基于肝窦毛细血管化探讨抗纤软肝方抗肝纤维化的作用机制

The anti-liver fibrosis effect of Kangxian Ruangan prescription: A cell study based on hepatic sinusoidal capillarization

目的 基于肝窦毛细血管化探讨抗纤软肝方抗肝纤维化的作用及机制。方法 将16只Wistar雄性大鼠分为抗纤软肝方组和对照组,每组各8只。抗纤软肝方组生药40 g/kg大鼠体质量灌胃,对照组用生理盐水给大鼠灌胃。将抗纤软肝方制备成含药血清与对照血清分别处理原代肝窦内皮细胞模型。采用Western-Blot检测血管内皮标志物分化抗原簇31(CD31)和血管性血友病因子(vWF)的蛋白表达水平;采用免疫细胞化学的方法检测细胞中CD31、vWF的细胞因子表达水平。扫描电镜检测肝窦内皮细胞窗孔的变化。计量资料两组间比较采用t检验。结果 抗纤软肝方对肝窦内皮细胞形态学无明显影响。对照组肝窦内皮细胞窗孔狭小,结构将近闭塞,数量较少;抗纤软肝方组肝窦内皮细胞窗孔结构清晰,窗孔增多增大,明显可见。 免疫细胞化学测定结果显示:与对照组比较,抗纤软肝方组CD31和vWF表达水平显著降低(CD31: 0.069±0.015 vs 0.115±0.011, t=4.257, P<0.05;vWF: 0.092±0.009 vs 0.132±0.014, t=4.274, P<0.05)。Western-Blot检测结果显示:与对照组相比,经抗纤软肝方组治疗后CD31、vWF的蛋白水平显著下降(CD31:0.334±0.029 vs 0.448±0.013, t=6.245, P<0.01;vWF: 0.560±0.047 vs 0.709±0.045, t=3.966, P<0.05)。结论 抗纤软肝方可明显抗肝窦毛细血管化,可以通过抗肝窦毛细血管化阻止肝纤维化的进程。

Objective To investigate the anti-liver fibrosis effect and mechanism of action of Kangxian Ruangan prescription based on hepatic sinusoidal capillarization. Methods A total of 16 male Wistar rats were divided into Kangxian Ruangan prescription group and control group, with 8 rats in each group. The rats in the Kangxian Ruangan prescription group were given crude drug 40 g/kg body weight by gavage, and those in the control group were given normal saline by gavage. Serum containing Kangxian Ruangan prescription was prepared, and then primary liver sinusoidal endothelial cells were treated with the serum containing Kangxian Ruangan prescription or the control serum. Western blot was used to measure the protein expression of the vascular endothelial markers cluster of differentiation 31 (CD31) and von Willebrand factor (vWF), and immunocytochemistry was used to measure the expression of CD31 and vWF in cells. Scanning electron microscopy was used to observe the change in the fenestrae of liver sinusoidal endothelial cells. The t-test was used for comparison of continuous data between two groups. Results Kangxian Ruangan prescription had no marked influence on the morphology of liver sinusoidal endothelial cells. The liver sinusoidal endothelial cells in the control group had a small number of narrow fenestrae with an occluded structure, while those in the Kangxian Ruangan prescription group had an increased number of large fenestrae with a clear structure. The results of immunocytochemistry showed that compared with the control group, the Kangxian Ruangan prescription group had significant reductions in the expression of CD31 (0.069±0.015 vs 0.115±0.011, t=4.257, P<0.05) and vWF (0.092±0.009 vs 0.132±0.014, t=4.274, P<0.05). The results of Western blot showed that compared with the control group, the Kangxian Ruangan prescription group had significant reductions in the protein expression of CD31 (0.334±0.029 vs 0.448±0.013, t=6.245, P<0.01) and vWF (0.560±0.047 vs 0.709±0.045, t=3.966, P<0.05). Conclusion Kangxian Ruangan prescription can significantly resist hepatic sinusoid capillarization and thus prevent the progression of liver fibrosis.