IBS-D肝郁脾虚型病证结合大鼠模型的建立与评价
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摘要
研究背景
肠易激综合征(irritable bowel syndrome, IBS)是一种以腹痛或腹部不适伴排便习惯改变和(或)粪便性状改变的功能性肠病,该病缺乏可解释症状的形态学改变和生化异常,其患病率逐年增高,与精神心理因素和感染因素密切相关。腹泻型肠易激综合征(IBS-D)是我国IBS最常见的类型,其发病机制复杂,包括内脏感觉过敏、肠道动力增强、脑肠作用异常、神经-内分泌-免疫网络异常等。近年来对IBS-D发病机制的研究逐渐深入到了分子水平,但还比较片面。中医学认为肝郁脾虚是其基本病机,肝郁脾虚证是其最主要的证型。西药作用靶点单一,难以解决IBS-D复杂的临床症状,中药复方具有多靶点作用整体治疗的优势。目前对IBS-D动物模型的研究尚处于探索阶段,其造模方法还不成熟,动物模型稳定性欠佳,深入全面研究IBS-D的发病机制需要建立一种稳定的重复性好的动物模型。进一步建立IBS-D肝郁脾虚型病证结合动物模型有利于研究中药复方的作用机理,对于开发新药,发挥中医药治疗IBS-D的优势具有推动作用。
第一部分:IBS-D大鼠模型制作方法的探索
目的：探索建立IBS-D大鼠模型的理想制作方法
材料和方法：本部分研究采用新生雌性SD大鼠造模,分为正常对照组、番泻叶高剂量组、中剂量组、低剂量组、高乳糖饲料组、醋酸灌肠组和5-HT腹腔注射组,每组10只。除正常组外,其余各组大鼠采用新生期母子分离法建立内脏高敏感模型,即出生后第2-14天,每天与母鼠分离3h,2月后选择体重大于250g的大鼠进行致泻造模。番泻叶高剂量组给予番泻叶煎剂4.5g/kg、中剂量组给予3g/kg、低剂量组给予2g/kg灌胃,灌胃体积为10ml/kg,连续7天,给予普通饲料喂养；高乳糖组采用高乳糖饲料喂养,7天后换用普通饲料；醋酸灌肠组给予4%的醋酸灌肠1ml,灌肠1次,普通饲料喂养,此后不做任何处理；5-HT腹腔注射组给予5-HT2.1mg/kg腹腔注射,连续7天,普通饲料喂养；正常组不给于任何处理。观察各组大鼠造模期间的腹泻率,造模后1周每天的大便积分(硬便1分,软便2分,不成形便3分),造模前后的体重增长情况以及近端结肠的病理组织学变化。
结果：(1)腹泻率：番泻叶高剂量组(4.5g/kg)、高乳糖饲料组和醋酸灌肠组大鼠腹泻率均为100%,其余各组均未出现腹泻。(2)体重增长量：与正常组大鼠相比,除番泻叶低剂量组大鼠体重无明显变化外,其余各组体重增长量均显著降低,具有显著性差异(P<0.01),其中高乳糖饲料组和醋酸灌肠组大鼠体重出现明显的负增长。(3)大便积分：造模后番泻叶高剂量组和醋酸灌肠组大鼠6天内的平均大便积分显著高于正常组,具有显著性差异(P<0.05,P<0.01)。(4)近端结肠病理：高乳糖组可见淋巴细胞、中性粒细胞和嗜酸性粒细胞浸润,细胞间质轻度水肿；醋酸灌肠组部分结肠与腹腔组织粘连、增厚,结肠缩短,近端结肠可见粘膜轻度充血,绒毛变钝,其余各组未见异常。
结论：(1)母子分离+适当剂量的番泻叶灌胃是复制IBS-D动物模型较为合理的造模方法。(2)基于大便基本特征和病理组织学改变是判定IBS-D动物模型是否成功的重要依据之一。
第二部分：IBS-D肝郁脾虚型病证结合大鼠模型的建立与评价
目的：建立IBS-D肝郁脾虚型病证结合大鼠模型。
材料和方法：本部分研究采用新生SD雄性大鼠造模,分为正常组、正常束缚组、母子分离组、分离+束缚组(模型组)和以方测证组,每组20只大鼠。基于第一部分研究结论选择母子分离+番泻叶灌胃复制IBS-D大鼠模型,采用慢性束缚应激复制肝郁脾虚证候模型,将二者叠加建立IBS-D肝郁脾虚型病证结合大鼠模型。分三步造模,第一步：正常组和正常束缚组大鼠新生期母子不分离,其余各组于出生后第2-14天,每天与母鼠分离3h；第二步：各组大鼠2月龄时,正常组和母子分离组不处理,其余各组采用自制束缚架,束缚肩部和腹部,使其固定不动,每天束缚3h,连续3周；第三步：束缚造模期第2周开始每天上午9点给予以方测证组大鼠灌服痛泻要方煎剂(3g/kg,10ml/kg),其余各组灌服生理盐水(10ml/kg),第3周开始,正常束缚组、母子分离组和模型组每天上午9点灌服生理盐水,下午4点灌服番泻叶煎剂(4.5g/kg,10ml/kg),乙方测证组上午灌服痛泻要方煎剂,下午灌服番泻叶煎剂,正常对照组上下午均灌服生理盐水。从宏观疾病特征、宏观证候特征和微观生物学指标三个方面对模型进行评价。宏观疾病特征评价方法：束缚造模前后检测各组大鼠的内脏敏感性(痛觉阈值)、测评束缚期间和造模后各组大鼠的大便积分(计分方法同前)。宏观证候特征评价方法：肝郁的评价于束缚造模期前后观察各组大鼠旷场行为学、糖水偏好率、悬尾不动时间,脾虚的评价通过观察各组大鼠在束缚期间的体重增长情况以及造模后的进食量。微观生物学指标评价：检测血清D-木糖、5-HT、BDNF、IgA、 IgG含量；采用流式细胞仪分析血液和胸腺组织的T淋巴细胞亚群分布,比较各组大鼠的脾脏指数；采用免疫组织化学的方法检测近端结肠和末端回肠组织中的肥大细胞和嗜铬细胞数目变化；评价近端结肠和末端回肠的病理组织学变化。
结果：(1)痛觉阈值：造模结束后,母子分离组和模型组大鼠痛觉阈值较正常组明显降低(均P<0.01),正常束缚组痛觉阈值与正常组无显著性差异,痛泻要方可提高模型组大鼠的痛觉阈值(P<0.01)。(2)大便情况：短期束缚应激可使大鼠排便粒数明显增加,长期束缚应激对大鼠排便粒数无明显影响,但可使其大便含水量增加,甚至不成形。造模结束后,5天内的平均大便积分模型组显著高于正常组、正常束缚组和母子分离组(均P<0.01),痛泻要方对模型组大鼠的腹泻情况无明显改善。(3)体重增长量：造模结束后,正常束缚组、母子分离组和模型组大鼠的体重增长量均较正常组降低(均P<0.01),痛泻要方对模型组大鼠的体重无明显影响。(4)旷场实验：造模结束后,正常束缚组、母子分离组、模型组大鼠的穿格数和站立数均较正常组减少(均P<0.05),痛泻要方可提高模型组大鼠的穿格数和站立数(P<0.05)。(5)糖水偏好率：造模结束后,正常束缚组、母子分离组和模型组的糖水偏好率均较正常组降低(P<0.01,P<0.05,P<0.01),痛泻要方可提高模型组大鼠的糖水偏好率(P<0.05)。(6)悬尾不动时间：造模结束后,正常束缚组、母子分离组和模型组大鼠的悬尾不动时间较正常组延长(P<0.01,P<0.05,P<0.05),痛泻要方可缩短模型组大鼠的悬尾不动时间(P<0.05)。(7)进食量：造模后正常束缚组、母子分离组和模型组大鼠平均进食量与正常组比较无明显变化,痛泻要方可提高模型组的进食量(P<0.05)。(8)血清指标：造模结束后,血清D-木糖含量正常束缚组、母子分离组和模型组较正常组明显降低(均P<0.01),痛泻要方对模型组大鼠的血清D-木糖含量无明显影响。血清5-HT正常束缚组与正常组比较无显著性差异,母子分离组和模型组较正常组升高(均P<0.01),痛泻要方可降低模型组大鼠的血清5-HT含量(P<0.01)。血清BDNF各组之间无显著性差异。血清IgA母子分离组和模型组较正常组升高(均P<0.05),正常束缚组和正常组无显著性差异；痛泻要方对模型组大鼠血清IgA水平无明显影响。血清IgG母子分离组和模型组与正常组无显著性差异,痛泻要方对模型组血清IgG无明显影响。(9)T淋巴细胞亚群比例：造模结束后,血液Th (CD3+CD4+CD8-)亚群比例正常束缚组、母子分离组和模型组较正常组升高(均P<0.05),Tc (CD3+CD4-CD8+)亚群比例较正常组降低(均P<0.01),胸腺T总细胞比例较正常组升高(均P<0.05),痛泻要方对模型组的血液和胸腺T淋巴细胞亚群比例无明显影响。(10)脾脏指数：母子分离组和模型组较正常组降低(均P<0.01),正常束缚组和正常组无显著性差异,痛泻要方对模型组脾脏指数无明显影响。(11)近端结肠肥大细胞数目：模型组较正常组增多(P<0.01),正常束缚组和母子分离组与正常组无显著性差异,痛泻要方可显著减少模型组大鼠近端结肠的肥大细胞数目(P<0.01)。(12)嗜铬细胞数目：正常束缚组、母子分离组和模型组近端结肠和末端回肠中的肥大细胞数目均较正常组增多(均P<0.01),痛泻要方对模型组大鼠近端结肠组织中的嗜铬细胞数目无明显影响。(13)组织病理：各组大鼠近端结肠和末端回肠病理检查未见明显异常。
