲佐菌素制备糖尿病大鼠模型;12周末,天狼星红试剂染色检测大鼠左室心肌胶原表达情况;Western blot检测大鼠左室心肌中α-SMA、CollagenⅠ、CollagenⅢ蛋白表达。SD大鼠乳鼠差速贴壁培养心肌成纤维细胞(cardiac fibroblast,CFs),不同浓度的SalB(12.5,25,50μmol·L^(-1))预处理CFs 1 h,高糖诱导确定最佳给药剂量;免疫荧光检测CFs内RhoA蛋白表达;Western blot检测CFs内α-SMA、CollagenⅠ、CollagenⅢ、RhoA、ROCK1蛋白表达。结果与正常对照组相比,糖尿病组大鼠左室心肌组织胶原表达明显增多,α-SMA、CollagenⅠ、CollagenⅢ表达明显升高,经SalB干预后,胶原表达明显减少,上述各蛋白表达明显降低(P<0.01)。25 mmol·L^(-1)高糖刺激CFs 24 h,α-SMA、CollagenⅠ、CollagenⅢ、RhoA、ROCK1表达明显升高,经SalB(25μmol·L^(-1))及抑制剂Y-27632(10μmol·L^(-1))预处理后,CFs活力明显受到抑制,上述各蛋白表达明显降低(P<0.01)。结论SalB可改善糖尿病大鼠心肌纤维化,对保护糖尿病大鼠心肌具有一定的作用,其机制可能是与抑制RhoA/ROCK1信号通路有关。

Objective To investigate the effect and mechanism of salvianolic acid B(SalB)on myocardial fibrosis in diabetic rats based on RhoA/ROCK1 signaling pathway.Methods Diabetic rat model was established by injecting streptozotocin into tail vein of healthy male SD rats.At the end of the 12th week,the collagen fiber expression in the left ventricular myocardium of rats was detected by Sirius Red reagent staining.The protein expressions ofα-SMA,CollagenⅠand CollagenⅢin left ventricular myocardium were detected by Western blot.Cardiac fibroblasts(CFs)were cultured in SD rats by differential adhesion,and CFs were pretreated with different concentrations of SalB(12.5,25,50μmol·L^(-1))for 1 h,and the optimal dose was determined by high glucose induction.RhoA protein expression in CFs was detected by immunofluorescence;the protein expressions ofα-SMA,CollagenⅠ,CollagenⅢ,RhoA and ROCK1 in CFs were detected by Western blot.Results Compared with the normal control group,the expression of collagen in left ventricular myocardium of diabetic rats increased significantly,and the expression ofα-SMA,CollagenⅠand CollagenⅢincreased significantly.After SalB intervention,the expression of collagen decreased significantly,and the expression of the above proteins decreased significantly(P<0.01).The expression ofα-SMA,CollagenⅠ,CollagenⅢ,RhoA and ROCK1 in CFs stimulated with 25 mmol·L^(-1)high glucose for 24 h was significantly increased.After pretreatment with SalB(25μmol·L^(-1))and inhibitor Y-27632(10μmol·L^(-1)),the activity of CFs was significantly inhibited,and the expression of these proteins was significantly decreased(P<0.01).Conclusion SalB can improve myocardial fibrosis in diabetic rats,and has a certain role in protecting the myocardium of diabetic rats.The mechanism may be related to the inhibition of RhoA/ROCK1 signaling pathway.