温肾健脾法干预IBS-D致脑-肠CRF系统神经可塑性改变的机制研究

Visceral hypersensitivity is the core of IBS pathogenesis mechanism, brain-gut nervous plasticity which includes neurotransmitters, synapses, and the change of the neural circuits, is one of the important link to internal high sensitive. The brain-gut CRF system has attracted much more attention. The last fund project found that: chronic stress composite building can affect neurotransmitters, regulation and control of the synaptic plasticity, Warming Kidney And Strengthening Spleen Method involved in regulation of IBS-D related peptides hormone and the plasticity of the mast cells,which is one of the mechanisms of Warming Kidney And Strengthening Spleen Method improving IBS symptoms.Also we observed that Warming Kidney And Strengthening Spleen Method can affect the change of the neural circuits in the CRF, elevate serum 5-HT. Based on previous study, we hypothesize that chronic stress composite building method can affect the IBS-D brain-gut CRF system(neurotransmitters and receptors and signaling pathways) neural plasticity, and method of Warming Kidney And Strengthening Spleen could improve the intestinal CRF system of neural plasticity of IBS-D. This project intends to apply methods of nerve electrophysiology and molecular biology to research how the method of Warming Kidney And Strengthening Spleen treat IBS-D relating to change of the neural circuits in the CRF. We want to interprets method of Warming Kidney And Strengthening Spleen treat visceral hypersensitivity syndroms by changing the brain-gut neural plasticity.

内脏高敏感是IBS发病的核心机制，脑-肠神经可塑性包括神经递质、神经突触及神经环路的改变，是致内脏高敏感的重要环节之一，其中，脑-肠CRF系统备受关注。上一个基金项目发现：慢性应激复合造模法可影响局部神经递质、神经突触可塑性调控，而温肾健脾法参与调控IBS-D相关肽类激素及肥大细胞的可塑性，是改善其内脏高敏症状的机制之一。同时观察到温肾健脾法可影响神经环路中CRF的改变，血清5-HT升高。基于既往研究，我们提出假说：温肾健脾法干预IBS-D是通过影响脑-肠神经CRF系统（包括：递质、受体以及信号通路）的神经可塑性，降低内脏高敏性而发挥治疗作用。本项目拟应用神经电生理、分子生物学等方法，研究温肾健脾法对IBS-D脑-肠CRF相关神经信号系统可塑性改变的影响，综合前期基础系统诠释温肾健脾法通过改变脑-肠神经可塑性治疗IBS-D内脏高敏反应症状的机制。项目实施将为中医药治疗IBS积累新思路。

目的：研究温肾健脾法对IBS-D脑-肠CRF相关神经信号系统可塑性改变的影响。方法：构建脾肾阳虚IBS-D动物模型及进行评价。并以该模型为研究对象，研究温肾健脾法干预CRF系统在5-HT信号通路神经可塑性改变的信号传导机制、温肾健脾致内脏敏感IBS-D大鼠CRF系统神经可塑性和SRF-IEG(Arc和c-fos)信号通路神经可塑性改变的机制。结果：①母婴分离+番泻叶灌胃及避水应激+番泻叶灌胃法是建立脾肾阳虚型IBS-D动物模型的可取方法，温肾健脾方可降低IBS-D大鼠内脏高敏感。②温肾健脾方可降低模型大鼠结肠黏膜5-HT、5-HT3R、5-HT4R的表达。③中药温肾健脾方高、中、低剂量可剂量依赖性的下调结肠CRF、CRF-R1、CRF mRNA的表达，上调CRF-R2表达；高剂量组可下调下丘脑CRF、CRF-R1的表达。④高剂量温肾健脾方可调节CREB/BDNF/TrkB通路上信号分子的异常表达及下调早期即刻基因的表达。结论：①番泻叶灌胃法（10g/kg‧d，4周）可建立脾肾阳虚型腹泻模型；番泻叶灌胃联合母婴分离、避水应激，可建立病证结合脾肾阳虚型IBS-D大鼠模型，并初步建立了“宏观体征-生物标志物-药物反证-造模因素反推”模型的四维评价体系。②脾肾阳虚型IBS-D大鼠模型结肠、下丘脑存在CRF-R1—CRF-R2失衡状态以及脑和肠不同层面上均有CREB/BDNF/TrkB信号通路的激活和早期即刻基因arc、c-fos介导的神经可塑性改变，从而导致了内脏高敏和肠道紊乱；中药温肾健脾方可调整CRF系统的失衡状态、影响5-HT及其受体5-HT3R、5-HT4R表达、调节CREB/BDNF/TrkB信号通路上信号分子的异常表达及下调早期即刻基因的表达，从而调节肠神经系统，降低肠道内脏高敏感，减弱肠道运动及分泌，达到治疗疾病的目的。