外源性硫化氫及KATP通道對(duì)大鼠慢性應(yīng)激結(jié)腸高動(dòng)力的調(diào)節(jié)*

劉 穎， 全曉靜， 夏 虹， 羅和生

(1桂林醫(yī)學(xué)院附屬醫(yī)院消化科，廣西 桂林 541001； 2武漢大學(xué)人民醫(yī)院消化科，湖北 武漢 430060)

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外源性硫化氫及KATP通道對(duì)大鼠慢性應(yīng)激結(jié)腸高動(dòng)力的調(diào)節(jié)*

劉 穎1△， 全曉靜2， 夏 虹2， 羅和生2

(1桂林醫(yī)學(xué)院附屬醫(yī)院消化科，廣西 桂林 541001；2武漢大學(xué)人民醫(yī)院消化科，湖北 武漢 430060)

目的： 研究外源性硫化氫(hydrogen sulfide，H2S)及ATP敏感性鉀通道(ATP-sensitive potassium channels，KATP)在慢性應(yīng)激結(jié)腸高動(dòng)力中的作用。方法: 制作慢性避水應(yīng)激(water avoidance stress，WAS) 和假避水應(yīng)激(sham water avoidance stress，SWAS)大鼠模型，觀察2組大鼠結(jié)腸肌條的收縮活性以及硫氫化鈉(NaHS)和格列本脲預(yù)處理后對(duì)2組大鼠結(jié)腸肌條收縮影響并計(jì)算NaHS的半數(shù)抑制濃度(half maximal inhibitory concentration，IC50)，使用免疫熒光及Western blotting法觀察KATP通道各亞基在結(jié)腸中的分布及表達(dá)。結(jié)果: WAS組結(jié)腸肌條收縮活性明顯高于SWAS組；NaHS濃度依賴(lài)性抑制2組大鼠縱行肌 (longitudinal muscle，LM)和環(huán)形肌(circular muscle，CM)的收縮；WAS組LM和CM的NaHS IC50分別為0.2033 mmol/L和0.1438 mmol/L，均明顯低于SWAS組(P<0.01)；格列本脲明顯增加2組大鼠肌條NaHS IC50(P<0.01)；Kir6.1、Kir6.2和SUR-2B在2組大鼠結(jié)腸固有肌細(xì)胞膜均有分布；WAS組(去除黏膜及黏膜下層后)Kir6.1和SUR2B蛋白表達(dá)高于SWAS組(P<0.01)。結(jié)論: H2S外源性供體NaHS對(duì)慢性應(yīng)激結(jié)腸高動(dòng)力具有潛在的治療作用。KATP通道亞基Kir6.1/SUR2B表達(dá)增加可能是慢性應(yīng)激結(jié)腸動(dòng)力紊亂的一種適應(yīng)性反應(yīng)。

慢性應(yīng)激； 高動(dòng)力； 硫化氫； ATP敏感性鉀通道

慢性應(yīng)激可導(dǎo)致結(jié)腸動(dòng)力增加[1-4]，從而誘發(fā)或加劇IBS和IBD胃腸道癥狀等[2, 5-6]。已證實(shí)硫化氫(H2S)對(duì)胃腸動(dòng)力具有重要的調(diào)節(jié)作用，主要表現(xiàn)為抑制腸道動(dòng)力[7-9]，H2S抑制機(jī)制主要與直接開(kāi)放位于平滑肌細(xì)胞上的ATP敏感性鉀通道 (ATP-sensitive potassium channels，KATP)有關(guān)[8, 10-14]。我們的前期研究發(fā)現(xiàn)慢性避水應(yīng)激(water avoidance stress，WAS)大鼠結(jié)腸動(dòng)力增加，而結(jié)腸局部?jī)?nèi)源性H2S合成酶表達(dá)下調(diào)、H2S產(chǎn)生減少可能是其動(dòng)力增加的原因之一[15]。但作為H2S的靶通道亦是與消化道平滑肌興奮性密切相關(guān)的通道，KATP通道的數(shù)量及功能在慢性WAS結(jié)腸動(dòng)力紊亂時(shí)變化如何仍不清楚；H2S外源性供體硫氫化鈉(NaHS)是否對(duì)WAS結(jié)腸高動(dòng)力具有潛在治療作用以及該作用是否通過(guò)KATP通道來(lái)介導(dǎo)亦不明了。本研究通過(guò)觀察慢性應(yīng)激時(shí)結(jié)腸平滑肌KATP通道各亞基分布表達(dá)變化以及H2S外源性供體NaHS對(duì)結(jié)腸高動(dòng)力的影響，以探討KATP通道在慢性應(yīng)激結(jié)腸動(dòng)力紊亂中的作用以及NaHS對(duì)其潛在的治療價(jià)值。

