ROCK阻断剂Y27632干预大鼠实验性肝纤维化的分子机制研究

Molecular mechanism of the effect of Y27632,an antagonist of ROCK,depressing liver fibrosis

目的观察ROCK阻断剂Y27632对实验性大鼠肝纤维化形成过程中组织病理学、细胞外基质成分及肝星状细胞中RhoA、Vinculin mRNA和RhoA、MLC蛋白质表达的影响,探讨Y27632治疗实验性肝纤维化的分子机制。方法雄性SD大鼠30只,随机分为对照组、模型组和治疗组。模型组和治疗组动物给予60%四氯化碳0.1 mL/100 g皮下注射,2次/周,对照组给予等量的生理盐水皮下注射。治疗组从第4周起给予Y27632口服灌胃（30 mg/Kg）,其余两组动物予以等量的生理盐水。于造模后第8周采集静脉血行放免法检测血清中PⅢP和Ⅳ型胶原的含量,取肝组织行组织病理学检查。另取雄性SD大鼠30只分为对照组（n=10）和模型组（n=20）,模型组造模方法同前。分离培养肝星状细胞,采用模型组星状细胞充分伸展贴壁时（培养8~10 d）为干预时间,并将模型组进一步分为非干预组和干预组,干预组采用终浓度为50μmol/L的Y27632干预,非干预组加入等量的生理盐水,1 h后观察细胞形态,收集各组细胞以RT-PCR检测RhoA和Vinculin mRNA表达,Western blotting检测RhoA和MLC蛋白的表达。结果与模型组相比,给予Y27632后组织病理学显示治疗组肝纤维化程度明显减轻,血清中PⅢP和Ⅳ型胶原的含量显著下降。细胞学试验显示,非干预组RhoA、Vinculin mRNA和RhoA蛋白的表达较对照组明显增强,给予Y27632干预后表达强度减弱。而对照组大鼠肝组织中MLC蛋白质呈阳性表达,非干预组其表达强度明显下调,给予Y27632后表达强度有所增加。结论 Y27632能有效干预实验性大鼠肝纤维化的形成,其分子机制可能与其调控星状细胞中RhoA和MLC有关。

Objective To discuss the molecular mechanism of Y27632,an antagonist of ROCK,depressing liver fibrosis through observing the expression of extracellular martrix,RhoA and Vinculin mRNA,RhoA and MLC protein.Methods Thirty male SD rats were randomly divided into control group,model group and treatment group.Liver fibrotic model was made by subcutaneous injection of CCl4,and Y27632（30 mg/Kg） was given orally everyday in treatment group after the 4th week.Liver tissue and blood samples were taken after the 8th week.The degree of liver fibrosis was evaluated by V-G staining,and the PⅢP and Ⅳ collagen were detected by RI.Other 30 male SD rats were divided into control group and model group.Hepatic stellate cells were isolated from the model group.The model group may be divided into non-intervention group（n=10） and intervention group（n=10）.Intervention group was given Y27632 which the final concentration was 50 μmol/L.Cell morphology was observed and all culture cells were harvested after 1 h.RT-PCR was used to detect the expression of Vinculin and RhoA mRNA,Western blotting was used to detect the expression of RhoA and MLC protein.Results Compared with model group, the organic pathology showed the degree of liver fibrosis alleviated in treatment group,contents of PⅢP and Ⅳ collagen decreased markedly in treatment group.Expression of RhoA,Vinculin mRNA and RhoA protein in non-intervention group were stronger than those in control group,and decreased markedly in intervention group.Expression of MLC protein decreased markedly in non-intervention group,and increased in intervention group.Conclusion Y27632 can alleviate the degree of liver fibrosis by blockade RhoA signaling transduction pathway.