⊙基础研究 BASIC RESEARCH⊙

乌司他丁对急性坏死型胰腺炎大鼠肺损伤的保护作用

尹  勇，高乃荣，李兆丽

尹勇, 广州中山医科大学附属第一医院胃肠外科  广东省广州市 510089
高乃荣,东南大学医学院附属中大医院普外科  江苏省南京市 210009
李兆丽,佛山市第一人民医院病理科  广东省佛山市 528000
尹勇,男,1973-05-22生, 内蒙古包头人, 回族, 中山医科大学普外科在读博士, 主要从事胃肠胰疾病的研究.
项目负责人  高乃荣, 210009, 江苏省南京市丁家桥87号, 东南大学医学院附属中大医院普外科.  yinyong1973@sohu.com
电话: 020-87334191
收稿日期  2002-02-25  接受日期  2002-03-05


Protective effects of ulinostatin on acute lung injury induced by acute necrotizing pancreatitis in rats

Yong Yin, Nai-Rong Gao, Zhao-Li Li

Yong Yin, Department of Gastrointestinal Surgery, the First Affiliated Hospital of SunYat-Sen University of Medical Sciences.Guangzhou 510089, Guangdong Province, China
Nai-Rong Gao, Department of General Surgery, ZhongDa Hospital, Southeast University. Nanjing 210009, Jiangsu Province, China
Zhao-Li Li, Department of Pathology, the First People, Hospital of Foshan, Foshan 528000, Guangdong Province, China
Correspondence to: Dr Yong Yin, Department of Gastrointestinal Surgery, the First Afiliated Hospital of SunYat-Sen University of Medical Sciences, Guangzhou 510089, Guangdong Province, China.  yinyong1973@sohu.com
Received  2002-02-25  Accepted  2002-03-05


Abstract
AIM:To investigate the protective effects of ulinostatin (UT) on acute lung injury in acute necrotizing pancreatitis (ANP) in rats.

METHODS:Seventy-two Sprague-Dawley rats were randomly divided into three groups: sham operation (SO) group, ANP group, ANP+UT group (UT:100KU/kg). ANP model was made by creating a closed duodenal loop in rats. Pulmonary ICAM-1 expression was detected by using ABC immunohistochemistry and the accumulation of polymorphonuclear leukocytes in lung tissue was determined by the myeloperoxidase (MPO) assay. Lung capillary permeability (LCP) and pathological changes of rat lung tissue were also observed.

RESULTS:UT treatment markedly downregulated ICAM-1 expression and pulmonary MPO activity of UT group was significantly decreased as compared with that of ANP group (6h: 3.0±1.0μmol/g vs 4.6±0.8μmol/g,  P <0.05; 16h: 3.5±1.1μmol/g vs 7.7±0.7μmol/g, P <0.01; 24h: 3.3±1.3μmol/g vs 7.2±1.0μmol/g, P <0.01), although it was still higher than that of SO group. LCP level of UT group was reduced as compared to ANP group（74.9±6.6μg/g vs 86.9±10.9μg/g, P <0.05）. Pathological changesof lung tissue revealed that lung injury was significantly improved with UT treatment.

CONCLUSION:Ulinostatin can reduce the accumulation and infiltration of polymorphonuclear leukocytes in lung tissue via downregulating of ICAM-1.Thus, this drug may exert a beneficial effect on acute lung injury induced by acute pancreatitis.

Yin Y, Gao NR, Li ZL. Protective effects of ulinostatin on acute lung injury induced by acute necrotizing pancreatitis in rats. Shijie Huaren Xiaohua Zazhi 2002;10(5):558-561


摘要
目的:观察乌司他丁（UT）对急性坏死型胰腺炎（ANP）大鼠肺损伤的保护作用.

方法:SD大鼠72只，随机分为3组：假手术组（SO）；ANP组；ANP+UT组（10万U/kg）. 通过结扎大鼠十二指肠使呈闭袢制备ANP模型. 免疫组织化学法观察ICAM-1蛋白的表达，测定肺脏髓过氧化物酶（MPO）活性，同时观察术后6h肺毛细血管通透性（LCP）的变化及肺组织的病理形态学改变.

结果:UT治疗组肺脏ICAM-1的表达下调，肺脏MPO活性虽明显高于SO组，但较ANP组明显降低（6h: 3.0±1.0μmol/g vs 4.6±0.8μmol/g, P <0.05；16h: 3.5±1.1μmol/g vs 7.7±0.7μmol/g, P <0.01； 24h: 3.3±1.3μmol/g vs 7.2±1.0μmol/g, P <0.01）. LCP水平也较ANP组明显降低（74.9±6.6μg/g vs 86.9±10.9μg/g, P <0.05），肺损伤程度较ANP组明显减轻.

结论:UT通过下调肺脏ICAM-1的表达，减少中性粒细胞在肺脏的聚集、浸润，对于急性坏死型胰腺炎大鼠的肺损伤具有一定保护作用.

