施新崗 許永春 滿曉華 金晶 龔燕芳 屠振興 李兆申

·論著·

細(xì)胞外調(diào)節(jié)蛋白激酶在大鼠急性壞死性胰腺炎發(fā)病機(jī)制中的作用

施新崗 許永春 滿曉華 金晶 龔燕芳 屠振興 李兆申

目的研究細(xì)胞外調(diào)節(jié)蛋白激酶(extracellular regulated kinase 1/2，ERK1/2)信號轉(zhuǎn)導(dǎo)通路在大鼠急性壞死性胰腺炎(ANP)時的變化規(guī)律，探討ERK1/2特異性抑制劑PD98059對ANP時胰腺的保護(hù)作用。方法以5%?；悄懰徕c胰膽管逆行注射建立ANP模型，5只大鼠作為正常組，余75只大鼠按數(shù)字表法隨機(jī)分為假手術(shù)組、ANP組及PD98059組，各25只。術(shù)后15 min、30 min、1 h、3 h和6 h分批處死動物，取血及胰腺組織。常規(guī)胰腺病理檢查并評分；ELISA方法檢測大鼠血清IL-1β、TNF-α水平；酶化學(xué)法檢測胰腺組織內(nèi)MPO活性；Western blotting法檢測胰腺組織磷酸化ERK1/2的表達(dá)量。結(jié)果假手術(shù)組胰腺無明顯病理改變；ANP組胰腺損傷明顯，3 h的病理評分為9.9±0.4，PD98059組胰腺損傷減輕，3 h病理評分為4.0±0.4，與ANP組相差顯著(Plt;0.05)。假手術(shù)組、ANP組和PD98059組大鼠3 h時血清TNF-α水平分別為(65.8±20.5)pg/ml、(286.5±50.3)pg/ml、(180.4±32.9)pg/ml； IL-1β水平為(85.8±25.5)pg/ml、(293.8±46.3)pg/ml、(200.5±33.6)pg/ml；胰腺MPO活性為(0.19±0.02)U/g、(0.61±0.05)U/g、(0.52±0.03)U/g。ANP組和PD98059組均顯著高于假手術(shù)組，而PD98059組又顯著低于ANP組(P值均lt;0.05)。正常大鼠胰腺磷酸化ERK1/2表達(dá)量為1100±141；ANP組15 min、30 min時磷酸化ERK1/2表達(dá)量分別為5300±486、5621±384，1 h開始下降，6 h時幾乎與假手術(shù)組相似；PD98059組造模后15 min、30 min的磷酸化ERK1/2表達(dá)量分別為4200±370、3600±290，顯著低于同時間點ANP組(P值均lt;0.01)。結(jié)論ERK1/2信號轉(zhuǎn)導(dǎo)通路參與大鼠ANP發(fā)病機(jī)制，PD98059干預(yù)可減少IL-1β、TNF-α的產(chǎn)生，降低胰腺組織MPO活性，改善胰腺的病理損傷程度。

胰腺炎，急性胰腺炎； 細(xì)胞外信號調(diào)節(jié)激酶； 胰腺損傷

迄今為止，重癥急性胰腺炎(SAP)的發(fā)病機(jī)制尚未完全闡明，臨床上又缺乏特異性的治療措施。近年研究表明，細(xì)胞外信號調(diào)節(jié)激酶(extracellular regulated kinase 1/2，ERK1/2)在蛙皮素誘導(dǎo)的大鼠水腫型急性胰腺炎的發(fā)病中起著重要作用。本研究檢測急性壞死性胰腺炎(ANP)大鼠胰腺組織ERK1/2磷酸化活化水平，探討ERK1/2信號轉(zhuǎn)導(dǎo)通路特異性抑制劑PD98059對ANP時胰腺組織的保護(hù)作用。

