殷濤 陳曉燕 丁佑銘 陳辰 王衛(wèi)星

·論著·

羅格列酮對(duì)急性壞死性胰腺炎大鼠肺損傷的保護(hù)機(jī)制

殷濤 陳曉燕 丁佑銘 陳辰 王衛(wèi)星

目的探討羅格列酮(ROSI)對(duì)急性壞死性胰腺炎(ANP)肺損傷的保護(hù)機(jī)制。方法雄性Wistar大鼠75只，按隨機(jī)表法分為假手術(shù)組、ANP組和羅格列酮處理組(ROSI組)。采用膽胰管逆行注射5%牛磺膽酸鈉制備ANP模型。ANP組在制模后30 min股靜脈注射10%二甲基亞砜(DMSO)0.2 ml/100 g體重。ROSI組在制模后30 min股靜脈注射10% DMSO溶解的ROSI 6 mg/kg體重。假手術(shù)組在膽胰管內(nèi)注入等容積生理鹽水，術(shù)后30 min股靜脈注射等量10% DMSO。術(shù)后3、6、12 h分批處死大鼠，取血檢測(cè)血清淀粉酶，測(cè)肺濕、干重比(W/D)，取肺組織行病理學(xué)檢查，蛋白質(zhì)印跡法檢測(cè)肺組織STAT1蛋白及磷酸化STAT1(p-STAT1)蛋白表達(dá)。結(jié)果ANP組血淀粉酶活性、肺W/D、肺組織病理評(píng)分均隨時(shí)間延長(zhǎng)逐漸升高，在各時(shí)點(diǎn)均顯著高于假手術(shù)組(P值均<0.05)，12 h時(shí)達(dá)峰值，分別為(5017±203)U/L、3.12±1.30、(3.33±0.18)分；STAT1蛋白表達(dá)無(wú)明顯變化，p-STAT1的表達(dá)水平則在3 h達(dá)到峰值，為0.87±0.06，以后逐漸下降，但仍顯著高于假手術(shù)組(P值均<0.01)。ROSI組12 h時(shí)的血淀粉酶活性、肺W/D、肺組織病理評(píng)分分別為(1912±164)U/L、1.83±1.26、(2.78±0.16)分，均顯著低于同時(shí)點(diǎn)的ANP組(P值均<0.05)；STAT1蛋白表達(dá)無(wú)明顯變化，3、6、12 h的p-STAT1表達(dá)量分別為0.41±0.04、0.22±0.05、0.15±0.03，均顯著低于同時(shí)點(diǎn)的ANP組(P值均<0.05)。結(jié)論羅格列酮對(duì)ANP大鼠的肺損傷具有保護(hù)作用，其機(jī)制可能與早期抑制肺組織STAT1蛋白磷酸化有關(guān)。

胰腺炎，急性壞死性； 過(guò)氧化物酶體增殖物激活受體； 羅格列酮； 肺損傷

重癥急性胰腺炎(severe acute pancreatitis，SAP)時(shí)常激活炎癥細(xì)胞釋放大量炎癥遞質(zhì)，導(dǎo)致全身炎癥反應(yīng)綜合征，累及多個(gè)胰外臟器損傷。肺臟是最常受累的胰外器官，急性胰腺炎肺損傷(acute pancreatitis associated-lung injury，APALI)是SAP患者早期病死的主要原因之一[1]。信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子(signal transducer and activator of transcription，STAT)是一種新型細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)和基因表達(dá)調(diào)控因子。細(xì)胞因子通過(guò)JAK/STATs途徑激活STATs而誘導(dǎo)目的基因的表達(dá)[2]。羅格列酮(rosiglitazone，ROSI) 屬于人工合成的高選擇性過(guò)氧化物酶體增殖物激活受體-γ(peroxisome proliferator-activated receptors-γ，PPAR-γ)的激動(dòng)劑，對(duì)急性胰腺炎(acute pancreatitis, AP)具有抗炎作用[3]。本研究探討羅格列酮對(duì)急性壞死性胰腺炎(acute necrotizing pancreatitis, ANP)抗炎作用的機(jī)制。

材料與方法

一、實(shí)驗(yàn)動(dòng)物與分組

76只SPF級(jí)雄性Wistar大鼠，體重200～250 g，由湖北省疾病預(yù)防控制中心提供。按隨機(jī)表法分為假手術(shù)組、ANP組和羅格列酮處理組(ROSI組)。假手術(shù)組15只大鼠，ANP組及ROSI組各30只大鼠。采用膽胰管逆行注射5%牛磺膽酸鈉(Sigma公司)0.1 ml/100 g體重的方法制備ANP模型。假手術(shù)組大鼠膽胰管內(nèi)注入等容積生理鹽水。ROSI組于造模后30 min經(jīng)股靜脈注射10% DMSO溶解的羅格列酮(Cayman公司)6 mg/kg體重，其他兩組注射等容積10% DMSO。術(shù)后3、6、12 h分批剖殺大鼠，取血及肺組織。

