黄芪汤通过VEGF/PI3K/Akt/eNOS通路对肝窦血管生成和重构的调节作用：门脉高压调控的新途径

Based on previous study results that Huangqi Decoction can effectively improve liver fibrosis stage in cirrhotic patients (S4) and the degree of gastro esophageal varices after 48 weeks treatment and these effects are associated with reduction of VEGF levels. Combined with the understanding that VEGF/PI3K/Akt/eNOS signaling pathway play essential roles in the regulation of hepatic sinusoidal capillarization and targeting this pathway is important in prevention and treatment of liver fibrosis, and decreasing portal vein pressure, we therefore proposed that Huangqi Decoction can alleviate liver cirrhosis, improve liver sinus vascular generation and reconstruction and hepatic sinusoidal capillarization, reduce light portal vein hypertension, and decrease the degree of esophageal varices through VEGF/PI3K/Akt/eNOS signaling pathway. In this study, the bile duct ligation cirrhosis model in rats was used to study the role of the decoction in hepatic sinusoidal capillarization, angiogenesis and remodeling, portal vein pressure. The liver sinusoidal endothelial cells (LSEC) is isolated and cultured to study the effect of Huangqi Decoction on structure and function of phenotype LSEC. By multiple experimental techniques, the effect of the decoction on regulatory effects VEGF/PI3K/Akt/eNOS pathway in LSEC was further investigated. Collectively, this study provides the foundation for developing the drugs with clear mechanism in prevention and treatment of liver cirrhosis with portal hypertension.

基于前期发现黄芪汤治疗48周能有效改善肝硬化患者(S4期)肝纤维化分期，改善食管胃底静脉曲张程度，该作用与降低VEGF水平相关，结合VEGF/PI3K/Akt/eNOS信号通路在调节肝窦毛细血管化机制中发挥重要作用，调控该通路是防治肝纤维化、降低肝硬化门脉压力的重要途径的认识，课题组提出假说：黄芪汤通过VEGF/PI3K/Akt/eNOS通路，影响肝硬化肝窦血管生成和重构，改善肝窦毛细血管化、减轻门静脉高压、改善食管静脉曲张的程度。本研究通过复制胆管结扎大鼠肝硬化门静脉高压模型，在整体动物水平研究该方对肝窦毛细血管化、血管生成和重构、门静脉压力的作用；体外分离培养肝窦内皮细胞(LSEC)，在细胞水平研究该方对LSEC表型形态功能的作用；采用分子生物学技术手段，阐明该方对LSEC VEGF/PI3K/Akt/eNOS通路的调控作用。通过本研究为开发机制明确的防治肝硬化门脉高压中药打下基础。

背景和目的：在前期临床研究发现黄芪汤治疗48周能有效改善肝硬化患者（S4期）肝纤维化分期、减轻食管胃底静脉曲张程度的基础上，进一步研究黄芪汤调控VEGF/PI3K/Akt/eNOS信号通路，对肝硬化门静脉高压、肝窦血管生成和重构影响及作用机制的整体动物模型和体外细胞研究。方法：复制胆管结扎大鼠肝硬化门静脉高压症模型，给予不同剂量黄芪汤药物处理4周。检测大鼠门静脉压力，血清生化和VEGF水平，肝组织行HE染色和天狼星红染色观察肝硬化程度，免疫组化染色检测I型胶原、vWF和CD31表达，观察肝窦毛细血管化的改变。体外分离培养大鼠肝窦内皮细胞（LSEC），给予黄芪汤药物血清等进行处理。采用MTT法、Transwell法和血管形成实验等检测LSEC的增殖、迁移和血管生成。定量PCR检测大鼠肝组织和体外分离LSEC的基因mRNA表达，Western blot检测VEGF、p-PI3K、PI3K、p-AKT、AKT、eNOS、CD31、LC3-II、LC3-I、Beclin-1和mTOR等蛋白表达。结果：黄芪汤显著降低大鼠肝脏门静脉压力，改善肝功能，减轻纤维化，减少肝组织胶原沉积，减轻肝窦内皮细胞毛细血管化的程度。降低外周血VEGF水平，抑制肝组织VEGF、PI3K和AKT蛋白磷酸化及eNOS蛋白的表达。黄芪汤药物血清抑制大鼠LSEC增殖、迁移和血管生成；减少CD31、eNOS和ET-1的表达；抑制AKT和mTOR蛋白的磷酸化以及LC3-II/LC3-I和Beclin-1蛋白的表达。结论：黄芪汤通过调控VEGF/PI3K/Akt/eNOS信号通路，显著减轻肝纤维化和肝窦毛细血管化，改善肝窦血管生成和重构，降低门静脉高压。