疏肝健脾补肾方对乙型肝炎病毒转基因小鼠脾T淋巴细胞及病毒载量的影响

目的观察疏肝健脾补肾方对乙型肝炎病毒(hepatitis B virus,HBV)转基因(transgenic,Tg)小鼠脾T淋巴细胞及病毒载量的影响,探讨其抗病毒疗效及作用机制。方法 60只SPF级BALB/C雄性小鼠,设10只HBV非Tg小鼠为正常对照组,50只HBVTg小鼠随机分为模型组、阿德福韦(ADV)组及中药小、中、大剂量组,每组10只。中药小、中、大剂量组分别给予疏肝健脾补肾方10、20、40g生药/kg灌胃;ADV组给予ADV50mg/kg灌胃;正常对照组和模型组以等量灭菌等渗生理盐水灌胃,均每天1次,连续21天。采用实时荧光PCR定量检测HBVTg小鼠给药前1天(T0)、给药后10天(T1)、21天(T2)、停药后3天(T3)血清HBVDNA;采用ELISA法检测其T3时血清HBsAg。流式细胞仪测定所有小鼠脾T淋巴细胞百分比。结果各给药组T0时血清HBsAg均为阳性,T3时ADV组HBsAg转阴数高于中药大剂量组,差异有统计学意义(P<0·01)。与本组T0时比较,给药后中药大剂量组可持续降低HBVDNA含量,差异均有统计学意义(P<0·01);ADV组HBVDNA含量亦逐渐下降(P<0·01),但在T3时有所回升。中药各剂量组及ADV组均可提高CD3+细胞百分比(P<0·05,P<0·01);中药各剂量组均可明显降低CD8+百分比(P<0·01)。与其他给药组比较,中药大剂量组CD4+、CD4+/CD8+升高,CD8+降低,差异均有统计学意义(P<0·01)。中药中剂量组CD3+及中药大剂量组CD4+、CD8+、CD4+/CD8+均与本组给药前后HBVDNA滴度减少呈显著正相关(r=0·654,P<0·05;r=0·53,r=0·79,r=0·80,P<0·01)。结论疏肝健脾补肾方具有抑制HBVTg小鼠HBVDNA复制的能力,改善T淋巴细胞亚群的异常,提高机体免疫功能可能是其抗HBV的机制之一。

英文摘要:

Objective To observe the effect of Shugan Jianpi Bushen Recipe(SJBR) on the splenic T lymphocytes and virus load in the hepatitis B virus(HBV) transgenic(Tg) mice,and to study its antiviral efficacy and mechanisms of action.Methods Sixty male BALB/C mice of SPF grade were included.Ten non-HBV Tg male mice were included as the normal control group.Fifty HBV Tg mice were randomly divided into five groups,i.e.,the model group,the adefovir(ADV) group,the low dose SJBR group,the middle dose SJBR group,and the high dose SJBR group,ten in each.10,20,and 40 g/kg SJBR crude drug was respectively given by gastrogavage to mice in the low dose SJBR group,the middle dose SJBR group,and the high dose SJBR group.ADV 50 mg/kg body weight was given by gastrogavage to mice in the ADV group.Equal volume of sterilized iso-osmia was given to mice in the normal group and the model group.The medication was performed once daily,totally for 21 successive days.The serum HBV DNA titers of HBV Tg mice were detected using Real-time fluorescent PCR one day before administration(T0),ten days after administration(T1),21 days after administration(T2),and three days after withdrawal(T3),respectively;the serum hepatitis B surface antigen(HBsAg) of HBV Tg mice on T3 was detected by ELISA.The splenic T lymphocyte percent of all mice was detected by flow cytometry.Results Serum HBsAg at T0 was positive in the high-,middle-,low-dose SJBR,and ADV groups.The HBsAg negative rate at T3 was lower in the high dose SJBR group than in the ADV group,showing statistical difference(P<0.01).Compared with T0 of the same group,the serum HBV DNA titers could be continually decreased by high dose SJBR,showing statistical difference(P<0.01).The serum HBV DNA titers also gradually decreased in the ADV group(P<0.01),but it somewhat increased at T3.The CD+3 cell percent could be elevated by high-,middle-,low-dose SJBR,and ADV groups(P<0.05,P<0.01).The CD+8T cell percent could also be obviously lowered by high-,middle-,and low-dose SJBR(P<0.01).Compared with the middle-,low-dose SJBR,and ADV groups,the CD+4 T cell percent and CD+4/CD+8 increased as well as CD+8 decreased in the high dose SJBR group,showing statistical difference(P<0.01).The CD+3 T cell percent was significantly positively correlated to the decrement of HBV DNA titers between the pre-treatment and post-treatment in the middle dose SJBR group(r=0.654,P<0.05).The percents of CD+4,CD+8 T cells and CD+4/CD+8 were significantly positively correlated to the decrement of HBV DNA titers between the pre-treatment and post-treatment in the high dose SJBR group(r=0.53,r=0.79,r=0.80,P<0.01).Conclusions SJBR were capable of inhibiting the HBV DNA duplication of HBV Tg mice.One of its anti-HBV mechanisms possibly be improving the the abnormality of T lymphocyte subsets and the immune function.

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