材 料 和 方 法

1 動(dòng)物、試劑與儀器

健康雄性Wistar大鼠，SPF級(jí)，體重180～220 g，購(gòu)自湖北省疾病預(yù)防控制中心，動(dòng)物許可證號(hào)：SCXK(鄂) 2008-0005。動(dòng)物用混合配方飼料喂養(yǎng)，自由進(jìn)食飲水，定期更換墊料，保持大鼠生活環(huán)境通風(fēng)和清潔衛(wèi)生。

NaHS及格列苯脲均購(gòu)自Sigma；羊抗大鼠Kir6.1、Kir6.2和SUR2B多克隆抗體均購(gòu)自Santa Cruz；羊抗大鼠GAPDH多克隆抗體、辣根過(guò)氧化物酶標(biāo)記兔抗羊IgG、Cy3標(biāo)記兔抗羊IgG、二甲基亞砜(dimethyl sulfoxide，DSMO)和DAB顯色劑均購(gòu)自北京中山生物技術(shù)有限公司；SDS-PAGE凝膠配制試劑盒、Western blotting轉(zhuǎn)膜液、封閉液、硝酸纖維膜和Bradford蛋白濃度測(cè)定試劑盒均購(gòu)自博士德公司。臺(tái)式液成分為(mmol/L)：氯化鈉 147.0, 氯化鉀 4.0, 氯化鈣 2.0, 磷酸二氫鈉 0.42, 磷酸氫二鈉 2.0, 氯化鎂 1.05, 葡萄糖 5.5，以氫氧化鈉調(diào)pH值至7.4。

解剖顯微鏡、熒光顯微鏡(Olympus)；張力換能器、RM6240多通道生理信號(hào)采集處理系統(tǒng)(成都儀器廠)；AB-104 電子天平(Mettler Toledo)；低溫離心機(jī)(Thermo Electron)；電泳儀(Bio-Rad)。

2 方法

2.1 大鼠分組及慢性避水應(yīng)激模型的制作 大鼠隨機(jī)分為慢性WAS組(n=10)和假避水應(yīng)激(sham water avoidance stress，SWAS)組(n=10)。WAS大鼠模型是國(guó)內(nèi)外普遍認(rèn)可并用于研究慢性應(yīng)激結(jié)腸高動(dòng)力相關(guān)機(jī)制的模型[1, 6]，模型制作方法按照文獻(xiàn)報(bào)道[1-2]進(jìn)行：大鼠放于周邊有水的8 cm×6 cm×6 cm的平臺(tái)上(水低于平臺(tái)1 cm)；SWAS組大鼠放于周?chē)鸁o(wú)水的平臺(tái)。2組大鼠均觀察1 h，每天1次，連續(xù)10 d，每日固定在8：00～13：00進(jìn)行實(shí)驗(yàn)。

2.2 結(jié)腸平滑肌條的制備及其自發(fā)性收縮的記錄 擊昏大鼠后頸動(dòng)脈放血處死，剖腹，在距回盲瓣1 cm處取約3 cm長(zhǎng)的結(jié)腸，置入通有95% O2和5% CO2混合氣體的臺(tái)氏液中，沿腸系膜處剪開(kāi)腸管并漂洗干凈，黏膜面朝上并固定于硅膠板上，解剖顯微鏡下迅速剝離黏膜和黏膜下層，沿腸管縱軸走向剪取縱行肌條(longitudinal muscle，LM)，沿橫軸走向剪取環(huán)形肌條(circular muscle，CM)。將肌條放置于盛有恒溫(37 ℃)臺(tái)式液的浴槽中(容積約6 mL)，并持續(xù)充有95% O2和5% CO2的混合氣體，肌條的一端固定在浴槽底部掛鉤，另一端用絲線(xiàn)固定于張力換能器上。給肌條1.0 g前負(fù)荷，孵育1 h，臺(tái)式液每隔15 min予以更換。每只大鼠選取出現(xiàn)規(guī)律收縮的LM和CM各 1 根記錄機(jī)械收縮信號(hào)，收縮強(qiáng)度以曲線(xiàn)下面積(area under the curve，AUC)表示。待結(jié)腸平滑肌條出現(xiàn)規(guī)律收縮后分別以累計(jì)加藥法加入不同濃度(0.01～1 mmol/L)的NaHS溶液，觀察肌條曲線(xiàn)下面積的變化；以格列苯脲(10 μmol/L)孵育肌條30 min后，再以累計(jì)加藥法加入不同濃度(0.01～1 mmol/L)的NaHS溶液，觀察肌條曲線(xiàn)下面積的變化，結(jié)果以肌條收縮AUC抑制率表示 [AUC抑制率(%)=(給藥前-給藥后)/給藥前×100%]。

