Cx43、NMDA與大鼠腸易激綜合征內(nèi)臟敏化的關(guān)系研究

張靜瑜，黃裕新，秦明，高會軍，竇維佳，王景杰

·基礎(chǔ)研究·

Cx43、NMDA與大鼠腸易激綜合征內(nèi)臟敏化的關(guān)系研究

張靜瑜，黃裕新，秦明，高會軍，竇維佳，王景杰

目的探討大鼠間隙連接蛋白43(Cx43)和骶髓N-甲基-D-天冬氨酸(NMDA)受體與腸易激綜合征(IBS)內(nèi)臟敏化的關(guān)系。方法雄性SD大鼠50只，隨機分為正常對照組、正常結(jié)腸擴張組、IBS組、IBS結(jié)腸擴張組及IBS結(jié)腸擴張+甲磺酸伊馬替尼(STI-571)干預(yù)組，每組10只。IBS組、IBS結(jié)腸擴張組和IBS結(jié)腸擴張并干預(yù)組大鼠采用旋毛蟲感染制作IBS模型。采用免疫熒光組織化學法觀察各組大鼠結(jié)腸Cx43和骶髓NMDA受體的表達。結(jié)果正常組、正常結(jié)腸擴張組、IBS組的結(jié)腸Cx43和骶髓NMDA表達水平差異無統(tǒng)計學意義(P>0.05)，IBS結(jié)腸擴張組結(jié)腸Cx43和骶髓NMDA表達水平與正常組、正常結(jié)腸擴張組、IBS組比較均顯著增強(P<0.01)，而STI-571干預(yù)后大鼠結(jié)腸Cx43和骶髓NMDA表達均有所下降(P<0.01)。結(jié)論Cx43和NMDA可能在IBS內(nèi)臟敏化機制中起關(guān)鍵作用。

腸易激綜合征；連接蛋白43；受體，N-甲基-D-天冬氨酸

近年來由于人們生活方式改變，腸易激綜合征(irritable bowel syndrome，IBS)的發(fā)病率不斷增加[1-2]，但發(fā)病機制卻并不十分清楚。近年研究發(fā)現(xiàn)，IBS與內(nèi)臟神經(jīng)敏化有密切關(guān)系[3-4]。間隙連接蛋白(connexin，Cx)在人體廣泛分布，其中Cx43是組成細胞間縫隙連接的主要成分[5]，其表達異常與多種疾病的發(fā)生有關(guān)[6-8]。N-甲基-D-天冬氨酸(NMDA)受體由NR1、NR2A-D及NR3A-B等7種亞單位組成，廣泛分布于中樞神經(jīng)系統(tǒng)，其中小鼠和人的NR2B同源基因已獲克隆。近年來研究發(fā)現(xiàn)，NR2B受體亞型在學習、記憶、進食行為、疼痛的產(chǎn)生及中樞性痛覺敏化形成等方面與人類多種神經(jīng)疾病相關(guān)[7,9-10]。本研究探討了Cx43和NMDA的表達與IBS的關(guān)系。

1 材料與方法

1.1 主要實驗材料及試劑 保蟲昆明鼠(旋毛蟲系云黑龍江株)購自河南省疾病預(yù)防控制中心，雄性SD大鼠購自第四軍醫(yī)大學實驗動物中心。甲磺酸伊馬替尼(STI-571，美國Abcam)，兔抗NMDA血清(1:3000，Novus公司)，兔抗Cx43血清(1:3000，CellSignaling公司)，羊抗兔IgG(1:500，Sigma公司)，ABC復(fù)合物(1:500，Sigma公司)。

1.2 實驗分組 50只SD大鼠隨機分為正常對照組、正常結(jié)腸擴張組、IBS組、IBS結(jié)腸擴張組及IBS結(jié)腸擴張+STI-571組，每組10只。結(jié)腸擴張條件選用本課題組既往實驗的高峰值，即球囊注水1.0ml[11]；STI-571采用腹腔注射(20mg/ml，0.5ml/kg)。

1.3 IBS動物模型制作 保蟲昆明鼠種鼠3只，脊柱脫臼法處死，剔除皮毛、內(nèi)臟及肌肉；將肌肉剪碎置于300ml含2.5%胃蛋白酶和0.5%鹽酸的消化液中，37℃水浴消化12～20h；經(jīng)篩過濾，用生理鹽水反復(fù)沉淀洗滌3～5次，收集旋毛蟲幼蟲囊包并計數(shù)。取昆明鼠20只，體重25g左右，灌胃法給予0.1ml含250～300條幼蟲的生理鹽水懸液感染大鼠；將傳代感染的昆明鼠以頸椎脫臼法處死，收集旋毛蟲(方法同前)，IBS組、IBS結(jié)腸擴張組及IBS結(jié)腸擴張+STI-571組大鼠采用灌胃法給予1ml含4000條幼蟲囊包的生理鹽水懸液，飼養(yǎng)8周后抽檢測定感染情況，確立模型制作成功[12]。

