生肌玉红膏抑制自噬对血栓闭塞性脉管炎大鼠凝血系统、EPCs分化及Rho激酶活性的影响

Effect of Shengjiyuhong ointment on coagulation system,EPCs differentiation and Rho kinase activity in rats with thromboangiitis obliterans by inhibiting autophagy

目的探究生肌玉红膏抑制自噬对血栓闭塞性脉管炎大鼠凝血系统、内皮祖细胞(EPCs)分化及Rho激酶活性的影响。方法采用左下肢动脉注射月桂酸钠溶液建立血栓闭塞性脉管炎大鼠模型。将40只SD大鼠随机分为假手术组、模型组、七叶皂苷钠凝胶组、生肌玉红膏组,每组10只。采用HE染色检测血管组织病理形态,免疫印迹法检测自噬相关蛋白表达,凝血检测仪检测凝血系统指标,流式细胞仪检测EPCs分化,免疫组化法检测Rho激酶活性。结果生肌玉红膏组可使血管组织中自噬标志蛋白微管相关蛋白轻链3Ⅱ(LC3Ⅱ)、苄氯素1(Beclin1)、Rho激酶蛋白表达、血浆纤维蛋白原、EPCs中CD34阳性表达率降低,血清中凝血酶原时间、国际标准比率、EPCs中血管性血友病因子阳性表达率、血管组织中P62表达升高(P<0.05);体外实验表明,生肌玉红膏组可降低损伤的内皮细胞中LC3Ⅱ/Ⅰ、Beclin1表达,升高P62表达(P<0.05)。结论生肌玉红膏可有效改善血栓闭塞性脉管炎大鼠凝血功能,促进EPCs分化,降低Rho激酶活性,其机制可能与抑制机体自噬有关。

Objective To investigate the effects of Shengjiyuhong ointment on coagulation system,endothelial progenitor cells(EPCs)differentiation and Rho kinase activity in rats with thromboangiitis obliterans by inhibiting autophagy.Methods The rat model of thromboangiitis obliterans was established by injecting sodium laurate solution into the left lower limb artery.40 SD rats were randomly divided into sham operation group,model group,compound sodium aescinate gel group and Shengjiyuhong ointment group,10 per group.The pathological morphology of vascular tissue was detected by HE staining.The expression of autophagy related proteins was detected by Western blot,coagulation system parameters were detected by coagulation detector,EPCs differentiation was detected by flow cytometry,and Rho kinase activity was detected by immunohistochemistry.Results The expression of autophagy marker protein LC3Ⅱ/Ⅰ,Beclin1,Rho kinase protein,the positive expression rate of CD34 in fibrinogen and EPCs of Shengjiyuhong ointment group were decreased,and the positive expression rate of serum prothrombin time,international normalized ratio,von Willebrand factor in EPCs and the expression of P62 in vascular tissue were increased(P<0.05).The in vitro experiment showed that Shengjiyuhong ointment could reduce the expression of LC3Ⅱ/Ⅰand Beclin1,and increase the expression of P62 protein in injured endothelial cells(P<0.05).Conclusion Shengjiyuhong ointment can effectively improve the coagulation function,promote the differentiation of EPCs and reduce the activity of Rho kinase in rats with thromboangⅡtis obliterans.The mechanism may be related to inhibition of autophagy.