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目的:探讨补肾益精法中药复方对系统性硬皮病血管内皮细胞内皮间质转化(endothelial to mesenchymal transition,End MT)的影响及机制。方法:采用博莱霉素建立硬皮病小鼠模型,造模同时补肾益精方软皮汤2号方(中药复方)进行干预,4周后HE染色观察小鼠皮肤组织病理、免疫组化法检测α-SMA表达水平;体外构建TGF-β1诱导的血管内皮细胞End MT模型,用补肾益精法含药血清处理48 h后,采用细胞免疫荧光方法检测血管内皮细胞α-SMA表达,Western Blot方法检测血管内皮细胞CD31、FSP1、Snail-1表达水平。结果:HE染色结果显示,模型组小鼠皮肤纤维化区域中,胶原纤维明显增多、胶原间隙变窄、毛细血管明显少于正常皮肤组织;与模型组比较,中药复方组小鼠皮肤胶原纤维较少、间隙较宽、血管数量较多;免疫组化染色检测结果显示,模型组小鼠皮肤组织中血管内皮细胞α-SMA表达高于正常对照组,中药复方组α-SMA阳性表达明显减少。免疫荧光检测结果显示,经TGF-β1诱导成功建立End MT模型,End MT模型组细胞α-SMA高表达,内皮细胞发生间质转化,经含药血清处理后,α-SMA表达明显减少。Western Blot检测结果显示,End MT模型组中Snail-1、FSP1均明显高于对照血清组,CD31明显低于对照血清组;含药血清处理组Snail-1、FSP1表达明显低于End MT模型组,CD31表达高于End MT模型组(P<0.05)。结论:补肾益精法对系统性硬皮病血管的保护作用,可能与通过某些途径拮抗Snail介导的End MT所参与的纤维增生性闭塞性血管损伤有关。
Objective: To investigate the effect and mechanism of Bushen Yijing Method on systemic endothelial cell endothelium (ECM) of systemic sclerosis. Methods: The scleroderma mouse model was established by bleomycin. At the same time, the model of Bushen Yijing Decoction Decoction No. 2 (TCM compound) was used for intervention. After 4 weeks, the pathological changes of the skin were observed by immunohistochemistry The expression of α-SMA in vascular endothelial cells was detected by ELISA. End MT model of endothelial cells induced by TGF-β1 was constructed in vitro. The expression of α-SMA in endothelial cells was detected by immunofluorescence with Bushenyijing method for 48 h. Blot method was used to detect the expression of CD31, FSP1 and Snail-1 in vascular endothelial cells. Results: The results of HE staining showed that the collagen fibers in the skin fibrosis area of the model group increased obviously, the collagen gap became narrower and the capillaries were obviously less than the normal skin tissue. Compared with the model group, The results of immunohistochemistry showed that the expression of α-SMA in vascular endothelial cells of model group was higher than that of normal control group, and the expression of α-SMA in compound Chinese herbs group was significantly decreased. The result of immunofluorescence showed that the End MT model was successfully established by TGF-β1 induction. The expression of α-SMA in End MT model group was highly expressed, and the endothelial cells were transformed in the endothelium. The results of Western Blot showed that the expression of Snail-1 and FSP1 in End MT model group was significantly higher than that in control serum group, and CD31 was significantly lower than that in control serum group. The expression of Snail-1 and FSP1 in serum-containing treatment group was significantly lower than that in End MT model group , CD31 expression was higher than End MT model group (P <0.05). Conclusion: Bushenyijing method can protect against systemic scleroderma vascular injury, which may be related to the inhibition of fibrinolytic occlusive vascular injury involved in Snail-mediated End MT through some pathways.