王慶剛 雷若慶 許志偉 李紅昌 韓天權(quán) 張圣道

·論著·

急性出血壞死性胰腺炎大鼠腸道黏膜屏障功能變化及己酮可可堿對其的保護(hù)作用

王慶剛 雷若慶 許志偉 李紅昌 韓天權(quán) 張圣道

目的研究急性壞死性胰腺炎(ANP)大鼠腸道屏障功能改變及己酮可可堿(pentoxifylline，PTX)對腸道屏障的保護(hù)作用。方法54只SD雄性大鼠按數(shù)字表法隨機(jī)分為ANP組、PTX組和假手術(shù)組。采用逆行胰膽管注射5%?；悄懰徕c建立ANP模型。假手術(shù)組只翻動(dòng)十二指腸。PTX組在制模后經(jīng)陰莖靜脈注射PTX 25 mg/kg體重。術(shù)后3、6、24 h分批處死大鼠。取血測淀粉酶、D-乳酸及TNF-α含量，取胰腺及末端回腸常規(guī)行病理學(xué)檢查，免疫組化法檢測回腸黏膜上皮緊密連接蛋白ZO-1的表達(dá)。結(jié)果建模后6 h，ANP組血清淀粉酶、TNF-α、D-乳酸含量分別為(9141±672)U/L、(347.96±79.47)pg/ml 和(10.21±1.08) mg/L，顯著高于假手術(shù)組的(1723±57)U/L、(134.09±31.36)pg/ml和(4.33±0.49)mg/L(P值均<0.01) ；PTX組血淀粉酶、TNF-α、D-乳酸分別為(7965±318)U/L、(238.48±44.35)pg/ml和(8.75±1.28) mg/L，較ANP組顯著降低，但仍顯著高于假手術(shù)組(P<0.05或<0.01)。假手術(shù)組大鼠腸黏膜上皮ZO-1陽性率為(3.29±0.36)%；ANP組為(1.91±0.32)%，較假手術(shù)組明顯減少(P<0.05)；PTX組為(2.53±0.43)%，較假手術(shù)組減少，但較ANP組明顯增加(P<0.05)。結(jié)論P(yáng)TX可減輕ANP大鼠腸黏膜屏障功能的損傷，其機(jī)制可能是通過減少腸黏膜上皮ZO-1的降解。

胰腺炎，急性壞死性； 連接蛋白類； 己酮可可堿； 腸道屏障

重癥急性胰腺炎(SAP)時(shí)腸道黏膜屏障在全身炎癥反應(yīng)綜合征的形成過程中起到重要作用[1]。己酮可可堿(pentoxifylline，PTX)是一種非特異性磷酸二酯酶抑制劑，臨床上作為血管擴(kuò)張劑用于治療周圍血管疾病，同時(shí)用于改善微循環(huán)及缺血組織的氧供應(yīng)[2]。近年研究發(fā)現(xiàn)，PTX能夠調(diào)節(jié)炎癥反應(yīng)，降低腸道促炎癥因子的合成[3-4]。本研究應(yīng)用PTX干預(yù)急性壞死性胰腺炎大鼠，觀察其對腸道黏膜屏障的保護(hù)作用。

材料與方法

一、動(dòng)物分組及模型制作

54只健康雄性SD大鼠，體重200～250 g，SPF級，由瑞金醫(yī)院實(shí)驗(yàn)動(dòng)物中心提供。按數(shù)字表法隨機(jī)分為ANP組、PTX組和假手術(shù)組，各18只。采用逆行胰膽管注射5%牛磺膽酸鈉(Sigma公司)建立ANP模型。假手術(shù)組只翻動(dòng)十二指腸后關(guān)腹。PTX組于制模后經(jīng)陰莖靜脈注射PTX 25 mg/kg體重，ANP組及假手術(shù)組注射等容積生理鹽水。術(shù)后3、6、24 h分批處死每組大鼠各6只。腹主動(dòng)脈取血，離心取血清，-80℃保存；取胰腺組織置4%多聚甲醛中固定；留取兩段末端回腸，一段置-80℃保存，另一段于4%多聚甲醛中固定。