2.3 免疫熒光染色檢測(cè)結(jié)腸SUR-2B、Kir6.1和Kir6.2的分布 取結(jié)腸標(biāo)本約1 cm，10%中性緩沖福爾馬林固定約24 h；沖洗后常規(guī)脫水、透明、浸蠟、包埋，石蠟包埋切片厚約5 μm；切片脫蠟至水并行抗原修復(fù)后，滴加3%牛封閉血清37 ℃濕盒中封閉1 h；分別加入 I 抗(SUR-2B 1∶250，Kir6.1 1∶300，Kir6.2 1∶300)，4 ℃濕盒中孵育過(guò)夜；PBS沖洗后，Cy3標(biāo)記兔抗羊IgG (1∶100)，37 ℃濕盒中避光孵育1 h；PBS沖洗后，甘油封片，熒光顯微鏡下觀察。

2.4 Western blotting檢測(cè)結(jié)腸(去除黏膜及黏膜下層)SUR-2B、Kir6.1和Kir6.2的表達(dá) 取大鼠近端結(jié)腸組織(去除黏膜及黏膜下層)約1 g，經(jīng)RIPA裂解液裂解后，勻漿液經(jīng)離心后上清分裝放入-80 ℃冰箱保存；使用BCA蛋白濃度測(cè)定試劑盒、紫外分光光度計(jì)測(cè)定蛋白濃度。取50 μg總蛋白進(jìn)行聚丙烯酰胺凝膠(SDS-PAGE，10%分離膠)電泳，將電泳后的蛋白質(zhì)電轉(zhuǎn)移至PVDF膜上，用5%脫脂奶粉搖床室溫封閉2 h，洗脫后分別加入 I 抗(SUR-2B 1∶400，Kir6.1 1∶400，Kir6.2 1∶400，GAPDH 1∶200)、4 ℃過(guò)夜，經(jīng)PBST 沖洗后以堿性磷酸酶標(biāo)記的 II抗(1∶10 000)室溫?fù)u床孵育2 h；經(jīng)TBST洗滌后滴加適量的ECL底物液，孵育數(shù)分鐘；待熒光帶明顯后，覆上保鮮膜，X光膠片壓片后行顯影、定影。使用BandScan分析膠片灰度值，將各組SUR-2B、Kir6.1和Kir6.2灰度值分別與GAPDH灰度值的比值作為相對(duì)表達(dá)量，統(tǒng)計(jì)分析各組表達(dá)量差異。

3 統(tǒng)計(jì)學(xué)處理

用GraghPad Prism 5.0軟件分析處理，數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，組間比較行兩獨(dú)立樣本t檢驗(yàn)；進(jìn)行曲線(xiàn)擬合并計(jì)算半數(shù)抑制濃度(half maximal inhibitory concentration，IC50)，單位換算成mmol/L；以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 WAS大鼠結(jié)腸肌條收縮活性明顯增高

WAS組大鼠LM 和CM的AUC均高于SWAS 組 [LM:(23.59±3.23) g/minvs(14.38±2.56) g/min；CM:(12.62±2.13) g/minvs(7.97±1.94) g/min, 均P<0.01),見(jiàn)圖1。

Figure 1.WAS increased contractile activity of colonic strips. Mean ± SD.n=10.**P< 0.01vsSWAS group.

