本文關(guān)鍵詞：基于腦—腸互動(dòng)溫腎健脾法干預(yù)腹瀉型腸易激綜合征的蛋白組學(xué)研究　出處：《北京中醫(yī)藥大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：研究背景腸易激綜合征(irritable bowel syndrome,IBS)為常見(jiàn)的功能性胃腸病(fuuctional gastrointestinal disorders,FGIDs),全球發(fā)病率為9.8%～12.8%,發(fā)病具有多因素性,機(jī)制假說(shuō)繁多,癥狀特異性差,確診困難。IBS患者常因腹部癥狀、糞便性狀改變、排便頻率異常等就診。在排除器質(zhì)性疾病的漫長(zhǎng)過(guò)程中,治療持續(xù)性差,導(dǎo)致癥狀反復(fù)、頻繁就醫(yī),嚴(yán)重影響患者生活質(zhì)量和心身健康。根據(jù)羅馬Ⅲ和羅馬Ⅳ的診斷,IBS主要包括 4 種亞型:便秘型 IBS(IBS-constipation,IBS-C),腹瀉型 IBS(IBS-diarrhea,IBS-D),混合型 IBS(IBS-mixed type,IBS-M)以及未確定型 IBS(IBS-unclassified,IBS-U),其中,IBS-D在我國(guó)發(fā)病率最高,是IBS最常見(jiàn)的類(lèi)型。中醫(yī)針對(duì)IBS-D治療具有一定優(yōu)勢(shì),并認(rèn)為其病因多集中于情志所傷、飲食不潔、素體虛弱等幾個(gè)方面,而參考病程漫長(zhǎng)、病情反復(fù)頻發(fā)的疾病特點(diǎn),久病及腎,導(dǎo)致脾腎陽(yáng)虛的證型變化,辨證論治采取溫腎健脾法進(jìn)行治療。進(jìn)一步針對(duì)溫腎健脾法治療IBS-D的隨機(jī)對(duì)照研究進(jìn)行meta分析,認(rèn)為本法在治療IBS-D整體療效方面確有優(yōu)勢(shì),同時(shí)安全性高、不良反應(yīng)少。溫腎健脾方是由治療五更瀉的經(jīng)典名方四神丸加減化裁而來(lái),臨證治療脾腎陽(yáng)虛證IBS-D獲得較好療效,并通過(guò)研究證實(shí)其可明顯緩解患者焦慮抑郁和腹痛腹瀉癥狀,考慮其治療可能是通過(guò)腦-腸互動(dòng)發(fā)揮了作用。蛋白組學(xué)作為從系統(tǒng)化的角度解讀疾病的研究方法,有助于從蛋白質(zhì)的表達(dá)中挖掘疾病特征和治療規(guī)律,而目前針對(duì)脾腎陽(yáng)虛證IBS-D,運(yùn)用蛋白組學(xué)方法進(jìn)行機(jī)制及療效探索尚屬空白。研究目的基于腦-腸互動(dòng),采用TMT蛋白組學(xué)技術(shù)探索溫腎健脾法干預(yù)脾腎陽(yáng)虛證IBS-D大鼠的作用機(jī)制。研究方法1.通過(guò)國(guó)內(nèi)外文獻(xiàn)分析,綜述IBS目前的中西醫(yī)病因病機(jī)、診治和蛋白組學(xué)研究進(jìn)展。2.采用meta分析,對(duì)溫腎健脾法治療IBS-D的臨床療效及安全性等進(jìn)行評(píng)估。3.基于前期動(dòng)物實(shí)驗(yàn)研究,對(duì)脾腎陽(yáng)虛證IBS-D大鼠進(jìn)行行為學(xué)觀察及腹瀉癥狀、內(nèi)臟敏感性和腸道動(dòng)力的療效評(píng)價(jià)。4.基于TMT蛋白組學(xué)技術(shù),分析溫腎健脾法干預(yù)脾腎陽(yáng)虛證IBS-D大鼠結(jié)腸組織和海馬組織重點(diǎn)差異蛋白的變化,并探索腦-腸互動(dòng)的作用途徑。