1.4 組織冰凍切片制作 各組大鼠進行灌注內(nèi)固定后立即剖腹，取乙狀結(jié)腸處0.4cm×0.2cm大小組織以及長約1cm的完整骶髓組織，放入4%多聚甲醛中于4℃固定6h，取出標本移入30%蔗糖液，4℃放置24h，超低溫切片機(美國Nuair公司)10μm切片待染。

1.5 雙重熒光免疫組織化學法檢測Cx43及NMDA表達 將標本置于0.3% H2O2-甲醇中浸泡30min封閉內(nèi)源性過氧化物酶，0.1% Triton浸泡30min，PBS漂洗；加入免疫血清稀釋的Cx43過夜(1:2000，約14h)；將切片和鋪片置于室溫下復(fù)溫1h，經(jīng)PBS漂洗后入1:60稀釋的四甲基異硫氰酸羅丹明(TMRITC)標記的抗兔IgG二抗及異硫氰酸熒光素(FITC)標記的抗小鼠IgG二抗，室溫孵育3h；PBS漂洗后將鋪片平置于載玻片上，與切片標本同時用60%緩沖甘油封片。NMDA制備方法同上。采用激光共聚焦顯微鏡采圖分析，每張切片取4～6個視野，將數(shù)字化圖像儲存于LSCM系統(tǒng)計算機，實驗結(jié)束后進行圖像分析。

1.6 統(tǒng)計學處理 采用SPSS 16. 0軟件進行統(tǒng)計分析。熒光半定量結(jié)果經(jīng)Image J 1.43軟件處理，所得到的熒光光度值差異采用Kruskal-WallisH檢驗及Nemenyi法檢驗進行比較。P<0.05為差異有統(tǒng)計學意義。

圖1 各組大鼠結(jié)腸Cx43表達情況(免疫組化檢測)Fig.1 Expression of Cx43 protein in colon of rats in each group (Immunohistochemistry)A. Normal group; B. Colon distension group; C. IBS group; D. IBS with colon distension group; E. STI-571 in IBS with colon distension group; (1)P<0.01 compared with normal group,colon distension group and IBS group; (2)P<0.01 compared with IBS with colon distension group

2 結(jié) 果

2.1 大鼠結(jié)腸Cx43表達情況 由圖1可見，正常狀態(tài)下大鼠結(jié)腸部位Cx43處于低水平表達，其熒光光度值為40±5。給予結(jié)腸插入球囊灌水擴張后，大鼠結(jié)腸部位Cx43表達水平有所增加，其熒光光度值為45±5，與正常對照組相比未見明顯差異(P>0.05)。IBS組大鼠結(jié)腸部位Cx43表達水平(熒光光度值48±5)與正常對照組及正常擴張組相比均未見明顯差異(P>0.05)。IBS組大鼠給予結(jié)腸擴張激后，其結(jié)腸部位Cx43表達水平顯著增高，熒光光度值為90±5，與正常結(jié)腸擴張組比較差異有統(tǒng)計學意義(P<0.01)，而給予STI-571腹腔注射干預(yù)的IBS大鼠結(jié)腸部位Cx43表達明顯降低，其熒光光度值(46±5)與IBS結(jié)腸擴張組相比差異有統(tǒng)計學意義(P<0.01)。

2.2 大鼠骶髓NMDA表達情況 正常狀態(tài)下大鼠結(jié)腸部位骶髓NMDA處于低水平表達，其熒光光度值為59±5，給予結(jié)腸插入球囊灌水擴張后，大鼠結(jié)腸部位NMDA表達水平有所增加，其熒光光度值為64±5，與正常對照組相比未見明顯差異(P>0.05)。IBS組大鼠結(jié)腸部位NMDA表達水平(熒光光度值68±5)與正常對照組及正常擴張組相比未見明顯差異(P>0.05)。IBS組大鼠給予結(jié)腸擴張后，其結(jié)腸部位NMDA表達水平顯著增高，熒光光度值為108±5，與正常擴張組比較有顯著差異(P<0.01)，而給予STI-571腹腔注射干預(yù)的IBS大鼠結(jié)腸部位NMDA表達明顯降低，其熒光光度值(62±5)與IBS結(jié)腸擴張組相比差異有統(tǒng)計學意義(P<0.01，圖2)。

圖2 各組大鼠骶髓NMDA表達情況(激光共聚焦顯微鏡)Fig.2 Expression of sacral NMDA of rats in each group (Laser confocal microscopy)A. Normal group; B. Colon distension group; C. IBS group; D. IBS with colon distension group; E. STI-571 in IBS with colon distension group; (1)P<0.01 compared with normal group,colon distension group and IBS group; (2)P<0.01 compared with IBS with colon distension group

3 討 論

IBS與腸道動力異常、內(nèi)臟感覺異常、免疫異常、炎癥反應(yīng)、腸道菌群失調(diào)、遺傳因素、食物因素、性別以及腦-腸相互作用、腸道神經(jīng)內(nèi)分泌網(wǎng)絡(luò)調(diào)控失常等存在著密切關(guān)系[13]。研究提示，IBS患者以腸道動力異常和內(nèi)臟感覺異常為主要表現(xiàn)，這些表現(xiàn)和變化均與神經(jīng)作用存在密切聯(lián)系[14]。

