补肾益精法对系统性硬皮病血管损伤的保护机制：拮抗Snail介导的EndMT所致血管损伤?

Tonifying kidney and Boosting essence treatment is the therapy for systemic sclerosis from Prof. Deng, who recommended treating this disease on the theory of “Lung-Spleen-Kidney Correlation”, and “tonifying kidney and boosting essence at the first place, strengthening spleen and nourishing lung at the supplement place”. In the previous projects on this therapy, we found the anti-fibrotic mechanism and its main gradient; meanwhile, we also discovered its protective role in proliferative vasculopathy of SSc: this therapy increased the number of capillaries in the fibrotic legion, and decreased the amount of α-SMA positive endothelial cells; in TGF-β induced endothelial to mesenchymal transition(EndMT) cell models, this treatment inhibited the transition from endothelial to mesenchymal cells, correlated with declined snail levels in the nuclei. Thus, having referred to the classical theory on the relation between kidney and vessel, we proposed the hypothesis: one of therapeutic mechanisms of Tonifying kidney and Boosting essence treatment on proliferative vasculopathy in systemic sclerosis could be that the Chinese medicine therapy might antagonize vascular injuries caused by the snail mediated EndMT? This project would be carried out in vivo and in vitro, by utilizing techniques such as immunofluorescence, PCR, Western Blot, gene over-expression or interference by RNA interference, cell lineage tracing, etc., to comprehensively study the mechanisms of the Tonifying kidney and Boosting essence treatment on vascular protection in systemic sclerosis, so as to provide substantial evidence for this classical Chinese therapy, to possibly suppose an EndMT-targeted strategy in the treatment of systemic sclerosis, and to strengthen the basis of Chinese medicine theory.

补肾益精法是邓铁涛教授辨治硬皮病的经验治法；主张从“肺脾肾相关理论” 辨治硬皮病、“补肾益精为主，健脾养肺为辅”。前期研究确定了抗纤维化的主要机制与有效成分；同时，也发现其对硬皮病血管的保护作用：补肾益精法可增加皮损区毛细血管数量，内皮细胞表达α-SMA减少；在体外可抑制血管内皮细胞-间质转化（EndMT）过程，且伴随核内Snail水平回落。然而，具体机制不明确。因此，结合传统中医肾与血脉相关理论，提出研究假说：补肾益精法对硬皮病血管损伤的保护作用，可能通过拮抗Snail介导的EndMT过程、抑制纤维增生性闭塞性血管损伤而产生？本项目拟在体外与体内两个层面，采用免疫荧光、PCR、Western Blot、质粒过表达、RNA干扰、细胞谱系示踪等技术，阐明补肾益精法对硬皮病血管保护作用的相关机制，为中医治疗疑难免疫性疾病提供科学依据，为硬皮病治疗提供新的干预策略，为传统中医理论夯实科学基础。

硬皮病是高致残率的自身免疫性疾病，补肾益精法是其重要的经验治法；本项目研究其血管保护作用与机制；内皮细胞-间质转化（EndMT）是硬皮病血管损伤的重要过程之一，通过TGF-β、博来霉素建立了血管内皮细胞EndMT模型和小鼠的硬皮病模型,用于补肾益精法血管保护作用相关研究：① 定位补肾益精法中药复方中血管保护作用的有效成分熊果酸，及有Snail共同作用通路的栀子苷分子；在体外血管内皮细胞EndMT模型中，发现了补肾益精法中药复方及有效成分通过Snail分子影响血管内皮细胞EndMT过程的机制，其主要作用与AKT-mTOR-Snail通路相关。② 在博来霉素诱导的硬皮病小鼠模型中，补肾益精法中药复方增加皮肤毛细血管数量，抑制血管内皮细胞EndMT过程，机制与Snail因子相关；Tie2-LacZ双转基因硬皮病小鼠模型研究中，内皮细胞示踪显示，向成纤维细胞分化的内皮细胞仍停留在血管及其周边；造模后皮肤有明显纤维化，肺组织呈炎症期伴早期纤维化，心与肾的损伤不明显。③ 在干预实验中，通过调控Snail的表达及入核，可影响血管内皮细胞EndMT过程，降低血管内皮细胞损伤；补肾益精法及熊果酸能通过一系列分子机制降低Snail表达，从而抑制EndMT，减轻血管损伤。本项目定位了补肾益精法对血管有保护作用的山萸肉单体--熊果酸；与熊果酸有相似作用通路的栀子苷；Snail因子是硬皮病EndMT介导血管损伤及纤维化的关键分子之一，调控Snail分子可成为减轻硬皮病血管EndMT损伤的潜在靶点；补肾益精法及单体熊果酸能减少Snail表达，抑制EndMT进程，从而产生血管保护作用；本项目阐明了补肾益精法的血管保护作用的机制，提供了硬皮病血管损伤治疗的新靶点，夯实了补肾益精法治疗硬皮病的科学基础。