结论：(1)采用母子分离+慢性束缚应激+番泻叶灌胃可成功建立IBS-D肝郁脾虚型病证结合大鼠模型。(2)采用慢性束缚联合母子分离法造模优于单一因素法。(3)本研究所建立的IBS-D肝郁脾虚型大鼠模型具有内脏高敏感性、肠道通透性增加、抑郁和免疫功能异常等多种特征。(4)根据IBS-D肝郁脾虚证患者的宏观疾病、证候特征和微观生物学指标评价动物模型是否成功的方法科学、合理,具有可行性。
第三部分：IBS-D肝郁脾虚型大鼠模型结肠和脑的蛋白质组学研究
目的：基于第二部分大鼠模型,从结肠和脑组织中筛选和鉴定与IBS-D发病相关的差异蛋白,并进行对比分析。
材料和方法：采用第二部分正常组、正常束缚组、母子分离组和模型组大鼠的近端和远端结肠和全脑组织样本进行蛋白质组学分析。利用iTRAQ技术筛选各组样本中的差异蛋白,采用Mascot软件对差异蛋白的质谱信息进行鉴定和定量分析,然后基于Uniprot和Gene Ontology数据库对鉴定到的差异蛋白进行功能注释,采用IPA软件分析各组差异蛋白涉及到的生物通路和相互作用关系。并采用实时荧光定量RT-PCR技术从基因水平对选定差异蛋白进行验证。
结果：(1)与正常组比较,在正常束缚组大鼠结肠组织中筛选和鉴定出了542个差异表达蛋白(差异倍数>1.2,P<0.05),其中309个上调,233个下调,脑组织中筛选和鉴定出了1884个差异蛋白,其中764个上调,1120个下调；母子分离组结肠组织中筛选和鉴定出了809个蛋白,其中415个上调,394个下调,脑组织筛选和鉴定出了2386个蛋白,其中1080个上调,1306个下调；模型组结肠组织中筛选和鉴定出了731个差异蛋白,其中424个上调,307个下调,脑组织中筛选和鉴定出了2567个差异蛋白,其中1187个上调,1380个下调。(2)正常束缚组、母子分离组和分离束缚组在结肠组织中有192个差异蛋白共同表达,在脑组织中有1501个差异蛋白共同表达。(3)在正常束缚组,有153个差异蛋白在脑和肠组织共同表达,母子分离组有280个差异蛋白在脑和肠组织共同表达,分离束缚组有239个蛋白在脑和肠组织中共同表达。三组均有55个差异蛋白在脑和肠组织中共同表达。(4)模型组结肠组织中差异倍数>3蛋白有6个,其中4个上调：S腺苷甲硫氨酸脱羧酶酶原、内凝集素蛋白、FH2区包含蛋白1、主要组织相溶性复合体Ⅱ类抗原；2个下调：磷脂酰肌醇特异的磷脂酶CX区包含蛋白3、血红蛋白α亚。这些蛋白主要参与了S腺苷甲硫氨酸合成、信号转导、肌动蛋白细胞骨架组装、免疫反应、脂质代谢、氧气转运过程。脑组织中差异倍数>4的蛋白有22个,其中4个上调：过氧化物酶膜蛋白PEX14、NADH泛醌氧化还原酶链2、唐氏综合症临界区域基因3、Lrba蛋白；18个下调：血红蛋白p亚基、胸腺旁腺素、神经生长因子、细胞色素C氧化酶6B1亚基、40s核糖体蛋白、ATP合成酶d亚基、线粒体输入内膜移位酶亚基Tim13、NADH脱氢酶黄素蛋白3、细胞色素C氧化酶5A亚基、浦肯野细胞蛋白4、ATP合酶藕联因子6、脂酰辅酶A结合蛋白、细胞色素C、金属硫蛋白3、肌动蛋白相互作用蛋白1、泛醌细胞色素C还原酶结合蛋白、甘油醛3磷酸脱氢酶、胸腺素p4。这些蛋白参与了蛋白质运输、电子传递、空泡运输、氧气转运、免疫、神经内分泌、rRNA转运、金属离子结合、肌动蛋白细胞骨架组装、核苷酸代谢等。在模型组肠组织中鉴定出的热休克蛋白27、免疫球蛋白J链表达上调、血红蛋白α亚基、血红蛋白β亚基表达下调,这些蛋白与临床报道一致。(5)模型组结肠组织的差异蛋白功能主要与细胞组织结构、细胞的功能和修复、细胞死亡和生存、细胞形态、组织发育、细胞发育、细胞的生长和增殖、神经系统发育和功能、细胞与细胞的信号传导和相互作用、小分子生物化学等有关,脑组织的差异蛋白功能主要与细胞的组织结构、细胞的功能和修复、细胞死亡和生存、细胞形态、小分子生物化学、神经系统发育和功能、小分子转运、细胞发育、组织发育和核苷酸代谢等有关。(6)模型组大鼠结肠组织中的差异蛋白主要涉及了整合素信号、5-HT降解信号、抗原呈递通路、白介素4信号等27个生物通路,脑组织的差异蛋白主要涉及了间隙连接信号、上皮黏着连接信号、线粒体功能障碍等26个生物通路。结肠组织中存在9类差异蛋白相互作用网络,脑组织中存在12类差异蛋白相互作用网络。(7)结肠组织AQP8mRNA、NHE3mRNA表达水平与蛋白表达水平一致,结肠和海马BDNFmRNA表达与蛋白表达水平不完全一致。
结论：(1)IBS-D肝郁脾虚证大鼠模型结肠和脑组织中存在大量差异表达的蛋白,部分蛋白与临床报道一致,该模型具备IBS-D的病理特征。(2) IBS-D肝郁脾虚证大鼠模型结肠和脑组织中存在大量共同表达的差异蛋白,表达方式多样,印证了IBS-D脑肠相互作用异常的机制。(3)慢性束缚应激、母子分离应激以及二者联合作用具有共同的分子生物学基础,二者联合对脑组织差异蛋白表达的影响具有协同作用。(4)IBS-D发病与多种蛋白质分子改变有关,深入研究各差异蛋白的功能可进一步阐明IBS-D的发病机制。
BACKGROUND:Irritable bowel syndrome(IBS) is a functional gastrointestinal disorder characterized by abdominal discomfort and altered bowel habits in the absence of structural or biochemical disorders to account for these symptoms. It's prevalence rate is increasing every year, which is related to psychological factor and infectious factor. Diarrhea-predominant IBS is the major type of IBS in China, the pathogenetic mechanisms of IBS-D are complicated, including visceral hyperesthesia, enhanced colonic motility, brain-gut dysfunction, abnormal neuro-immuno-endocrine network and so on. Recently, some molecular mechanisms are studied. Liver depression and spleen deficiency is the basic pathogenesis of IBS-D in traditional Chinese medicine, liver depression and spleen deficiency syndrome is the main syndrome. There is no an effective drug to treatment all the symptoms of IBS-D in the western medicine, TCM has advantages in comprehensive treatment. Now, the research on the model of IBS-D is still at a groping stage. A reliable and repeatable animal model of IBS-D is needed to study it's pathogenesis. The establishment of an animal model of IBS-D with liver depression and spleen deficiency syndrome is necessary for the research on mechanisms of Chinese herbal compound prescription, which will promote the development of new drugs.
