高迁移率族蛋白1在急性坏死性胰腺炎伴器官功能损害中的作用及其抗体的保护作用

2013-10-19 00:52夏敏张婷郭继中陈卫昌
中华胰腺病杂志 2013年1期
关键词:胰腺炎胰腺肝脏

夏敏 张婷 郭继中 陈卫昌

·论著·

高迁移率族蛋白1在急性坏死性胰腺炎伴器官功能损害中的作用及其抗体的保护作用

夏敏 张婷 郭继中 陈卫昌

目的探讨高迁移率族蛋白1(HMGB1)在急性坏死性胰腺炎(ANP)伴器官功能受损发生过程中的作用。方法雄性ICR小鼠按数字表法随机分为对照组、ANP组和HMGB1单抗处理组(抗体组)。采用20% L-精氨酸腹腔注射制备小鼠ANP模型,抗体组于建模成功后即刻给予腹腔内注射HMGB1单抗200 μg。制模后12、24、48 h分批处死小鼠,取血检测血清淀粉酶活性及肝、肾功能指标,ELASA法检测血HMGB1水平,取胰腺及肝脏组织行常规病理检查并评分,实时荧光定量PCR法检测胰腺和肝脏组织HMGB1 mRNA表达。结果对照组、ANP组、抗体组12 h时的血清HMGB1水平分别为(9.09±1.03)、(25.04±4.30)、(16.84±4.27)μg/L;胰腺病理评分为(1.50±0.55)、(4.33±0.52)、(3.03±0.32)分;胰腺组织HMGB1 mRNA表达量为0.48±0.18、7.53±2.71、3.26±2.33;肝脏组织HMGB1 mRNA表达量为-1.23±0.37、0.15±0.65、-1.27±0.72。ANP组的上述指标均显著高于对照组(P值均<0.05),而抗体组均显著低于ANP组(P值均<0.05)。造模后24 h血清HMGB1水平与胰腺病理评分呈线性正相关(r=0.768,P<0.05)。此外,各组血清淀粉酶、谷草转氨酶、谷丙转氨酶、乳酸脱氢酶、尿素氮、肌酐水平的变化趋势同血清HMGB1的变化,血清HMGB1水平与肌酐、尿素氮、谷丙转氨酶水平呈线性正相关(r值分别为0.824、0.719、0.590,P值均<0.05)。结论HMGB1可能是ANP小鼠体内炎症反应和器官功能损害的关键因子,外源性给予其抗体可减轻ANP时胰腺及其他器官功能受损程度。

胰腺炎,急性坏死性; 高迁移率族蛋白质类; 抗体,单克隆

高迁移率族蛋白1(high mobility group box-1 protein, HMGB1)是30年前发现的一种DNA核捆绑蛋白,近来发现其在脓毒症中具有晚期炎症介质的作用。脓毒症[1]、出血性休克[2]、急性肺损伤[3]、类风湿关节炎[4]及弥散性血管内凝血[5]患者的血清HMGB1浓度均升高。新近发现重症急性胰腺炎患者血清HMGB1水平升高,且与疾病的严重程度有关[6]。本研究应用HMGB1抗体干预急性坏死性胰腺炎(ANP)小鼠,探讨HMGB1在ANP器官功能损害发生中的作用机制。

材料与方法

一、实验动物及分组

雄性ICR小鼠,体重为20~25 g,由江苏省血吸虫病防治研究所提供。按数字表法随机分为对照组、ANP组和HMGB1单抗组(抗体组),每组36只。参照文献[7],采取腹腔注射20% L-精氨酸200 mg/100 g体重两次、间隔1 h的方法制备小鼠ANP模型。抗体组于建模后即刻每只小鼠给予腹腔内注射HMGB1单抗200 μg[8],对照组腹腔注射等容积无菌生理盐水。制模后12、24、48 h分别处死12只小鼠,眼球摘除采血,分离血清待测。取新鲜胰腺、肝脏组织,部分甲醛固定,部分液氮保存。

二、检测指标及方法

1.血清HMGB1浓度测定:采用ELISA法,按试剂盒说明书操作。

2.胰腺和肝脏组织HMGB1 mRNA检测:应用Trizol提取小鼠新鲜胰腺和肝脏组织RNA,逆转录合成cDNA。实时荧光定量PCR法检测组织中HMGB1 mRNA的表达,以GAPDH作为内参对照。