資料和方法

一、實驗動物及分組

SD大鼠80只，清潔級，雄性，體重250～300 g，購自第二軍醫(yī)大學(xué)實驗動物中心。按數(shù)字表法隨機(jī)分為假手術(shù)組、ANP組及PD98059干預(yù)組，每組25只。以0.2 ml/min速度于膽胰管逆行注入5%?；悄懰徕c(Simga公司)1 ml/kg體重的方法制備ANP模型。假手術(shù)組開腹后僅翻動十二指腸并觸摸胰腺數(shù)次。PD98059組于造模前30 min經(jīng)尾靜脈注入ERK1/2抑制劑PD98059(Calbiochem公司)10 mg/kg體重；ANP組注入等體積雙蒸水。各組于術(shù)后15 min、30 min、1 h、3 h、6 h分批腹主動脈取血處死5只大鼠，并迅速取胰腺組織。5只大鼠先處死作為正常對照。

二、觀察項目

1．胰腺病理檢查及評分：胰腺組織常規(guī)切片，HE染色，由?？漆t(yī)師光鏡下觀察胰腺病理變化，并參照Rongione等[1]標(biāo)準(zhǔn)進(jìn)行評分。

2．血清TNF-α、IL-1β的測定：采用ELISA方法測定。試劑盒購自上海增健生物科技公司。

3．胰腺組織髓過氧化酶(MPO)含量測定：采用酶化學(xué)法。檢測試劑盒購自南京建成生物工程研究所。

4．胰腺組織磷酸化ERK1/2表達(dá)的檢測：采用Western blotting方法[2]。稱取100 mg胰腺組織標(biāo)本，剪碎后于液氮中研磨，應(yīng)用胞質(zhì)蛋白試劑盒(NE-PER，Pierce公司)抽提胞質(zhì)蛋白，采用BCA法定量。取40 g樣品常規(guī)行Western blotting?？沽姿峄疎RK1/2單抗購自Cell Signaling公司，山羊抗小鼠IgG-HRP購自Immuclub Labs公司。ECL發(fā)光。應(yīng)用Fluors Mutilmager圖像分析儀(Bio-Rad公司)掃描，采用Quantity One 4.1版圖像分析軟件分析，以相應(yīng)蛋白條帶的平均光密度值來表示ERK1/2磷酸化活化水平的相對強(qiáng)度。

三、統(tǒng)計學(xué)處理

結(jié) 果

一、胰腺組織病理改變

假手術(shù)組胰腺無明顯病理改變。ANP組3、6 h時胰腺組織間質(zhì)水腫，小葉間隙增大，腺泡腫脹，并可見片狀出血壞死和腺泡小葉結(jié)構(gòu)破壞，有大量嗜中性白細(xì)胞及單核細(xì)胞浸潤。PD98059組也可見較明顯的胰腺病理改變，但較ANP組減輕，病理評分顯著降低(Plt;0.05，表1)。

二、血清TNF-α、IL-1β水平變化

正常大鼠血清TNF-α、IL-1β水平分別為(60.3±15.2)pg/ml和(90.3±20.2)pg/ml。假手術(shù)組術(shù)后3h及6h的血清TNF-α、IL-1β水平在正常范圍。ANP組及PD98059組血清TNF-α、IL-1β水平隨造模后時間的延長而逐漸升高，均顯著高于同時間點的假手術(shù)組，而PD98059組又均顯著低于同時間點的ANP組(P值均lt;0.05，表1)。

三、胰腺組織MPO水平變化

正常大鼠胰腺組織MPO為(0.21±0.03)U/g 。假手術(shù)組MPO水平與正常值接近。ANP組及PD98059組MPO水平隨造模后時間的延長而逐漸升高，均顯著高于同時間點的假手術(shù)組，而PD98059組又均顯著低于同時間點的ANP組(P值均lt;0.05，表1)。

四、胰腺組織磷酸化ERK1/2表達(dá)量的變化

正常大鼠胰腺磷酸化ERK1/2相對表達(dá)量為1100±141。ANP組于造模后15 min時磷酸化ERK1/2顯著增加，30 min達(dá)到峰值，1 h后開始下降，6 h時磷酸化ERK1/2幾乎與造模前相似(圖1)。PD98059組于造模后15 min、30 min時胰腺組織磷酸化ERK1/2水平較同時間點ANP組顯著下降(P值均lt;0.01，表2)。

