基于肝脏免疫微环境发现恩替卡韦联合扶正化瘀片有效逆转乙肝肝纤维化的目标人群特征与主要作用机制

On the basis of therapeutic advantages of integrative medicine on HBV liver fibrosis, we utilize the biochemical samples from two cohort of HBV cirrhosis established by national key grant, and combine the animal and cell culture experiments in the study focusing on liver immune microenvironment which involve hepatic stellate cell, NK cell, hepatic macrophage and retinoid metabolism. Firstly the baseline data was analyzed according to the stratification of endpoints, and the characteristic of target population with hepatic immune microenvironment and TCM pattern. Then the efficacy and hepatic micro-immune were compared between the before and after treatment, and between the 2 groups, to understand the main action mechanisms of the combination of entacavir and Fuzheng Huayu Tablet on the regression of HBV induced hepatic fibrosis. The liver fibrotic animal models with HBV background were established, treated with entacavir and FZHY, the phenotype and functions of above 3 cells and their relationship were assayed, to investigate the regulation of integrative medicine on liver immunological disorder. Lastly, the cirrhosis model were induced with dimethylnitrosamine, and human liver cells isolated, and treated with FZHY, to study the FZHY effect on retinoid metabolism and liver immune cells interaction. The hypothesis will be testified as follows: the hepatic immune disorder including cells interaction and retinoid metabolism, in particular activated HSC negative regulation of NK cells, affect the regression of advanced liver fibrosis, and be main mechanism of integrative medicine reversing liver fibrosis. The work will promote the practice of precision medicine and progress of basic science in the filed of liver disease with integrative medicine.

立足于中西医结合抗乙肝肝纤维化疗效优势，借助传染病重大科技专项乙肝肝硬化临床研究生物样本，结合动物与细胞实验，围绕肝纤维化免疫微环境中肝星状细胞(HSC)、NK细胞与肝巨噬细胞的相互联系及其视黄醇代谢通路，验证“调节肝脏免疫微环境、恢复NK细胞对活化HSC的免疫杀伤功能是恩替卡韦联合扶正化瘀片逆转乙肝高度肝纤维化的主要机制”科学假说。首先基于疗效分层与回归分析，发现中西医结合治疗有效人群主要生物学与中医证候特征，并通过组内、组间比较分析阐明治疗方案的主要作用机制；其次，复制乙肝病毒背景的肝纤维化动物模型，体内给药，检测细胞表型功能及相互关系，探讨中西药物联用对肝免疫病理调控机制；第三，复制肝硬化动物模型，分离培养人肝脏细胞，阐明中药影响活化HSC对NK细胞负调控等细胞联系的作用特点；最终发现有效方案目标人群特征，阐明其主要治疗机制与中药作用特点，促进中西医结合肝病的精准医疗与基础研究发展。

我们前期工作发现，扶正化瘀片+恩替卡韦(entecavir, ETV)的中西药联合方案可明显提高ETV的乙肝肝纤维化逆转率。这种中西药联用方案的作用机制及其中药的作用特点如何？其效应目标人群特征是什么？等等问题尚不清楚，即是本课题拟解决的两个关键科学问题。我们围绕肝脏纤维化免疫微环境，临床生物样本分析与动物细胞实验相结合，一方面表征中西药联用方案的有效人群特征，另一方面解析该方案的作用机制及其中药作用特点。通过研究，①提出“肝纤维化免疫微环境”的新概念，并阐明其主要特征在于：慢性肝损伤时NK细胞活性下降、肝巨噬细胞促炎性极化、肝星状细胞(Hepatic Stellate Cells，HSC)的视黄醇代谢紊乱与活化、肝细胞的消亡再生、肝窦内皮细胞去分化等，这些细胞变化相互联系，最终形成肝纤维化；调控其中主要环节可以促进肝纤维化逆转；②发现扶正化瘀片+ETV联用方案的有效人群的生物标志物为谷氨酰胺、花生四烯酸等；而其效应人群中医证候特点为“肝郁脾虚”；③发现该联用方案可通过促进小鼠纤维化肝脏NK细胞活性，杀伤HSC从而发挥抗肝纤维化作用；其中中药扶正化瘀方调节HSCs视黄醇代谢，并通过NKG2D-RAE1通路，促进NK细胞对HSC的杀伤作用；④发现CX3CR1+骨髓来源这一群特殊巨噬细胞具有治疗肝纤维化的作用；该中西药联用方案调节肝脏巨噬细胞的募集与极化而抗肝纤维化，扶正化瘀方的作用特点在于影响巨噬细胞CCL2/CX3CL1 轴、促进其从M1向M2型极化；⑤发现扶正化瘀方调节肝细胞TOMM34而减少慢乙肝的HBeAg分泌，主要活性成分包括槲皮素与虫草素等；调节肝窦内皮细胞VEGF信号通路与Wnt2外分泌，而改善该细胞的去分化及其肝脏血管新生、减少肝细胞的消亡；⑥发现扶正化瘀方君药—丹参调节NK细胞与巨噬细胞功能的抗肝纤维化作用机制，发现其主要活性成分如丹参酚酸B、丹参酮IIA等。以上结果揭示了扶正化瘀片+ETV中西药结合方案的主要效应人群特征、作用机制及其中药的作用特点，为中西医结合的精准医疗与作用原理阐明等提供了新依据。