桂枝茯苓丸抗大鼠肝纤维化作用及其机制研究

Study on Inhibition and Mechanism of Guizhi Fuling Wan on Hepatic Fibrosis Rats

目的:观察桂枝茯苓丸对大鼠肝纤维化的拮抗作用,并探讨可能的作用机制。方法:按照随机数字表将60只Wistar大鼠分为正常组8只(A组)和造模组52只,正常组sc予生理盐水3 mL.kg-1,造模组采用等量40%四氯化碳(carbontetrachloride,CCl4)复制肝纤维化模型,共6周。再随机将造模组大鼠分为模型组(B组)、桂枝茯苓丸低、中、高剂量组(C,D,E组)每组7只大鼠。A组与B组大鼠均予生理盐水9.0 mL.kg-1ig,C,D,E组分别给予桂枝茯苓丸0.45,0.9,1.8 g.kg-1.d-1 ig治疗,共4周,并在治疗期间继续使用CCl4维持。采用免疫组化(immunohistochemistry,IH)法及实时定量PCR(real-timequantitative PCR,Real-time PCR)法检测各组大鼠肝组织α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、转化生长因子β1(transforming growing factorβ1,TGF-β1)、结缔组织生长因子(connective tissue grower factor,CTGF)、Ⅰ型胶原(CollagenⅠ,C-Ⅰ)及Ⅲ型胶原(CollagenⅢ,C-Ⅲ)的蛋白及基因表达量。结果:模型组大鼠肝组织α-SMA,TGF-β1,CTGF,C-Ⅰ,C-Ⅲ蛋白和mRNA的表达较正常组显著增多(P<0.01),治疗组均能下调大鼠肝组织α-SMA,TGF-β1,CTGF,C-Ⅰ以及C-Ⅲ蛋白和mRNA的表达,其中以D组,E组减少α-SMA,C-Ⅰ,C-Ⅲ蛋白的表达明显(P<0.05或P<0.01),以E组降低TGF-β1,CTGF蛋白表达最为显著(P<0.05),以D组,E组降低α-SMA,TGF-β1,CTGF mRNA的表达显著(P<0.01),以E组减少C-Ⅰ,C-ⅢmRNA的表达最明显(P<0.01)。结论:桂枝茯苓丸混悬液可有效减少肝组织α-SMA,TGF-β1,CTGF,C-Ⅰ,C-Ⅲ蛋白和mRNA表达量,有良好的抗肝纤维化作用。

Objective： Guizhi Fuling Wan which inhibited hepatic fibrosis in rats was observed and its possible mechanism of action was explored.Method： According to the random-number table,wistar rats of 60 were divided into control group of 8（group A） and model group of 52.The control group was injected subcutaneously with normal saline by a dose of 3 mL·kg^-1,model group was injected subcutaneously with 40% CCl4 to make rat liver fibrosis model by a dose of 3 mL·kg^-1 for the consecutive 6 weeks.And according to the random-number table again,model group rats were divided into the model group（group B） and the treatment group which comprised low dose（group C）,middle dose（group D） and high dose（group E）.Group A and group B were given normal saline by a dose of 9.0 mL·kg^-1.Group C,group D and group E were given Guizhi Fuling Wan by the dose of 0.45,0.9,1.8 g·kg^-1·d-1respectively,and CCl4 would be maintained subcutaneously during period of 4 weeks.After 4 weeks of treatment,general condition,body weight,liver and spleen weight and their coefficient of rats were compared in each group;rats of pathological changes in each group were determined by naked eye,light microscopy and electron microscopy,rspectively.The protein expression of α-SMA,TGF-β1,CTGF,Co-Ⅰ and Co-Ⅲ was detected by using immunohistochemical stains in each group.The mRNA genetic expression of α-SMA,TGF-β1,CTGF,Co-Ⅰ and Co-Ⅲ was detected by using Real-time quantitative PCR in each group.Result： Immunohistochemical stains and real-time quantitative PCR： In model group,protein and mRNA genetic expression of α-SMA,TGF-β1,CTGF,Co-Ⅰ and Co-Ⅲ was significantly increased in liver tissue（P〈0.01,vs control group）.All the treatment group could bring down protein and mRNA genetic expression of α-SMA,TGF-β1,CTGF,Co-Ⅰ and Co-Ⅲ in liver tissue of rats.Among the treatment group,group D and group E decreased mRNA genetic expression of α-SMA,Co-Ⅰ and Co-Ⅲ significantly（P〈0.05 or P〈0.01 vs model group）,and Group Elessened protein expression of TGF-β1,CTGF obviously（P〈0.05 vs model group）.Among the treatment group,group D and group E cut down mRNA genetic expression of α-SMA,TGF-β1,CTGF significantly（P〈0.01 vs model group）,and group E of all the treatment group reduced evidently genetic mRNA expression of Co-Ⅰ and Co-Ⅲ（P 0.01 vs model group）.Conclusion： Guizhi Fuling Wan suspension can effectively inhibit liver α-SMA,TGF-β1,CTGF,Co-Ⅰ and Co-Ⅲ gene and protein expression,have a good anti-liver fibrosis.