巨噬细胞M2极化在情志应激诱发小鼠NASH相关肝纤维化中的作用研究

The classical theory from traditional Chinese Medicine (TCM) indicates that emotional stress affects the development of functional diseases. On the basis of the findings that emotional stress induced nonalcoholic steatohepatitis (NASH) in mice, the applicant demonstrated that NASH-related hepatic fibrosis, induced by emotional stress, was related to excess medium- and short-chain acylcarnitines and macrophages accumulation. The accumulation of macrophages in the liver could improve M2 macrophages polarization, one of the critical mechanisms involving hepatic fibrosis formation. Expect for cytokines, macrophage polarization could be induced by fatty acid oxidation. Thus, the study hypothesized that emotional stress-induced hepatic fibrosis in NASH was involved M2 macrophages’ promoting acylcarnitines oxidation. In this study, the emotional stress-induced NASH mice model was used to confirm the positive effects of emotional stress on M2 macrophages polarization. In addition, the in vitro macrophages polarization models were applied to explore whether the M2 macrophages polarization was dependent on acylcarnitines oxidation. Subsequently, the mechanism of M2 macrophages on acylcarnitines oxidation was investigated by13C-labeled metabolic flux analysis. The effects and mechanisms were confirmed by pathway inhibition and gene silencing methods. The results were expected to elucidate the pathogenesis of NASH-related hepatic fibrosis induced by emotional stress, thereby promoting the scientific connotation of emotional stress-induced functional diseases in TCM. The findings also contributed to establish suitable models for the evaluation of anti-stagnation of liver-qi effects of TCM.

中医“七情内伤”与疾病的发生密切相关。申请人在已发现情志应激能诱发小鼠非酒精性脂肪肝炎（NASH）的基础上，观察到情志应激诱发NASH相关肝纤维化与中、短链脂酰肉碱过度堆积及大量巨噬细胞聚集有关。肝脏巨噬细胞长期聚集并发生M2极化是肝纤维化发生的重要机制之一。巨噬细胞极化除了依赖于细胞因子外，还与脂肪酸氧化相关，提示了情志应激诱发NASH相关肝纤维化与M2型巨噬细胞摄取脂酰肉碱进行氧化代谢有关。本项目拟采用情志应激诱发NASH小鼠模型确认情志应激诱导肝脏巨噬细胞M2极化；采用巨噬细胞极化的体外模型确认其M2极化依赖于脂酰肉碱氧化代谢；利用13C标记的代谢流分析探究M2型巨噬细胞脂酰肉碱氧化代谢的作用机制。本项目采取通路抑制、基因沉默技术等确证作用和机制。研究结果有望阐明情志应激诱发NASH相关肝纤维化的发病机制，提升情志致病理论的科学内涵，建立与拓展适合疏肝解郁中药的药效评价方法和模型。

中医“七情内伤”与非酒精性脂肪肝炎（NASH）发病密切相关。目前情志应激诱发NASH相关肝纤维化的生物学机制仍不清楚。该项目基于促进巨噬细胞M2极化这一角度探究情志应激诱发NASH相关肝纤维化的作用机制，并且针对调控巨噬细胞M2极化相关靶点对疏肝解郁方剂及中药成分进行评价。该项目取得了以下研究成果：首先，建立两种NASH小鼠模型，给予拘束应激负荷以模拟情志应激，确认了情志应激诱导NASH相关纤维化与巨噬细胞M2极化有关。另外，拘束应激上调的短链脂肪酸能抑制巨噬细胞M1极化并促进M2极化，特别是丙酸的作用显著。此外，转录组及代谢组学分析发现拘束应激相关NASH肝纤维化中核受体、胆汁酸及脂肪酸代谢产物改变。进一步研究发现拘束应激调控法尼酯x受体 (FXR)/shp通路促进了JAK/STAT通路介导的巨噬细胞M2极化，并且依赖于代谢酶APOE。FXR激动剂逆转了巨噬细胞M2极化。针对筛选的靶点，我们评价了逍遥散、鹰嘴豆芽素A等中药及其成分对NASH的改善作用。本研究结果为情志因素诱发NASH相关肝纤维化的发病机制提供新的理论依据，为建立与拓展适合疏肝解郁中药的药效评价方法和评价模型提供参考，为情志致病理论内涵提供了科学依据。