圖1 WAS增加大鼠結(jié)腸肌條收縮活性

2 外源性NaHS抑制大鼠結(jié)腸平滑肌條自發(fā)性收縮

NaHS(0.01～1 mmol/L)對(duì)SWAS和WAS大鼠的LM和CM自發(fā)性收縮均產(chǎn)生濃度依賴(lài)性抑制效應(yīng), NaHS為 1 mmol/L時(shí)產(chǎn)生100%抑制。WAS大鼠LM的NaHS IC50為0.203 mmol/L(95%可信區(qū)間 IC500.187～0.221, logIC50=-3.692±0.015), 明顯低于SWAS組(IC50=0.305 mmol/L, 95%可信區(qū)間IC500.271～0.345, logIC50=-3.515±0.020,P<0.01)。WAS大鼠CM的 NaHS IC50為0.144 mmol/L(95%可信區(qū)間 IC500.127～0.162, logIC50=-3.842±0.021)，亦明顯低于SWAS組(IC50=0.210 mmol/L, 95% 可信區(qū)間 IC500.181～0.244，logIC50=-3.678±0.025,P<0.01)，見(jiàn)圖2。

3 KATP通道阻斷劑減弱NaHS的抑制作用

KATP通道阻斷劑格列本脲明顯減低NaHS的抑制作用。加入格列本脲(10 μmol/L)孵育肌條30 min后，NaHS(1 mmol/L)的抑制效應(yīng)僅達(dá)原來(lái)的30%～45%。SWAS組，格列本脲增加LM的NaHS IC50，NaHS IC50從0.305 mmol/L增加至2.102 mmol/L(95%可信區(qū)間IC50為 1.622～2.725，logIC50=-2.677±0.044，P<0.01)； 格列本脲亦增加CM的NaHS IC50，NaHS IC50從0.210 mmol/L增加至1.484 mmol/L(95% 可信區(qū)間IC50為0.991～2.224，logIC50=-2.828±0.068，P<0.01)。WAS組, 格列本脲增加LM的NaHS的IC50，NaHS IC50從0.203 mmol/L增加至1.756 mmol/L(95% 可信區(qū)間 IC501.235～2.496，logIC50=-2.756±0.059，P<0.01)； 格列本脲亦增加CM的NaHS IC50，NaHS IC50從0.144增加至1.231 mmol/L(95%可信區(qū)間IC500.900～1.682，logIC50=-2.910±0.053，P<0.01)，見(jiàn)圖3。

4 KATP通道各亞基在大鼠結(jié)腸中的分布

在2組大鼠的結(jié)腸組織中，Kir6.1、Kir6.2和SUR-2B在LM和CM細(xì)胞膜均有分布；Kir6.1和SUR-2B表達(dá)較強(qiáng)，而Kir6.2表達(dá)較弱，見(jiàn)圖4。

5 WAS增加KATP通道亞基蛋白的表達(dá)

Western blotting實(shí)驗(yàn)結(jié)果顯示W(wǎng)AS組大鼠的Kir6.1和SUR2B蛋白表達(dá)高于SWAS組(均P<0.01)，而Kir6.2的表達(dá)無(wú)顯著差異，見(jiàn)圖5。

討 論

KATP通道是H2S主要的靶通道[8, 14, 16-17]，H2S通過(guò)開(kāi)放KATP通道，增加KATP電流，使細(xì)胞膜超極化，進(jìn)而舒張平滑肌，調(diào)節(jié)消化道動(dòng)力。本實(shí)驗(yàn)制作WAS大鼠模型并通過(guò)肌條實(shí)驗(yàn)證實(shí)結(jié)腸LM及CM收縮活性均高于對(duì)照組；而WAS結(jié)腸動(dòng)力增加與局部腸道內(nèi)源性H2S合成減少與結(jié)腸高動(dòng)力相關(guān)[15]。使用H2S外源性供體NaHS作用于大鼠肌條以觀察外源性H2S對(duì)WAS結(jié)腸高動(dòng)力的影響，結(jié)果顯示NaHS均抑制SWAS和WAS大鼠結(jié)腸LM和CM自發(fā)性收縮，抑制效應(yīng)呈濃度依賴(lài)性，這與既往的研究一致[7-8, 11, 18-19]；而使用KATP通道阻斷劑格列苯脲預(yù)處理后，NaHS 的抑制效應(yīng)明顯減弱，進(jìn)一步證實(shí)NaHS的抑制作用通過(guò)KATP通道介導(dǎo)。

Figure 2.NaHS inhibited contractile activity of colonic strips. A, B: representative effects of different concentrations of NaHS on spontaneous contraction of colonic smooth muscle strips in the SWAS and WAS rats; C, D: the concentration-dependent curve fitting showing that NaHS inhibited contractile activity of LM and CM in both SWAS and WAS rats. IC50values of NaHS for the WAS rats were lower than those for the SWAS rats. Mean±SD.n=10.

