基于CRF/CRF1系统探讨痛泻要方治疗肝郁脾虚型肠易激综合征内脏高敏感的中枢机制

Visceral hypersensitivity has been supposed to be one of the key pathophysiological characteristics in irritable bowel syndrome (IBS). Corticotropin releasing factor (CRF), one of the key hormones in hypothalamic-pituitary-adrenal axis (HPA)-mediated stress responses, is involved in stress-induced visceral hypersensitivity through central CRF/CRF1 pathway. Tongxie Yaofang decoction (TYD) as a classical prescription of traditional Chinese medicine is used for treating IBS with alleviating pain and anti-diarrheal effect. Previous studies showed that TYD regulates a variety of brain gut peptides secretion to maintaining the balance of brain-gut axis. However, its mechanism of action is still unclear. In view of a close relationship between central CRF/CRF1 and visceral hypersensitivity, the present study is designed to explore the mechanism of action of TYD in alleviating visceral hyperalgesia. Firstly, the effect of TYD on visceral hypersensitivity is evaluated in neonatal maternal separated male rats by colorectal distention. The c-fos and p-CREB expression in central tissue including brain and spine cord is tested to assure the central zone, of which takes part in the hyperalgesia. Meanwhile, the targeted tissue of TYD exerting lower visceral hypersensitivity is also explored. The expression of CRF/CRF1, GRK3/6 and β-arrestin1/2, and the level of AC, cAMP and PKA in targeted tissues are explored to demonstrate that TYD relieves visceral hypersensitivity of irritable bowel syndrome through central CRF/CRF1 signaling pathway. What is more, this project provides option for the research of traditional Chinese medicine on treating IBS.

内脏高敏感是肠易激综合征（IBS）的主要病理生理特征之一，促肾上腺皮质激素释放因子（CRF）作为下丘脑-垂体-肾上腺轴参与应激反应的关键调节因子，中枢CRF通过激活其受体CRF1参与应激诱导的内脏高敏感。痛泻要方作为中医临床治疗肝郁脾虚型IBS的经典方剂具有明确缓痛功效，既往研究显示其可调节脑-肠轴中多种脑-肠肽的分泌，但其作用机制仍不明确。本项目从脑肠轴出发以CRF为切入点，围绕中枢CRF/CRF1参与肠易激综合征内脏高敏感发生这一科学认识，利用母子分离联合慢性束缚建立肝郁脾虚型IBS大鼠模型，以即早基因c-fos探索参与内脏高敏感的中枢区域并分析痛泻要方靶向部位，重点研究靶向组织CRF、CRF1、GRK3/6及β-arrestin1/2的表达变化，并检测AC、cAMP及PKA含量，基于CRF/CRF1系统阐明痛泻要方临床缓痛的中枢机制，为治疗IBS中药的作用机制研究提供思路。

内脏高敏感是肠易激综合征（IBS）的主要病理生理特征之一。痛泻要方作为中医临床治疗肝郁脾虚型IBS的经典方剂具有明确缓痛效果。本课题通过母子分离联合慢性束缚应激诱导肝郁脾虚型内脏高敏感大鼠模型，从一般状况、进食量、体重增加、焦虑和抑郁行为学、粪便含水量、结直肠扩张诱导的腹壁撤退反射评分和腹壁肌电增幅等方面评价痛泻要方对模型大鼠肝郁脾虚证候和内脏高敏感的影响，结果表明痛泻要方灌胃大鼠在一般状况、进食量和体重增加方面有所改善，尤其以高剂量可明显增加大鼠体重；痛泻要方可改善旷场实验和高架十字迷宫实验中各项焦虑指标，也可明显改善强迫游泳中不动时间和糖水消耗量的抑郁指标，同时减少结直肠扩张诱导的腹壁回撤反射评分和腹壁肌电增幅。另外痛泻要方可降低模型大鼠小肠推进率，这可能与改善粪便含水量相关。阳性对照Astressin可改善模型大鼠焦虑与抑郁行为，降低小肠推进率，但对模型大鼠体重没有明显影响。在整体动物研究基础上，采集标本进行机制探索研究，结果显示痛泻要方可降低模型大鼠脑脊液CRF、血清ACTH和CORT含量，大脑皮层c-FOS阳性神经元数目减少，同时一方面通过降低杏仁核CRF和CRF1基因mRNA和蛋白表达，另一方面降低β-arrestin-1和β-arrestin-2表达调节CRF1受体脱敏，从而干预持续活化CRF/CRF1通路发挥调节内脏高敏感。本研究继续采用网络药理学探索痛泻要方调控内脏高敏感作用机理，方中58个活性化合物以协同和相加的方式作用于102个潜在靶点。相关性分析显示其中18个核心靶点集中在炎症反应、神经内分泌调节、肌肉收缩和激素调节。此外，13条关键通路也集中在炎症反应、神经内分泌功能、肌肉收缩和激素调节方面。其中对于神经内分泌功能的调节进一步丰富了痛泻要方治疗肠易激综合征的中枢作用机制。