圖1 ANP組(a)、PD98059組(b)磷酸化ERK1/2表達(dá)

討 論

近年來絲裂原活化蛋白激酶(MAPK)信號轉(zhuǎn)導(dǎo)通路在SAP發(fā)病機(jī)制中的作用日益引起研究者們的重視。MAPK信號轉(zhuǎn)導(dǎo)通路在腫瘤發(fā)生、應(yīng)激反應(yīng)、炎癥、神經(jīng)系統(tǒng)發(fā)育、缺血再灌注損傷、免疫調(diào)控等領(lǐng)域發(fā)揮極其重要的作用[3-4]。其中ERK1/2信號轉(zhuǎn)導(dǎo)通路研究最為透徹，它是細(xì)胞外信號轉(zhuǎn)導(dǎo)至核內(nèi)的重要通路之一[5]。ERK主要參與細(xì)胞增殖、轉(zhuǎn)化和分化，還參與應(yīng)激刺激、細(xì)菌產(chǎn)物、炎癥介質(zhì)等引起的細(xì)胞反應(yīng)[6-7]。許多絲裂原,如表皮生長因子、血小板衍生生長因子、血栓素A2 、血管緊張素Ⅱ、轉(zhuǎn)化生長因子、胰島素、單核細(xì)胞和內(nèi)皮細(xì)胞的黏附素等均可激活ERK1/2級聯(lián)反應(yīng)。

ERK有ERK1(p44)和ERK2(p42)兩種同工型。在蛙皮素誘導(dǎo)的急性水腫性胰腺炎(AEP)模型，

表1 各組大鼠胰腺組織病理評分、血清TNF-α和IL-1β水平及胰腺組織MPO變化

注：與假手術(shù)組比較，aPlt;0.05；與ANP組比較，bPlt;0.05

表2 兩組胰腺組織磷酸化ERK1/2的相對表達(dá)量

注：與ANP組比較，aPlt;0.01

造模后2 min分離培養(yǎng)的胰腺腺泡細(xì)胞中ERK1/2被大量激活；造模后5 min胰腺組織內(nèi)ERK1/2被大量激活，約為正常對照組的9倍；15 min后磷酸化活性顯著下降為正常對照組的2倍。提示ERK1/2信號轉(zhuǎn)導(dǎo)通路在急性胰腺炎早期病理機(jī)制中具有重要作用，可能是胰腺對應(yīng)激發(fā)生全身炎癥反應(yīng)的重要機(jī)制之一。Clemons等[8]用單次蛙皮素注射誘導(dǎo)AEP大鼠，造模后30 min磷酸化ERK1/2達(dá)峰值，而多次注射蛙皮素造模，磷酸化ERK1/2在3 h后更加升高，并且活化時間持續(xù)更長。本實驗結(jié)果顯示，在5%?；悄懰徕c的大鼠ANP模型中，造模后15 min ERK1/2磷酸化即達(dá)到峰值，維持約1 h后開始下降，于6h時磷酸化ERK1/2幾乎與造模前相似，提示ERK1/2信號轉(zhuǎn)導(dǎo)通路在ANP病程早期即被大量、快速激活，與ANP的病理機(jī)制有關(guān)。

Yubero等[9]研究發(fā)現(xiàn)，抑制ERK信號轉(zhuǎn)導(dǎo)通路可下調(diào)炎癥趨化因子單核細(xì)胞化學(xué)吸引蛋白質(zhì)1(monocyte chemoattractant protein-1,MCP-1)、細(xì)胞因子誘導(dǎo)中性粒細(xì)胞化學(xué)趨化因子(cytokine-induced neutrophil chemoattractant，CINC)及IL-1β等水平，從而改善ANP的嚴(yán)重程度。ERK1/2特異性抑制劑PD98059通過特異性地抑制ERK1/2上游激酶MEK1/2而阻斷ERK通路的激活。在蛙皮素誘導(dǎo)的AEP體內(nèi)、外模型中,外源性給予PD98059可改善AEP病變的嚴(yán)重程度[10-11]。本研究結(jié)果顯示，給予PD98059能顯著降低ANP大鼠胰腺內(nèi)MPO活性、降低血漿炎性細(xì)胞因子IL-1β及TNF-α的水平,從而改善胰腺組織損傷的嚴(yán)重程度。

