内皮素-1特异性抗体对应激性胃黏膜损伤的保护作用

摘要

目的

探讨内皮素-1抗体(ET-1Ab)对应激性胃黏膜损伤(SU)的保护作用．

方法

建立SD大鼠冷束缚应激性(CRS)胃溃疡模型, 观察静脉应用ET-1 Ab对冷束缚应激大鼠血浆、胃黏膜组织ET-1含量、胃黏膜血流量(GMBF)及胃黏膜损伤指数(UI)等变化的影响．

结果

(1)与正常对照组相比, 各应激组血浆胃黏膜ET-1水平和UI明显升高(116.2±4.7 mv and 125.1±4.2 mv vs 49.1±9.7 mv, 113.8±9.3 mv and 122.9±19.6 mv vs 52.3±10.3 mv, 28.6±1.85 mv and 51.2±5.93 mv vs 0, P<0.01), GMBF明显下降(227.8±13.5 mv and 150.8±11.5 mv vs 405.8±23.3 mv, P<0.01); ET-1水平与UI呈显著正相关(r = 0.96, P<0.01), 与GMBF呈显著负相关(r = -0. 91, P<0.01)．(2)与单纯应激组相比, ET-1Ab应激组ET-1水平和UI明显下降(69.2±7.3 mv vs 116.2±24.7 mv, 80.6±12.3 mv vs 125.1±24.2 mv, 58.5±6.3 mv vs 113.8±29.3 mv, 68.9±9.6 mvvs 122.9±19.6 mv, 13.2±2.05 mv vs 28.6±1.85 mv, 25.8±3.62 mv vs 51.2±5.93 mv, P<0.01), GMBF明显回升(329.8±16.3 mv vs 227.8±13.5 mv, 251.9±11.3 mv vs 150.8±1.5 mv, P<0.01), 且ET-1Ab可呈剂量依赖性地显著降低ET-1水平、增加GMBF和降低UI．

结论

在冷束缚应激诱发大鼠SU的过程中, 血浆胃黏膜组织ET-1水平显著升高, 并可能通过其缩血管效应, 引起GMBF显著下降, 从而导致急性胃黏膜损伤. ET-1Ab可呈剂量依赖性地显著减轻应激性胃黏膜损伤程度, 对SU具有一定的防治作用．

关键词: N/A

Protective effects of endothelin-1 antibody on stress induced lesion of gastric mucosa in rats

Yi-Min Duan, Zhao-Shen Li, Xian-Bao Zhan, Yan-Fang Gong, Guo-Ming Xu

Yi-Min Duan, Zhao-Shen Li, Xian-Bao Zhan, Yan-Fang Gong, Guo-Ming Xu, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China

Supported by: the Tenth-Five-Year Plan's Military Medical Scientific Foundation of PLA, No. 01Z059.

Correspondence to: Dr.Zhao-Shen Li, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, 174 Changhai Road, Shanghai 200433, China. yixb@sh163.net

Received: November 5, 2002
Revised: November 20, 2002
Accepted: November 25, 2002
Published online: July 15, 2003

Abstract

AIM

To explore the protective effects of endothelin-1 antibody (ET-1Ab) on acute gastric mucosa lesions induced by stress in rats.

METHODS

Stress ulcer induced by cold-restraint-stress(CRS) was used as model in this study, different dose ET-1Ab were administered by left femoral vein prior to stress so as to observe the effects of the drug on ET-1 levels in plasma and gastric mucosa, gastric mucosa blood flow(GMBF)and ulcer index(UI) of CRS rats.

