调肝健脾解毒方通过Bcl-6/Blimp-1轴调控滤泡性T细胞功能促进慢性乙肝HBeAg转阴的机制研究

Hepatitis B e antigen (HBeAg) loss is one of the decisive indicators of competent immune control on Chronic Hepatitis B (CHB). Previous clinical trials had confirmed therapeutic effects of "TiaoGanJianPiJieDu" (TGJPJD) formula on HBeAg loss in CHB patients, the mechanism is to be investigated. Latest literatures suggests functions of follicular T cells, which regulated by Bcl-6 / Blimp-1 axis, are essential for the immune control in chronical virus infections. Inspiringly, we observed regulated Bcl-6 & Blimp-1 expression alongside increased follicular T cell frequency and elevated downstream cytokines in peripheral blood of patients received TGJPJD formula. Based on these findings, we hypothesized that therapeutic outcomes of TGJPJD formula is partially dependent on follicular T cell behaviors, which was potentially regulated by Bcl-6 / Blimp-1 axis. Herein, we will confirm the hypothesis by inspecting Bcl-6 / Blimp-1 expression and follicular T cell frequency / phenotypes / functions among CHB patients by flow cytometry, multi analyte profiling, RT-PCR and immunohistochemistry. The results would be cross-analyzed with clinical assessments. Thus, to explore the therapeutic mechanisms of TJGPJD formula along this regulatory pathway. This study could provide clues on the targets of TGJPJD formula and further establish a foundation to optimize the composition.

乙肝e抗原(HBeAg)转阴是慢性乙型肝炎(CHB)获得免疫控制的重要指标之一。既往研究证实“调肝健脾解毒方”可提高CHB患者HBeAg转阴率，但机制未明。当前的研究发现Bcl-6/Blimp-1轴调控的滤泡性T细胞功能改变对慢性病毒感染的免疫清除有重要作用。我们前期研究发现调肝健脾解毒方可调节CHB患者外周血Bcl-6/Blimp-1轴，并促进滤泡性T细胞频率与下游效应因子的增加。由此提出该方基于Bcl-6/Blimp-1轴调控滤泡性T细胞功能以实现HBeAg转阴的假说。本项目将依托临床队列，通过流式细胞分析、血清多因子检测、RT-PCR及免疫组化等手段分析该方对CHB患者外周血与肝内Bcl-6、Blimp-1表达的影响及其对滤泡性T细胞表型、功能的调控作用。结合临床指标，探讨调肝健脾解毒方基于该调控通路的疗效机制。为阐明该方的作用靶点，进一步优化处方奠定基础。

背景：乙肝e抗原(HBeAg)转阴是慢性乙型肝炎(CHB)获得免疫控制的重要指标之一。既往研究证实“调肝健脾解毒方”可提高CHB患者HBeAg转阴率，但机制未明。当前的研究发现Bcl-6/Blimp-1轴调控的滤泡性T细胞功能改变对慢性病毒感染的免疫清除有重要作用。.方法：基于临床队列，通过流式细胞分析、血清多因子检测、RT-PCR分析调肝健脾解毒方对CHB患者外周血Bcl-6、Blimp-1表达的影响及其对滤泡性T细胞表型、功能的调控作用。.结果：调肝健脾解毒方可以提高CHB患者外周血Tfh细胞频率及表面分子ICOS、Tfc细胞、CD4+T细胞、CD8+T细胞以及CD8+CD127+T细胞的表达水平，组内有统计学差异（P<0.05）；其中，CD8+T细胞在第24周具有组间差异（P<0.05）。调肝健脾解毒方可以上调CHB患者Bcl-6 mRNA表达，与对照组相比，组间具有显著统计学差异（96周，P<0.05）；调肝健脾解毒方可以明显抑制CHB患者Blimp-1 mRNA表达，与对照组相比，组间具有显著统计学差异（144周，P<0.01）。.结论：我们推测调肝健脾解毒方早期可能调控HBV特异性CD8+T细胞，通过细胞杀伤机制产生抗病毒效应；治疗中后期调控Bcl-6和Blimp-1表达，促进CXCR5+T细胞分化增殖，激活机体免疫应答，促进HBeAg的清除；而在治疗后期也能促进干扰素分泌，抑制病毒复制，并激活下游的免疫细胞分化，通过非细胞毒机制清除感染细胞。研究结果揭示了调肝健脾解毒方促进慢性乙肝HBeAg转阴的机制，为中药治疗慢性乙型肝炎提供了理论依据。