紀(jì)濤 湯志剛 邱陸軍 黃強(qiáng) 李建生 許戈良

烏司他定對急性壞死性胰腺炎大鼠合并肺損傷的影響

紀(jì)濤 湯志剛 邱陸軍 黃強(qiáng) 李建生 許戈良

目的探討烏司他定(UTI)對急性壞死性胰腺炎大鼠(ANP)合并肺損傷時肺內(nèi)ET-1和NF-κB表達(dá)及肺損傷的影響。方法60只SD大鼠按隨機(jī)數(shù)字法分成假手術(shù)組、ANP組和UTI組，各20只。采用膽胰管逆行注射5%牛磺膽酸鈉溶液1 ml/kg體重制備ANP模型，假手術(shù)組胰管注射等量生理鹽水，UTI組在ANP制模成功后即從大鼠尾靜脈注射UTI 10 000 U/kg體重。24 h后處死動物，測血清淀粉酶、TNF-α、肺組織濕/干重比，免疫組化法檢測肺組織NF-κB和 ET-1蛋白表達(dá)以及使用TUNEL法檢測細(xì)胞凋亡。結(jié)果UTI組術(shù)后24 h血清淀粉酶、TNF-α和肺濕/干重比分別為(5 648±378)U/L、(89.19±3.54)ng/L和4.55±0.07，較ANP組的(6 799±437)U/L、 (183.30±8.18) ng/L和4.89±0.20顯著降低(Plt;0.05)。假手術(shù)組未見NF-κB和ET-1表達(dá)，未見凋亡細(xì)胞。UTI組NF-κB和ET-1陽性表達(dá)率分別為(19±3)%和(8±1)%，較ANP組的(25±2)%和(13±1)%顯著降低(Plt;0.05)。UTI組細(xì)胞凋亡指數(shù)為13.75±1.25，較ANP組的6.90±0.85顯著升高(Plt;0.05)。結(jié)論ANP時肺組織NF-κB和ET-1的高表達(dá)可能導(dǎo)致肺損傷。UTI能改善肺微循環(huán)，減輕肺炎癥性損傷。

胰腺炎，急性壞死性； 急性肺損傷； 烏司他定

重癥急性胰腺炎(SAP)病程進(jìn)展兇險，易發(fā)生多器官功能障礙，其中肺臟最易受累。研究證實，炎癥反應(yīng)和微循環(huán)障礙參與SAP合并肺損傷的發(fā)生、發(fā)展[1]。本實驗通過檢測急性壞死性胰腺炎(ANP)大鼠肺組織ET-1和NF-κB的表達(dá)，探討烏司他定(UTI)對ANP合并肺損傷的影響機(jī)制。

材料與方法

一、實驗動物與分組

清潔級健康成年SD大鼠，體重250～300 g，雌雄不拘，由安徽醫(yī)科大學(xué)實驗動物中心提供。按隨機(jī)數(shù)字法分為假手術(shù)(SO)組、ANP組和UTI組，每組20只。參考Ellison等[2]的方法，經(jīng)膽胰管逆行注射5%?；悄懰徕c1 ml/kg體重制備ANP模型。SO組胰管注射等量生理鹽水。UTI組于ANP模型制作成功后從大鼠尾靜脈注射UTI 10 000 U/kg體重。各組動物于模型建立后24 h處死。

二、觀測指標(biāo)及檢測方法

1．血清淀粉酶、TNF-α檢測： 血清淀粉酶采用BeckmanCX9 全自動生化分析儀檢測，TNF-α采用ELASA法檢測，嚴(yán)格按照試劑盒說明書操作。

2．肺組織濕/干比：開胸后立即取出肺右葉，用電子天平稱濕重后置80℃烤箱烤24 h至恒重，計算肺濕/干重比值。

3．肺組織病理學(xué)檢查：肺組織常規(guī)甲醛固定、石蠟包埋、切片、HE染色，光學(xué)顯微鏡下觀察。

4．NF-κB和ET-1蛋白檢測：采用免疫組化法。用PBS代替一抗作陰性對照，用已知陽性標(biāo)本作陽性對照。胞質(zhì)內(nèi)出現(xiàn)棕褐色顆粒為陽性表達(dá)。計算5個高倍視野內(nèi)陽性細(xì)胞占總細(xì)胞數(shù)的百分率。

5．細(xì)胞凋亡檢測：采用原位末端標(biāo)記TUNEL法。按說明書操作。顯微鏡下見細(xì)胞核中有棕褐顆粒，即為凋亡細(xì)胞。計算5個高倍視野100個胰腺細(xì)胞，以染色陽性細(xì)胞占總細(xì)胞數(shù)的百分率為凋亡指數(shù)(AI)。