HMGB1引物序列上游: 5′-ACAGCCATTGCAGTACATTGAG-3′,下游:5′-TTGCCCATGTTTAGTTGA-TTTTCC-3′,荧光探针: 5′-(FAM)AGAGTCGCC-CAGTGCCCGTCCG (Eclipse)-3′,扩增片段113 bp。PCR反应条件:95℃ 30 s;95℃ 5 s、58℃ 30 s,40个循环,最后72℃ 1 min。使用LightCycler荧光定量PCR仪,计算荧光信号达到指数扩增时的循环周期数(Ct),同时与内参GAPDH的Ct值相比较,用2-△△Ct计算mRNA的表达量。

3.胰腺组织病理检查:胰腺组织常规切片、HE染色,由病理科医师盲法读片,并参照Schmidt、Pozsar方法从水肿、坏死、出血及炎细胞浸润4项对胰腺损伤进行评分,分值之和为总评分。每张切片计数5个视野,取均值。

4.血肝、肾功能检测:采用自动生化分析仪测定血清淀粉酶(AMY)、谷草转氨酶(AST)、谷丙转氨酶(ALT)、乳酸脱氢酶(LDH)、尿素氮(BUN)、肌酐(Cr)的含量。

三、统计学处理

结 果

一、HMGB1单抗对血清HMGB1水平的影响

ANP组小鼠血清HMGB1水平显著高于对照组,抗体组小鼠血清HMGB1水平显著低于ANP组,但仍显著高于对照组(P值均<0.05,表1)。

二、HMGB1单抗对胰腺、肝脏HMGB1 mRNA表达的影响

ANP组胰腺、肝脏组织HMGB1 mRNA较对照组显著增加,抗体组的表达较ANP组显著下降(P值均<0.05,表1);抗体组胰腺HMGB1 mRNA水平仍显著高于对照组(P<0.05),而肝脏HMGB1 mRNA水平与对照组差异无统计学意义。

组别只数血清HMGB1水平(μg/L)12h24h48h对照组369.09±1.035.98±1.973.58±0.67ANP组3625.04±4.30a48.56±9.10a47.18±3.18a抗体组3616.84±4.27ab19.09±1.70ab15.98±1.94ab组别只数胰腺HMGB1mRNA12h24h48h对照组360.48±0.180.30±0.21-0.16±0.44ANP组367.53±2.71a9.63±2.52a9.68±2.53a抗体组363.26±2.33ab4.30±1.13ab3.03±2.11ab组别只数肝脏HMGB1mRNA12h24h48h对照组36-1.23±0.37-2.90±0.97-4.98±1.08ANP组360.15±0.65a0.28±0.49a0.62±0.23a抗体组36-1.27±0.72b-2.61±0.54b-2.81±0.08b

注:与对照组比较,aP<0.05;与ANP组比较,bP<0.05;t值范围1.823~2.156、1.758~2.304、1.698~1.947

三、胰腺和肝脏组织学改变

对照组小鼠胰腺间质轻度水肿,少许炎性细胞浸润;ANP组小鼠24 h时见胰腺间质充血水肿,中性粒细胞和单核细胞浸润,腺泡细胞坏死,部分腺叶结构消失,病理评分升高;抗体组小鼠24 h时的胰腺损伤较ANP组明显减轻(表2,图1上)。

对照组小鼠肝组织未见明显变化;ANP组小鼠24 h时肝表面可见散在出血点,镜下见肝窦淤血,肝细胞水肿,散在坏死,肝细胞索排列紊乱,肝小叶失去正常结构,汇管区及肝小叶内炎症细胞浸润, 48 h时肝损伤较前加重,病理评分显著升高;抗体组小鼠24 h时肝脏病变程度较ANP组明显减轻(图1下)。

四、血清HMGB1水平与胰腺病理评分的相关性

ANP造模后24 h血清HMGB1水平与胰腺病理评分呈线性正相关(r=0.768,P<0.05,图2)。

表2 各组胰腺组织病理评分的比较

注:与对照组比较,aP<0.05;与ANP组比较,bP<0.05;t值范围1.744~2.239

图1ANP组(a)、抗体组(b)小鼠胰腺(上)及肝脏(下)的病理学改变(HE ×200)