Part1:Study of methods of establishing an ideal rat model of IBS-D
Aims:To explore a reliable method of establishing a rat model of IBS-D
Materials and methods:The Sprague Dawley(SD) female rats were used in this part and were divided into7groups:normal group, high dose senna group, middle dose senna group, low dose senna group, lactose group, acetic acid group,5-HT group, there are10rats in each group. Pups were exposed to a3h period of daily maternal separation on postnatal day2-14except the normal group. The following treatments were performed at2month of age(>250g). The high dose senna group was given4.5g/kg Senna decoction by gavage for7days, the middle dose senna group was given3g/kg Senna decoction, low dose senna group was given2g/kg Senna decoction, the lactose group was given lactose enriched diet for7days, the acetic acid group was given4%acetic acid1ml by anus once, the5-HT group was given2.1mg/kg5-HT by intraperitoneal injection for7days. The diarrhea rate was evaluated during the process, the stool consistency was observed and the fecal score was calculated after the modeling for1week, score of stool consistency was classified as follows:normal1, soft2, watery3. Body weight was evaluated before and after the modeling, the histopathologic evaluation of the proximal colon was did after the modeling.
Results:(1) Diarrhea rate:the diarrhea rate of the high dose senna group, lactose group and acetic acid group was100%, there was no diarrhea in the other groups.(2) Weight growth:Except the low dose senna group, the weight growth of all the other groups decreased significantly compared to the normal group(P<0.01).Negative growth was observed in the lactose group and acetic acid group.(3) Fecal score:the average fecal score of the high dose senna group and acetic acid group was significantly higher compared to the normal group after the modeling (P<0.05, P<0.01).(4) Histopathologic evaluation:the infiltration of lymphocytes, neutrophils, Eosnophils and interstitial edema were observed in colon of the lactose group, tissue adhesions and proliferation were observed in the acetic acid group. The other groups had no abnormal changes.
Conclusions:(1) Maternal separation and senna by gavage were reliable to duplicate a rat model of IBS-D.(2) The character of stools and histopathologic evaluation of the colon were important to judge the rationality of methods to establish the animal model of IBS-D.
Part2:Establishment and evaluation of a rat model of IBS-D with liver depression and spleen deficiency syndrome
Aims:To establish a rat model of IBS-D with liver depression and spleen deficiency syndrome
Materials and methods:The male SD rats were used in this part and were divided into5groups. The normal group (N), chronic restraint group(R), maternal separation group(S), maternal separation and chronic restraint (SR), Tongxieyaofang group (TX), each group has20rats. The establishment of IBS-D is based on the part 1.There were three steps in the process of modeling. Firstly, the pups in the S, SR and TX group were exposed to a3h period of daily maternal separation on postnatal day2-14, the N and R group didn't exposed to maternal separation. Secondly, the R, SR and TX group were exposed to chronic restraint stress by specially designed restraint frame for3weeks at2month of ages, the N and S group received no treatment. Thirdly, from the second week of the restraint period, the TX group was given Tongxieyaofang decoction (3g/kg) by gavage on9:00am for2weeks, the other groups were given normal saline (10ml/kg) instead, on the last week of the restraint period, all the groups were given Senna decoction(4.5g/kg) by gavage on the4:00pm except the normal group. The models were evaluated by the features of IBS-D, liver depression and spleen deficiency syndrome, some biomarkers also were tested. The visceral sensitivity was tested before and after the restraint stress; the fecal scores were calculated during the restraint period and after the modeling. The liver depression syndrome was evaluated by open field test, sucrose preference test and tail suspension test. The spleen deficiency syndrome was evaluated by body weight and food consumption. The level of serum D-xylose,5-HT, BDNF, IgA, IgG were tested after the modeling. T lymphocyte subpopulation analysis of blood and thymus were did after the modeling by flow cytometry. The number of mast cells(MC) and enterochromaffin cell(EC) of colon and small intestinal was tested by immunohisto-chemistry. The histopathologic evaluation of the proximal colon and distal ileum was done after the modeling.
