针灸对染色质重塑介导的克罗恩病自噬的调节机制研究

Based on a large number of the previous clinical and basic studies,which are confirmed that acupuncture and moxibustion can effectively control or decrease intestinal inflammatory and regulate the abnormal immunity in Crohn's disease (CD). Combined with the current international frontier theory that autophagy abnormalities is one of the important links during the occurrence and development of CD. The reseach will adopt the related technologies of molecular biology and epigenetics, use the bioinformatics analysis, and start the research from the point of autophagy gene transcriptions and abnormal expressions regulated by chromatin remodeling-related enzymes and histone H3 lysine modification. It intend to confirm from clinical and animal studies that: acupuncture and moxibustion may affect autophagy of CD by regulating the abnormal expressions of chromatin remodeling-related enzymes Brg1, LSD1, HDACs and hMOF, thus regulate the modification levels of H3K27me3, H3K4me2 and H3K27ac, and then affect the concentrations in autophagy-related target genes enhancer or promoter region, thereby regulate the expressions and transcriptions of autophagy-related genes and factors ATG16L1, IRGM and IL-18. Aim to clarify preliminarily the epigenetic regulatory mechanisms of acupuncture and moxibustion regulating CD autophagy mediated by chromatin remodeling, and provide the scientific and theoretical bases for further revealing the effect mechanism of acupuncture and moxibustion on CD.

在课题组前期大量临床与基础研究证实针灸能有效控制或缓解克罗恩病(Crohn’s disease,CD)肠道炎症和调节免疫异常的基础上，结合目前国际前沿“自噬异常是导致CD发生与发展的重要环节之一”理论认识，采用分子生物学与表观遗传学相关技术，结合生物信息学分析，以染色质重塑相关酶及组蛋白H3赖氨酸修饰所调控的自噬相关靶基因转录与表达异常为切入点，拟从临床与动物研究二方面证实：针灸调节CD自噬效应可能是通过调节CD染色质重塑相关酶Brg1、LSD1、HDACs、hMOF的异常表达，继而调控组蛋白H3K27me3、H3K4me2和H3K27ac修饰水平，影响其在自噬相关靶基因增强子或启动子区域的富集，由此调控自噬相关基因/因子ATG16L1、IRGM、IL-18的转录与表达，初步阐明针灸调节染色质重塑介导的CD自噬的表观遗传调控机制，以期为深入揭示针灸治疗CD的效应机制提供科学依据和理论基础。

本项目以染色质重塑相关酶及组蛋白H3赖氨酸修饰所调控的自噬相关靶基因转录与表达异常为切入点，拟从临床研究入手，比较隔药灸结合针刺组与隔麸灸结合安慰针刺组治疗CD的临床疗效，并提取外周血PBMCs进行RNA-seq检测其全基因转录组水平和ChIP-seq检测、ChIP-assay验证H3K27me3、H3K4me2和H3K27ac在自噬相关靶基因启动子和/或增强子区域的富集情况，通过生物信息学分析探索针灸疗效相关的自噬相关靶基因表达与染色质重塑的关系。再从实验研究入手，在明确针灸临床治疗CD有效的基础上，探讨染色质重塑相关酶Brg1、LSD1、HDAC3、HDAC9、hMOF与组蛋白H3K27me3、H3K4me2和H3K27ac修饰之间的关系以及对自噬相关基因/因子ATG16L1、IRGM、IL-18表达的调控作用。结果显示：（1）针灸(隔药灸结合针刺)治疗能够有效缓解CD肠组织炎症；显著降低自噬相关因子ATG16L1、IRGM、IL-18表达水平。（2）染色质重塑相关酶Brg1、LSD1、HDAC3、HDAC9、hMOF表达异常可通过改变组蛋白H3K27me3、H3K4me2和H3K27ac的修饰水平，继而影响CD自噬相关基因/因子ATG16L1、IRGM、IL-18增强子或启动子的转录与表达，是染色质重塑介导CD自噬的重要调控蛋白。（3）针灸(隔药灸结合针刺)治疗可通过对CD外周血和结肠上皮组织染色质重塑相关酶 Brg1、LSD1、HDAC3、HDAC9、hMOF的异常表达进行调节，继而调控组蛋白H3K27me3、H3K4me2和H3K27ac修饰水平，影响其在自噬相关靶基因增强子或启动子区域的富集，由此调控自噬相关基因/因子ATG16L1、IRGM、IL-18的转录与表达，达到调节CD自噬异常之效应。本项目初步阐释了针灸调节染色质重塑介导CD自噬的表观遗传调控机制。