研究結(jié)果1 中西醫(yī)研究進(jìn)展的文獻(xiàn)綜述IBS的發(fā)病發(fā)展為多因素共同作用的結(jié)果,其中腦-腸互動(dòng)異常為其重要病機(jī)。現(xiàn)代醫(yī)學(xué)治療手段主要包括藥物治療、飲食和心理治療等,但往往難以充分緩解患者癥狀、控制病情反復(fù)。作為最常見(jiàn)的臨床亞型,IBS-D的蛋白組學(xué)研究較少,與脾腎陽(yáng)虛證相關(guān)的蛋白組學(xué)研究尚屬空白。中醫(yī)治療IBS-D從癥狀、體質(zhì)入手,以整體觀、恒動(dòng)觀的角度進(jìn)行分析,認(rèn)為脾腎陽(yáng)虛證為本病的常見(jiàn)證型之一,并通常運(yùn)用溫腎健脾法進(jìn)行治療,療效較為滿意,但中醫(yī)藥的治療機(jī)制尚未明確,值得進(jìn)一步研究。2 溫腎健脾法治療IBS-D的薈萃分析檢索 Cochrane Library、Pub Med、Embase、CNKI、CBM、萬(wàn)方數(shù)據(jù)庫(kù)和維普中文科技期刊數(shù)據(jù)庫(kù)自建庫(kù)至2017年01月20日關(guān)于溫腎健脾法治療脾腎陽(yáng)虛證IBS-D的臨床隨機(jī)對(duì)照試驗(yàn)(Randomized Controlled Trial,RCT),共納入10個(gè)RCTs。分析后發(fā)現(xiàn):主要結(jié)局指標(biāo)評(píng)定中,溫腎健脾法治療脾腎陽(yáng)虛證IBS-D的整體療效較西藥常規(guī)療法體現(xiàn)出了優(yōu)勢(shì),在腹痛、腹瀉癥狀改善方面二者無(wú)明顯差異;中醫(yī)證候療效評(píng)價(jià)方面,由于只有4項(xiàng)RCTs報(bào)告了中醫(yī)證候療效,樣本量過(guò)小,無(wú)法判斷是否有統(tǒng)計(jì)學(xué)差異(P=0.05);復(fù)發(fā)率、不良反應(yīng)方面,溫腎健脾法較常規(guī)西藥治療的復(fù)發(fā)率低、不良反應(yīng)少,但由于評(píng)價(jià)的樣本量小,結(jié)果的普遍性較局限。研究證實(shí)溫腎健脾法指導(dǎo)了多種方藥用于脾腎陽(yáng)虛證IBS-D的臨證治療并具有一定優(yōu)勢(shì)。3 溫腎健脾法治療脾腎陽(yáng)虛證IBS-D大鼠的療效評(píng)價(jià)基于前期研究基礎(chǔ),進(jìn)行溫腎健脾方治療脾腎陽(yáng)虛證IBS-D大鼠的療效評(píng)價(jià)后發(fā)現(xiàn):溫腎健脾方中劑量(1.55g/kg/d)和高劑量(3.10g/kg/d)在腹瀉癥狀、內(nèi)臟敏感性和腸道動(dòng)力方面的改善較為明顯,并可達(dá)到與匹維溴銨相似的療效;此外,溫腎健脾方中劑量治療脾腎陽(yáng)虛證IBS-D大鼠,在行為學(xué)改善方面有一定優(yōu)勢(shì)。4 溫腎健脾法干預(yù)脾腎陽(yáng)虛證IBS-D大鼠腦-腸互動(dòng)蛋白組學(xué)研究篩選脾腎陽(yáng)虛證IBS-D模型大鼠與正常組大鼠結(jié)腸組織的差異蛋白,并分析不同治療組的蛋白表達(dá)改變,確定了 3個(gè)主要蛋白(Wbp11、Gsk3b和Tgm2)和3個(gè)次要蛋白(Cox6b1、Acss2 和 Mrpl12),并發(fā)現(xiàn)存在 Metabolic pathways、Oxidative phosphorylation、Ribosome、Alzheimer's disease、Huntington's disease 和 Spliceosome 6 個(gè)信號(hào)通路的改變。