Results:(1)Pain threshold:after the modeling, the pain threshold of the S group and SR group was lower compared to the N group(P<0.01),there was no significant difference between the R and N group,Tongxieyaofang could enhance the pain threshold of the model(P<0.01).(2)Fecal score:acute restraint stress could increase the number of fecal pellet output, chronic restraint stress didn't influence the number of fecal pellet output, but could increase the water content. The average fecal score of the SR group was significantly higher than the N,R, and S group(P<0.01), Tongxieyaofang had no obvious effect on improving the diarrhea.(3)Weight growth:the weight growth of the R, S and SR was lower compared to the N group(P<0.01). Tongxieyaofang had no obvious effect on the weight growth.(4) Open field test:the horizontal and vertical motion of the rats in the R, S and SR group were decreased compared to the N group(P<0.05). Tongxieyaofang could improve this presentation of the rat model.(5) Sucrose preference test:the sucrose preference rate of the rats in the R, S and SR group was decreased compared to the N group(P<0.01, P<0.05, P<0.01). Tongxieyaofang could increase the sucrose preference rate of the rat model (P<0.05).(6) Tail suspension test:the still time in the R, S and SR group was longer compared to the N group(P<0.01, P<0.05, P<0.05).Tongxieyaofang could shorten the still time of the rat model(P<0.05).(7) Food consumption:There was no significant difference of the food consumption in the SR group compared to the normal group. Tongxieyaofang could increase the food consumption of the rat model(P<0.05).(8)Serum markers:The level of serum D-xylose decreased in the R,S and SR group compared to the N group(P<0.01).Tongxieyaofang had no effect on the serum D-xylose.The level of serum5-HT increased in the S and SR group compared to the N group(P<0.01).Tongxieyaofang could decrease the serum5-HT of the rat model(P<0.01). The level of serum IgA increased in the S and SR group compared to the N group(P<0.05). Tongxieyaofang had no effect on the serum IgA.There was no significant change in the level of serum BDNF, IgG in the SR group.(9)The proportion of T lymphocyte subpopulation:the proportion of blood CD3+CD4+CD8-increased, and the subpopulation CD3+CD4-CD8+decreased in the R,S and SR group compared to the N group(P<0.01). The proportion of thymus total T lymphocytes increased in the R,S and SR group compared to the N group(P<0.05). Tongxieyaofang had no significant effect on the proportion of T lymphocyte subpopulation.(10)Spleen index:the spleen index in the S and SR group decreased compared to the N group(P<0.01). Tongxieyaofang had no effect on the spleen index of the rat model.(11)The number of MC:the number of the MC increased significantly in the SR group compared to the N group(p<0.01), tongxieyaofang could decrease the number of MC of the rat model(P<0.01).(12) The number of EC:the number of EC in the R,S and SR group increased significantly compared to the N group (P<0.01). Tongxieyaofang had no effect on the number of EC of the rat model.(13)There was no obvious abnormal change of proximal colon and distal ileum in all the groups.
Conclusions:(1)The maternal separation stress, chronic restraint stress and senna decoction by gavage could duplicate the IBS-D rat model with liver depression and spleen deficiency.(2)The combination of chronic restraint stress and maternal separation was superior to one factor.(3)The model was characterized by visceral hypersensitivity, increased permeability of intestinal epithelial cell, depression and immune dysfunction.(3) It's scientific, reasonable and feasible to evaluate the rat model according to the character of IBS-D patients.
Part3:Proteomic analysis of colon and brain in the rat model of IBS-D with liver depression and spleen deficiency syndrome
Aims:To screen the differential proteins in the colon and brain based on the rat models in the part2, contrastive analysis was did between the two tissues.
Materials and methods:The colon and brain tissue of the part2rats were used to analysis. The differential proteins were screened based on the technology of isobaric tags for relative and absolute quantitation (iTRAQ). Protein identification was performed by mass spectrum. Mascot software was used to search the related information of the differential proteins based on the Uniprot and Gene Ontology database. The pathways and functional networks of the differential proteins were analysed by the ingenuity pathway analysis software(IPA).Selected proteins were validated by realtime RT-PCR.
Results:(1) In the chronic restraint stress group(R),542proteins were identified in the colon tissue(ratio>1.2, P<0.05),309proteins were significantly up-regulated and233proteins were significantly down-regulated,1884proteins were identified in the brain tissue,764proteins were significantly up-regulated and1120proteins were significantly down-regulated. In the maternal separation group(S),809proteins were identified in the colon tissue,415proteins were significantly up-regulated and394proteins were significantly down-regulated,2386proteins were identified in the brain tissue,1080proteins were significantly up-regulated and1306proteins were significantly down-regulated. In the maternal separation and chronic restraint group (SR),731proteins were identified in the colon tissue,424proteins were significantly up-regulated and307proteins were significantly down-regulated,2567proteins were identified in the brain tissue,1187proteins were significantly up-regulated and1380 proteins were significantly down-regulated.(2) In the three groups,192proteins co-expressed in the colon tissue,1501proteins co-expressed in the brain tissue.(3)153proteins co-expressed in the colon and brain tissue in the R group,280proteins co-expressed in the colon and brain tissue in the S group, and239proteins co-expressed in the colon and brain tissue.in the SR group.55proteins of the colon and brain tissue co-expressed in the three groups.(4) In the SR group, there were6proteins differentially expressing with the the ratio>3in the colon tissue,4proteins were up-regulated:S-adenosylmethionine decarboxylase proenzyme, protein Itlnl, FH2domain-containing protein1, MHC class Ⅱ antigen;2proteins were down-regulated:PI-PLC X domain-containing protein3, Hemoglobin subunit alpha. They involved in the S-adenosylmethioninamine biosynthetic process, signal transduction, actin cytoskeleton organization, immune response, lipid metabolic process, oxygen transport. There were22proteins differentially expressing with the the ratio>3in the brain tissue.4proteins were up-regulated:peroxisomal membrane protein PEX14, NADH-ubiquinone oxidoreductase chain2, down syndrome critical region gene3, protein Lrba;18proteins were down-regulated:protein Hbb-bl, parathymosin, neurosecretory protein VGF, Cytochrome c oxidase subunit6B1,40S ribosomal protein S28, ATP synthase subunit d, mitochondrial import inner membrane translocase subunit Tim13, NADH dehydrogenase flavoprotein3, cytochrome c oxidase subunit5A, Pcp411protein, ATP synthase-coupling factor6, Acyl-CoA-binding protein, cytochrome c, metallothionein-3, AIP1, protein uqcrb, glyceraldehyde-3-phosphate dehydrogenase, thymosin beta4. They involved in protein transport, electron transport, vacuolar transport, oxygen transport, immunity, rRNA export from nucleus, hydrogen ion transport, protein import into mitochondrial inner membrane, cadmium ion binding, endosomal transport, actin cytoskeleton organization. Heat shock27kDa protein1and immunoglobulin joining chain were up-regulated, hemoglobin subunit alpha and protein Hbb-b1were down-regulated in the colon of the model rat, which were in accord with the clinical report.(5) The differential proteins in the colon tissue of the SR group mainly involved in cellular assembly and organization, cellular function and maintenance, cell death and survival, cell morphology, tissue development, cellular development, cellular growth and proliferation, nervous system development and function, cell to cell signaling and interaction, small molecule biochemistry.The differential proteins in the brain tissue of the SR group mainly involved in cellular assembly and organization, cellular function and maintenance, cell death and survival, cell morphology, small molecule biochemistry, nervous system development and function, molecular transport, cellular development, tissue development, nucleic acid metabolism.(6) The differential proteins in the colon tissue of the SR group could be assigned to27pathways, including integrin signaling, serotonin degradation, antigen presentation pathway, IL-4signaling, and so on. The differential proteins in the brain tissue involved26pathways, including gap junction siganling, epithelia adherens junction signling, mitochondrial dysfuncion, etc. The differential proteins in the colon tissue of the SR group included9interaction networks, and12interaction networks in the brain tissue.(7) The expression of AQP8mRNA and Na+/H+exchanger3(NHE3) mRNA in the colon tissue were in accordance with the expression of homologous proteins, while the expression of brain-derived neurotrophic factor (BDNF) mRNA in the colon and hippocampus tissue were different from the expression of homologous protein.
Conclusions:(1) There were a large number of differential proteins in the colon and brain tissue of the rat model of IBS-D with liver depression and spleen deficiency syndrome, some of them were in accord with the clinical report.(2) The rat model of IBS-D had some proteins co-expressed in the colon and brain tissue, which indicated abnormal interaction between colon and brain.(3) The models induced by chronic restraint stress, maternal separation stress, and the combination of the two factors had the same molecule biological basis, mental stimulation mainly changed the proteins expression in the brain tissue.(4) The pathogenesis of IBS-D was related to changes of a number of proteins, studying the function of the differential proteins could gain a deeper understanding in the pathogenesis of IBS-D.
