补肾健脾方对乙肝病毒感染者免疫功能的影响

Effects of Bushen Jianpi Formula(补肾健脾方)on Immune Function in HBV Infected Patients

目的:观察乙型肝炎病毒(Hepatitis B Virus,HBV)感染对机体免疫功能的影响,研究补肾健脾方对HBV感染者免疫功能的影响。方法:(1)将招募的慢性HBV感染者及健康志愿者分为实验组和对照组,采集各组研究对象的外周血,分离血清,ELISA法检测两组血清中γ干扰素(IFN-γ)、α干扰素(IFN-α)、白细胞介素-2(IL-2)、IL-12、肿瘤坏死因子α(TNF-α)含量;密度梯度离心法分离外周血单个核细胞(PBMC),流式细胞术(FCM)检测两组的PBMC中自然杀伤细胞(NK)、辅助性T细胞1(Th1)、细胞毒性T淋巴细胞(CTL)数量及胞内IFN-γ、IFN-α、IL-2、IL-12、TNF-α、干扰素基因刺激蛋白(STING)、T盒子转录因子(T-BET)、信号传导转录激活因子4(STAT4)蛋白表达水平。(2)制备大鼠含药血清,通过WST-1细胞增殖及细胞毒性检测试剂盒筛选含药血清干预PBMC的安全剂量;以HBV感染者PBMC为实验对象将其分为3个组,空白对照组、空白血清组和含药血清组,干预48 h后,ELISA法检测细胞培养上清中细胞因子IFN-γ、IFN-α、IL-2、IL-12、TNF-α含量,FCM检测各组PBMC中CTL、Th1、NK细胞数量及胞内细胞因子水平;RT-qPCR法检测各组细胞因子mRNA表达。结果:(1)与健康志愿者相比,HBV感染者血清中IFN-γ、IL-12含量和PBMC中CTL、Th1、NK细胞数量明显降低(P<0.05);HBV感染者PBMC胞内IFN-γ、IL-12、TNF-α、STAT4、T-BET较健康志愿者合成减少(P<0.05)。(2)与空白血清组相比,补肾健脾方含药血清组PBMC细胞培养上清中IFN-γ、IL-12含量明显升高(P<0.05);补肾健脾方含药血清组PBMC中CTL、Th1、NK细胞数量以及胞内IFN-γ蛋白表达水平较空白血清组明显升高(P<0.05);与空白血清组相比,补肾健脾方含药血清可明显上调Ifnγ、IL2、Il12、TnfαmRNA表达(P<0.05)。结论:补肾健脾方可通过上调CTL、Th1、NK细胞数量,促进IL-12分泌,进而促进IFN-γ的合成与分泌,从而恢复HBV感染者PBMC的免疫功能。

Objective:To observe the effect of hepatitis B virus(HBV)infection on the immune function associated with interferon(IFN)-γand to study the therapeutic effect of Bushen Jianpi Formula(补肾健脾方).Methods:First,chronic HBV-infected patients and healthy volunteers were recruited and assigned into experimental and control groups.Peripheral blood was collected for the separation of the serum,and the serum levels of IFN-γ,IFN-α,interleukin(IL)-2,IL-12,and tumor necrosis factor(TNF)-αin both groups were measured by enzyme-linked immunosorbent assay(ELISA).Peripheral blood mononuclear cells(PBMCs)were separated by density gradient centrifugation,and flow cytometry(FCM)was employed to examine the number of natural killer(NK)cells,T helper 1(Th1)cells,and cytotoxic T lymphocytes(CTLs).The intracellular protein levels of IFN-γ,IFN-α,IL-2,IL-12,TNF-α,stimulator of interferon genes(STING),T-box expressed in T cell(T-BET),and signal transducer and activator of transcription 4(STAT4)were determined.Second,the drug(Bushen Jianpi Formula)-containing serum of rats was prepared,and the safe dose of the drug-containing serum for PBMC intervention was screened by the WST-1 method and cytotoxicity assay kit.The PBMCs from HBV-infected patients were classified into 3 groups,control,blank serum,and drug-containing serum groups.After 48 h of intervention,ELISA was employed to measure the levels of IFN-γ,IFN-α,IL-2,IL-12,and TNF-αin the cell culture supernatant.FCM was employed to count CTLs,Th1 cells,and NK cells and examine the cytokine levels in PBMCs of each group.RT-qPCR was performed to determine the mRNA levels of cytokines in each group.Results:Compared with healthy volunteers,the HBV-infected patients showed lowered serum levels of IFN-γand IL-12,decreased CTLs,Th1 cells,and NK cells,and reduced synthesis of intracellular IFN-γ,IL-12,TNF-α,STAT4,and T-BET(P<0.05).Compared with the blank serum,the drug-containing drug serum elevated the levels of IFN-γand IL-12 in the PBMC culture supernatant,increased CTLs,Th1 cells,and NK cells,up-regulated the intracellular protein level of IFN-γand the mRNA levels of Ifn-γ,Il-2,Il-12,and Tnf-αgenes(P<0.05).Conclusion:By increasing CTLs,Th1 cells,and NK cells and promoting the secretion of IL-12 and the synthesis and secretion of IFN-γ,Bushen Jianpi Formula can restore the immune function of PBMCs in HBV-infected patients.