三、統(tǒng)計學(xué)處理

結(jié) 果

一、血清淀粉酶、TNF-α含量的變化

SO組、ANP組和UTI組的血清淀粉酶含量分別為(862±43)U/L、(6 799±437)U/L和(5 648±378)U/L；TNF-α水平分別為(9.72±0.85)ng/L、(183.30±8.18)ng/L和(89.19±3.54)ng/L。ANP組和UTI組較SO組明顯升高，UTI組又低于ANP組，差異具有統(tǒng)計學(xué)意義(Plt;0.05)。

二、肺組織濕/干重比值和病理改變

SO組、ANP組和UTI組的肺組織濕/干重比值分別為4.33±0.04、4.89±0.20和4.55±0.07。ANP組和UTI組較SO組明顯升高，UTI組又低于ANP組，差異具有統(tǒng)計學(xué)意義(Plt;0.05)。

SO組肺組織未見明顯異常；ANP組部分肺泡腔內(nèi)有少量滲出合并出血，肺泡大小不一，有少許塌陷，肺泡壁明顯增厚，毛細(xì)血管高度充血，胸腔內(nèi)少量積液呈血性或乳糜性；UTI組肺泡壁輕度增厚，間質(zhì)毛細(xì)血管充血、擴(kuò)張，少量炎性細(xì)胞浸潤，支氣管管壁輕度水腫。

三、肺組織NF-κB和ET-1蛋白的表達(dá)

SO組肺組織無NF-κB和ET-1蛋白表達(dá)。ANP組和UTI組NF-κB和ET-1蛋白主要表達(dá)于肺泡及其周圍的間質(zhì)組織(圖1)。ANP組和UTI組的NF-κB陽性表達(dá)率分別為(25±2)%和(19±3)%；ET-1陽性表達(dá)率分別為(13±1)%和(8±1)%。兩組間差異均顯著(Plt;0.05)。

四、肺組織細(xì)胞AI

SO組未見明顯凋亡細(xì)胞，ANP組和UTI組凋亡細(xì)胞明顯多于SO組(圖1)。3組AI分別為0、6.90±0.85和13.75±1.25。UTI組的AI明顯高于ANP組，差異有統(tǒng)計學(xué)意義(Plt;0.05)。

討 論

SAP時各種激活的胰酶和過度激活的炎癥介質(zhì)釋放入血，損傷胰腺外重要器官。由于肺的特殊解剖學(xué)結(jié)構(gòu)，首當(dāng)其沖成為胰酶和細(xì)胞因子損傷的重要器官[3]。因此調(diào)節(jié)和抑制酶及炎癥介質(zhì)的釋放對治療SAP合并肺損傷具有重要意義。

圖1SO組(A)、ANP組(B)和UTI組(C)的肺組織NF-κB(上)、ET-1(中)表達(dá)和細(xì)胞凋亡(下)的變化(免疫組化、TUNEL ×100)

TNF-α在炎癥反應(yīng)中最早升高，并且處于始動地位，可以誘發(fā)中性粒細(xì)胞在肺組織內(nèi)大量聚集，并釋放炎性介質(zhì)，引起肺損傷。NF-κB作為調(diào)控諸多炎性細(xì)胞因子的樞紐與其他轉(zhuǎn)錄因子共同作用參與炎癥介質(zhì)的誘導(dǎo)表達(dá)[4]。進(jìn)一步研究發(fā)現(xiàn)，NF-κB的活化與細(xì)胞因子的表達(dá)及肺損傷之間呈正相關(guān)[5-6]。本實驗動物在ANP制模后血淀粉酶、TNF-α水平及肺組織NF-κB表達(dá)、肺濕/干重比值均增加，表明ANP制模成功，肺組織受損害。

UTI是從人尿中提取制成的一種相對分子質(zhì)量為67 000的糖蛋白，可抑制胰蛋白酶、胰淀粉酶、糜蛋白酶等活性，減輕各種胰液對胰腺組織自身的損傷。本實驗給予UTI后TNF-α含量下降，肺組織NF-κB蛋白表達(dá)減少，肺濕/干重比值降低，肺組織炎癥反應(yīng)明顯減輕，提示UTI可能通過抑制NF-κB活化、減少炎癥介質(zhì)釋放而改善肺組織的損害。

ET-1是迄今為止發(fā)現(xiàn)的最強(qiáng)烈的血管收縮因子，ET與其受體結(jié)合將大大降低血流量，造成組織缺血、壞死、功能障礙甚至衰竭。Paulino等[7]報道，SAP患者血漿ET增高，本實驗在ANP制模后，肺組織ET-1表達(dá)增加，結(jié)果一致。應(yīng)用UTI處理后，肺組織ET-1表達(dá)較ANP組降低，肺濕/干重比值也低于ANP組，可見UTI的應(yīng)用能明顯改善肺組織的微循環(huán)，減少滲出，減輕ANP合并的肺損傷。

研究發(fā)現(xiàn)，凋亡參與了SAP發(fā)生發(fā)展的全過程[8]。由于凋亡過程中細(xì)胞膜保持完整，不伴有炎癥介質(zhì)和溶酶體釋放，因此對機(jī)體具有保護(hù)效應(yīng)。Koh等[9]報道，凋亡對急性肺損傷有明顯的保護(hù)作用。本實驗結(jié)果也顯示，UTI組肺組織細(xì)胞的凋亡指數(shù)明顯高于ANP組，表明UTI在抑制炎癥反應(yīng)的同時也能促進(jìn)細(xì)胞凋亡。

[1] Surbatovic M,Jovanovic K,Radakovic S,et al.Pathophysiological aspects of severe acute pancreatitis-associated lung injury.Srp Arh Celok Lek,2005,133:76-81.

