替加色羅對結(jié)腸炎大鼠內(nèi)臟敏感性的影響及其與結(jié)腸內(nèi)P物質(zhì)和降鈣素基因相關(guān)肽表達(dá)的關(guān)系

1、替加色羅對結(jié)腸炎大鼠內(nèi)臟敏感性的影響及其與結(jié)腸內(nèi)P物質(zhì)和降鈣素基因相關(guān)肽表達(dá)的關(guān)系         08-01-15 10:22:00     編輯：studa20                    作者：孫怡寧羅金燕蘭莉饒志仁 【摘要】  目的 評價替加色羅對結(jié)腸炎大鼠內(nèi)臟

2、敏感性的影響，并探討其調(diào)節(jié)內(nèi)臟敏感性的作用途徑。方法 成年雄性SD大鼠42只，經(jīng)三硝基苯磺酸(TNBS)灌腸誘導(dǎo)結(jié)腸炎后隨機分為4個實驗組和4個對照組：其中3個結(jié)直腸擴張(CRD)實驗組(n=6)及3個CRD對照組(n=4)分別給大鼠替加色羅和生理鹽水灌胃，在灌胃3、7、14d后記錄腹壁肌電活動；1個免疫組化(IH)實驗組(n=6)及其對照組(n=6)分別在替加色羅和生理鹽水灌胃7d后，采用免疫組織化學(xué)法觀察大鼠結(jié)腸內(nèi)P物質(zhì)(SP)和降鈣素基因相關(guān)肽(CGRP)的表達(dá)。結(jié)果 替加色羅灌胃3d后，在1.2、1.6mL擴張容量下，大鼠腹肌收縮次數(shù)(6.00±1.10，3.17±

3、0.98)較對照組明顯減少(9.50±2.52，13.0±2.31，P<0.05)。替加色羅連續(xù)灌胃7d和14d后，在0.4、0.8、1.2、1.6mL擴張容量下，大鼠腹肌收縮次數(shù)均明顯少于對照組(P<0.01)。替加色羅灌胃7d后，結(jié)腸內(nèi)SP染色評分下降，而CGRP染色強度與對照組比較無明顯變化。結(jié)論 替加色羅可以明顯降低結(jié)腸炎大鼠對結(jié)腸球囊擴張刺激的敏感性，其降低內(nèi)臟敏感性的作用可能與其抑制胃腸道內(nèi)臟感覺傳入神經(jīng)表達(dá)SP有關(guān)。 【關(guān)鍵詞】  替加色羅；內(nèi)臟敏感性；腸易激綜合征；P物質(zhì)；降鈣素基因相關(guān)肽Effects of tegaserod on

4、visceral sensitivity and expression of SPand CGRP in the rat colonABSTRACT: Objective  To investigate the effects of tegaserod on visceral sensitivity and explore the regulating mechanism. Methods  Fortytwo male SpragreDawley rats, which were induced colonic inflammation by intraluminal ad

5、ministration of trinitrobenzenesulfonic acid (TNBS), were randomly divided into eight groups. In the three colorectal distention (CRD) treated groups (n=6), abdominal contractions were recorded after 3, 7 and 14 days of intragastric administration of tegaserod 2mg/kg d. In the three CRD control grou

6、ps (n=4), abdominal contractions were recorded after 3, 7 and 14 days of intragastric injection of saline 2.0mL/d. In immunohistochemistry (IH) treated group (n=6) and IH control group (n=6), samples of colon were removed and processed for SP and CGRP immunohistochemistry after 7 days of intragastri

7、c administration of tegaserod and saline, respectively. Results  Abdominal contractions induced by colonic distention decreased significantly at 1.2mL and 1.6mL distention volume after 3 days of tegaserod administration (P<0.05). After 7 and 14 days of intragastric administration of tegasero

8、d, abdominal contractions decreased significantly at 0.4, 0.8, 1.2mL and 1.6mL distention volume compared with those of the control rats (P<0.01). In the rats treated with tegaserod for 7 days, the scoring of substance P in the colon reduced significantly. However, staining of CGRP in the colon d

