羧甲基茯苓多糖抗乙型肝炎病毒的体内与体外研究

Study on effect of carboxymethyl-pachymaran on anti-hepatitis virus in vivo and in vitro

目的探讨羧甲基茯苓多糖(carboxymethyl-pachymaran,CMP)抗HBV、调节免疫、抗肝纤维化的作用及其机制。方法体外实验:人肝癌Hep G 2.2.15细胞株作为细胞模型,分为阴性对照组、CMP(1.5、3.0、6.0、12.0 mg/m L)组和阳性对照阿昔洛韦(aciclovir,ACV,浓度分别为0.5、1.0、1.5 mg/m L)组,MTT法观察CMP的细胞毒作用,ELISA法测定2.2.15细胞上清中HBs Ag和HBe Ag的含量。体内实验:采用四氯化碳复合因素法制作SD大鼠肝纤维化模型,分为正常对照组、模型组、CMP组,香菇多糖组及秋水仙碱组,每组10只,免疫组织化学法观察CMP对转化生长因子β(TGF-β)、细胞凋亡相关因子caspase-3、Ⅲ型胶原(collagen-III)、层粘蛋白(laminin)的影响,Western blot检测Smad3、Smad7的表达。结果体外实验:CMP对Hep G2.2.15细胞株的半数毒性浓度(TC50)为13.61 mg/m L,对Hep G2.2.15细胞株HBs Ag、HBe Ag分泌的半数有效浓度(IC50)分别为4.38 mg/m L、5.78 mg/m L,治疗指数(TI)值分别为3.15和2.45,CMP对HBs Ag和HBe Ag有抑制作用,并且抑制HBs Ag和HBe Ag分泌的作用优于ACV。模型组与TGF-β、caspase-3、Ⅲ型胶原、层粘蛋白的表达高于正常对照组(P<0.01);CMP,香菇多糖,秋水仙碱能够降低肝纤维化大鼠肝脏中TGF-β、caspase-3、Ⅲ型胶原、层粘蛋白的表达(P<0.05,P<0.01)。模型组Smad-3的表达高于对照组、Smad-7的表达低于对照组(均P<0.01);CMP,香菇多糖,秋水仙碱能够降低肝纤维化大鼠Smad-3的表达、升高Smad-7的表达(均P<0.01)。CMP显示了较好的抗纤维化作用,与香菇多糖组比较无统计学差异。结论通过体内和体外实验,证明CMP具有抑制HBV,调节TGF-β/Smad信号转导通路等作用,本研究为CMP在慢性乙型肝炎临床的应用提供了重要的理论和实验依据。

Objective To study carboxymethyl-pachymaran＇s effect of anti-hepatitis virus in vivo and in vitro and its mechanism. Methods In vitro,Hep G2. 2. 15 cells was chosen as the cell model,and divided into three groups： negative control group,CMP（ 1. 5,3. 0,6. 0,12. 0 mg / m L） groups and ACV group（ acyclovir,0. 5,1. 0,1. 5 mg / m L）. The cytotoxicity of CMP was observed by MTT method. The content of HBs Ag and HBe Ag in the culture supernatant was determined by ELISA. In vivo,the model of hepatic fibrosis was induced by CCl4 composite method in SD rats. The rats were divided into five groups： normal control group,model group,CMP group,lentinan group and colchicines group,10 rats in each group. The levels of transformation growth factor β（ TGF-β）,caspase-3,collagen-III and laminin were determined with immunohistochemical method. The expressions of Smad3 and Smad7 were detected by Western blot. Results In vitro,the TC50 value of CMP on Hep G2. 2. 15 cell line was 13. 61 mg / m L,the IC50 values of CMP on HBs Ag,HBe Ag secreted by Hep G2. 2. 15 were 4. 38 mg / m L,5. 78 mg / m L,and the therapeutic index（ TI） values were 3. 15,2. 45. CMP had some inhibitory effect on HBs Ag,HBe Ag,and the inhibitions of HBs Ag,HBe Ag secretion were better than ACV. The levels of TGF-β,collagen-III,laminin and caspase-3 of model group were higher than those of normal control group（ P〈 0. 01）,and the CMP,lentinan and colchicines decreased the above indexes of hepatic fibrosis rats（ P〈 0. 05,P〈 0. 01）. The expression of Smad-3 was higher,Smad-7 was lower in model group than that in control group,respectively（ P〈 0. 01）,and the CMP,lentinan and colchicines decreased Smad-3 level and increased Smad-7 level of hepatic fibrosis rats（ P〈 0. 01）. CMP showed the good antifibrosis effect,and had no significant difference compared with lentinan group. Conclusion In vivo and in vitro,it is confirmed that CMP could inhibit HBV,regulate TGF-β / Smad signal transduction pathway,etc. The study provides important academic and experimental evidence for the application of CMP in the treatment of chronic hepatitis B.