温肾健脾法干预脑-肠互动腹泻型肠易激综合征大鼠蛋白质组学研究

Diarrhea-predominant Irritable bowel syndrome (IBS-D) is a common functional bowel disease.In Traditional Chinese Medicine(TCM),it belongs to "diarrhoea","abdominal pain",etc.In recent years,the brain-gut axis(BGA) and proteomics are hot topics to probe into the cause of IBS.In the previous fund,research findings were that:Warming Kidney and Tonifying Spleen had an important part in regulating nerve,endocrine,immune of IBS-D rats'colonic mucosa,but targets were not sure,considered more points, pathways and effects.Based on the previous studies,we propose a hypothesis:Mechanism of Warming Kidney and Tonifying Spleen interventions on IBS-D may be by intervening key proteins in different levels of BGA.Our research plan to study systematically on proteomics of IBS-D in different levels of BGA:Observing key proteins of IBS-D rats’ colon in brain-gut interaction by two-dimensional gel electrophoresis,aim at key proteins, testing the expression in Western-blot and immunohistochemical staining,and also the effect of Warming Kidney and Tonifying Spleen interventions on IBS-D,to deeply explore the mechanism of Warming Kidney and Tonifying Spleen interventions and provide a new data for TCM research on IBS-D.

腹泻型肠易激综合征（IBS-D）是常见的功能性肠病，属中医“泄泻”、“腹痛”等范畴。脑-肠轴与蛋白质组学为近年来IBS发生机制研究的热点。前期研究发现温肾健脾法在调控IBS-D大鼠结肠神经、内分泌、免疫等方面具有重要作用，但中药复方作用靶点并不肯定，目前认为是多靶点、多途径、多效应的。基于既往研究，而以脑-肠轴为靶点系统探寻中医药对IBS-D蛋白质组学研究较少。我们提出假说: 温肾健脾法对IBS-D的疗效机制可能为通过干预脑-肠轴不同水平的差异蛋白而产生综合效应。拟从脑-脊髓-肠不同层次探索IBS-D蛋白质组学机制：采用双向凝胶电泳质谱分析技术筛选脑-肠互动IBS-D大鼠结肠组织的重点差异蛋白；针对重点差异蛋白，通过W-blot和免疫组化法分别在脑实质、脊髓进行蛋白质组学的验证，从而深入探讨温肾健脾法的疗效机制，为中医药治疗IBS-D的研究提供新的依据。

中医针对IBS-D治疗具有一定优势，并认为其病因多集中于情志所伤、饮食不洁、素体虚弱等几个方面，而参考病程漫长、病情反复频发的疾病特点，久病及肾，导致脾肾阳虚的证型变化，辨证论治采取温肾健脾法进行治疗。研究目的：基于脑-肠互动，采用TMT蛋白组学技术探索温肾健脾法干预脾肾阳虚证IBS-D大鼠的作用机制。研究内容：通过国内外文献分析，综述IBS目前的中西医病因病机、诊治和蛋白组学研究进展；采用meta分析，对温肾健脾法治疗IBS-D的临床疗效及安全性等进行评估；基于前期动物实验研究，对脾肾阳虚证IBS-D大鼠进行行为学观察及腹泻症状、内脏敏感性和肠道动力的疗效评价；基于TMT蛋白组学技术，分析温肾健脾法干预脾肾阳虚证IBS-D大鼠结肠组织和海马组织重点差异蛋白的变化，并探索脑-肠互动的作用途径。研究结论：1.文献综述及Meta分析发现，中西医对IBS-D的疾病理解有异同之处，而以温肾健脾法指导中药临证治疗IBS-D的整体疗效突出；2.温肾健脾方中剂量（1.55g/kg/d）和高剂量（3.10g/kg/d）对脾肾阳虚证IBS-D大鼠的腹泻症状、内脏敏感性和肠道动力等有明显改善作用；同时，温肾健脾方中剂量在行为学改善方面有一定治疗优势；3.蛋白组学研究发现，脾肾阳虚证IBS-D在肠组织及海马组织中均存在调节肠道粘膜与菌群环境、动力、炎症、免疫和神经等的蛋白表达异常；海马组织中Slc4a7与结肠组织中Tgm2的表达上调可能诱发了脾肾阳虚证IBS-D的脑-肠互动异常；温肾健脾方参与调节海马及结肠蛋白的表达，可能通过降低应激改变和内脏敏感性、维持神经稳态、调节肠道动力和免疫等发挥了治疗作用。