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⊙研究原著⊙

环氧合酶-2在急性肝损害中的作用机制

田  耕，于皆平，罗和生，余保平，李建英

田耕，于皆平，罗和生，余保平，李建英，武汉大学人民医院消化内科  湖北省武汉市 430060
田耕，男，1970-09-09生，山西人，汉族，1992年山西医科大学毕业，1996年山西医科大学肝病研究所硕士，现为武汉大学人民医院消化内科博士研究生.发表论文及综述10篇.
项目负责人  于皆平，430060,武汉大学人民医院消化内科,湖北省武汉市解放路238号.tg3030330@sina.com
电话: 027-88077184
收稿日期  2001-09-25  接受日期  2001-10-20


The expression and effect of cycloox ygenases-2 in acute hepatic injury

Geng Tian, Jie-Ping Yu, He-Sheng Luo, Bao-Ping Yu, Jian-Ying Li

Geng Tian, Jie-Ping Yu, He-Sheng Luo, Bao-Ping Yu, Jian-Ying Li. Digestive department. Renmin hospital of Wuhan university, 238 Jie-fang Road, W uhan 430060,Hubei Province, China
Correspondence to:  Pro Jie-Ping Yu, Digestive department. Renm in hospital of Wuhan university, 238 Jie-fang Road, Wuhan 430060,Hubei Province , China.tg3030330@sina.com
Received  2001-09-25  Accepted  2001-10-20


Abstract
AIM:To study the expression of cyclooxygenases-2 and its re lationship with acute hepatic injury.

METHODS:Fifty Wistar rats were divided into five groups: normal group 10 rats, control group 10 rats receiving ip CCL4 1mL·kg-1, and t hree model groups (10 rats in each group)ip CCL4 1mL·kg-1,and after tw o hours ig Celecoxib 20mg·kg-1,40mg·kg-1,80mg·kg-1 were administered,respectively.

RESULTS:The levels of serum ALT,AST and LPS in the control group  were higher than these in normal group (ALT: 1392±16 vs 624±5,AST:1803±1 8 vs 748±6,LPS:124±61 vs 60±50, P<0.05). These indices also showe d a significant difference between control and model groups(ALT: 1790±13, 3301 ±20, 4500±32 vs 1392±16,AST: 2183±21, 3946±30, 4903±23 vs. 1803±1 8,LPS: 143±37,186±77,232±35 vs 124±61. P<0.05). HE stains showed hep atic cell swelling, vacuolation and steatosis in control and model groups. Immun ohistochemical stain showed that expression of COX-2 and NF-kBp65 in model gro ups were higher than these in control group(NF-kBp65: 0.175±0.045, 0.196±0.02 4, 0.217±0.029 vs. 0.217±0.029 P<0.05; COX-2: 0.148±0.025, 0.158±0 .030, 0.169±0.017 vs 0.146±0.024 P<0.05).

CONCLUSIONS:Hepatic injury induced the expression of COX-2 in h epatocytes which might be a protective role against injury by the organs. The le vels of NF-kBP65 rose with the increase of COX-2 levels.

Tian G, Yu JP, Luo HS, Yu BP， Li JY.The expression and effect of cyclo oxygenases-2 in acute hepatic injury.
Shijie Huaren Xiaohua Zazhi 2002;10(1):24 -27


摘  要
目的:观察COX-2在急性肝损伤中的表达，探讨COX-2与急性肝损伤的 关系.

方法:将50只♂Wistar大鼠随机分为正常组10只，对照组10只腹腔注射CCL 4原液1mL·kg-1、实验组30只分3组腹腔注射CCL4原液1mL·kg-1，2h后 每组分别给予20mg·kg-1、40mg·kg-1和80mg·kg-1塞来昔布灌胃.

结果:血清学检测结果显示CCL4组和CCL4+Celecoxib组大鼠血清ALT， AST和LPS均较正常大鼠组明显增高（ALT:1392±16 vs 624±5,AST:1803±18 vs 7 48±6,LPS:124±61 vs 60±50 P<0.05），CCL4组与CCL4+Celecoxib组大鼠血 清ALT、AST和LPS水平存在显著差异（ALT: 1790±13, 3301±20, 4500±32 vs 1392± 16,AST: 2183±21, 3946±30, 4903±23 vs 1803±18,LPS: 143±37,186±77,232±3 5 vs 124±61. P<0.05）.HE染色光镜下示CCL4组和CCL4+Celecoxib组大鼠大 量肝细胞肿胀、空泡变性和脂肪变性，部分肝细胞坏死.免疫组织化学检测结果显示NF-k BP65在部分正常肝组织弱阳性表达，CCL4组与CCL4+Celecoxib组大鼠均有NF-kBP65的 阳性表达，且两组NF-kBp65的表达存在显著差异（0.175±0.045, 0.196±0.024, 0.217± 0.029 vs 0.217±0.029  P<0.05）；COX-2在正常肝组织无表达，CCL4+Celeco xib组大鼠较CCL4组大鼠COX-2表达明显增高（0.148±0.025, 0.158±0.030, 0.169±0. 017 vs 0.146±0.024 P<0.05）.