RESULTS

(1) Compared with the normal control group, ET-1 levels of plasma and gastric mucosa and UI were increased significantly in the stress groups (116.2±4.7 mv and 125.1±4.2 mv vs 49.1±9.7 mv, 113.8±9.3 mv and 122.9±19.6 mv vs 52.3±10.3 mv, 28.6±1.85 mv and 51.2±5.93 mv vs 0, P<0.01), while GMBF were decreased markedly in stress groups (227.8±13.5 mv and 150.8±11.5 mv vs 405.8±23.3 mv, P<0.01).There was significantly positive correlation between ET-1 and UI (r = 0.96, P<0.01), and significantly negative correlation between ET-1 and GMBF(r = -0.91, P<0.01). (2) However, compared with the single-stress groups, ET-1 levels of plasma and gastric mucosa and UI were decreased significantly in ET-1Ab-stress groups (69.2±7.3 mv vs 116.2±24.7 mv, 80.6±12.3 mv vs 125.1±24.2 mv, 58.5±6.3 mv vs 113.8±29.3 mv, 68.9±9.6 mvvs 122.9±19.6 mv, 13.2±2.05 mv vs 28.6±1.85 mv, 25.8±3.62 mv vs 51.2±5.93 mv, P<0.01), GMBF were increased dramatically (329.8±16.3 mv vs 227.8±13.5 mv, 251.9±11.3 mv vs 150.8±1.5 mv, P<0.01), ET-1Ab can dose-dependently reduce levels of the ET-1 and UI, and improve GMBF significantly in cold-restraint-stress.

CONCLUSION

In the course of CRS-induced ulcers, endogenous ET-1 levels of plasma and gastric mucosa were increased significantly, and there were close relationship between ET-1 levels and UI. The increased ET-1 may involve in the pathogenesis of CRS-induced ulcers in rats by its vasoconstriction and marked reduction of GMBF. ET-1Ab can dose-dependently attenuate the degree of gastric mucosa lesions induced by CRS significantly. So it may have therapeutic effect for SU.

Key Words: N/A

0 引言

应激性溃疡(stress ulcer, SU), 又称急性胃黏膜损伤, 是临床危重疾病的常见而严重的并发症, 具有较高的发病率和死亡率, 其发生机制尚不清楚, 防治效果也不佳.近年国外有研究报道内源性强缩血管生物活性多肽内皮素(endothelin ET)-1[1]与急性胃黏膜损伤关系密切[2-6].由于ET-1是通过与其特异性受体结合后发挥生物学效应的[1], 因此, 如用ET-1生物效应拮抗剂减少或抑制ET-1与ETR的结合, 就有可能削弱ET-1生物学效应, 从而减轻与ET-1升高相关的急性胃黏膜损伤程度, 目前国外这方面的研究还较少, 而国内尚无这方面的研究, 为此, 本研究应用内皮素-1抗体(endothelin-1 antibody ET-1Ab)来观察其对应激状态下内源性ET-1、GMBF及UI的影响, 从而为临床防治SU探索新的方法．

1 材料和方法

1.1 材料

ET-1放免试剂盒及ET-1Ab (北京东亚免疫技术研究所). LDF-Ⅲ型微机化激光多普勒血流仪(天津南开大学电子仪器厂). SD♂大鼠(上海西普尔－必凯实验动物有限公司)．

1.2 方法

1.2.1 动物模型与分组 采用SD大鼠冷束缚应激性(CRS)胃溃疡为模型, 将SD♂健康大鼠禁食24 h, 可自由饮水, 活动不受限, 实验前1 h禁水, 称重, 乙醚轻度麻醉, 束缚四肢于自制木板上后, 待其清醒后, 置于4±1 ℃冷室内, 经过3 h后胃部即可出现点线状出血或直径1-2 mm表浅性圆形溃疡．取SD♂健康大鼠36只, 体重200±20 g, 随机分为单纯应激组(12只)、1 mL ET-1Ab应激组(12只)、2 mL ET-1Ab应激组(12只), 每组又分为应激1 h、3 h两个时相, 每时相6只大鼠. 每次实验前预先用含1∶12 500 IU肝素钠带4号半针头的输液管行左股静脉保留插管, 于冷束缚应激开始后自左股静脉以0.5 mL.kg^-1.min^-1的速度恒速补充生理盐水10 min, ET-1Ab应激组在补充的盐水中按1 mL.kg^-1或2 mL.kg^-1加入滴度为1∶1000的ET-1Ab, 而单纯应激组只补充生理盐水, 另取6只同样大鼠进行同样操作但不应激、不补液, 作为正常对照组. 分别于应激1 h、3 h两时相点检测GMBF、UI及ET-1等指标．

1.2.2 ET-1含量放免测定[7] (a)取颈静脉血2 mL注入含10% EDTA-Na2及抑肽酶的试管中混匀, 4 ℃, 3000 rpm×10 min, 取上清; (b)取新鲜受损胃黏膜组织, 真空冷冻干燥、称重匀浆, 4 ℃, 3000 rpm×15 min, 取上清. 按试剂盒步骤测ET-1含量．

