白头翁汤通过HMGB1介导肠道固有免疫发挥抗炎作用的机制研究

A number of experiments and clinical researches have confirmed that Pulsatilla decoction has a definite therapeutic effect on ulcerative colitis, but few studies have investigated the mechanism. Innate immunity dysfunction in the pathogenesis of ulcerative colitis is gaining more and more attention.HMGB1 is an important inflammatory cytokine found in recent years and first activates the innate immune system.The preliminary study of the subject found that the effect of Pulsatilla Decoction is closely related with HMGB1, which has a clear inhibition of NF-κB signaling pathway and significant anti-inflammatory effect.In view of this, this project intends to study the intestinal anti-inflammatory mechanism of HMGB1 in the innate immune system from both overall animal and cell levels.Through serum pharmacology, western blot, laser scanning confocal microscopy and chromatin immunoprecipitation, we can reveal the anti-inflammatory effect of HMGB1 mediated innate immunity and clarify the intestinal anti-inflammatory mechanism from two receptor angles of TLR4 and RAGE.The implementation of the project is expected to preliminarily identify the role of HMGB1 in the innate immune system of the intestine and reveal the molecular immune mechanism of traditional Chinese medicine prescription in the treatment of ulcerative colitis innate immune system.It provides a new target for clinical drug intervention and clinical diagnosis, and provides a theoretical basis for the study of traditional Chinese medicine for the treatment of ulcerative colitis.

多项实验和临床研究证实白头翁汤对于溃疡性结肠炎具有明确的治疗作用，但鲜有研究探讨其作用机制。固有免疫功能障碍在溃疡性结肠炎发病中的作用越来越得到重视。HMGB1是近年发现的重要炎症细胞因子，并首先激活固有免疫系统。本课题前期研究发现，白头翁汤作用与HMGB1关系密切，具有明确的抑制NF-κB信号通路的作用，抗炎作用显著。鉴于此，本项目拟从整体动物和细胞两种水平，研究HMGB1在固有免疫系统中肠道抗炎机制。通过血清药理学、免疫印迹法、激光共聚焦和染色质免疫共沉淀方法等实验从TLR4和RAGE两个受体角度，揭示HMGB1介导固有免疫发挥抗炎作用，阐明其肠道抗炎机制。本项目的实施，预期将初步鉴定HMGB1在肠道固有免疫系统中的作用，揭示中医经典方剂治疗溃疡性结肠炎固有免疫系统分子免疫机制。为临床药物干预及临床诊断提供新的靶点，为研究传统中医药治疗溃疡性结肠炎提供理论依据。

溃疡性结肠炎(ulcerative colitis, UC)是一种非特异性、病因未明的结肠黏膜慢性炎症性疾病,因其病程长,病变程度轻重各异,反复发作而治愈难度大,被世界卫生组织列为现代难治病之一。溃疡性结肠炎发病机制目前仍不甚清楚，一般认为与遗传、环境、感染及免疫等多因素有关，近年来固有免疫功能障碍在溃疡性结肠炎发病中的作用越来越得到重视。HMGB1 是近年发现的重要炎症细胞因子，本研究利用两批动物实验，先后明确 HMGB1 在溃疡性结肠炎国有免疫过程中的抗炎作用。从大体情况，病理切片，细胞因子分析，目标蛋白的检测明确了白头翁汤对于葡聚糖硫酸钠盐(DSS)导致的肠道炎症动物模型的炎症状态改善作用，同时发现HMGB1水平显著下降。另外白头翁汤可以通过提高脱乙酰化酶水平，降低HMGB1的分泌，减轻炎症反应。另外我们通过细胞实验进一步验证，白头翁汤可以通过HMGB1调控TLR4受体，导致内质网应激下降，减少单核细胞产生多种细胞因子，如 IL-1β、IL-2、IL-6、IL-8、TNF-α的分泌，从而下调NF-KB信号通路，阻碍炎症急性发作过程。我们发现白头翁汤还可以促进与RAGE 受体结合下调HMGB1的含量，p-ERK，p-P38表达水平上升，p-jnk表达水平下降。调控中性粒细胞表面整合素家族黏附分子-1（mac-1）和晚期糖基化终末产物受体（receptor for advanced glycation end products, RAGE）的相互作用，阻碍炎症慢性发展过程。从而明确HMGB1了调控 TLR4 和 RAGE两个受体调控 NF-κB和MAPKs等相关信号通路调控在固有免疫抗炎作用及临床意义，为临床上中医方剂治疗溃疡性结肠炎提供新的干预靶点提供理论依据。