结论:急性肝损伤后诱导COX-2表达，可能是机体对抗损伤的一种保护性 机制.使用COX-2抑制剂肝损伤加重，NF-kBp65的表达增高，随着NF-kBP65表达增高，COX -2表达存在相应增高的趋势.

田耕,于皆平,罗和生,余保平,李建英.环氧合酶-2在急性肝损害中的作用机制.世 界华人消化杂志 2002;10(1):24-27


0  引言
环氧合酶（cyclooxygenases COX）是花生四烯酸合成前列腺素的限速酶， 是非甾体类药物 发挥抗炎、退热和镇痛作用的主要靶点.环氧合酶包括两种亚型COX-1和COX-2，COX-1由M iyamoto et al［1］1976发现，COX-2于1989年由Simmons et al［2]报道.许多流行病学和临床资料表明非甾体类抗炎药可降低结直肠、食管等部位的肿瘤的 发生率，某些实验室和临床资料也报道非甾体类抗炎药可以降低胃癌、胰腺癌和肝癌的发病 率，抑制肿瘤细胞的增殖，诱导肿瘤细胞的凋亡［3-43］.而非甾体类抗炎药的作用 ，主要与抑制肿瘤细胞表达COX-2有关.COX2在炎症、癌前病变以及肿瘤中的作用已成为研 究的热点.COX-2在炎症区域表达增高［44-45］.而环氧合酶-2在急性肝损害中的 表达与作用则知之较少.

1  材料和方法
1.1  材料 ♂Wistar大鼠（100±10）g 50只，购自武 汉大学医学院实验动物中心.塞来昔布，购自美国Sigma公司.CCL4购自上海生化试剂公司.  NF-кВ P65 和 COX-2多克隆抗体购自Santa Cruz公司，免疫组化染色试剂盒购自北京中山生物技术有限公司.血清谷丙转氨酶（ALT）、谷草转氨酶（AST）试剂盒购自南 京建成生化试剂公司.鲎试剂盒，购自上海医学化验所.
1.2  方法 将50只大鼠随机分为正常组10只，对照组10 只腹腔注射CCL4原液1mL·kg-1.实验组30只分3组，每组各10只，腹腔注射CCL4 原液1mL·kg-1，2h后分别给予20mg·kg-1，40mg·kg-1和80mg·kg -1塞来昔布灌胃.24h后腹主动脉取血.取肝右叶约10g，100mL·L-1福尔马林 固定进行组织学与免疫组织化学检查.血清谷丙转氨酶（ALT），谷草转氨酶（AST）按试剂 盒说明操作.血清内毒素检测：鲎试剂法.NF-кВ P65和COX-2免疫组织化学检测：肝 组织切片脱蜡至水，30mL·L-1H2O2封闭内源性过氧化物酶，室温10min，组织 切片PBS冲洗，置0.01mol·L-1枸橼酸盐缓冲液中，微波修复抗原10min，组织切片 蒸馏水冲洗，置于PBS液4min，然后与封闭用山羊血清室温孵育15min封闭非特异抗原，使用 羊抗鼠的单克隆抗体NF-кВ P65 和COX-2多克隆抗体分别1∶100,37℃孵育2h.PBS冲洗， 生物素标记二抗IgG抗体37℃孵育15min.PBS冲洗，辣根酶标记链霉卵白素37℃孵育15min，P BS冲洗，DAB显色，自来水充分冲洗，苏木素复染，封片.阴性染色采用略去一抗.结果判断 ： NF-кВ P65和COX-2阳性细胞均为胞质或胞膜染成棕黄色.采用HIPAS-2000型计算机 图象分析系统（同济千屏影象工程公司产品），通过显微摄影系统放大400倍摄取图象，输 入图象分析系统，对图象进行灰度变换，使染色阳性面积与背景分开，自动记录吸光度A值. 每张切片随机选取5个视野，取A均值（x±s）.
    统计学处理  所取数据以±s表示，做方差齐性检验 、方差分析，以P<0.05为差异有显著性.