二、檢測項(xiàng)目及方法

1．血淀粉酶、D-乳酸測定：淀粉酶由Beckmancoulter SYNCHRON CX4 PRO全自動(dòng)生化儀檢測。D-乳酸采用改良的酶學(xué)分光光度法測定[5]。

2．胰腺及末端回腸組織病理學(xué)檢查：取甲醛固定的胰腺及回腸組織，常規(guī)石蠟包埋、切片、HE染色，由病理科醫(yī)師閱片。

3．血清及回腸組織TNF-α濃度檢測：回腸組織按每克加入5 ml PBS制成組織勻漿。TNF-α濃度采用ELISA法檢測。

4．緊密連接蛋白ZO-1檢測：取24 h處死大鼠的末端回腸組織，采用常規(guī)免疫組化SP法檢測腸黏膜ZO-1蛋白表達(dá)。每張切片于光學(xué)顯微鏡下觀察3個(gè)視野，應(yīng)用Image-Pro Plus圖片處理軟件(Media Cybernetics公司)計(jì)算陽性表達(dá)百分率。

三、統(tǒng)計(jì)學(xué)方法

結(jié) 果

一、血淀粉酶、D-乳酸及TNF-α含量變化

ANP組大鼠血清淀粉酶、D-乳酸及TNF-α含量均較假手術(shù)組明顯升高(P值均<0.01)。PTX組大鼠血淀粉酶、D-乳酸及TNF-α含量較ANP組明顯降低，但仍較假手術(shù)組高(P<0.05或<0.01，表1)。

二、腸道組織TNF-α含量變化

ANP組大鼠腸道組織TNF-α含量較假手術(shù)組明顯升高(P<0.01)，而PTX組較ANP組明顯下降，但仍較假手術(shù)組高(P值均<0.01，表1)。

表1 各組大鼠血淀粉酶、D-乳酸、TNF-α含量及回腸組織TNF-α含量的變化

注：與假手術(shù)組比較，aP<0.01；與ANP組比較，bP<0.05，cP<0.01

三、胰腺及腸道組織的病理變化

假手術(shù)組大鼠胰腺組織結(jié)構(gòu)完整；ANP組大鼠胰腺小葉結(jié)構(gòu)模糊，呈片狀壞死，間質(zhì)內(nèi)出血和大量炎細(xì)胞浸潤；PTX組胰腺水腫，點(diǎn)片狀壞死，較ANP組病理損傷明顯減輕(圖1上)。

假手術(shù)組大鼠回腸黏膜絨毛完整，排列整齊；ANP組回腸黏膜絨毛的完整性被破壞，部分固有層潰瘍出血；PTX組回腸組織損傷較輕，僅表現(xiàn)為絨毛變短、變鈍，絨毛結(jié)構(gòu)完整(圖1下)。

圖1假手術(shù)組(左列)、ANP組(中列)、PTX組(右列)胰腺(上)及末端回腸(下)的病理學(xué)變化(HE ×100)

四、腸道黏膜上皮ZO-1蛋白表達(dá)的變化

腸道黏膜上皮ZO-1蛋白主要位于上皮細(xì)胞的邊緣、細(xì)胞膜頂端，沿絨毛下方均勻連續(xù)分布，表達(dá)呈棕褐色。假手術(shù)組大鼠腸黏膜上皮染色較強(qiáng)，陽性表達(dá)率為(3.29±0.36)%；ANP組大鼠腸黏膜上皮染色較淺，分布不均，陽性表達(dá)率為(1.91±0.32)%(P<0.05)；PTX組大鼠ZO-1陽性表達(dá)率為(2.53±0.43)%，較假手術(shù)組減少，但較ANP組有明顯增加(P<0.05，圖2)。

圖2假手術(shù)組(a)、ANP組(b)、PTX組(c)腸道上皮緊密連接蛋白ZO-1的表達(dá)(免疫組化 ×100)

討 論

腸道是人體的一個(gè)重要免疫器官，大約60%的T細(xì)胞位于小腸，同時(shí)腸道內(nèi)也存在著大量的細(xì)菌及其產(chǎn)生的內(nèi)毒素。生理情況下，機(jī)體憑借完整的腸黏膜屏障可以阻止這些細(xì)菌及內(nèi)毒素進(jìn)入體內(nèi)。SAP早期往往伴有腸黏膜屏障損傷，上皮通透性增加，細(xì)菌及內(nèi)毒素易位，從而加重急性胰腺炎病情[6]。

