基于脑—肠互动温肾健脾法干预腹泻型肠易激综合征的蛋白组学研究

发布时间：2017-12-26 18:33

  本文关键词：基于脑—肠互动温肾健脾法干预腹泻型肠易激综合征的蛋白组学研究　出处：《北京中医药大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 蛋白组学 腹泻型肠易激综合征 脑肠互动 温肾健脾法

【摘要】：研究背景肠易激综合征(irritable bowel syndrome,IBS)为常见的功能性胃肠病(fuuctional gastrointestinal disorders,FGIDs),全球发病率为9.8%～12.8%,发病具有多因素性,机制假说繁多,症状特异性差,确诊困难。IBS患者常因腹部症状、粪便性状改变、排便频率异常等就诊。在排除器质性疾病的漫长过程中,治疗持续性差,导致症状反复、频繁就医,严重影响患者生活质量和心身健康。根据罗马Ⅲ和罗马Ⅳ的诊断,IBS主要包括 4 种亚型:便秘型 IBS(IBS-constipation,IBS-C),腹泻型 IBS(IBS-diarrhea,IBS-D),混合型 IBS(IBS-mixed type,IBS-M)以及未确定型 IBS(IBS-unclassified,IBS-U),其中,IBS-D在我国发病率最高,是IBS最常见的类型。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。进一步针对温肾健脾法治疗IBS-D的随机对照研究进行meta分析,认为本法在治疗IBS-D整体疗效方面确有优势,同时安全性高、不良反应少。温肾健脾方是由治疗五更泻的经典名方四神丸加减化裁而来,临证治疗脾肾阳虚证IBS-D获得较好疗效,并通过研究证实其可明显缓解患者焦虑抑郁和腹痛腹泻症状,考虑其治疗可能是通过脑-肠互动发挥了作用。蛋白组学作为从系统化的角度解读疾病的研究方法,有助于从蛋白质的表达中挖掘疾病特征和治疗规律,而目前针对脾肾阳虚证IBS-D,运用蛋白组学方法进行机制及疗效探索尚属空白。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。研究方法1.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。2.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。3.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。4.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。研究结果1 中西医研究进展的文献综述IBS的发病发展为多因素共同作用的结果,其中脑-肠互动异常为其重要病机。现代医学治疗手段主要包括药物治疗、饮食和心理治疗等,但往往难以充分缓解患者症状、控制病情反复。作为最常见的临床亚型,IBS-D的蛋白组学研究较少,与脾肾阳虚证相关的蛋白组学研究尚属空白。中医治疗IBS-D从症状、体质入手,以整体观、恒动观的角度进行分析,认为脾肾阳虚证为本病的常见证型之一,并通常运用温肾健脾法进行治疗,疗效较为满意,但中医药的治疗机制尚未明确,值得进一步研究。2 温肾健脾法治疗IBS-D的荟萃分析检索 Cochrane Library、Pub Med、Embase、CNKI、CBM、万方数据库和维普中文科技期刊数据库自建库至2017年01月20日关于温肾健脾法治疗脾肾阳虚证IBS-D的临床随机对照试验(Randomized Controlled Trial,RCT),共纳入10个RCTs。分析后发现:主要结局指标评定中,温肾健脾法治疗脾肾阳虚证IBS-D的整体疗效较西药常规疗法体现出了优势,在腹痛、腹泻症状改善方面二者无明显差异;中医证候疗效评价方面,由于只有4项RCTs报告了中医证候疗效,样本量过小,无法判断是否有统计学差异(P=0.05);复发率、不良反应方面,温肾健脾法较常规西药治疗的复发率低、不良反应少,但由于评价的样本量小,结果的普遍性较局限。研究证实温肾健脾法指导了多种方药用于脾肾阳虚证IBS-D的临证治疗并具有一定优势。3 温肾健脾法治疗脾肾阳虚证IBS-D大鼠的疗效评价基于前期研究基础,进行温肾健脾方治疗脾肾阳虚证IBS-D大鼠的疗效评价后发现:温肾健脾方中剂量(1.55g/kg/d)和高剂量(3.10g/kg/d)在腹泻症状、内脏敏感性和肠道动力方面的改善较为明显,并可达到与匹维溴铵相似的疗效;此外,温肾健脾方中剂量治疗脾肾阳虚证IBS-D大鼠,在行为学改善方面有一定优势。4 温肾健脾法干预脾肾阳虚证IBS-D大鼠脑-肠互动蛋白组学研究筛选脾肾阳虚证IBS-D模型大鼠与正常组大鼠结肠组织的差异蛋白,并分析不同治疗组的蛋白表达改变,确定了 3个主要蛋白(Wbp11、Gsk3b和Tgm2)和3个次要蛋白(Cox6b1、Acss2 和 Mrpl12),并发现存在 Metabolic pathways、Oxidative phosphorylation、Ribosome、Alzheimer's disease、Huntington's disease 和 Spliceosome 6 个信号通路的改变。在不同方向提示了脾肾阳虚证IBS发病与肠道环境、动力、炎症、免疫及神经调节等改变相关,而温肾健脾方治疗后蛋白表达回归,提示了其在以上方面有调节作用,并于维持神经稳态方面可能存在优势。筛选脾肾阳虚证IBS-D模型大鼠与正常组大鼠海马组织的差异蛋白,并分析不同治疗组的蛋白表达改变,确定了 1个主要差异蛋白(Slc4a7)和3个次要差异蛋白(Cacnb2、Rbp4和Rgs14)。我们发现海马体中差异蛋白Slc4a7与结肠组织中差异蛋白Tgm2在IBS-D发病的作用机制均涉及神经递质谷氨酸和谷氨酰胺,二者于造模后共同呈现上调趋势,推测二者在IBS-D发病过程中引起了内脏高敏感、肠道免疫和神经稳态状态改变,且二者的相关性可能是基于脑-肠互动实现的;进一步行ELISA验证,确定结果与质谱分析具有一致性。此外,差异蛋白Cacnb2、Rbp4和Rgs14在中枢系统和肠道存在双重作用,推测其可能参与了IBS-D的脑-肠互动过程。温肾健脾方影响了海马蛋白的表达,可能通过参与以上蛋白的调节过程,降低了应激改变和内脏敏感性,调节了神经稳态、肠道动力和免疫等,从而发挥了治疗作用。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。
[Abstract]:Background: irritable bowel syndrome (IBS) is a common functional gastrointestinal disease (fuuctional gastrointestinal disorders, FGIDs). The incidence of the disease is 9.8% to 12.8%. The pathogenesis is multifactorial. The mechanism is various, and the symptom is poor. It is difficult to diagnose accurately. IBS patients were often diagnosed with abdominal symptoms, changes in stool traits and abnormal frequency of defecation. In the long process of eliminating organic diseases, the persistent poor treatment leads to repeated symptoms and frequent medical