2  结果
2.1  四氯化碳组大鼠与使用塞来昔布和四氯化碳组大鼠较正常 大鼠摄食明显减少，活动减少，对声光反应迟钝.使用塞来昔布和四氯化碳80mg·kg-1组两只大鼠在用药10h后死亡.
2.2  生化指标测定结果 对照组ALT，AST和LPS水平较正 常组明显增高，差异具有显著性（P<0.05）.实验组ALT，AST和LPS水平较对照组明显增 高，差异具有显著性（P<0.05），且随着塞来昔布浓度的增高，这些指标也明显增高（ 表1）.
2.3  各组肝损伤病理组织学变化 在对照组和实验组小叶的全部，特别在小叶中央区的肝细胞肿胀变大，细胞胞质疏松成网状，在细胞质内可见空 泡.有的细胞肿大，呈气球样，其胞质颗粒大部分消失，只在细胞核周围存留有较多 颗粒，细胞核仍完整，核仁常明显增大.同时小叶内散在肝细胞嗜酸性变和坏死肝细胞（图1 ，2）.
2.4  NF-кВp65 与 Cox2 表达 正常肝组织 NF-кВ 弱阳性表达，未见COX-2表达.肝损伤后 NF-кВ 在小叶中央静脉周围有脂肪变性细胞强 阳性表达，COX-2在肝损伤后呈阳性表达（图3，4 ，表2）.

表1  各组血清ALT,AST和内毒素含量变化  （x±s）

分组 Dosemg·k-1 n ALT(nkat·L-1)  AST/U(nkat·L-1) Endotoxin(Eu·L-1)  
正常组 - 10  624±5 748±6 60±50
CCL4组 - 10 1392±16a 1803±18a 124±61a
CCL4+塞来昔布组 20 10 1790±13c 2183±21c 143±37c
CCL4+塞来昔布组 40 10 3301±20c 3946±30c 186±77c
CCL4+塞来昔布组 80 8 4500±32c 4903±23c 232±35c

aP<0.05，vs正常组; cP<0.05 vs CCL4组.

表2  NF-кВ P65和COX-2 在各组的表达（x±s，A）

分组 Dose（mg·k-1） n NF-кВ COX-2
正常组 - 10 0.046±0.037 0
CCL4 - 10 0.163±0.031a 0.146±0.024a
CCL4+塞来昔布 20 10 0.175±0.045c 0.148±0.025c
CCL4+塞来昔布 40 10 0.196±0.024c 0.158±0.030c
CCL4+塞来昔布 80 8 0.217±0.029c 0.169±0.017c

aP<0.05 vs正常组; cP<0.05 vs CCL4组

图1 对照组大鼠肝组织病理切片  HE×100

图2 模型组（CCL4+Celecoxib 80mg·kg-1）大鼠肝组织  HE×100

图3 COX-2在模型组（CCL4+Celecoxib 80mg·kg-1）大鼠肝 组织表达 ×400

图4 NF-кВ P65在模型组（CCL4+Celecoxib 80mg·kg-1）大鼠肝组织表达 ×400


3  讨论
环氧合酶是花生四烯酸合成前列腺素的限速酶，COX至少有两种亚型，即COX-1和COX-2，COX-1属结构型酶，在多种组织中微量恒定表达，其催化产物参与维持细胞结构的完整性和机体正常的生理活动；COX-2属诱导型酶，在正常组织中无表达，当细胞受到各种刺激因素包括生长因子、细胞因子、炎性递质、促癌剂、细菌内毒素和激素等作用时，COX-2的表达迅速上调.我们的实验证实大鼠急性肝损害后，血清转氨酶和内毒素水平增高，免疫组织化学检测COX-2表达较正常组大鼠明显增高，即炎症时COX-2的表达增加.同时在肝损害使用COX-2抑制剂后，肝组织COX-2表达较对照组显著增高，且呈剂量依赖关系.COX-2蛋白堆积的机制可能涉及抑制剂引起的酶的稳定性和前列腺素缺乏引起的酶上调有关［46］.
      急性肝损伤后，有许多炎性递质、细胞因子及内毒素等参与了肝损伤过程，他们与COX-2及其代谢产物的相互作用是复杂的.其中NF-кВ是一个多功能的核转录因子，激活后可促进细胞因子、粘附分子、趋化因子等基因转录，在炎症反应中具有重要作用.我们观察到大鼠急性肝损伤后，NF-кВ表达增高，使用COX-2抑制剂后NF-кВ表达较对照组显著增高，随着NF-кВ表达增高COX-2表达存在增高趋势，其机制可能与COX-2的启动子区域含有NF-кВP65结合位点有关［47，48］.
    COX-2的表达可能是机体对抗损伤的一种保护性机制.因为我们在使用COX-2抑制剂塞来昔布后，观察到肝损害较对照组加重，Tsuksda et al［49］研究COX-2在出血性休克（Hemorrhagic shock HS）导致肝、结肠损伤中的作用时发现，♂SD大鼠致失代偿HS后进行复苏.此时检测休克终末期、复苏1h后血清转氨酶、组织学变化和COX-2 mRNA在肝表达.COX-2 mRNA的表达上调，同时使用选择性COX-2抑制剂NS398后，肝损伤加重.在临床使用选择性COX-2抑制剂时也有致肝损伤的报道［50，51］.其机制是否与COX-2抑制剂的代谢后产物的毒性作用还是与其他机制有关待进一步深入研究.

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