基于Fli1调节的补肾益精法对硬皮病小鼠EndMT的影响

Effect of tonifying kidney and strengthening essence methods on endothlial-mesenchymal transition scleroderma mice based on regulation of Fli1

目的研究补肾益精法中药复方通过干预Fli1表达调控TGF-β信号,抑制系统性硬皮病血管内皮细胞内皮间质转化(endothelial to mesenchymal transition,End MT)的机制。方法采用博来霉素构建硬皮病小鼠模型,分为正常组、模型组、黄芪甲苷组、中药复方组、Fli1 siRNA+中药复方组,其中正常组不予造模。分别采用HE、Masson染色检测小鼠皮肤血管与胶原,采用实时荧光定量聚合酶链式反应法检测各组小鼠皮肤组织Fli1、VE-cadherin和FSP-1 mRNA表达水平;采用蛋白印迹法(Western Blot)检测各组小鼠皮肤组织Fli1、VE-cadherin、FSP-1蛋白表达水平。结果成功建立硬皮病小鼠模型,HE染色与Masson染色结果显示:中药复方组显著降低小鼠皮肤厚度(P<0.01),胶原纤维减少,血管增多,提示中药复方能改善小鼠皮肤纤维化程度及保护血管; QPCR结果显示:补肾益精中药复方能提高Fli1 mRNA、VE-cadherin mRNA表达,降低FSP-1 mRNA表达。Western Blot结果显示:补肾益精中药复方能提高Fli1、VE-cadherin蛋白表达,降低FSP-1蛋白表达。结论补肾益精法中药复方通过上调Fli1表达,抑制血管内皮间质转化。

Objective To study the mechanism of tonifying kidney and strengthening essence methods interfering the Fli1’s expression to regulate TGF-β signal, and to inhibit systemic scleroderma endothelial to mesenchymal transition （EndMT）. Methods Bleomycin was used to construct mice model, and they were randomly divided them into 5 groups, normal group, model group, astragaloside IV group, Traditional Chinese Medicine （TCM） formula group and FLi1 siRNA ＋ TCM formula group. And the mice in normal group was not given modeling. Mice’s skin blood vessels were detected with HE staining method, and collagen in mice were detected with Masson staining method. Gene expression of Fli1, VE-cadherin and FSP-1 mRNA in skin tissue of each group were detected by quantitative polymerase chain reaction （QPCR） method. Protein expression of Fli1, VE-cadherin and FSP-1 were detected by western blotting （WB）. Results Mice model of scleroderma were successfully established. In TCM formula group, the HE and Masson staining results showed that the thickness of skin was reduced （ P 〈0.01）, and the number of collagenous fibers reduced and the number of vessel increased. These results suggest that TCM formula can reduce the degree dermal fibrosis and protect vascular in model mice. And the QPCR results showed that TCM formula of tonifying kidney and strengthening essence could increase the level of mRNA expression of FLi1 and VE-cadherin and decrease the level of mRNA expression of FSP-1. Western Bolt results showed that TCM formula of tonifying kidney and strengthening essence could increase the level FLi1 and VE-cadherin protein expression and decrease the level of protein expression of FSP-1. Conclusion TCM formula of tonifying kidney and strengthening essence methods can interact the Fli1’s expression to inhibit EndMT.