尹勇，高乃荣，李兆丽. 乌司他丁对急性坏死型胰腺炎大鼠肺损伤的保护作用. 世界华人消化杂志 2002;10(5): 558-561


0  引言
中性粒细胞过度激活聚集于肺组织是导致急性坏死型胰腺炎（ANP）早期合并肺损伤的重要因素^［1-3］，其分子基础在于中性粒细胞-内皮细胞的相互作用即细胞黏附过程^［4］. 我们探讨丝氨酸蛋白酶抑制剂乌司他丁（UT）对ANP大鼠肺脏中性粒细胞聚集和细胞间黏附分子-1（ICAM-1）表达的影响以及对急性肺损伤（ALI）的防治作用.

1  材料和方法
1.1  材料  健康SD大鼠(东南大学医学院实验动物中心提供)72只，雄雌不拘，身体重量180～250g，随机分为3组：假手术组（SO）；ANP组；ANP+UT组. 采用“十二指肠闭袢法”制备ANP模型. 实验动物术前禁食24h，自由饮水，30g/L戊巴比妥钠(Serva进口分装批号84-06-12)按30mg/kg ip麻醉，入腹后经胃窦切口将一根长3cm，内径0.4cm无菌塑料管置入十二指肠，于胆胰管开口上、下各1cm处围绕塑料管结扎十二指肠使呈闭袢，袢长2cm，缝合胃窦切口，逐层关腹. SO组仅作胃窦切开术. UT治疗组在模型建立后5min尾静脉穿刺，连接微量输液泵，乌司他丁（广东天普生物化学制药有限公司批号X1990133）剂量按10万U/kg、容积按12.5ml/kg持续静脉推注45min. 其他两组代以等量生理盐水.每组各18只大鼠于术后6,16,24h分批处死，取一块肺组织迅速置于-70℃冻存，待测肺组织髓过氧化物酶活性；另取一块肺组织固定于40g/L甲醛溶液中，包埋制片用作免疫组织化学检测，HE染色进行光镜观察. 各组其余6只大鼠用于观察术后6h肺毛细血管通透性的变化.
1.2  方法
1.2.1  肺组织ICAM-1的表达  肺组织石蜡切片采用免疫组织细胞化学ABC法检测ICAM-1的表达，一抗（小鼠抗大鼠ICAM-1mAb，工作浓度为1∶50）、即用型SABC免疫组化检测试剂盒及DAB显色试剂盒均购自武汉博士德生物工程有限公司. 所有操作步骤严格按照试剂盒说明书进行，以PBS液代替一抗孵育作为阴性对照. 判断标准：ICAM-1是一种膜抗原，主要表达在肺泡上皮细胞和血管内皮细胞表面，采用盲法请有经验的病理科医生对切片进行观察并作半定量分析，根据细胞染色的深浅评分：无染色-0分，淡黄色-1分，棕黄色-2分，深棕黄色-3分；再以视野中显色细胞所占的比例（取10个视野的平均值）评分：<20%-0分，20～40%-1分，40～60%-2分，>60%-3分.将两项评分的乘积作为积分数，根据积分将ICAM-1表达分为四级：0分-阴性，1～3分-弱阳性，4～6分-阳性，>6分-强阳性.
1.2.2  肺脏MPO活性的测定  MPO(髓过氧化物酶)活性可反映组织中性粒细胞的聚集浸润程度，试剂盒购自南京建成生物工程研究所，酶活力单位定义：每克组织蛋白在37℃的反应体系中H₂O₂分解1μmol为1个酶活力单位.
1.2.3  LCP的测定  采用伊文思蓝(Fluka公司)浸入法，以单位组织中染料提取量反映LCP(肺脏毛细血管通透性)水平，以组织湿重μg/g为单位.
      统计学处理  数据以均数±标准差表示，用SPSS软件进行统计学分析，组间比较采用t检验，ICAM-1表达的积分数比较采用秩和检验.

2  结果
采用盲法请有经验的病理科医生对各组大鼠肺组织切片进行光镜观察. SO组肺泡结构清晰，肺泡隔无增厚，间质血管无扩张，无中性粒细胞浸润. ANP组6h肺泡壁塌陷，间质毛细血管扩张、充血，肺泡隔中性粒细胞浸润明显，部分中性粒细胞游走到肺泡腔内；16h间质血管扩张、充血明显，部分肺泡腔有红细胞漏出，肺泡隔增厚伴大量中性粒细胞浸润；24h出血明显加重，肺泡腔内可见淡红色液体渗出（肺水肿），与16h比较中性粒细胞有所减少. UT治疗组各时相点肺间质血管充血、出血，肺泡隔增厚、中性粒细胞浸润及肺水肿等均较ANP组明显减轻.
2.1  肺组织ICAM-1的表达  SO组肺泡上皮细胞和肺泡隔血管内皮细胞表面均无染色，ICAM-1的表达为阴性. ANP组术后6h部分肺泡隔血管内皮细胞表面有淡黄色或棕黄色颗粒，少数肺泡上皮细胞也有轻度染色，肺组织ICAM-1蛋白表达为弱阳性；术后16h肺泡隔血管内皮细胞表面出现棕黄色或深棕黄色颗粒，部分肺泡Ⅰ,Ⅱ型上皮细胞表面也可见黄色颗粒，ICAM-1表达为阳性或强阳性；至24h上述细胞ICAM-1的表达呈现强阳性(图1). UT治疗组肺组织ICAM-1表达(图2)的分布与ANP组基本一致，但其表达积分数较ANP组明显减低（6h: P <0.05; 16, 24h: P <0.01）.