肠易激综合征(irritable bowel syndrome, IBS)是一种以腹痛或腹部不适伴排便习惯改变和(或)粪便性状改变的功能性肠病,该病缺乏可解释症状的形态学改变和生化异常,其患病率逐年增高,与精神心理因素和感染因素密切相关。腹泻型肠易激综合征(IBS-D)是我国IBS最常见的类型,其发病机制复杂,包括内脏感觉过敏、肠道动力增强、脑肠作用异常、神经-内分泌-免疫网络异常等。近年来对IBS-D发病机制的研究逐渐深入到了分子水平,但还比较片面。中医学认为肝郁脾虚是其基本病机,肝郁脾虚证是其最主要的证型。西药作用靶点单一,难以解决IBS-D复杂的临床症状,中药复方具有多靶点作用整体治疗的优势。目前对IBS-D动物模型的研究尚处于探索阶段,其造模方法还不成熟,动物模型稳定性欠佳,深入全面研究IBS-D的发病机制需要建立一种稳定的重复性好的动物模型。进一步建立IBS-D肝郁脾虚型病证结合动物模型有利于研究中药复方的作用机理,对于开发新药,发挥中医药治疗IBS-D的优势具有推动作用。
第一部分:IBS-D大鼠模型制作方法的探索
目的：探索建立IBS-D大鼠模型的理想制作方法
材料和方法：本部分研究采用新生雌性SD大鼠造模,分为正常对照组、番泻叶高剂量组、中剂量组、低剂量组、高乳糖饲料组、醋酸灌肠组和5-HT腹腔注射组,每组10只。除正常组外,其余各组大鼠采用新生期母子分离法建立内脏高敏感模型,即出生后第2-14天,每天与母鼠分离3h,2月后选择体重大于250g的大鼠进行致泻造模。番泻叶高剂量组给予番泻叶煎剂4.5g/kg、中剂量组给予3g/kg、低剂量组给予2g/kg灌胃,灌胃体积为10ml/kg,连续7天,给予普通饲料喂养；高乳糖组采用高乳糖饲料喂养,7天后换用普通饲料；醋酸灌肠组给予4%的醋酸灌肠1ml,灌肠1次,普通饲料喂养,此后不做任何处理；5-HT腹腔注射组给予5-HT2.1mg/kg腹腔注射,连续7天,普通饲料喂养；正常组不给于任何处理。观察各组大鼠造模期间的腹泻率,造模后1周每天的大便积分(硬便1分,软便2分,不成形便3分),造模前后的体重增长情况以及近端结肠的病理组织学变化。
结果：(1)腹泻率：番泻叶高剂量组(4.5g/kg)、高乳糖饲料组和醋酸灌肠组大鼠腹泻率均为100%,其余各组均未出现腹泻。(2)体重增长量：与正常组大鼠相比,除番泻叶低剂量组大鼠体重无明显变化外,其余各组体重增长量均显著降低,具有显著性差异(P<0.01),其中高乳糖饲料组和醋酸灌肠组大鼠体重出现明显的负增长。(3)大便积分：造模后番泻叶高剂量组和醋酸灌肠组大鼠6天内的平均大便积分显著高于正常组,具有显著性差异(P<0.05,P<0.01)。(4)近端结肠病理：高乳糖组可见淋巴细胞、中性粒细胞和嗜酸性粒细胞浸润,细胞间质轻度水肿；醋酸灌肠组部分结肠与腹腔组织粘连、增厚,结肠缩短,近端结肠可见粘膜轻度充血,绒毛变钝,其余各组未见异常。
结论：(1)母子分离+适当剂量的番泻叶灌胃是复制IBS-D动物模型较为合理的造模方法。(2)基于大便基本特征和病理组织学改变是判定IBS-D动物模型是否成功的重要依据之一。
第二部分：IBS-D肝郁脾虚型病证结合大鼠模型的建立与评价
目的：建立IBS-D肝郁脾虚型病证结合大鼠模型。
材料和方法：本部分研究采用新生SD雄性大鼠造模,分为正常组、正常束缚组、母子分离组、分离+束缚组(模型组)和以方测证组,每组20只大鼠。基于第一部分研究结论选择母子分离+番泻叶灌胃复制IBS-D大鼠模型,采用慢性束缚应激复制肝郁脾虚证候模型,将二者叠加建立IBS-D肝郁脾虚型病证结合大鼠模型。分三步造模,第一步：正常组和正常束缚组大鼠新生期母子不分离,其余各组于出生后第2-14天,每天与母鼠分离3h；第二步：各组大鼠2月龄时,正常组和母子分离组不处理,其余各组采用自制束缚架,束缚肩部和腹部,使其固定不动,每天束缚3h,连续3周；第三步：束缚造模期第2周开始每天上午9点给予以方测证组大鼠灌服痛泻要方煎剂(3g/kg,10ml/kg),其余各组灌服生理盐水(10ml/kg),第3周开始,正常束缚组、母子分离组和模型组每天上午9点灌服生理盐水,下午4点灌服番泻叶煎剂(4.5g/kg,10ml/kg),乙方测证组上午灌服痛泻要方煎剂,下午灌服番泻叶煎剂,正常对照组上下午均灌服生理盐水。从宏观疾病特征、宏观证候特征和微观生物学指标三个方面对模型进行评价。宏观疾病特征评价方法：束缚造模前后检测各组大鼠的内脏敏感性(痛觉阈值)、测评束缚期间和造模后各组大鼠的大便积分(计分方法同前)。宏观证候特征评价方法：肝郁的评价于束缚造模期前后观察各组大鼠旷场行为学、糖水偏好率、悬尾不动时间,脾虚的评价通过观察各组大鼠在束缚期间的体重增长情况以及造模后的进食量。微观生物学指标评价：检测血清D-木糖、5-HT、BDNF、IgA、 IgG含量；采用流式细胞仪分析血液和胸腺组织的T淋巴细胞亚群分布,比较各组大鼠的脾脏指数；采用免疫组织化学的方法检测近端结肠和末端回肠组织中的肥大细胞和嗜铬细胞数目变化；评价近端结肠和末端回肠的病理组织学变化。
结果：(1)痛觉阈值：造模结束后,母子分离组和模型组大鼠痛觉阈值较正常组明显降低(均P<0.01),正常束缚组痛觉阈值与正常组无显著性差异,痛泻要方可提高模型组大鼠的痛觉阈值(P<0.01)。(2)大便情况：短期束缚应激可使大鼠排便粒数明显增加,长期束缚应激对大鼠排便粒数无明显影响,但可使其大便含水量增加,甚至不成形。造模结束后,5天内的平均大便积分模型组显著高于正常组、正常束缚组和母子分离组(均P<0.01),痛泻要方对模型组大鼠的腹泻情况无明显改善。(3)体重增长量：造模结束后,正常束缚组、母子分离组和模型组大鼠的体重增长量均较正常组降低(均P<0.01),痛泻要方对模型组大鼠的体重无明显影响。(4)旷场实验：造模结束后,正常束缚组、母子分离组、模型组大鼠的穿格数和站立数均较正常组减少(均P<0.05),痛泻要方可提高模型组大鼠的穿格数和站立数(P<0.05)。(5)糖水偏好率：造模结束后,正常束缚组、母子分离组和模型组的糖水偏好率均较正常组降低(P<0.01,P<0.05,P<0.01),痛泻要方可提高模型组大鼠的糖水偏好率(P<0.05)。(6)悬尾不动时间：造模结束后,正常束缚组、母子分离组和模型组大鼠的悬尾不动时间较正常组延长(P<0.01,P<0.05,P<0.05),痛泻要方可缩短模型组大鼠的悬尾不动时间(P<0.05)。(7)进食量：造模后正常束缚组、母子分离组和模型组大鼠平均进食量与正常组比较无明显变化,痛泻要方可提高模型组的进食量(P<0.05)。(8)血清指标：造模结束后,血清D-木糖含量正常束缚组、母子分离组和模型组较正常组明显降低(均P<0.01),痛泻要方对模型组大鼠的血清D-木糖含量无明显影响。血清5-HT正常束缚组与正常组比较无显著性差异,母子分离组和模型组较正常组升高(均P<0.01),痛泻要方可降低模型组大鼠的血清5-HT含量(P<0.01)。血清BDNF各组之间无显著性差异。血清IgA母子分离组和模型组较正常组升高(均P<0.05),正常束缚组和正常组无显著性差异；痛泻要方对模型组大鼠血清IgA水平无明显影响。血清IgG母子分离组和模型组与正常组无显著性差异,痛泻要方对模型组血清IgG无明显影响。(9)T淋巴细胞亚群比例：造模结束后,血液Th (CD3+CD4+CD8-)亚群比例正常束缚组、母子分离组和模型组较正常组升高(均P<0.05),Tc (CD3+CD4-CD8+)亚群比例较正常组降低(均P<0.01),胸腺T总细胞比例较正常组升高(均P<0.05),痛泻要方对模型组的血液和胸腺T淋巴细胞亚群比例无明显影响。(10)脾脏指数：母子分离组和模型组较正常组降低(均P<0.01),正常束缚组和正常组无显著性差异,痛泻要方对模型组脾脏指数无明显影响。(11)近端结肠肥大细胞数目：模型组较正常组增多(P<0.01),正常束缚组和母子分离组与正常组无显著性差异,痛泻要方可显著减少模型组大鼠近端结肠的肥大细胞数目(P<0.01)。(12)嗜铬细胞数目：正常束缚组、母子分离组和模型组近端结肠和末端回肠中的肥大细胞数目均较正常组增多(均P<0.01),痛泻要方对模型组大鼠近端结肠组织中的嗜铬细胞数目无明显影响。(13)组织病理：各组大鼠近端结肠和末端回肠病理检查未见明显异常。