treatment, which seriously affects the quality of life and physical and mental health of the patients. According to the diagnosis of Rome and Rome III IV, IBS includes 4 subtypes: constipation type IBS (IBS-constipation, IBS-C), IBS (IBS-diarrhea, IBS-D type of diarrhea), hybrid IBS (IBS-mixed type IBS-M) and unidentified type IBS (IBS-unclassified, IBS-U), among them, IBS-D was the highest incidence in our country IBS is the most common type. Traditional Chinese medicine for IBS-D treatment has certain advantages, and that the cause of injury, mostly to emotional eating unclean, debility etc., disease characteristics and reference the long duration of disease and frequent repeated illness, chronic illness and kidney, cause different syndromes of spleen kidney yang deficiency, syndrome differentiation and treatment to take the method of warming kidney and invigorating spleen treatment. Further, meta analysis was conducted for randomized controlled trials of warming kidney and invigorating spleen for treatment of IBS-D. It is believed that this method has advantages in treating IBS-D overall efficacy, and has high safety and fewer side effects. Wenshenjianpi recipe by treatment of even purging the classical prescription pill chmical process and clinical effect in treating spleen kidney yang deficiency syndrome of IBS-D, and through the study confirmed that it can significantly relieve the anxiety and depression of patients with abdominal pain and diarrhea, the treatment may be through brain gut interaction play a role. Proteomics, as a research method for interpreting diseases from a systematic perspective, helps to discover disease characteristics and treatment rules from protein expression. At present, it is still a blank for us to explore the mechanism and efficacy of proteomics based on IBS-D. The purpose of this study is to explore the mechanism of the intervention of the spleen kidney yang deficiency syndrome (IBS-D) rats by using the TMT proteomics technique based on the interaction of brain and intestine. Analysis of 1. research methods through literature, Chinese and Western medicine review IBS's current due to illness, diagnosis and proteomics research progress. 2. the clinical efficacy and safety of IBS-D with warm kidney and spleen strengthening method were evaluated by meta analysis. 3. on the basis of early animal experiment, the behavioral observation of IBS-D rats with spleen kidney yang deficiency syndrome and the evaluation of the effect of diarrhea symptoms, visceral sensitivity and intestinal motility were evaluated. 4., based on TMT proteomics technology, we analyzed the changes of key differentially expressed proteins in colonic tissues and hippocampus tissues of IBS-D rats, and explored the way of interaction between brain and intestine by warming kidney and invigorating spleen. Research results 1 literature review on the progress of Chinese and Western medicine research. The development of IBS is the result of multiple factors, in which the abnormal brain intestinal interaction is the important pathogenesis. Modern medical treatment means mainly including drug treatment, diet and psychotherapy, but it is often difficult to relieve the symptoms and control the relapse of the disease. As the most common subtype of clinical subtype, the proteomics research of IBS-D is less, and the study of