解毒凉血健脾方调节腹腔巨噬细胞M2c型极化治疗急性肝衰竭的机制研究

In acute liver failure, the polarization direction of macrophages is critical to the degree of liver inflammation and tissue repair. Excessive activation of M1 macrophages is an important mechanism of hepatic immune injury, while M2 macrophages can inhibit the liver inflammation and accelerate tissue repair. The latest studies have found that peritoneal macrophages rapidly move to the liver during acute liver injury, which is the main source of M2 macrophages. Jiedu-Liangxue-Jianpi Decoction is an empirical formula in treating liver failure.On the basis of the previous study, the applicant found that this decotion could not only inhibit monocytes to M1 polarization,but also promote M2c polarization.Based on the results mentioned above,the applicant proposed a hypothesis that promoting rapid infiltration of peritoneal macrophage and M2c polarization is an important mechanism in treating acute liver failure with Jiedu-Liangxue-Jianpi Decoction.The aim of this study is:1)to evaluate the curative effect of Jiedu-Liangxue-Jianpi Decoction in treating acute liver failure with ConA model;2)to confirm the key role of peritoneal macrophage in the treatment of acute liver failure through cell clearance and transplantation experiments;3)to define the regulating effect of the decotion on M2c polarization in vitro with cell models;4)to further reveal the molecular mechanism of the polarization regulation by sequencing and validating experiments,finally to provide experimental evidence for the treatment of acute liver failure with traditional Chinese medicine.

急性肝衰竭时，巨噬细胞极化方向对肝脏炎症损伤程度和组织修复至关重要。M1型巨噬细胞过度活化导致肝脏免疫损伤，M2型巨噬细胞可抑制肝脏炎症反应，促进组织修复。最新研究发现腹腔巨噬细胞在急性肝损伤时迅速迁至肝脏，是M2型巨噬细胞的主要来源。解毒凉血健脾方是治疗肝衰竭的临床经验方，申请人在前一项国自然的基础上，发现该方不仅能抑制巨噬细胞向M1极化，而且能促进M2c极化，据此提出：促进腹腔巨噬细胞快速浸润并向M2c极化，是解毒凉血健脾方治疗急性肝衰竭的重要机制。本项目拟1）采用ConA模型评价解毒凉血健脾方对急性肝衰竭的疗效；2）结合细胞清除、移植实验从正反两方面确证腹腔巨噬细胞在治疗急性肝衰竭中的关键作用；3）采用细胞模型体外明确该方对M2c极化的调节作用；4）通过表达谱测序及验证实验，进一步揭示该方调节极化的分子机制，为中医药治疗急性肝衰竭提供实验依据。

急性肝衰竭是指既往无肝病者突然出现大量肝细胞凋亡、坏死，肝功能异常，短期内迅速进展至肝性脑病的一种严重综合征，病死率可达80%以上。但目前尚无明确的发病机制。而且由于病情凶险，救治困难，缺乏阻断急性、大面积肝细胞坏死的有效药物,我国每年因此而死亡的人数也高达10-20万。因此及时准确地对患者进行药物干预就成为了治疗的关键之一。解毒凉血健脾方是治疗急性肝衰竭的临床经验方，在以往的研究中也发现该方能够抑制机体免疫损伤的循环放大，发挥肝保护作用。因此，本研究采用ConA模型评价解毒凉血健脾方对急性肝衰竭的疗效，发现解毒凉血健脾方能显著提高ConA模型小鼠的生存率；并从该中药复方中筛选出的两种单体成分也能减轻小鼠肝细胞损伤。通过解离ConA模型小鼠肝脏，对其肝单细胞悬液进行单细胞转录组测序分析，发现ConA致小鼠发生急性肝衰竭与中性粒细胞、淋巴细胞、单核细胞等炎症性细胞相关。同时我们也发现单体1和单体2均能够阻止中性粒细胞向肝脏转移，并可同时提高肝脏中淋巴细胞和单核细胞水平发挥肝保护作用。本研究采用单细胞转录组测序技术，将现代化的研究方法应用于中药研究，有助于进一步阐明中医药如何在治疗急性肝衰竭中发挥作用。