在不同方向提示了脾腎陽(yáng)虛證IBS發(fā)病與腸道環(huán)境、動(dòng)力、炎癥、免疫及神經(jīng)調(diào)節(jié)等改變相關(guān),而溫腎健脾方治療后蛋白表達(dá)回歸,提示了其在以上方面有調(diào)節(jié)作用,并于維持神經(jīng)穩(wěn)態(tài)方面可能存在優(yōu)勢(shì)。篩選脾腎陽(yáng)虛證IBS-D模型大鼠與正常組大鼠海馬組織的差異蛋白,并分析不同治療組的蛋白表達(dá)改變,確定了 1個(gè)主要差異蛋白(Slc4a7)和3個(gè)次要差異蛋白(Cacnb2、Rbp4和Rgs14)。我們發(fā)現(xiàn)海馬體中差異蛋白Slc4a7與結(jié)腸組織中差異蛋白Tgm2在IBS-D發(fā)病的作用機(jī)制均涉及神經(jīng)遞質(zhì)谷氨酸和谷氨酰胺,二者于造模后共同呈現(xiàn)上調(diào)趨勢(shì),推測(cè)二者在IBS-D發(fā)病過(guò)程中引起了內(nèi)臟高敏感、腸道免疫和神經(jīng)穩(wěn)態(tài)狀態(tài)改變,且二者的相關(guān)性可能是基于腦-腸互動(dòng)實(shí)現(xiàn)的;進(jìn)一步行ELISA驗(yàn)證,確定結(jié)果與質(zhì)譜分析具有一致性。此外,差異蛋白Cacnb2、Rbp4和Rgs14在中樞系統(tǒng)和腸道存在雙重作用,推測(cè)其可能參與了IBS-D的腦-腸互動(dòng)過(guò)程。溫腎健脾方影響了海馬蛋白的表達(dá),可能通過(guò)參與以上蛋白的調(diào)節(jié)過(guò)程,降低了應(yīng)激改變和內(nèi)臟敏感性,調(diào)節(jié)了神經(jīng)穩(wěn)態(tài)、腸道動(dòng)力和免疫等,從而發(fā)揮了治療作用。研究結(jié)論1.文獻(xiàn)綜述及Meta分析發(fā)現(xiàn),中西醫(yī)對(duì)IBS-D的疾病理解有異同之處,而以溫腎健脾法指導(dǎo)中藥臨證治療IBS-D的整體療效突出。2.溫腎健脾方中劑量(1.55g/kg/d)和高劑量(3.10g/kg/d)對(duì)脾腎陽(yáng)虛證IBS-D大鼠的腹瀉癥狀、內(nèi)臟敏感性和腸道動(dòng)力等有明顯改善作用;同時(shí),溫腎健脾方中劑量在行為學(xué)改善方面有一定治療優(yōu)勢(shì)。3.蛋白組學(xué)研究發(fā)現(xiàn),脾腎陽(yáng)虛證IBS-D在腸組織及海馬組織中均存在調(diào)節(jié)腸道粘膜與菌群環(huán)境、動(dòng)力、炎癥、免疫和神經(jīng)等的蛋白表達(dá)異常;海馬組織中Slc4a7與結(jié)腸組織中Tgm2的表達(dá)上調(diào)可能誘發(fā)了脾腎陽(yáng)虛證IBS-D的腦-腸互動(dòng)異常;溫腎健脾方參與調(diào)節(jié)海馬及結(jié)腸蛋白的表達(dá),可能通過(guò)降低應(yīng)激改變和內(nèi)臟敏感性、維持神經(jīng)穩(wěn)態(tài)、調(diào)節(jié)腸道動(dòng)力和免疫等發(fā)揮了治療作用。
[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
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R259
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本文編號(hào)：1338378