结论：(1)采用母子分离+慢性束缚应激+番泻叶灌胃可成功建立IBS-D肝郁脾虚型病证结合大鼠模型。(2)采用慢性束缚联合母子分离法造模优于单一因素法。(3)本研究所建立的IBS-D肝郁脾虚型大鼠模型具有内脏高敏感性、肠道通透性增加、抑郁和免疫功能异常等多种特征。(4)根据IBS-D肝郁脾虚证患者的宏观疾病、证候特征和微观生物学指标评价动物模型是否成功的方法科学、合理,具有可行性。
第三部分：IBS-D肝郁脾虚型大鼠模型结肠和脑的蛋白质组学研究
目的：基于第二部分大鼠模型,从结肠和脑组织中筛选和鉴定与IBS-D发病相关的差异蛋白,并进行对比分析。
材料和方法：采用第二部分正常组、正常束缚组、母子分离组和模型组大鼠的近端和远端结肠和全脑组织样本进行蛋白质组学分析。利用iTRAQ技术筛选各组样本中的差异蛋白,采用Mascot软件对差异蛋白的质谱信息进行鉴定和定量分析,然后基于Uniprot和Gene Ontology数据库对鉴定到的差异蛋白进行功能注释,采用IPA软件分析各组差异蛋白涉及到的生物通路和相互作用关系。并采用实时荧光定量RT-PCR技术从基因水平对选定差异蛋白进行验证。
结果：(1)与正常组比较,在正常束缚组大鼠结肠组织中筛选和鉴定出了542个差异表达蛋白(差异倍数>1.2,P<0.05),其中309个上调,233个下调,脑组织中筛选和鉴定出了1884个差异蛋白,其中764个上调,1120个下调；母子分离组结肠组织中筛选和鉴定出了809个蛋白,其中415个上调,394个下调,脑组织筛选和鉴定出了2386个蛋白,其中1080个上调,1306个下调；模型组结肠组织中筛选和鉴定出了731个差异蛋白,其中424个上调,307个下调,脑组织中筛选和鉴定出了2567个差异蛋白,其中1187个上调,1380个下调。(2)正常束缚组、母子分离组和分离束缚组在结肠组织中有192个差异蛋白共同表达,在脑组织中有1501个差异蛋白共同表达。(3)在正常束缚组,有153个差异蛋白在脑和肠组织共同表达,母子分离组有280个差异蛋白在脑和肠组织共同表达,分离束缚组有239个蛋白在脑和肠组织中共同表达。三组均有55个差异蛋白在脑和肠组织中共同表达。(4)模型组结肠组织中差异倍数>3蛋白有6个,其中4个上调：S腺苷甲硫氨酸脱羧酶酶原、内凝集素蛋白、FH2区包含蛋白1、主要组织相溶性复合体Ⅱ类抗原；2个下调：磷脂酰肌醇特异的磷脂酶CX区包含蛋白3、血红蛋白α亚。这些蛋白主要参与了S腺苷甲硫氨酸合成、信号转导、肌动蛋白细胞骨架组装、免疫反应、脂质代谢、氧气转运过程。脑组织中差异倍数>4的蛋白有22个,其中4个上调：过氧化物酶膜蛋白PEX14、NADH泛醌氧化还原酶链2、唐氏综合症临界区域基因3、Lrba蛋白；18个下调：血红蛋白p亚基、胸腺旁腺素、神经生长因子、细胞色素C氧化酶6B1亚基、40s核糖体蛋白、ATP合成酶d亚基、线粒体输入内膜移位酶亚基Tim13、NADH脱氢酶黄素蛋白3、细胞色素C氧化酶5A亚基、浦肯野细胞蛋白4、ATP合酶藕联因子6、脂酰辅酶A结合蛋白、细胞色素C、金属硫蛋白3、肌动蛋白相互作用蛋白1、泛醌细胞色素C还原酶结合蛋白、甘油醛3磷酸脱氢酶、胸腺素p4。这些蛋白参与了蛋白质运输、电子传递、空泡运输、氧气转运、免疫、神经内分泌、rRNA转运、金属离子结合、肌动蛋白细胞骨架组装、核苷酸代谢等。在模型组肠组织中鉴定出的热休克蛋白27、免疫球蛋白J链表达上调、血红蛋白α亚基、血红蛋白β亚基表达下调,这些蛋白与临床报道一致。(5)模型组结肠组织的差异蛋白功能主要与细胞组织结构、细胞的功能和修复、细胞死亡和生存、细胞形态、组织发育、细胞发育、细胞的生长和增殖、神经系统发育和功能、细胞与细胞的信号传导和相互作用、小分子生物化学等有关,脑组织的差异蛋白功能主要与细胞的组织结构、细胞的功能和修复、细胞死亡和生存、细胞形态、小分子生物化学、神经系统发育和功能、小分子转运、细胞发育、组织发育和核苷酸代谢等有关。(6)模型组大鼠结肠组织中的差异蛋白主要涉及了整合素信号、5-HT降解信号、抗原呈递通路、白介素4信号等27个生物通路,脑组织的差异蛋白主要涉及了间隙连接信号、上皮黏着连接信号、线粒体功能障碍等26个生物通路。结肠组织中存在9类差异蛋白相互作用网络,脑组织中存在12类差异蛋白相互作用网络。(7)结肠组织AQP8mRNA、NHE3mRNA表达水平与蛋白表达水平一致,结肠和海马BDNFmRNA表达与蛋白表达水平不完全一致。
结论：(1)IBS-D肝郁脾虚证大鼠模型结肠和脑组织中存在大量差异表达的蛋白,部分蛋白与临床报道一致,该模型具备IBS-D的病理特征。(2) IBS-D肝郁脾虚证大鼠模型结肠和脑组织中存在大量共同表达的差异蛋白,表达方式多样,印证了IBS-D脑肠相互作用异常的机制。(3)慢性束缚应激、母子分离应激以及二者联合作用具有共同的分子生物学基础,二者联合对脑组织差异蛋白表达的影响具有协同作用。(4)IBS-D发病与多种蛋白质分子改变有关,深入研究各差异蛋白的功能可进一步阐明IBS-D的发病机制。
BACKGROUND:Irritable bowel syndrome(IBS) is a functional gastrointestinal disorder characterized by abdominal discomfort and altered bowel habits in the absence of structural or biochemical disorders to account for these symptoms. It's prevalence rate is increasing every year, which is related to psychological factor and infectious factor. Diarrhea-predominant IBS is the major type of IBS in China, the pathogenetic mechanisms of IBS-D are complicated, including visceral hyperesthesia, enhanced colonic motility, brain-gut dysfunction, abnormal neuro-immuno-endocrine network and so on. Recently, some molecular mechanisms are studied. Liver depression and spleen deficiency is the basic pathogenesis of IBS-D in traditional Chinese medicine, liver depression and spleen deficiency syndrome is the main syndrome. There is no an effective drug to treatment all the symptoms of IBS-D in the western medicine, TCM has advantages in comprehensive treatment. Now, the research on the model of IBS-D is still at a groping stage. A reliable and repeatable animal model of IBS-D is needed to study it's pathogenesis. The establishment of an animal model of IBS-D with liver depression and spleen deficiency syndrome is necessary for the research on mechanisms of Chinese herbal compound prescription, which will promote the development of new drugs.