图2 血清HMGB1水平与胰腺病理评分的关系

五、各组小鼠血肝、肾功能变化

ANP组小鼠24 h时的血AMY、AST、ALT、LDH、BUN、Cr水平均较对照组显著升高(P值均<0.05),抗体组则均较ANP组显著下降(P值均<0.05,表3)。血清HMGB1水平与Cr、BUN、ALT水平呈线性正相关(r值分别为0.824、0.719、0.590,P值均<0.05) 。

表3 建模后24 h各组小鼠血肝、肾功能变化

注:与对照组比较,aP<0.05;与ANP组比较,bP<0.05,t值范围1.836~3.398

讨 论

细胞外HMGB1是近来发现的晚期炎症介质,是脓毒症致死性多器官功能不全发生的重要介质[9]。既往研究表明,HMGB1与重症急性胰腺炎(SAP)的发生及疾病的严重程度有关,并与SAP时多脏器功能受损相关。Luan等[10]报道,HMGB1参与了ANP大鼠肠道屏障功能受损的过程。体外实验发现,HMGB1以及B-box可能增加Caco-2肠黏膜上皮细胞的通透性,损害小鼠肠黏膜,导致肠道菌群易位,去除HMGB1蛋白可逆转这种作用[11];进一步的实验发现,给健康和内毒素血症小鼠体内注射HMGB1均可引起肠黏膜通透性增加、细菌易位和胃肠道屏障功能障碍[11],给予HMGBl抗体则可减轻这种屏障功能受损的程度。ANP急性肺损伤大鼠的肺组织HMGB-1 mRNA表达明显升高,动脉血氧分压进行性降低,应用HMGB1抑制剂丙酮酸乙酯治疗后大鼠肺组织HMGB-1 mRNA表达下降,动脉血氧分压升高,肺组织受损减轻[12]。

本研究采用腹腔内注射L-精氨酸制备小鼠ANP模型,制模后小鼠血清HMGB1水平明显升高,同时血清AMY、AST、ALT、LDH、BUN、Cr水平明显升高,且血清HMGB1水平与血Cr、BUN、ALT呈线性正相关。此外,胰腺和肝脏组织HMGB1 mRNA表达增加,胰腺组织病理评分与相应血清HMGB1水平呈线性正相关。提示HMGB1参与了胰腺炎症反应,也参与了组织受损和器官功能障碍的过程。应用抗HMGB1单抗干预后小鼠血清HMGB1水平明显下降,血清AMY、AST、ALT、BUN、Cr水平也明显下降,胰腺组织损伤病理评分降低,胰腺及肝脏组织HMGB1 mRNA的表达显著减少,提示HMGB1抗体可有效地拮抗HMGB1的促炎作用,减轻了ANP小鼠胰、肝、肾的病理损伤,与陈瑜等[13]报道的HMGB1抗体可明显缓解肝脏缺血再灌注损伤,Ueno等[3]报道的减轻LPS诱导的大鼠损伤,Ogawa等[14]报道的过度通气诱发小鼠的急性肺损伤的结果基本一致。这些研究进一步证实HMGB1可能是器官功能损害发生、发展中的恶化因子之一。

[1] Sundén-Cullberg J, Norrby-Teglund A, Rouhiainen A, et al. Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit Care Med, 2005, 33: 564-573.

[2] Ombrellino M, Wang H, Ajemian MS, et al. Increased serum concentrations of high-mobility-group protein 1 in haemorrhagic shock. Lancet,1999, 354: 1446-1447.

[3] Ueno H, Matsuda T, Hashimoto S, et al. Contributions of high mobility group box protein in experimental and clinical acute lung injury. Am J Respir Crit Care Med, 2004, 170: 1310-1316.

[4] Taniguchi N, Kawahara K, Yone K, et al. High mobility group box chromosomal protein 1 plays a role in the pathogenesis of rheumatoid arthritis as a novel cytokine. Arthritis Rheum, 2003, 48: 971-981.

[5] Hatada T, Wada H, Nobori T, et al. Plasma concentrations and importance of High Mobility Group Box protein in the prognosis of organ failure in patients with disseminated intravascular coagulation. Thromb Haemost, 2005, 94: 975-979.

[6] Yasuda T, Ueda T, Takeyama Y, et al. Significant increase of serum high-mobility group box chromosomal protein 1 levels in patients with severe acute pancreatitis. Pancreas, 2006, 33: 359-363.

[7] 赵秋玲,黄承钰,徐家玉,等.L-精氨酸诱导小鼠急性坏死性胰腺炎模型的建立.疾病控制杂志,2004,8:141-143.

