烏司他定對急性壞死性胰腺炎大鼠合并肺損傷的影響

紀濤 湯志剛 邱陸軍 黃強 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

紀濤 湯志剛 邱陸軍 黃強 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ET-1和NF-κB表達及肺損傷的影響。方法60只SD大鼠按隨機數字法分成假手術組、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蛋白表達以及使用TUNEL法檢測細胞凋亡。結果UTI組術后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)。假手術組未見NF-κB和ET-1表達，未見凋亡細胞。UTI組NF-κB和ET-1陽性表達率分別為(19±3)%和(8±1)%，較ANP組的(25±2)%和(13±1)%顯著降低(Plt;0.05)。UTI組細胞凋亡指數為13.75±1.25，較ANP組的6.90±0.85顯著升高(Plt;0.05)。結論ANP時肺組織NF-κB和ET-1的高表達可能導致肺損傷。UTI能改善肺微循環，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)病程進展兇險，易發生多器官功能障礙，其中肺臟最易受累。研究證實，炎癥反應和微循環障礙參與SAP合并肺損傷的發生、發展[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，探討烏司他定(UTI)對ANP合并肺損傷的影響機制。

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，由安徽醫科大學實驗動物中心提供。按隨機數字法分為假手術(SO)組、ANP組和UTI組，每組20只。參考Ellison等[2]的方法，經膽胰管逆行注射5%牛磺膽酸鈉1 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．肺組織病理學檢查：肺組織常規甲醛固定、石蠟包埋、切片、HE染色，光學顯微鏡下觀察。

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，用已知陽性標本作陽性對照。胞質內出現棕褐色顆粒為陽性表達。計算5個高倍視野內陽性細胞占總細胞數的百分率。

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

三、統計學處理

結 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，差異具有統計學意義(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，差異具有統計學意義(Plt;0.05)。

SO組肺組織未見明顯異常；ANP組部分肺泡腔內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，毛細血管高度充血，胸腔內少量積液呈血性或乳糜性；UTI組肺泡壁輕度增厚，間質毛細血管充血、擴張，少量炎性細胞浸潤，支氣管管壁輕度水腫。

三、肺組織NF-κB和ET-1蛋白的表達

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

四、肺組織細胞AI

SO組未見明顯凋亡細胞，ANP組和UTI組凋亡細胞明顯多于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，差異有統計學意義(Plt;0.05)。

討 論

SAP時各種激活的胰酶和過度激活的炎癥介質釋放入血，損傷胰腺外重要器官。由于肺的特殊解剖學結構，首當其沖成為胰酶和細胞因子損傷的重要器官[3]。因此調節和抑制酶及炎癥介質的釋放對治療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)表達和細胞凋亡(下)的變化(免疫組化、TUNEL ×100)

TNF-α在炎癥反應中最早升高，并且處于始動地位，可以誘發中性粒細胞在肺組織內大量聚集，并釋放炎性介質，引起肺損傷。NF-κB作為調控諸多炎性細胞因子的樞紐與其他轉錄因子共同作用參與炎癥介質的誘導表達[4]。進一步研究發現，NF-κB的活化與細胞因子的表達及肺損傷之間呈正相關[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、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蛋白表達減少，肺濕/干重比值降低，肺組織炎癥反應明顯減輕，提示UTI可能通過抑制NF-κB活化、減少炎癥介質釋放而改善肺組織的損害。

ET-1是迄今為止發現的最強烈的血管收縮因子，ET與其受體結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表達增加，結果一致。應用UTI處理后，肺組織ET-1表達較ANP組降低，肺濕/干重比值也低于ANP組，可見UTI的應用能明顯改善肺組織的微循環，減少滲出，減輕ANP合并的肺損傷。

研究發現，凋亡參與了SAP發生發展的全過程[8]。由于凋亡過程中細胞膜保持完整，不伴有炎癥介質和溶酶體釋放，因此對機體具有保護效應。Koh等[9]報道，凋亡對急性肺損傷有明顯的保護作用。本實驗結果也顯示，UTI組肺組織細胞的凋亡指數明顯高于ANP組，表明UTI在抑制炎癥反應的同時也能促進細胞凋亡。

[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制.胰腺病學，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

安徽省衛生廳基金(05A004)

230001 合肥，安徽醫科大學附屬省立醫院普外科

共同第一作者：湯志剛

湯志剛，Email:tzg7031@163.com