1.2.3 GMBF的测定[8] SD大鼠用20%乌拉坦(5 mL.kg^-1)腹腔内注射麻醉, 用恒温水浴装置使胃在整个测量过程中浸浴在37 ℃生理平衡液内, 于剑突下正中切开腹前壁, 轻轻分离暴露胃体并固定腺胃, 于腺胃前壁近大弯侧少血管区作一长度约0.5 cm的横行切口, 于切开胃后5-20 min内经此切口插入血流计探头, 轻轻接触胃黏膜, 并与胃黏膜表面保持垂直, 待血流稳定后, 在腺胃窦大小弯、胃体大小弯取四点测定GMBF, 取其平均值, 结果以激光普勒信号电压值(mV)表示血流量的相对数值．

1.2.4 按Guth标准评定 UI斑点糜烂为1分; 糜烂长度<1 mm为2分; 1-2 mm为3分; 2-3 mm为4分; >3 mm为5分．

统计学处理 所有实验数据均采用mean±SD表示, 统计学处理采用方差分析, 两样本间均数比较用t检验．

2 结果

2.1 ET-1Ab对SU时血浆、胃黏膜ET-1的影响

与单纯应激组相比, 各ET-1Ab应激组血浆、胃黏膜组织ET-1水平随着ET-1Ab使用剂量的增加呈剂量依赖性地显著下降(P<0.01). 1 mLET-1Ab+应激1 h组血浆、胃黏膜ET-1水平分别下降了40.5%和48.6%, 2mLET-1Ab+应激1 h组分别下降了53.7%和56.2%; 1mLET-1Ab+应激3 h组血浆胃黏膜ET-1水平分别下降了27.5%和43.9%, 2mLET-1Ab+应激3h组分别下降了49.3%和55.2%．ET-1水平与UI呈显著正相关(r = 0.96, P<0.01, 见表1).

表1 ET-1Ab对CRS大鼠血浆、胃黏膜ET-1、GMBF及UI的影响.

组别	血浆ET-1(mv)	胃黏膜组ET-1(ng/g.tissue)	GMBF(mV)	UI
正常对照组	49.08±9.73	52.37±10.35	405.8±23.36	0
单纯应激1 h组	116.2±24.69a	113.8±29.28a	227.8±13.53a	28.6±1.85a
1 ml ET-1Ab+应激1 h组	69.18±7.32b	58.52±6.38b	329.8±16.32b	1 3.2±2.05b
2 ml ET-1Ab+应激1 h组	52.56±13.75b	49.81±6.18b	383.2±13.98b	5.20±1.48b
单纯应激3 h组	125.1±24.18a	122.9±19.64a	150.8±11.53a	51.2±5.93a
1 ml ET-1Ab+应激3 h组	80.65±12.26b	68.91±9.55b	251.9±11.38b	25.8±3.62b
2 ml ET-1Ab+应激3 h组	63.53±8.54b	55.09±10.92b	339.2±15.31b	10.6±2.07b

^aP<0.01 vs 正常组,

^bP<0.01 vs 单纯应激组.

2.2 ET-1Ab对SU时胃黏膜GMBF的影响

与单纯应激组相比, 各ET-1Ab应激组GMBF明显回升(P<0.01), 而且随着ET-1Ab使用剂量的增加, GMBF的回升幅度也相应增大. 1 mLET-1Ab+应激1 h组GMBF回升了25.2%, 达到正常对照的81.3%, 2 mL ET-1Ab +应激1 h组GMBF回升了38.3%, 达到正常对照的93.5%; 1 mLET-1Ab+应激3 h组GMBF回升了24.9%, 达到正常对照的62.1%, 2mLET-1Ab+应激3 h组GMBF回升了46.2%, 达到正常对照的83.6%. ET-1水平与GMBF呈显著负相关(r = -0. 91, P<0.01, 见表1).