proteomics related to spleen and kidney yang deficiency is still a blank. Chinese medicine treatment of IBS-D symptoms, starting from the constitution, analyzed in the view of the whole, Hengdong view that spleen kidney yang deficiency syndrome is one of the common syndromes of this disease, and often use of warming kidney and invigorating spleen method treatment, curative effect is satisfactory, but the treatment mechanism of Chinese medicine is not yet clear, worthy of further study. Cochrane Library Pub, meta analysis retrieval Med, Embase, CNKI, CBM, Wanfang database and VIP database from Chinese journals up to 2017 01 20 randomized clinical trials on treatment of warming kidney and invigorating spleen yang deficiency of spleen and kidney IBS-D 2 warming kidney and strengthening spleen treatment of IBS-D (Randomized Controlled Trial, RCT). A total of 10 RCTs. The analysis found that: the primary outcome evaluation, the overall efficacy of warming kidney and invigorating spleen method in treatment of spleen kidney yang deficiency IBS-D compared with conventional Western therapy showed advantages in abdominal pain and diarrhea symptoms two have no obvious difference; evaluation of TCM syndromes, with only 4 RCTs reports of TCM syndrome, the sample size is too small, unable to determine whether there is a significant difference (P=0.05); the recurrence rate and adverse reactions, warming kidney and invigorating spleen method than the conventional western medicine treatment with low recurrence rate, less adverse reaction, but the evaluation of small sample size, the universality is limited. The study confirmed that the method of warming kidney and invigorating spleen has guided many kinds of prescription drugs in the treatment of spleen kidney yang deficiency syndrome IBS-D, and has some advantages. Evaluation of the curative effect of 3 treatment method of warming kidney and invigorating spleen yang deficiency of spleen and kidney of IBS-D rats on the basis of previous research, evaluation of curative effect of Jianpi Wenshen Decoction in the treatment of spleen kidney yang deficiency IBS-D rats were found: dose of wenshenjianpi recipe in (1.55g/kg/d) and high dose (3.10g/kg/d) in diarrhea and intestinal motility, visceral sensitivity the improvement is more obvious, and can achieve a similar effect with pinaverium bromide; in addition, wenshenjianpi recipe in dose treatment of spleen kidney yang deficiency IBS-D rats, there are certain advantages in behavioral improvement. The 4 intervention method of warming kidney and invigorating spleen yang deficiency of spleen and kidney of IBS-D rat brain gut interaction protein group study screening model of Yang deficiency of spleen and kidney of IBS-D rats and normal rats colon tissue protein, and analysis of different treatment groups the protein expression changes, identified 3 main proteins (Wbp11, Gsk3b and Tgm2) and 3 minor proteins (Cox6b1, Acss2 and Mrpl12), pathways, Oxidative and Metabolic found phosphorylation, Ribosome, Alzheimer's disease, Huntington's disease and Spliceosome 6 signaling pathway. In different directions, the pathogenesis of spleen kidney yang deficiency syndrome IBS and the intestinal environment, motive force, inflammation and immunity
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R259
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