圖2 NaHS抑制大鼠結(jié)腸平滑肌條收縮活性

KATP通道是由Kir亞基和SUR亞基組成，Kir和SUR按1∶1比例相連，以四聚體模式構(gòu)成八聚體(SUR/Kir6.X)4復(fù)合物。Kir構(gòu)成KATP穿通孔道，具有內(nèi)向整流的特性；SUR是KATP通道的調(diào)節(jié)亞基，是KATP通道開(kāi)放劑或抑制劑作用靶點(diǎn)。不同KATP通道亞基組成形式體現(xiàn)不同生物物理學(xué)、藥理學(xué)以及新陳代謝特性。Kir6.1/SUR2B和Kir6.2/SUR2B是胃腸平滑肌細(xì)胞KATP通道的主要構(gòu)型[20-21]。表達(dá)Kir6.1的KATP通道電導(dǎo)為35 pS, 表達(dá)Kir6.2的為80 pS，而消化道平滑肌KATP單通道電導(dǎo)為42 pS，提示Kir6.1是胃腸平滑肌KATP通道主要的成孔亞基[20]。本實(shí)驗(yàn)證實(shí)大鼠結(jié)腸平滑肌有Kir6.1、Kir 6.2和SUR2B的分布。

本研究發(fā)現(xiàn)WAS組結(jié)腸肌條收縮活性高于對(duì)照組，但WAS組NaHS IC50明顯低于SWAS組，提示W(wǎng)AS組肌條對(duì)NaHS的抑制作用具有更高的敏感性，其是否通過(guò)KATP通道的可塑性變化實(shí)現(xiàn)，如可增加KATP通道數(shù)量或提高該通道功能(如開(kāi)放頻率)等；Western blotting結(jié)果顯示W(wǎng)AS大鼠Kir6.1和SUR2B蛋白表達(dá)增加，Kir6.2表達(dá)無(wú)改變，提示W(wǎng)AS可引起KATP通道數(shù)量增加。Kir6.1/SUR2B表達(dá)增加的原因仍不清楚，這種可塑性變化可能為動(dòng)物對(duì)應(yīng)激原的一種適應(yīng)性反應(yīng)，通過(guò)提高KATP通道數(shù)量從而增加對(duì)開(kāi)放劑(如H2S)的敏感性，從而糾正腸道功能紊亂并改善癥狀。本實(shí)驗(yàn)尚未對(duì)KATP通道功能進(jìn)行研究，WAS時(shí)KATP通道功能是否存在變化有待于膜片鉗等電生理實(shí)驗(yàn)進(jìn)一步證實(shí)。

Figure 3.Inhibitory effect of NaHS was reversed by glibenclamide. A, C: representative effects of different concentrations of NaHS on spontaneous contraction of colonic smooth muscle strips in the SWAS and WAS rats after the incubation with glfibenclamide (10 μmol/L); B, D: the concentration-dependent curve tting showing that glybenclamide signi cantly reduced the inhibitory effect induced by H2S. NaHS (1 mmol/L) only caused 30%～45% of inhibition with previous addition of gli-benclamide. Mean±SD.n=10.

圖3 格列本脲減弱NaHS對(duì)大鼠結(jié)腸平滑肌收縮的抑制效應(yīng)

Figure 4.Distribution of subunits of KATPchannels (immunohistochemical staining, scale bar=20 μm).

圖4 KATP通道各亞基在大鼠結(jié)腸中的分布

H2S在消化道具有保護(hù)黏膜、調(diào)節(jié)白細(xì)胞黏附和聚集、抗炎等作用，對(duì)多種消化道疾病具有潛在的治療作用，如H2S供體或H2S合成的底物L(fēng)-半胱氨酸可維持胃黏膜完整、促進(jìn)大鼠胃潰瘍愈合[22]；抑制H2S的合成可使腸道環(huán)氧化酶2的mRNA表達(dá)和前列腺素的合成下調(diào)，導(dǎo)致結(jié)腸炎癥和黏膜損傷，結(jié)腸內(nèi)灌入H2S供體明顯減輕結(jié)腸炎嚴(yán)重性和降低炎癥介質(zhì)如腫瘤壞死因子α mRNA的表達(dá)[23-24]。本實(shí)驗(yàn)體外研究顯示W(wǎng)AS大鼠NaHS的IC50明顯低于對(duì)照組，可能源于平滑肌細(xì)胞上Kir6.1和SUR2B表達(dá)上調(diào)，提示W(wǎng)AS大鼠結(jié)腸對(duì)NaHS具有更高的敏感性，外源性的H2S對(duì)改善慢性應(yīng)激引起的結(jié)腸高動(dòng)力具有潛在的治療價(jià)值。