2.3 ET-1Ab对SU时胃黏膜UI的影响

与单纯应激组相比, ET-1Ab应激组UI随着ET-1Ab使用剂量的增加呈剂量依赖性地显著减小(P<0.01). 1 mLET-1Ab +应激1 h组UI下降了53.8%, 2 mL ET-1Ab +应激1 h组UI下降了81.8%; 1 mL ET-1Ab +应激3 h组UI下降了49.6%, 2 mLET-1Ab+应激3 h组UI下降了79.3%. UI与ET-1水平呈显著正相关(r = 0.96, P<0.01, 见表1)．

3 讨论

内皮素(ET)-1是近年发现的一种具有强大缩血管作用的内源性生物活性多肽, 全身多种组织器官都有ET-1的合成分泌及其受体的分布[1]. 生理条件下, 他在调节血管张力和局部组织微循环灌流中起着非常重要的作用[9-12]. 而在许多病理情况下, 多种病理应激因素可刺激机体大量合成释放ET-1, 参与机体的多种病理生理过程[13-20]．

近年有研究提示ET-1与应激性溃疡关系密切, 可能在应激性胃黏膜损伤中起着重要的致病作用[21-26]. ET-1抗血清是用特异的大鼠ET-1抗原静脉注入兔体内致敏, 获取的特异性兔抗鼠ET-1免疫血清, 他可与体内ET-1结合形成抗原-抗体复合物, 显著降低血浆与组织ET-1水平, 减少ET-1与其特异性受体ETR结合的数量, 起到拮抗ET-1病理效应的作用. 因此, 我们推测若能应用特异性ET-1抗血清预处理冷束缚应激大鼠中和过多的ET-1以拮抗其生物学效应, 就有可能减轻与ET-1相关的应激胃黏膜组织损伤．

本研究观察到在大鼠冷束缚应激开始后各时相, 单纯应激组大鼠血浆、胃黏膜组织ET-1水平均较正常对照组明显升高且随着应激时间的延长而升高. 与此同时伴有GMBF的不断降低和UI不断增加. 而预用ET-1特异性抗血清处理的应激组大鼠与单纯应激组相比较, 其血浆和胃黏膜组织ET-1水平则显著降低, 且伴随着GMBF显著回升与胃黏膜损伤指数UI明显下降. 相关性分析显示ET-1水平与UI呈显著正相关, 与GMBF呈显著负相关, 这些不但表明ET-1抗血清可显著降低ET-1水平, 改善胃黏膜组织血液灌流(GMBF)与减轻胃黏膜组织损伤程度, 而且提示显著降低GMBF可能是ET-1诱发应激性胃黏膜损伤的重要机制之一.

国外一些学者在研究乙醇及缺血-再灌注等因素诱发胃黏膜损伤时, 发现ET-1特异性抗血清也有类似的作用. Kitajima et al[27]报道缺血-再灌注诱发胃黏膜损伤时, 血浆胃黏膜组织ET-1浓度明显升高．而预用ET-1单克隆抗体对抗中和ET-1, 则可预防其血液动力学紊乱及显著减轻胃黏膜组织损伤程度与损伤总面积. Szabo et al[28]研究报道向大鼠胃灌注乙醇后, 在发生胃黏膜出血性损伤之前, 会快速引起ET-1随时间依赖性向体循环释放及GMBF的不断下降, 随着所用预处理的ET-1 抗体剂量的增大呈剂量依赖性地减轻黏膜损伤与GMBF下降的程度, 表明ET-1可能参与了胃黏膜组织损伤的病理生理过程以及GMBF下降可能是其重要的作用机制之一, 而特异性ET-1抗体对其血液动力学效应与损伤效应有明显的拮抗作用．

另有研究表明ET-1还可通过收缩血管导致引起组织缺血、缺氧, 激发PKC, 抑制K+-ATP通道, 促进体内脂质过氧化物反应, 引起氧自由基及MDA大量产生, 二者对组织细胞有较强的攻击损伤作用, 给予ET-1Ab特异性抗血清可明显减少氧自由基生成和显著降低组织MDA的含量, 减轻损伤[29-32]．

总之, 本研究提示内源性ET-1参与了应激性溃疡的病理生理过程, 可能是胃黏膜组织损伤的重要致病因子, 而特异性ET-1抗体可有效地减轻胃黏膜组织损伤, 起到保护胃黏膜组织的作用. 当然, 目前制备的ET-1抗体还是多克隆抗体, 其作用时间较短、效果也不甚满意, 相信随着研究的深入和技术的提高, 有望研究出效果满意、高度特异的ET-1单克隆抗体, 为防治SU提供新的方法．

备注

$[Footnotes]

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