腸道黏膜上皮的通透性主要受腸道上皮細(xì)胞頂端的緊密連接控制，緊密連接蛋白ZO-1起著重要作用[7]。本實(shí)驗(yàn)結(jié)果顯示，ANP大鼠腸黏膜上皮ZO-1蛋白表達(dá)較假手術(shù)組明顯減少，而血清D-乳酸濃度較假手術(shù)組明顯升高。說明ANP大鼠出現(xiàn)腸道屏障功能障礙。PTX組腸黏膜上皮ZO-1表達(dá)較ANP組明顯改善，血D-乳酸濃度明顯降低，說明PTX能夠減輕ANP大鼠的腸黏膜損傷。

PTX能夠調(diào)節(jié)炎癥反應(yīng)，降低促炎癥因子的合成。TNF-α是一種促炎癥介質(zhì)，參與全身炎癥反應(yīng)綜合征的發(fā)生，并且能夠加速ZO-1的降解[8]。本結(jié)果顯示，PTX組大鼠血清及腸道組織TNF-α濃度較ANP組顯著降低，提示PTX降低血及腸道組織TNF-α濃度是其保護(hù)腸黏膜屏障的機(jī)制之一。

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2010-09-16)

(本文編輯：呂芳萍)

Changeofintestinalbarrierfunctioninacutenecrotizingpancreatitis(ANP)ratsandpentoxifylline′sprotectiveeffects

WANGQing-gang,LEIRuo-qing,XUZhi-wei,LIHong-chang,HANTian-quan,ZHANGSheng-dao.

DepartmentofSurgery,RuijinHospital,MedicineSchool,ShanghaiJiaotongUniversity,Shanghai20025,China

LEIRuo-qing,Email：ruoqinglei@yahoo.com.cn

ObjectiveTo investigate the change of intestinal barrier function and the protection of pentoxifylline (PTX) to intestinal barrier.MethodsFifty-four SD male rats were randomly divided into 3 groups, including sham operation group, ANP group, PTX group. ANP rat model were induced by retrograde injection of 5% sodium taurocholate into pancreatic and bile duct. Rats in sham operation group underwent operation without injection of taurocholate. After ANP induction, the rats in PTX group

PTX at a dose of 25 mg/kg weight via penis vein. The rats were sacrificed 3, 6, 24 h after operation, the serum levels of amylase, D-lactic acid, TNF-α were determined. The pancreas tissue and terminal ileum were harvested for pathological examination; ZO-1 levels of ileum epithelial tight junction were analyzed by immunohistochemistry.ResultsSix hours after induction, the serum levels of amylase, TNF-α, D-lactic acid in ANP group were (9141±672)U/L,(347.96±79.47)pg/ml and (10.21±1.08)mg/L, which were significantly higher than those in sham operation group [(1723±57)U/L, (134.09±31.36)pg/ml and (4.33±0.49)mg/L,P<0.01]. The serum levels of amylase, TNF-α, D-lactic acid in PTX group were (7965±318)U/L, (238.48±44.35)pg/ml and (8.75±1.28)mg/L, which were significantly lower than those in ANP group, but they were significantly higher than those in fsham group (P<0.05 or <0.01). The positive rate of ZO-1 was (3.29±0.36)% in sham operation group, and it was (1.91±0.32)% in ANP group, which was significantly lower than that in sham operation group (P<0.05); and the value was (2.53±0.43)% in PTX group, which was lower than that in sham group, but it was higher than that in ANP group (P<0.05).ConclusionsPTX may attenuate intestinal barrier function injury by decreasing the breakdown of intestinal ZO-1.

Pancreatitis,acute necrotizing; Connexins; Pentoxifylline; Enteval barrier

10.3760/cma.j.issn.1674-1935.2011.02.013

上海市重點(diǎn)學(xué)科——外科學(xué)(S30204)

200025 上海，上海交通大學(xué)醫(yī)學(xué)院附屬瑞金醫(yī)院膽胰外科

雷若慶，Email：ruoqinglei@yahoo.com.cn