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2010-01-19)

(本文編輯：呂芳萍)

本刊2010年2月第10卷第1期《慢性胰腺炎的內(nèi)鏡治療》一文應(yīng)放在“綜述”欄目。特此補(bǔ)充說明。

《中華胰腺病雜志》編輯部

Roleofextracellularregulatedkinasesignaltransductionpathwayinthepathogenesisofacutenecrotizingpancreatitis

SHIXin-gang,XUYong-chun,MANXiao-hua,JINjing,GONGYan-fang,TUZhen-xing,LIZhao-shen.

DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

LIZhao-shen,Email:zhsli@81890.net

ObjectiveTo investigate the changes of extracellular regulated kinase 1/2 (ERK1/2) phosphorylation and assess the effects of blocking the ERK1/2 phosphorylation on rats with acute necrotizing pancreatitis (ANP).MethodsThe ANP model was induced by retrograde injection of 5% sodium taurocholate into the biliary and pancreatic duct. 5 rats were treated as normal control. Other 75 Sprague-Dawley (SD) rats were randomly divided into sham operations(SO) group (n=25), ANP group (n=25) and PD98059 group (n=25). The rats were sacrificed at 15 min, 30 min, 1 h, 3 h and 6 h after ANP induction, the blood and pancreatic sample were taken. Pathological changes of pancreas were observed with light microscope and scored. The serum level of TNF-α and IL-1β was determined by ELISA. MPO activities in pancreas were measured by enzyme chemistry assay. Western blotting was performed to determine the phosphorylations of ERK1/2 in the pancreas homogenates.ResultsThere was no significant pathologic changes in rats of SO group; but significant injuries occurred in ANP group, the pathologic score at 3 h was 9.9±0.4; the extent of injuries attenuated in PD98059 group, the pathologic score at 3 h was 4.0±0.4 (Plt;0.05). The serum levels of TNF-α at 3 h in SO, ANP and PD98059 groups were (65.8±20.5) pg/ml, (286.5±50.3)pg/ml, (180.4±32.9)pg/ml, respectively; the serum levels of IL-1β at 3 h in SO, ANP and PD98059 groups were (85.8±25.5)pg/ml, (293.8±46.3) pg/ml, (200.5±33.6) pg/ml, respectively; MPO activities in pancreas were (0.19±0.02)U/g, (0.61±0.05)U/g, (0.52±0.03) U/g, and the values in ANP and PD98059 groups were significantly higher than those in SO group, while the values in PD98059 group were significantly lower than those in ANP group (Plt;0.01). The expression of ERK1/2 phosphorylation in normal pancreas was 1100±141, the expressions of ERK1/2 phosphorylation in ANP group at 15 min, 30 min were 5300±486, 5621±384, respectively; the expressions began to decrease 1 h later and returned the similar level as SO group at 6 h; the expressions of ERK1/2 phosphorylation in PD98059 group at 15 min, 30 min were 4200±370, 3600±290, respectively; which were significantly lower than those in ANP group (allPvaluelt;0.01).ConclusionsThe ERK1/2 signal transduction pathway plays an important role in the pathogenesis of ANP. Inhibition of ERK1/2 phosphorylation by PD98059 may decrease the production of IL-1β, TNF-α and pancreatic MPO, attenuate the extent of pancreatic pathologic injuries.

Pancreatitis,acute necrotizing; Extracellular signal-regulated kinases; Pancreatic injury

10.3760/cma.j.issn.1674-1935.2010.02.014

200433 上海，第二軍醫(yī)大學(xué)長海醫(yī)院消化內(nèi)科

李兆申，Email：zhsli@81890.net