图1  ANP组24h肺脏血管内皮细胞和肺泡上皮细胞表面ICAM-1强阳性表达(免疫组化ABC×400)

图2  UT：治疗组24h肺脏ICAM-1的表达较ANP组减低(免疫组化ABC×400)

2.2  肺组织MPO活性的变化  ANP组术后6h肺组织MPO水平即明显高于SO组（P <0.01），16h达峰值（7.7±0.7）μmol/g ，24h有所下降，但仍显著高于SO组. UT治疗组各时相点MPO的活性明显高于SO组（P <0.01），但其活性升高幅度较ANP组相应时相点显著降低（表1）.

表1  肺组织髓过氧化物酶（MPO）活性的变化（μmol/g）

^bP <0.01 vs SO组; ^aP <0.05 vs ANP组; ^dP <0.01 vs ANP组

2.3  LCP变化  SO组LCP值为组织湿重（42.9±7.9）μg/g，ANP组和UT治疗组LCP值分别为（86.9±10.9）μg/g和（74.9±6.6）μg/g，显著高于SO组（P <0.01），但UT治疗组LCP值较ANP组显著降低（P <0.05）.


3  讨论
急性呼吸窘迫综合征（ARDS）是导致ANP早期病情危重和死亡的主要原因^［5,6］. 在发病7d内的死亡病例中有60%与急性肺损伤（ALI）关系密切^［7］. 其主要病理改变为肺泡隔毛细血管高度扩张、充血，肺间质和肺泡腔内有大量含蛋白质液体渗出（肺水肿），肺间质中还可发生点状出血、灶性坏死以及淋巴管扩张. 目前尚缺乏有效的治疗手段，因此探索新的治疗途径十分必要.
      研究证明，ANP时大量中性粒细胞聚集于肺组织是ALI早期的基本病理改变，也是导致ALI的重要因素^{［1-3,8,9］}， 中性粒细胞跨内皮细胞游走、浸润，一方面直接释放大量细胞毒性物质如弹性蛋白酶、胶原酶、蛋白水解酶、磷脂酶A₂、活性氧代谢产物、血小板激活因子及促炎细胞因子，发挥细胞毒性作用，使肺内皮细胞损伤、变性，血管通透性增加，肺间质水肿，氧交换障碍^［10-12］；另一方面，聚集的中性粒细胞机械堵塞毛细血管致微循环障碍，中性粒细胞和内皮细胞还可以产生血小板激活因子、血栓素A₂，二者均是强有力的缩血管物质，使肺血管收缩，产生肺动脉高压，导致通气/血流比例失调、低氧血症，发生ARDS^［13-15］. 在中性粒细胞跨越内皮细胞屏障、聚集浸润于局部受损区域之前，首先须完成与血管内皮细胞的黏附. 近年来研究表明，活化的中性粒细胞表面的整合素CD11b/CD18β₂复合体与血管内皮细胞表面的细胞间黏附分子-1（ICAM-1）以受体-配体的形式相结合是细胞黏附过程中的关键环节^［16-20］. 一些学者还发现急性胰腺炎时ICAM-1等黏附分子的异常表达较细胞因子出现晚，中性粒细胞浸润和器官损伤往往发生在ICAM-1表达上调之后，认为细胞黏附在治疗中是可以阻断的步骤^{［1, 2, 21-25］}.
      乌司他丁（UT）是一种从新鲜人尿中提取的丝氨酸蛋白酶抑制剂，其具有抑制多种胰酶活性和抑制炎症递质释放的药理作用. Okumuma et al ^［26］ (J Gastroenterol, 1995; 30: 379-386)在体外实验中发现，UT可以抑制中性粒细胞与人脐静脉内皮细胞的黏附. 我们发现，经UT治疗后大鼠各时相点肺组织中ICAM-1的表达明显减低，MPO（中性粒细胞标志酶）活性的升高幅度也较ANP组相应时相点显著降低，同时发现UT治疗后大鼠术后6h肺毛细血管通透性明显降低，病理切片观察示肺间质毛细血管扩张、充血及出血、水肿等改变较ANP组减轻. 据以上发现我们认为，UT通过下调大鼠肺脏ICAM-1的表达，从而减少中性粒细胞在肺组织中活化、黏附和聚集，中性粒细胞所释放的各种有毒炎性递质也将随之减少，对肺泡上皮细胞和肺泡隔血管内皮细胞具有一定的保护作用，UT的抗黏附作用为ANP合并肺损伤提供了一条新的治疗途径. 同时也提示丝氨酸蛋白酶可能参与了肺血管内皮细胞表面ICAM-1的表达，但其机制有待深入研究.

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