9、id not significantly decrease. Conclusion  Tegaserod can significantly reduce visceral sensitivity to colonic distention in conscious rats. It may reduce visceral sensitivity by inhibiting SP expression of the primary visceral afferents in the colon.KEY WORDS: tegaserod; visceral sensitivity; i

10、rritable bowel syndrome; substance P; calcitonin generelated peptide (CGRP)替加色羅(tegaserod)是一種新型選擇性5HT4受體部分激動劑，由于靜脈和口服給藥不能通過血腦屏障，因而主要作用于胃腸道5HT4受體。臨床流行病學(xué)研究發(fā)現(xiàn)，口服替加色羅可以明顯改善腸易激綜合征(irritable bowel syndrome, IBS)患者，尤其是女性IBS患者的腹部不適和腹痛癥狀，提示替加色羅可以降低IBS患者的內(nèi)臟高敏感性12。但是，替加色羅調(diào)節(jié)內(nèi)臟感覺的機制目前仍不清楚。在本研究中我們在三硝基苯磺酸(trinitro

11、benzenesulfonic acid, TNBS)灌腸建立大鼠內(nèi)臟高敏感性模型的基礎(chǔ)上，通過記錄結(jié)腸球囊擴張時大鼠腹壁肌電活動，評價替加色羅對大鼠內(nèi)臟敏感性的影響；采用免疫組織化學(xué)法觀察大鼠結(jié)腸P物質(zhì)(SP)和降鈣素基因相關(guān)肽(CGRP)的表達(dá)，探討替加色羅調(diào)節(jié)內(nèi)臟敏感性的作用途徑。1  材料與方法1.1  實驗材料和試劑  銀制雙極電極購自上海諾城電氣有限公司；動脈栓子清除術(shù)導(dǎo)管(6F，氣囊長2cm，直徑2mm，F(xiàn)ogarty，Edwards)購自西安歐萊科貿(mào)有限公司；替加色羅(批號：2000011001)由北京諾華制藥有限公司惠贈；兔抗SP抗體、兔抗CGR

12、P抗體、生物素標(biāo)記的羊抗兔IgG和ABC復(fù)合物均購自武漢博士德生物工程公司。1.2  實驗動物及分組  成年雄性SD大鼠42只，體重220-250g，由第四軍醫(yī)大學(xué)實驗動物中心提供。大鼠置于安靜、溫暖(18-20)、避強光的環(huán)境中喂養(yǎng)，自由飲水、攝食。隨機分為4個實驗組和4個對照組。實驗組和對照組大鼠在TNBS灌腸后24h開始分別給替加色羅和生理鹽水灌胃(表1)。表1  各組大鼠灌胃劑量和時間（略）Table 1  The dosage and duration of intragastric administration in rats of diff

13、erent groupsIH: immunohistochemistry; CRD: colorectal distention1.3  方法2  結(jié)果表2  不同容量結(jié)腸球囊擴張下大鼠腹肌收縮的次數(shù)（略）Table 2  The number of abdominal contractions at various volumes of colonic distension in CRD groups (per 5min)*P<0.05, P<0.01 vs. the control group圖1  1.2mL擴張容量下記錄的大

14、鼠腹肌收縮圖（略）Fig.1 Records of the abdominal contractions in rats at the volume of distension of 1.2mLA: only single abdominal contractions occurred in rats treated with tegaserod for 14 days; B: consecutive abdominal spike bursts occurred in rats treated with saline. (Each square represents 1mV in the vertical lines. Abdominal contractions were recorded at a paper speed of 4.5×10-3m/s)2.2  替加色羅對結(jié)腸SP和CGRP表達(dá)的影響  對照組大鼠結(jié)腸內(nèi)SP和CGRP陽性細(xì)胞數(shù)量多，分布密集，染色深，呈棕黃色，評分為-。替加色羅灌胃7d后，結(jié)腸內(nèi)SP陽性細(xì)胞減少，染色明顯變淺，呈淡黃色，評分為+-。CGRP染色強度與對照組比較無明顯變化(表3、圖2)。3  討論