二、方法

1．血清淀粉酶測(cè)定：采用全自動(dòng)生化分析儀測(cè)定。

2．肺濕、干重比：取右肺中葉，拭去肺組織表面血跡及水分后稱(chēng)濕重，置70℃烘箱24 h后稱(chēng)干重，計(jì)算肺濕、干重比(W/D)。

3．肺組織病理學(xué)檢查：取右下肺組織置4%多聚甲醛固定，常規(guī)石蠟包埋、切片、HE染色，光鏡下觀察，參照文獻(xiàn)[4]對(duì)肺組織行病理學(xué)評(píng)分。

4．肺組織STAT1蛋白和磷酸化STAT1(p-STAT-1)蛋白檢測(cè)：取新鮮液氮凍存后置-80℃冰箱保存的肺組織，融化后制備組織勻漿，蛋白定量后常規(guī)行蛋白質(zhì)印跡法檢測(cè)STAT1和p-STAT1表達(dá)。兔抗大鼠p-STAT1多抗購(gòu)自Cell Signaling公司，工作濃度1∶600；兔抗大鼠STAT1抗體購(gòu)自美國(guó)Santa Cruz公司，工作濃度1∶3000； HRP-山羊抗兔IgG二抗購(gòu)自美國(guó)Pierce公司，工作濃度1∶5000；ECL化學(xué)發(fā)光顯色試劑盒購(gòu)自Millipore公司。以β-actin為內(nèi)參。凝膠成像系統(tǒng)掃描條帶灰度值，以目的條帶與β-actin條帶灰度比值代表蛋白的表達(dá)水平。

三、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、血清淀粉酶活性及肺W/D的變化

ANP組大鼠血清淀粉酶活性及肺W/D比隨著時(shí)間的延長(zhǎng)逐漸升高，12 h達(dá)到峰值；而ROSI大鼠的血清淀粉酶活性、肺W/D比較ANP組顯著下降，差異有統(tǒng)計(jì)學(xué)意義(t值5.31～24.26，F(xiàn)=83.581，P值均<0.05，表1)。

二、肺組織病理學(xué)改變

假手術(shù)組大鼠肺組織結(jié)構(gòu)清晰，肺泡壁完整，無(wú)明顯水腫和滲出。ANP組大鼠肺組織間質(zhì)明顯充血、水腫，細(xì)胞浸潤(rùn)，間質(zhì)明顯增寬，隨著時(shí)間延長(zhǎng)肺損傷加重，肺組織病理評(píng)分逐漸增高。ROSI組大鼠肺組織間質(zhì)充血、水腫和浸潤(rùn)程度較ANP組減輕，病理評(píng)分下降，其中3、6、12 h的病理評(píng)分與同時(shí)間點(diǎn)ANP組的差異有統(tǒng)計(jì)學(xué)意義(P值均<0.05)，與假手術(shù)組比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義(圖1，表1)。

圖1假手術(shù)組(a)、ANP組(b)、ROSI組(c) 3 h時(shí)大鼠肺組織病理改變(HE ×200)

三、肺組織STAT1及p-STAT1蛋白的表達(dá)

假手術(shù)組、ANP組、ROSI組STAT1蛋白表達(dá)在各時(shí)間點(diǎn)差異均無(wú)統(tǒng)計(jì)學(xué)意義。ANP組p-STAT1蛋白表達(dá)水平于3 h時(shí)達(dá)峰值，6 h時(shí)開(kāi)始下降，12 h時(shí)明顯下降，但均顯著高于假手術(shù)組(P值均<0.01)。ROSI組p-STAT1蛋白在3、6、12 h時(shí)均較同時(shí)點(diǎn)ANP組顯著下降，差異有統(tǒng)計(jì)學(xué)意義(F=26.317，P值均<0.05，圖2，表1)。

圖2假手術(shù)組(1)、ANP組(2)、ROSI組(3)3、6、12 h(a、b、c)肺組織STAT1及p-STAT1蛋白表達(dá)