縫隙連接蛋白在人體廣泛分布，Cx43是組成細胞間縫隙連接的主要成分。近年大量研究表明，胃腸運動受腸神經(jīng)系統(tǒng)和Cajal間質(zhì)細胞(interstitial cells of Cajal，ICC)共同調(diào)控，ICC連接平滑肌與神經(jīng)細胞，三者之間廣泛存在縫隙連接并形成網(wǎng)絡(luò)樣結(jié)構(gòu)。通過縫隙連接，ICC為神經(jīng)細胞和平滑肌細胞之間提供了重要的中介通道，從而更有效地傳遞電、神經(jīng)信號以刺激平滑肌細胞，實現(xiàn)其起搏、舒縮功能[15]。本課題組前期實驗發(fā)現(xiàn)IBS大鼠中ICC表達顯著增高[16]，本實驗結(jié)果表明IBS擴張大鼠Cx43表達明顯增高，推測IBS內(nèi)臟敏化是通過ICC的過度敏化，而其間的縫隙連接蛋白發(fā)揮著重要作用。

近期研究發(fā)現(xiàn)，NMDA受體在內(nèi)臟傷害性信息轉(zhuǎn)導(dǎo)中具有重要作用，結(jié)腸炎恢復(fù)后脊髓背角NMDAR1和NMDAR2A/B的同步上調(diào)是導(dǎo)致內(nèi)臟敏感性持續(xù)增高的原因[17]。正常NMDA受體被鎂離子阻滯，外周神經(jīng)損傷后，去極化導(dǎo)致鎂離子從通道上移開，引起NMDA活化，進而促使大量鈣離子內(nèi)流，使突觸后神經(jīng)元興奮性增高；細胞鈣離子濃度增加引起轉(zhuǎn)錄因子c-fos、c-jun活化，最終活化許多下游基因；轉(zhuǎn)錄的改變最終引起細胞內(nèi)和細胞表面受體蛋白表達的改變；蛋白激酶(包括PKC)在維持中樞敏化中發(fā)揮重要作用[18-21]。本研究采用旋毛蟲感染制備大鼠IBS模型，給予結(jié)腸擴張刺激后可表現(xiàn)為IBS的內(nèi)臟高敏化[12]，本課題組曾采用STI-571阻斷ICC，其后IBS的內(nèi)臟敏化明顯下降[16]，而本實驗的各項結(jié)果提示，IBS結(jié)腸擴張組的Cx43和NMDA表達與比正常對照組、正常結(jié)腸擴張組、IBS組和IBS結(jié)腸擴張+STI-571組比較均顯著增強，表明Cx43和NMDA與IBS的內(nèi)臟敏化可能有著密切聯(lián)系。

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The relation of Cx43 and NMDA to visceral sensitization in rats with irritable bowel syndrome

ZHANG Jing-yu,HUANG Yu-xin,QIN Ming,GAO Hui-jun,DOU Wei-jia,WANG Jing-jie*
Department of Gastroenterology,Tangdu Hospital,Fourth Military Medical University,Xi’an 710038,China
*< class="emphasis_italic">Corresponding author,E-mail: jingyu1104@163.com

,E-mail: jingyu1104@163.com
This work was supported by the National Natural Science Foundation of China (81070306)

ObjectiveTo study the relationship between connexin 43 (Cx43) and N-methyl-D-aspartate (NMDA) receptors and visceral sensitization in the rats with irritable bowel syndrome (IBS).MethodsThirty rats were gavaged withTriehinella spiralisto reproduce the IBS model. These rats were randomly divided into IBS group,IBS+colon distension group,and IBS+STI-571+colon distension group,and other groups of normal rats were randomized into normal group and normal+colon distension group,with 10 rats in each group. Immunofluorescent double staining were used to observe the expressions of intestine Cx43 and sacral NMDA receptors of rats in all the groups.ResultsThe Cx43 and sacral NMDA expressions in the normal group,normal+colon distension group and IBS group showed no significant changes (P>0.05),however,Cx43 and sacral NMDA expressions were significantly higher in IBS rats with colon distension as compared with those in normal group,normal+colon distension group,and IBS group (P<0.05),while they were significantly lower in the IBS+STI-571+colon distension group after STI-571 intervention (P<0.05).ConclusionCx43 and sacral NMDA may be the most important factor of visceral sensitization in IBS rats.

irritable bowel syndrome; connexin 43; receptors,N-methyl-D-aspartate

R574.4

A

0577-7402(2015)12-0946-04

10.11855/j.issn.0577-7402.2015.12.02

2015-06-28；

2015-09-05)

(責任編輯：熊曉然)

國家自然科學基金(81070306)

張靜瑜，碩士研究生。主要從事胃腸道動力及機制方面的研究

710038 西安 第四軍醫(yī)大學唐都醫(yī)院消化內(nèi)科(張靜瑜、黃裕新、秦明、高會軍、竇維佳、王景杰)

王景杰，E-mail：jingyu1104@163.com