[2] Ellison EC,Pappas TN,Johnson JA,et al.Demonstration and characterization of the hemoconcentrating effect of ascitic fluid that accumulates during hemorrhagic pancreatitis.J Surg Res,1981,30:241-248.

[3] 黃曉麗，劉順英，王國品.急性胰腺炎合并肺損傷的機(jī)制.胰腺病學(xué)，2007，7：64-66.

[4] Gulcubuk A,Altunatmaz K,Sonmez K,et al.Effects of curcumin on tumour necrosis factor-α and interleukin-6 in the late phase of experimental acute pancreatitis.J Vet Med A Physiol Pathol Clin Med,2006,53:49-54.

[5] Everhart MB,Han W,Sherrill TP,et al. Duration and intensity of NF-κB activity determine the severity of endotoxin-induced acute lung injury.J Immunol,2006,176:4995-5005.

[6] Abraham E,Nick JA,Azam T,et al.Peripheral blood neutrophil activation patterns are associated with pulmonary inflammatory responses to lipopolysaccharide in humans.J Immunol,2006,176:7753-7760.

[7] Paulino EC,de Souza LJ,Molan NA,et al.Neutrophils from acute pancreatitis patients cause more severe in vitro endothelial damage compared with neutrophils from healthy donors and are differently regulated by endothelins.Pancreas,2007,35:37-41.

[8] Yasuda T,Takeyama Y,Ueda T,et al.Breakdown of intestinal mucosa via accelerated apoptosis increases intestinal permeability in experimental severe acute pancreatitis.J Surg Res,2006,135:18-26.

[9] Koh H,Tasaka S,Hasegawa N,et al.Protective role of vascular endothelial growth factor in endotoxin-induced acute lung injury in mice.Respir Res,2007,25:60-73.

2008-08-04)

(本文編輯：呂芳萍)

Influenceofulinastatinonratswithacutenecrotizingpancreatitisassociatedlunginjury

JITao,TANGZhi-gang,QIULu-jun,HUANGQiang,LIJian-sheng,XUGe-liang.

DepartmentofGeneralSurgery,ProvincialHospitalofAnhui,AnhuiMedicalUniversity,Hefei230001,China

TANGZhi-gang,Email:tzg7031@163.com

Pancreatitis, acute necrotizing; Acute lung injury; Ulinastatia

AbatractObjectiveTo investigate the effects of Ulinastatin (UTI) on the expression of NF-κB and ET-1 in rats with acute necrotizing pancreatitis (ANP)-associated lung injury and morphology of lung tissue.Methods60 Sprague-Dawley rats were randomly divided into 3 groups with 20 rats in each group, including sham operation(SO) group, ANP group and UTI group. ANP was induced by injection of 5% sodium taurocholate (1 ml/kg) into pancreatic duct; normal saline was injected for SO group with same amount. UTI was injected for UTI group with the amount of 10 000 U/L via tail vein after ANP induction. The rats were sacrificed 24 h later. The contents of serum amylase, TNF-α and wet/dry weight ratio of the lung were measured. The expression of NF-κB and ET-1 protein were detected by immunohistochemical method. The level of apoptosis was detected by TUNEL.ResultsThe contents of serum amylase, TNF-α, and wet/dry weight ratio of the lungin in UTI group at 24 hours were (5 648±378)IU/L, (89.19±3.54)ng/L and 4.55±0.07, respectively; which were significantly lower than the corresponding (6 799±437)IU/L, (183.30±8.18)ng/L and 4.89±0.20 in ANP group (Plt;0.05). There was no NF-κB and ET-1 expression, and no apoptosis was present in SO group. The positive rates of NF-κB and ET-1 in UTI group were (19±3)% and (8±1)%, respectively, the corresponding values in ANP group were (25±2)% and (13±1)%, respectively (Plt;0.05). The level of apoptotic index in UTI group was 13.75±1.25, which was higher than that (6.90±0.85) in ANP group (Plt;0.05).ConclusionsThe high expression of NF-κB and ET-1 in lung tissue may cause lung injury. UTI could ameliorate the microcirculation and lung injury caused by inflammation.

10.3760/cma.j.issn.1674-1935.2009.02.009

安徽省衛(wèi)生廳基金(05A004)

230001 合肥，安徽醫(yī)科大學(xué)附屬省立醫(yī)院普外科

共同第一作者：湯志剛

湯志剛，Email:tzg7031@163.com