表1 各組大鼠血淀粉酶活性、肺W/D、肺組織病理評(píng)分及STAT1、p-STAT1表達(dá)的變化

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

討 論

炎癥遞質(zhì)的過(guò)度表達(dá)是很多炎癥性疾病發(fā)生、發(fā)展的作用機(jī)制之一。目前，細(xì)胞信號(hào)通路與炎癥發(fā)生機(jī)制之間的關(guān)系是研究的重點(diǎn)。信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子(STAT)是一類(lèi)DNA結(jié)合蛋白，作為JAK的底物與酪氨酸磷酸化耦聯(lián)從而發(fā)揮轉(zhuǎn)錄調(diào)控作用，介導(dǎo)細(xì)胞因子的多種生物學(xué)效應(yīng)。

炎癥過(guò)程中，大量細(xì)胞因子和炎癥遞質(zhì)的釋放，引起JAK活化并使其受體磷酸化，然后與STATs結(jié)構(gòu)中的SH2功能結(jié)構(gòu)域相互作用，使其早期磷酸化而被激活，進(jìn)入細(xì)胞核與相應(yīng)的靶基因啟動(dòng)子結(jié)合激活相應(yīng)基因的轉(zhuǎn)錄和表達(dá)。不同的STATs蛋白具有不同的功能[5]。有研究顯示，抑制STATs蛋白的表達(dá)對(duì)AP大鼠肺損傷具有保護(hù)作用[6]。而STAT1目的基因被證實(shí)具有促進(jìn)炎癥發(fā)生和抵抗細(xì)胞增殖作用[7]。在膿毒癥大鼠肺損傷模型中，抑制STAT1活化可以減輕膿毒癥所致肺組織損傷[8]。

羅格列酮屬于噻唑烷二酮(TZD)類(lèi)，其發(fā)揮抗炎作用的機(jī)制尚未完全明確。Cuzzocrea等[9]研究發(fā)現(xiàn)，羅格列酮能減少中性粒細(xì)胞浸潤(rùn)和細(xì)胞間黏附分子的表達(dá)，從而減輕蛙皮素誘導(dǎo)的AP各臟器損傷程度。PPAR-γ激動(dòng)劑的抗炎機(jī)制可能通過(guò)抑制JAK/STAT1信號(hào)通道的炎癥轉(zhuǎn)錄因子，如STAT1，從而抑制下游炎癥遞質(zhì)TNF-α、ICAM-1 mRNA的表達(dá)，進(jìn)一步減輕炎癥反應(yīng)[10-12]，表明羅格列酮能減輕ANP大鼠的肺損傷，

本研究結(jié)果顯示，各組大鼠STAT1蛋白表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義，但ANP組 STAT1蛋白在1 h時(shí)即被磷酸化而活化，3 h時(shí)達(dá)到高峰， 6 h后明顯下降，12 h表達(dá)最低，與Severgnini等[13]的報(bào)道一致。應(yīng)用羅格列酮干預(yù)后大鼠胰腺組織p-STAT1蛋白表達(dá)降低，且血清淀粉酶及肺濕、干重比和肺組織病理學(xué)評(píng)分均較ANP組顯著下降，其機(jī)制可能是通過(guò)抑制STAT1蛋白的磷酸化進(jìn)而下調(diào)炎癥因子如TNF-α、IL-6和細(xì)胞間黏附分子等促炎遞質(zhì)的表達(dá)，改善AP大鼠的臟器損傷[9-10,12,14]。

[1] Bhatia M. Acute pancreatitis as a model of SIRS. Front Biosci, 2009, 14: 2042-2050.

[2] Schindle CW. Series introduction. JAK/STAT signaling in human disease. J Clin Invest,2002,109:1133-1137.

[3] Rollins MD, Sudarshan S, Firpo MA, et al. Anti-inflammatory effects of PPAR-gamma agonists directly correlate with PPAR-gamma expression during acute pancreatitis. J Gastrointest Surg, 2006,10:1120-1130.

[4] 程石, 何三光, 張佳林. 肺泡巨噬細(xì)胞活化在急性壞死性胰腺炎大鼠肺損傷中的作用. 中華外科雜志, 2002, 40:609-612.

[5] Ivashkiv LB, Hu X. Signaling by STATs. Arthritis Res Ther, 2004,6:159-168.

[6] Simovic MO, Ballard BR, Gray KD, et al. The STAT4 and STAT6 pathways in pancreatitis-associated lung injury. J Surg Res, 2007, 137:10-15.

[7] Schindler C, Levy DE, Decker T, et al. JAK-STAT Signaling:from interferonsto cytokines. J Biol Chem, 2007,13;282:20059-20063.

