下瘀血汤调控ACE2介导的RAS自稳抑制肝纤维化实验研究

目的探讨下瘀血汤是否通过恢复或重建肾素-血管紧张素系统(renin-angiotensin system,RAS)的自稳平衡抑制肝纤维化进展。方法将27只Wistar雄性大鼠随机分为5组,即正常组、模型组、去除病因组、下瘀血汤组和氯沙坦钾组。除正常组外,各实验组大鼠背部皮下注射2.5 mL/kg 40%四氯化碳橄榄油混悬液造模,并以下瘀血汤及氯沙坦钾干预肝纤维化进展,应用HE染色、Masson三色胶原染色和超微病理学方法观察肝脏组织结构的改变,采用免疫组织化学染色法检测各组AT1R、TGF-β1表达,采用RT-PCR及Western-blot法检测RAS相关因子的基因和蛋白表达。结果①HE染色、Masson三色胶原染色和超微病理学结果均显示去除病因组、下瘀血汤组、氯沙坦钾组和模型组肝纤维化程度依次加重。②RT-PCR及Western-blot结果显示,与正常组相比,其他组RAS相关分子ACE2、ACE、AngⅡ、AT1R及TGF-β1的mRNA及蛋白表达均升高;除去除病因组TGF-β1基因表达差异无统计学意义(P>0.05)之外,其他组间比较均具有统计学意义(P<0.05)。与模型组相比,去除病因组、下瘀血汤组、氯沙坦钾组RAS相关分子ACE2、ACE、AngⅡ、AT1R及TGF-β1的mRNA及蛋白表达均显著降低(P<0.05)。③与氯沙坦钾组比较,下瘀血汤组ACE2的mRNA及蛋白表达显著升高(P<0.05),而AngⅡ及TGF-β1的mRNA及蛋白表达显著降低(P<0.05)。结论下瘀血汤可能通过增加ACE2的表达,抑制ACE、AngⅡ和TGF-β1的表达,使RAS中促进肝纤维化的经典通路ACE-AngⅡ-AT1R转变为抑制肝纤维化的ACE2-Ang(1-7)-Mas受体轴为主导,从而抑制肝纤维化。

Experimental Study of Xiayuxue Decoction on Self-regulation of RAS by Targeting ACE2 with Hepatic Fibrosis

Objective To explore whether Xiayuxue decoction attenuates hepatic fibrosis via recovering or rebuilding the self-regulation and balance of the renin-angiotensin system(RAS).Methods Twenty-seven SPF male Wistar rats were randomly divided into normal group,model group,removing disease cause group,Xiayuxue decoction group and the losartan potassium group.Hepatic fibrosis was induced in rats with subcutaneously injection of 40%CCl4 dissolved in olive oil(2.5 mL/kg).Liver pathology was detected using HE staining,Masson trichromatic collagen staining and electron microscope.The expression of AngⅡ,AT1R,TGF-β1,ACE and ACE2 in the liver tissue was evaluated with immunohistochemical staining,RT-PCR and Western-blot,respectively.Results①In terms of the results of HE staining,Masson trichromatic collagen staining and ultrastructural pathology,the degree of liver fibrosis was gradually aggravated in the removing disease cause group,Xiayuxue decoction group,losartan potassium group and model group.②The results of RT-PCR and Western-blot showed that the mRNA and protein expression of RAS related molecular ACE2,ACE,AngⅡ,AT1R and TGF-β1 in other groups were significantly higher than those in the normal group(P<0.05),except for no significant difference in gene expression of TGF-β1 in the removing disease cause group(P>0.05).Compared with the model group,the mRNA and protein expression of ACE2,ACE,AngⅡ,AT1R and TGF-β1 are significantly decreased in removing disease cause group,Xiayuxue decoction group and losartan potassium group(P<0.05).③Compared with the losartan potassium group,the mRNA and protein expression of ACE2 in Xiayuxue decoction group were significantly increased(P<0.05),while the mRNA and protein expression of AngⅡand TGF-β1 were significantly decreased(P<0.05).Conclusion Xiayuxue decoction may upgrade the expression of ACE2,inhibit the expression of ACE,AngⅡand TGF-β1,and convert the classical pathway ACE-AngⅡ-AT1R promoting liver fibrosis to ACE2-Ang(1-7)-Mas receptor axis,thus recovering and rebuilding the self-regulation and balance of RAS and exerting the anti-hepatic fibrosis effects.