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目的:探讨下瘀血汤对四氯化碳(carbon tetrachloride,CCl4)诱导的大鼠肝硬化形成过程中肝脏血管新生的影响。方法:采用50%CCl4-橄榄油溶液1mL/kg剂量腹腔注射,每周2次,共9周,制备大鼠肝硬化模型。造模3周和6周后,随机抽取正常及模型大鼠各6只,处死作动态观察,其余模型大鼠随机分为模型对照组(15只)和下瘀血汤组(11只),同龄正常大鼠6只设为正常对照。自第7周开始灌胃给予下瘀血汤干预治疗3周,于第9周末取材,苏木精和伊红染色及天狼星红染色观察肝组织病理学变化,免疫荧光法和蛋白质印迹法检测肝组织中血小板内皮细胞黏附分子1(platelet endothelial cell adhesion molecule-1,CD31)、第Ⅷ因子相关抗原(von Willebrandfactor,v WF)的表达,蛋白质印迹法检测肝组织中血管内皮生长因子(vascular endothelialgrowth factor,VEGF)、血管内皮生长因子受体2(VEGF receptor-2,VEGFR2)、补体衰变加速因子(complement decay-accelerating factor,DAF)和α平滑肌肌动蛋白(α-smooth actin,α-SMA)的表达,明胶酶谱法检测肝组织中基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)和MMP-9的活性。结果:与正常组比较,模型大鼠肝组织受损,脂肪变性显著,胶原纤维沉积严重,随造模时间的推移,肝组织CD31、v WF、VEGF、VEGFR2、DAF和α-SMA蛋白表达逐渐升高,MMP-2、MMP-9活性显著增强(P<0.01)。与9周模型组比较,下瘀血汤显著改善肝组织损伤、脂肪变性及胶原纤维的沉积,降低肝组织CD31、v WF、VEGF、VEGFR2、DAF和α-SMA蛋白的表达及MMP-2、MMP-9的活性(P<0.01)。结论:下瘀血汤可有效地抑制CCl4诱导的大鼠肝硬化形成过程中的血管新生,其作用机制主要在于抑制肝星状细胞的活化,下调MMP-2、MMP-9的活性以及消解新生血管的完整性。
Objective: To investigate the effect of Xiayuxue decoction on hepatic angiogenesis induced by carbon tetrachloride (CCl4) in rat cirrhosis. Methods: The rat model of liver cirrhosis was established by intraperitoneal injection of 1% (1 mL / kg) 50% CCl4-olive oil solution twice a week for 9 weeks. After 3 weeks and 6 weeks of modeling, 6 normal rats and 6 rats were randomly selected and sacrificed for dynamic observation. The rest rats were randomly divided into model control group (n = 15) and Xiaoyu Xue Tang Decoction group (n = 11) 6 normal rats of the same age were set as normal control. From the 7th week, the patients were treated with Xiayuxue Decoction by intragastric administration for 3 weeks. At the end of the 9th week, hematoxylin and eosin staining and Sirius red staining were used to observe the pathological changes of the liver. Immunofluorescence and Western blotting were used to detect the liver The expressions of platelet endothelial cell adhesion molecule-1 (CD31) and von Willebrand factor (v WF) in the liver tissues were detected by Western blotting. The expressions of vascular endothelial growth factor , VEGF, VEGF receptor-2 (VEGFR2), complement decay-accelerating factor (DAF) and α-smooth actin (α-SMA) Expression and gelatin zymography were used to detect the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 in liver tissue. Results: Compared with the normal group, the model rats had impaired hepatic tissue, significant fatty degeneration and severe collagen deposition. The expression of CD31, vWF, VEGF, VEGFR2, DAF and α-SMA gradually increased with the time of modeling The activity of MMP-2 and MMP-9 were significantly increased (P <0.01). Compared with the 9-week model group, Xiayu Decoction significantly improved the hepatic tissue injury, steatosis and deposition of collagen fibers, decreased the expression of CD31, vWF, VEGF, VEGFR2, DAF and α-SMA and the expression of MMP- MMP-9 activity (P <0.01). Conclusion: Xiayuxue Decoction can effectively inhibit angiogenesis during CCl4-induced cirrhosis in rats, and its mechanism of action is mainly to inhibit the activation of hepatic stellate cells, down-regulate the activity of MMP-2, MMP-9 and digestion of newborn Blood vessel integrity.