[8] 王松柏，翟秀珍，劉俊堂，等. 信號(hào)轉(zhuǎn)導(dǎo)子和轉(zhuǎn)錄激活子-1對(duì)膿毒癥大鼠肺損傷的影響.中華急診醫(yī)學(xué)雜志, 2007, 16:807-810.

[9] Cuzzocrea S, Pisano B, Dugo L, et al. Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor-gamma, reduces acute pancreatitis induced by cerulein. Intensive Care Med, 2004,30:951-956.

[10] Ricote M, Li AC, Willson TM, et al. The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation. Nature, 1998,391:79-82.

[11] 黃玲，高峻，蔣斐，等.過(guò)氧化物酶體增殖物激活受體-γ在大鼠慢性胰腺炎進(jìn)程中的表達(dá)及意義.中華胰腺病雜志，2011，11，426-429.

[12] Rollins MD, Sudarshan S, Firpo MA, et al. Anti-inflammatory effects of PPAR-gamma agonists directly correlate with PPAR-gamma expression during acute pancreatitis. J Gastrointest Surg, 2006, 10:1120-1130.

[13] Severgnini M, Takahashi S, Rozo LM, et al. Activation of the STAT pathway in acute lung injury. Am J Physiol Lung Cell Mol Physiol, 2004,286:L1282-L1292.

[14] 徐萍，李清華，王靜，等.吡格列酮對(duì)急性壞死性胰腺炎大鼠胰腺PPAR-γmRNA表達(dá)的影響.中華胰腺病雜志，2010,10:352-354.

Protectivemechanismofrosiglitazoneonacutenecrotizingpancreatitisassociatedlunginjuryinrats

YINTao,CHENXiao-yan,DINGYou-ming,CHENChen,WANGWei-xing.

DepartmentofHepatobiliaryPancreasSurgary,CancerHospitalofHubeiProvince,Wuhan430079,China

Correspondingauthor:WANGWei-xing,Email：sate.llite@163.com

ObjectiveTo investigate the protective effect of rosiglitazone on acute necrotizing pancreatitis (ANP) associated lung injury in rats.MethodsSeventy-five male Wistar rats were randomly divided into sham operation group (SO group), acute necrotizing pancreatitis group (ANP group) and rosiglitazone pretreatment group (ROSI group). ANP model was induced by retrograde infusion of 5% sodium taurocholate into the biliopancreatic duct. Thirty minutes after ANP induction, ANP groups were injected with 10% DMSO (0.2 ml/100 g) through femoral vein, and ROSI group were injected with ROSI dissolved with 10% DMSO (6 mg/kg) through femoral vein, while SO group was injected with normal saline, and 30 minutes later was injected with same amount of 10% DMSO. Rats were sacrificed at 3 h, 6 h and 12 h after the operation. Serum amylase and lung wet/dry weight ratio (W/D) were measured, lung tissues were harvested for pathologic examinations. STAT1 protein and phosphorylation-STAT1 protein (p-STAT1) expression were detected by Western blot.ResultsThe serum levels of amylase, lung W/D, pathologic score of lung tissues in ANP group were increased with time, and reached the peak at 12 h, which were (5017±203)U/L, 3.12±1.30, (3.33±0.18) score, and these were significantly higher than those in SO group (P<0.05 or 0.01), the expression of STAT1 protein was not statistically significant, but the expression of p-STAT1 reached the peak at 3 h (5.23±0.03), then it gradually decreased, but it was still significantly higher than that in SO group (0.16±0.04,p<0.01). The serum levels of amylase, lung W/D, pathologic score of lung tissues in ROSI group at 12 h were (1912±164) U/L, 1.83±1.26, (2.78±0.16), which were significantly lower than those in ANP group (P<0.05). The expression of STAT1 protein was not statistically significant, and the expressions of p-STAT1 at 3 h, 6 h, 12 h was 0.41±0.04, 0.22±0.05, 0.15±0.03, which were significantly lower than those in ANP group (P<0.05).ConclusionsRosiglitazone has the protective effect on ANP associated lung injury by inhibition of phosphorylation-STAT1 protein expression in the early phase.

Pancreatitis, acute necrotizing; Peroxisome proliferator-activated receptors; Rosiglitazone; Lung injury

2012-10-18)

(本文編輯：屠振興)

通知

10.3760/cma.j.issn.1674-1935.2013.04.013

湖北武漢，湖北省腫瘤醫(yī)院肝膽胰外科(殷濤)；武漢大學(xué)人民醫(yī)院肝膽腔鏡外科(殷濤、陳曉燕、丁佑銘、陳辰、王衛(wèi)星)

王衛(wèi)星，Email：sate.llite@163.com

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