Part1:Study of methods of establishing an ideal rat model of IBS-D
Aims:To explore a reliable method of establishing a rat model of IBS-D
Materials and methods:The Sprague Dawley(SD) female rats were used in this part and were divided into7groups:normal group, high dose senna group, middle dose senna group, low dose senna group, lactose group, acetic acid group,5-HT group, there are10rats in each group. Pups were exposed to a3h period of daily maternal separation on postnatal day2-14except the normal group. The following treatments were performed at2month of age(>250g). The high dose senna group was given4.5g/kg Senna decoction by gavage for7days, the middle dose senna group was given3g/kg Senna decoction, low dose senna group was given2g/kg Senna decoction, the lactose group was given lactose enriched diet for7days, the acetic acid group was given4%acetic acid1ml by anus once, the5-HT group was given2.1mg/kg5-HT by intraperitoneal injection for7days. The diarrhea rate was evaluated during the process, the stool consistency was observed and the fecal score was calculated after the modeling for1week, score of stool consistency was classified as follows:normal1, soft2, watery3. Body weight was evaluated before and after the modeling, the histopathologic evaluation of the proximal colon was did after the modeling.
Results:(1) Diarrhea rate:the diarrhea rate of the high dose senna group, lactose group and acetic acid group was100%, there was no diarrhea in the other groups.(2) Weight growth:Except the low dose senna group, the weight growth of all the other groups decreased significantly compared to the normal group(P<0.01).Negative growth was observed in the lactose group and acetic acid group.(3) Fecal score:the average fecal score of the high dose senna group and acetic acid group was significantly higher compared to the normal group after the modeling (P<0.05, P<0.01).(4) Histopathologic evaluation:the infiltration of lymphocytes, neutrophils, Eosnophils and interstitial edema were observed in colon of the lactose group, tissue adhesions and proliferation were observed in the acetic acid group. The other groups had no abnormal changes.
Conclusions:(1) Maternal separation and senna by gavage were reliable to duplicate a rat model of IBS-D.(2) The character of stools and histopathologic evaluation of the colon were important to judge the rationality of methods to establish the animal model of IBS-D.
Part2:Establishment and evaluation of a rat model of IBS-D with liver depression and spleen deficiency syndrome
Aims:To establish a rat model of IBS-D with liver depression and spleen deficiency syndrome
Materials and methods:The male SD rats were used in this part and were divided into5groups. The normal group (N), chronic restraint group(R), maternal separation group(S), maternal separation and chronic restraint (SR), Tongxieyaofang group (TX), each group has20rats. The establishment of IBS-D is based on the part 1.There were three steps in the process of modeling. Firstly, the pups in the S, SR and TX group were exposed to a3h period of daily maternal separation on postnatal day2-14, the N and R group didn't exposed to maternal separation. Secondly, the R, SR and TX group were exposed to chronic restraint stress by specially designed restraint frame for3weeks at2month of ages, the N and S group received no treatment. Thirdly, from the second week of the restraint period, the TX group was given Tongxieyaofang decoction (3g/kg) by gavage on9:00am for2weeks, the other groups were given normal saline (10ml/kg) instead, on the last week of the restraint period, all the groups were given Senna decoction(4.5g/kg) by gavage on the4:00pm except the normal group. The models were evaluated by the features of IBS-D, liver depression and spleen deficiency syndrome, some biomarkers also were tested. The visceral sensitivity was tested before and after the restraint stress; the fecal scores were calculated during the restraint period and after the modeling. The liver depression syndrome was evaluated by open field test, sucrose preference test and tail suspension test. The spleen deficiency syndrome was evaluated by body weight and food consumption. The level of serum D-xylose,5-HT, BDNF, IgA, IgG were tested after the modeling. T lymphocyte subpopulation analysis of blood and thymus were did after the modeling by flow cytometry. The number of mast cells(MC) and enterochromaffin cell(EC) of colon and small intestinal was tested by immunohisto-chemistry. The histopathologic evaluation of the proximal colon and distal ileum was done after the modeling.
