目的 通过研究肝纤维化大鼠缺氧诱导因子-1α（HIF-1α）介导的血管内皮细胞生长因子（VEGF）mRNA表达及膈下逐瘀汤对其的影响，探讨膈下逐瘀汤改善肝纤维化血管新生的机制。方法 108只Wistar大鼠随机分为对照组、模型组、N-乙酰半胱氨酸（NAC）组及膈下逐瘀汤高（GD）、中（GZ）、低（GX）剂量组，每组18只。大鼠ip 50%四氯化碳-橄榄油溶液1 mL/kg制备肝纤维化模型，每周2次，共9周。各组于造模同时给药。对照组及模型组ig无菌水10 mL/kg，NAC组ig给予NAC 0.1 g/kg；GD、GZ、GX组分别ig给予膈下逐瘀汤生药饮片浓煎液26.0、7.8、3.9 g/kg，每日给药1次。在第3、6、9周时间点，各组分别随机选取大鼠处死，Masson染色制作病理标本；免疫组织化学染色半定量分析IV型胶原（collage type IV，Col-IV）及层黏连蛋白（laminin，LN）表达水平；Real-time PCR检测HIF-1a、VEGF mRNA的相对表达量；蛋白免疫印迹法（Western blotting）检测VEGF、VEGFR2蛋白表达水平。结果 与模型组比较，NAC、GD在给药第9周时均能有效抑制大鼠肝细胞外基质LN表达（P<0.05）；在给药第6、9周，NAC、GD均能有效抑制大鼠肝细胞外基质Col-IV表达（P<0.05），NAC、GD及GZ均能有效抑制大鼠肝组织HIF-1α表达（P<0.05）；在给药第6、9周，NAC和GD能有效抑制大鼠肝组织VEGF mRNA表达（P<0.05）；膈下逐瘀汤各剂量和NAC均能抑制大鼠肝组织VEGF、VEGFR2的蛋白表达（P<0.05）。结论 膈下逐瘀汤对HIF-1α介导的VEGF mRNA表达有调控作用，可能是其抗肝纤维化血管新生的作用机制之一。

Objective To explore the mechanism of improving angiogenesis of hepatic fibrosis by Gexia Zhuyu Decoction (GZD) through the regulation of the mRNA expression of VEGF mediated by HIF-1α. Methods A total of 108 Wistar rats were randomly divided into normal group (n=18), model group (n=18), N-acetylcysteine (NAC) group (n=18), high-dose GZD group (GD, n=18), middle-dose GZD group (GZ, n=18), and low-dose GZD group (GX, n=18). Hepatic fibrosis model was established by intraperitoneal injection of 50% CCl₄-olive oil solution (1 mL/kg) twice a week for nine weeks. Each group was administered while model established, until the rats were sacrificed. Normal group and model group were ig given sterile water 10 mL/(kg·d), NAC group was ig given NAC 0.1 g/(kg·d), GD, GZ, GX groups were given 26, 7.8, and 3.9 g/(kg·d) GZD by oral gavage. At 3, 6, and 9 weeks, rats in the corresponding groups were randomly sacrificed. Masson staining was used to make pathological specimens, immunohistochemical analysis of Col-IV and laminin was also performed, and real-time PCR was used to detect the mRNA expression of HIF-1α and VEGF. Western blotting was used to detect the protein expression levels of VEGF and VEGFR2. Results Compared with model group, NAC group and GD group significantly inhibited the expression of LN in the extracellular matrix at 9 weeks (P<0.05). Both NAC and GD groups significantly inhibited the expression of extracellular matrix Col-IV, especially at 6 weeks and 9 weeks. NAC group, GD group, and GZ group can significantly inhibit the high expression of HIF-1α in liver tissue of rats with liver fibrosis (P<0.05). At 6 weeks and 9 weeks of administration, NAC and GD groups significantly inhibited the high expression of VEGF mRNA in liver tissue (P<0.05). Both GZD and NAC could inhibit the protein expression of VEGF and VEGFR2 in liver tissue. Conclusion GZD can regulate the expression of VEGF mRNA mediated by HIF-1α, which may be one of the key mechanisms of its anti-angiogenesis for hepatic fibrosis.

国家自然科学基金青年基金项目（81202633）