基于“肝开窍于目”理论探讨“肝郁”对EAU大鼠Treg/Th17的影响

The theory that “liver opening at eye” is one of the most important theories of traditional Chinese medicine, which is usually thought that the eye is directly related to the function of liver. Uveitis, a common cause of vision loss and autoimmune disorder is the major cause for the occurrence of uveitis. The syndrome of “liver depression” plays a central role in liver syndrome, which can affect the development and favorable recovery of multiple liver diseases. However, it is still unknown that how the syndrome of liver depression affects the development of autoimmune uveitis. A number of groups have recently shown that both T regulatory cells (Treg) and T helper cells (Th17) play an important role in the development and maintenance of EAU. However, the out-of-balance between Treg and Th17 will influence the occurrence and recurrence of autoimmune uveitis. Based on the theory of the syndrome of liver depression, an experimental autoimmune rat model with the syndrome of liver depression will be set up, and the model will be further evaluated by using behaviors and the determination of monoamine neurotransmitters. In the meantime, the effects of the syndrome of liver depression on the occurrence of EAU and on the ratio of Treg/Th17 cells in rats will be further investigated by means of flow cytometry, real-time quantitative PCR, western blot, ELISA techniques. Based on those results, the mechanisms of the effect of the syndrome of liver depression on the occurrence and development of EAU will be elucidated, and it will provide the thought and support for the understanding of the theory that “liver opening at eye”.

“肝开窍于目”是中医最重要理论学说之一，认为肝与目的功能状态直接相联。葡萄膜炎是常见、反复发作的致盲性眼病，自身免疫的紊乱是其发病的重要原因。肝郁证是肝病症候的核心，可影响多种自身免疫病的发展与转归，而从中医证候学角度研究肝郁证对葡萄膜炎影响的研究尚属空白。Treg/Th17细胞是自身免疫性葡萄膜炎致病过程中主要的免疫细胞，正常情况下两者处于一种效应平衡状态，两者的失衡对其发病与转归具有重要影响。本课题在中医肝郁证基础上，建立肝郁型EAU病证结合模型，采用行为学观察及单胺类神经递质检测对模型进行评价，并采用流式细胞术、荧光定量PCR、蛋白免疫印迹法、酶联免疫吸附试验等方法，探讨“肝郁”对EAU大鼠的发病及Treg/Th17细胞表达的影响，阐释“肝郁”对EAU影响的分子机制，为深入认识“肝开窍于目”理论奠定基础。

项目的背景:“肝开窍于目”是中医最重要理论学说之一，认为肝与目的功能状态直接相联。葡萄膜炎是常见、反复发作的致盲性眼病，自身免疫的紊乱是其发病的重要原因。Treg/Th17细胞是自身免疫性葡萄膜炎致病过程中主要的免疫细胞，正常情况下两者处于一种效应平衡状态，两者的失衡对其发病与转归具有重要影响。本课题在中医肝郁证基础上，建立肝郁型EAU病证结合模型，探讨“肝郁”对EAU大鼠的发病及Treg/Th17细胞表达的影响。主要研究内容:①EAU大鼠模型建立;②EAU大鼠Th17/Treg细胞及相关因子表达的动态研究；③肝郁EAU大鼠复合模型构建与评价；④肝郁对EAU大鼠Th17/Treg细胞及相关因子表达的影响；重要结果:①成功建立了EAU动物模型：免疫后第8天左右开始出现炎症，第13天炎症评分最高，然后眼部表现逐步缓解，至23天眼部炎症消退；②EAU模型大鼠Th17/Treg细胞及相关因子表达：EAU大鼠第18天、23天Treg/Th17比值分别为（3.240.49、6.401.52），与正常组相比差异有统计学意义（P=0.043、P=0.000），28天达到高峰（9.132.90），第35天比值与高峰相比出现有统计学意义的下降（P=0.047），其比值为（6.631.92），直至第48天依然维持在较高水平（5.521.18），与正常大鼠比较差异具有显著统计学意义（P=0.001）；③成功建立了肝郁EAU大鼠复合模型：造模第4周HPLC-EC检测各组大鼠海马组织抑制性神经递质（5-HT、DA、NE）含量与对照组相比，NE、DA、5-HT含量均明显下降，差异具有统计学意义；④肝郁对EAU大鼠Th17/Treg细胞及相关因子表达：肝郁整体上对EAU临床表现呈现抑制状态，免疫后第14天，两组Th17细胞达到高峰，应激EAU组略高，Treg细胞14天应激EAU组低于EAU组，随后细胞比例升高，28天达到高峰，高于EAU组。实时荧光PCR显示，RORγt与Foxp3变化趋势与流式检测细胞比例变化趋势基本一致；关键数据及其科学意义:①明确了Treg/Th17 失衡在EAU发病过程中的意义；②首次建立了肝郁型EAU模型，并对模型进行系统评价；③揭示“肝郁”影响EAU大鼠的病情的发展及转归的分子机制。