H2S的生理、藥理及毒副作用受到廣泛關(guān)注。人體中H2S的血清濃度為10～100 μmol/L[25]，在生理濃度時(shí)H2S對(duì)維持內(nèi)環(huán)境的穩(wěn)態(tài)具有一定作用，但一旦超過(guò)生理濃度H2S具有潛在的毒性作用，如誘發(fā)DNA損傷和抑制線(xiàn)粒體細(xì)胞色素C氧化酶等[26-27]；毫摩爾濃度的H2S對(duì)結(jié)腸細(xì)胞即有害作用[28]；而且H2S的生理和毒性作用間的濃度梯度非常小[26]；此外除腸道組織本身外，腸腔內(nèi)還有大量細(xì)菌來(lái)源的H2S并影響著局部腸道的生理功能，對(duì)腸道某些病理生理狀態(tài)起著保護(hù)抑或損傷作用。體外實(shí)驗(yàn)發(fā)現(xiàn)在慢性WAS結(jié)腸動(dòng)力增高時(shí)，NaHS抑制結(jié)腸LM和CM收縮的IC50分別為0.203 mmol/L、0.144 mmol/L，此種濃度在發(fā)揮改善動(dòng)力紊亂作用的同時(shí)是否存在對(duì)結(jié)腸組織的毒副作用有待于進(jìn)一步的研究。

Figure 5.Protein expression of subunits of KATPchannels in colonic tissues. Mean±SD.n=10.**P<0.01vsSWAS group.

圖5 2組大鼠結(jié)腸KATP通道各亞基蛋白表達(dá)

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Effect of exogenous H2S and ATP-sensitive potassium channels on colonic hypermotility in a rat model of chronic stress

LIU Ying1, QUAN Xiao-jing2, XIA Hong2, LUO He-sheng2

(1DepartmentofGastroenterology,TheAffiliatedHospitalofGuilinMedicalCollege,Guilin541001,China;2DepartmentofGastroenterology,RenminHospitalofWuhanUniversity,Wuhan430060,China.E-mail:liuy1009@sina.com)

AIM: To investigate the potential role of exogenous hydrogen sulfide (H2S) and ATP-sensitive potassium (KATP) channels in chronic stress-induced colonic hypermotility. METHODS: Male Wistar rats were divided into water avoidance stress (WAS) group and sham WAS (SWAS) group. Organ bath recordings were used to test the contractile activity of colonic strips. The effects of H2S donor NaHS and pretreatment with glibenclamide on the contractions of colonic smooth muscle were studied and the IC50of NaHS was calculated. The localization and expression of the subunits of KATPchannels were determined by the methods of immunohistochemistry and Western blotting. RESULTS: WAS increased contractile activity of colonic strips. NaHS concentration-dependently inhibited the spontaneous contractions of strips from the SWAS and WAS rats. The IC50of NaHS for longitudinal muscle (LM) and circular muscle (CM) of the WAS rats was 0.2033 mmol/L and 0.1438 mmol/L, significantly lower than those of the SWAS rats. Glibenclamide significantly increased the IC50of NaHS for LM and CM from the SWAS and WAS rats. In both SWAS and WAS rat colon, Kir6.1, Kir6.2 and SUR2B were expressed on the plasma membrane of the smooth muscle cells. WAS treatment resulted in up-regulation of the expression of Kir6.1 and SUR2B in the colon devoid of mucosa and submucosa. CONCLUSION: The increased expression of Kir 6.1 and SUR2B in colonic smooth muscle cells may be a defensive response to chronic WAS. H2S donors may have potential clinical effect on treating chronic stress-induced colonic hypermotility.

Chronic stress; Hypermotility; Hydrogen sulfide; ATP-sensitive potassium channels

1000- 4718(2015)04- 0725- 07

2014- 11- 05

2015- 01- 08

國(guó)家自然科學(xué)基金資助項(xiàng)目 (No. 81460111)；廣西自然科學(xué)基金資助項(xiàng)目(No. 2014GXNSFAA118166)；廣西壯族自治區(qū)衛(wèi)生廳計(jì)劃課題(No. Z2012408)

R333.3; R363.2

A

10.3969/j.issn.1000- 4718.2015.04.027

△通訊作者 Tel： 0773-2823740； E-mail： liuy1009@sina.com