Results:(1)Pain threshold:after the modeling, the pain threshold of the S group and SR group was lower compared to the N group(P<0.01),there was no significant difference between the R and N group,Tongxieyaofang could enhance the pain threshold of the model(P<0.01).(2)Fecal score:acute restraint stress could increase the number of fecal pellet output, chronic restraint stress didn't influence the number of fecal pellet output, but could increase the water content. The average fecal score of the SR group was significantly higher than the N,R, and S group(P<0.01), Tongxieyaofang had no obvious effect on improving the diarrhea.(3)Weight growth:the weight growth of the R, S and SR was lower compared to the N group(P<0.01). Tongxieyaofang had no obvious effect on the weight growth.(4) Open field test:the horizontal and vertical motion of the rats in the R, S and SR group were decreased compared to the N group(P<0.05). Tongxieyaofang could improve this presentation of the rat model.(5) Sucrose preference test:the sucrose preference rate of the rats in the R, S and SR group was decreased compared to the N group(P<0.01, P<0.05, P<0.01). Tongxieyaofang could increase the sucrose preference rate of the rat model (P<0.05).(6) Tail suspension test:the still time in the R, S and SR group was longer compared to the N group(P<0.01, P<0.05, P<0.05).Tongxieyaofang could shorten the still time of the rat model(P<0.05).(7) Food consumption:There was no significant difference of the food consumption in the SR group compared to the normal group. Tongxieyaofang could increase the food consumption of the rat model(P<0.05).(8)Serum markers:The level of serum D-xylose decreased in the R,S and SR group compared to the N group(P<0.01).Tongxieyaofang had no effect on the serum D-xylose.The level of serum5-HT increased in the S and SR group compared to the N group(P<0.01).Tongxieyaofang could decrease the serum5-HT of the rat model(P<0.01). The level of serum IgA increased in the S and SR group compared to the N group(P<0.05). Tongxieyaofang had no effect on the serum IgA.There was no significant change in the level of serum BDNF, IgG in the SR group.(9)The proportion of T lymphocyte subpopulation:the proportion of blood CD3+CD4+CD8-increased, and the subpopulation CD3+CD4-CD8+decreased in the R,S and SR group compared to the N group(P<0.01). The proportion of thymus total T lymphocytes increased in the R,S and SR group compared to the N group(P<0.05). Tongxieyaofang had no significant effect on the proportion of T lymphocyte subpopulation.(10)Spleen index:the spleen index in the S and SR group decreased compared to the N group(P<0.01). Tongxieyaofang had no effect on the spleen index of the rat model.(11)The number of MC:the number of the MC increased significantly in the SR group compared to the N group(p<0.01), tongxieyaofang could decrease the number of MC of the rat model(P<0.01).(12) The number of EC:the number of EC in the R,S and SR group increased significantly compared to the N group (P<0.01). Tongxieyaofang had no effect on the number of EC of the rat model.(13)There was no obvious abnormal change of proximal colon and distal ileum in all the groups.
Conclusions:(1)The maternal separation stress, chronic restraint stress and senna decoction by gavage could duplicate the IBS-D rat model with liver depression and spleen deficiency.(2)The combination of chronic restraint stress and maternal separation was superior to one factor.(3)The model was characterized by visceral hypersensitivity, increased permeability of intestinal epithelial cell, depression and immune dysfunction.(3) It's scientific, reasonable and feasible to evaluate the rat model according to the character of IBS-D patients.
Part3:Proteomic analysis of colon and brain in the rat model of IBS-D with liver depression and spleen deficiency syndrome
Aims:To screen the differential proteins in the colon and brain based on the rat models in the part2, contrastive analysis was did between the two tissues.
Materials and methods:The colon and brain tissue of the part2rats were used to analysis. The differential proteins were screened based on the technology of isobaric tags for relative and absolute quantitation (iTRAQ). Protein identification was performed by mass spectrum. Mascot software was used to search the related information of the differential proteins based on the Uniprot and Gene Ontology database. The pathways and functional networks of the differential proteins were analysed by the ingenuity pathway analysis software(IPA).Selected proteins were validated by realtime RT-PCR.
Results:(1) In the chronic restraint stress group(R),542proteins were identified in the colon tissue(ratio>1.2, P<0.05),309proteins were significantly up-regulated and233proteins were significantly down-regulated,1884proteins were identified in the brain tissue,764proteins were significantly up-regulated and1120proteins were significantly down-regulated. In the maternal separation group(S),809proteins were identified in the colon tissue,415proteins were significantly up-regulated and394proteins were significantly down-regulated,2386proteins were identified in the brain tissue,1080proteins were significantly up-regulated and1306proteins were significantly down-regulated. In the maternal separation and chronic restraint group (SR),731proteins were identified in the colon tissue,424proteins were significantly up-regulated and307proteins were significantly down-regulated,2567proteins were identified in the brain tissue,1187proteins were significantly up-regulated and1380 proteins were significantly down-regulated.(2) In the three groups,192proteins co-expressed in the colon tissue,1501proteins co-expressed in the brain tissue.(3)153proteins co-expressed in the colon and brain tissue in the R group,280proteins co-expressed in the colon and brain tissue in the S group, and239proteins co-expressed in the colon and brain tissue.in the SR group.55proteins of the colon and brain tissue co-expressed in the three groups.(4) In the SR group, there were6proteins differentially expressing with the the ratio>3in the colon tissue,4proteins were up-regulated:S-adenosylmethionine decarboxylase proenzyme, protein Itlnl, FH2domain-containing protein1, MHC class Ⅱ antigen;2proteins were down-regulated:PI-PLC X domain-containing protein3, Hemoglobin subunit alpha. They involved in the S-adenosylmethioninamine biosynthetic process, signal transduction, actin cytoskeleton organization, immune response, lipid metabolic process, oxygen transport. There were22proteins differentially expressing with the the ratio>3in the brain tissue.4proteins were up-regulated:peroxisomal membrane protein PEX14, NADH-ubiquinone oxidoreductase chain2, down syndrome critical region gene3, protein Lrba;18proteins were down-regulated:protein Hbb-bl, parathymosin, neurosecretory protein VGF, Cytochrome c oxidase subunit6B1,40S ribosomal protein S28, ATP synthase subunit d, mitochondrial import inner membrane translocase subunit Tim13, NADH dehydrogenase flavoprotein3, cytochrome c oxidase subunit5A, Pcp411protein, ATP synthase-coupling factor6, Acyl-CoA-binding protein, cytochrome c, metallothionein-3, AIP1, protein uqcrb, glyceraldehyde-3-phosphate dehydrogenase, thymosin beta4. They involved in protein transport, electron transport, vacuolar transport, oxygen transport, immunity, rRNA export from nucleus, hydrogen ion transport, protein import into mitochondrial inner membrane, cadmium ion binding, endosomal transport, actin cytoskeleton organization. Heat shock27kDa protein1and immunoglobulin joining chain were up-regulated, hemoglobin subunit alpha and protein Hbb-b1were down-regulated in the colon of the model rat, which were in accord with the clinical report.(5) The differential proteins in the colon tissue of the SR group mainly involved in cellular assembly and organization, cellular function and maintenance, cell death and survival, cell morphology, tissue development, cellular development, cellular growth and proliferation, nervous system development and function, cell to cell signaling and interaction, small molecule biochemistry.The differential proteins in the brain tissue of the SR group mainly involved in cellular assembly and organization, cellular function and maintenance, cell death and survival, cell morphology, small molecule biochemistry, nervous system development and function, molecular transport, cellular development, tissue development, nucleic acid metabolism.(6) The differential proteins in the colon tissue of the SR group could be assigned to27pathways, including integrin signaling, serotonin degradation, antigen presentation pathway, IL-4signaling, and so on. The differential proteins in the brain tissue involved26pathways, including gap junction siganling, epithelia adherens junction signling, mitochondrial dysfuncion, etc. The differential proteins in the colon tissue of the SR group included9interaction networks, and12interaction networks in the brain tissue.(7) The expression of AQP8mRNA and Na+/H+exchanger3(NHE3) mRNA in the colon tissue were in accordance with the expression of homologous proteins, while the expression of brain-derived neurotrophic factor (BDNF) mRNA in the colon and hippocampus tissue were different from the expression of homologous protein.
Conclusions:(1) There were a large number of differential proteins in the colon and brain tissue of the rat model of IBS-D with liver depression and spleen deficiency syndrome, some of them were in accord with the clinical report.(2) The rat model of IBS-D had some proteins co-expressed in the colon and brain tissue, which indicated abnormal interaction between colon and brain.(3) The models induced by chronic restraint stress, maternal separation stress, and the combination of the two factors had the same molecule biological basis, mental stimulation mainly changed the proteins expression in the brain tissue.(4) The pathogenesis of IBS-D was related to changes of a number of proteins, studying the function of the differential proteins could gain a deeper understanding in the pathogenesis of IBS-D.
引文
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