[8] Sawa H, Ueda T, Takeyama Y, et al. Blockade of high mobility group box-1 protein attenuates experimental severe acute pancreatits. World J Gastroenterol, 2006, 12: 7666-7670.

[9] Wang H, Czura CJ, Tracey KJ. Lipid unites disparate syndromes of sepsis. Nat Med, 2004,10:124-125.

[10] Luan ZG, Zhang H, Ma XC, et al. Role of High-mobility group box 1 protein in the pathogenesis of intestinal barrier injury in rats with severe acute pancreatitis. Pancreas, 2010, 39:216-223.

[11] Sappington PL, Yang R, Yang H, et al. HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice. Gastroenterology, 2002, 123:790-802.

[12] Tsung A, Sahai R, Tanaka H, et al. The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J Exp Med, 2005, 201: 1135-1143.

[13] 陈瑜,黄中伟,沈雁波.急性坏死性胰腺炎肺损伤大鼠高迁移率族蛋白B1的表达及意义.交通医学,2010,24:483-485.

[14] Ogawa EN,Ishizaka A,Tasaka S,et al.Contribution of high-mobility group box-1 to the development of ventilator-induced lung injury.Am J Respir Crit Care Med,2006,174:400-407.

(本文编辑:屠振兴)

RoleofHMGB1inorganinjuryduringacutenecrotizingpancreatitisandprotectiveeffectofHMGB1monoclonalantibody

XIAMin,ZHANGTing,GUOJi-zhong,CHENWei-chang.

DepartmentofGastroenterology,WuxiPeople′sHospital,NanjingMedicalUniveisuty,Wuxi214043,China

XIAMin,Email:xmzb1013@163.com

ObjectiveTo investigate the role of HMGB1 in the pathogenesis of organ injury of acute necrotizing pancreatitis (ANP).MethodsMale ICR mice were randomly allocated into control group, ANP group and HMGB1 monoclonal antibody group. ANP model was induced by intraperitoneal injection of 20% L-arginine. Mice in HMGB1 monoclonal antibody group were given intraperitoneal injection of 200 μg of HMGB1 monoclonal antibody immediately after the induction of the ANP model. All the mice were sacrificed at 12, 24, and 48 h after ANP induction. Serum level of amylase and liver, renal function were determined, level of serum HMGB1 was measured by using enzyme-linked immunosorbent assay ,and then the pathologic changes of pancreas and liver were routinely observed and scored. The HMGB1 mRNA levels in the liver and pancreas were studied by real time fluorescence quantitative PCR.ResultsThe serum levels of HMGB1 at 12 h in control group, ANP group and HMGB1 monoclonal antibody group were (9.09±1.03), (25.04±4.30),

(16.84±4.27)μg/L; and pathological scores of pancreatic tissue were (1.50±0.55), (4.33±0.52), (3.03±0.32) points; and HMGB1 mRNA expressions in pancreas were 0.48±0.18, 7.53±2.71, 3.26±2.33; HMGB1 mRNA expressions in liver were -1.23±0.37, 0.15±0.65, -1.27±0.72. The corresponding values in ANP group were significantly higher than those in control group (P<0.05). While the corresponding values in HMGB1 monoclonal antibody group were significantly lower than those in ANP group (P<0.05). There was a positive linear relationship between serum HMGB1 level and pancreatic pathological scores 24 h after ANP induction (r=0.768,P<0.05). In addition, the serum levels of AMY, AST, ALT, LDH, BUN, Cr showed a similar trend as that of serum level of HMGB1, and the serum level of HMGB1 was positively associated with serum levels of Cr, BUN and ALT (r=0.824, 0.719, 0.590,P<0.05).ConclusionsHMGB1 may be a key factor of inflammatory response and organ dysfunction of ANP in mice, and extrinsic supply of its monoclonal antibody may decrease the injuries of pancreas and other organs during ANP.

Pancreatits, acute necrotizing; High mobility group proteins; Antibodies, monoclonal

10.3760/cma.j.issn.1674-1935.2013.01.010

214023 无锡,南京医科大学附属无锡人民医院消化科(夏敏、郭继中);江苏省中医院消化科(张婷);苏州大学附属第一医院消化科(陈卫昌)

夏敏,Email: xmzb1013@163.com

2012-08-09)

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