基础研究 Open Access
Copyright ©The Author(s) 2005. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2005-07-28; 13(14): 1717-1720
在线出版日期: 2005-07-28. doi: 10.11569/wcjd.v13.i14.1717
消炎痛和阿斯匹林对C57BL/6和Balb/c小鼠胃酸分泌的效应
王昌成
王昌成, 淮安市第二人民医院消化科 江苏省淮安市 223002
王昌成, 男, 1964－03－06生, 江苏省泗阳县人, 汉族, 1991年上海第二医科大学消化内科硕士, 主任医师, 主要从事消化系疾病的基础和临床研究.
通讯作者: 王昌成, 223002, 江苏省淮安市第二人民医院消化科. wangchangcheng8001@163.com
收稿日期: 2005-04-26
修回日期: 2005-05-05
接受日期: 2005-05-15
在线出版日期: 2005-07-28

目的: 观察非甾体类抗炎药消炎痛和阿斯匹林对C57BL/6和Balb/c小鼠胃酸分泌的影响及胃黏膜损害的效应.

方法: 应用幽门结扎术, 测定消炎痛(40 mg/kg)和阿斯匹林 (250 mg/kg) i.p后C57BL/6和Balb/c小鼠总胃酸分泌量(mL), 胃酸度([H+] mmol/L)以及3 h总酸度(mmol/h)并应用Lanza score评分观察药物对胃黏膜的损害程度. 同时应用[14C]-氨基比林蓄积试验观察2种小鼠离体胃对组胺刺激的效应.

结果: 在基础状态下, C57BL/6和Balb/c小鼠的总胃酸分泌量和总酸度显著不同(0.82±0.06 vs 2.32±0.18; P<0.0 001; 2.9±0.4 vs 9.7±1.6, P = 0.0 005), 而胃酸度没有显著差别(P = 0.377). 在C57BL/6小鼠, 腹腔内应用消炎痛后总胃酸分泌量和总酸度与基础水平相比差别显著(2.08±0.16 vs 0.82±0.06, P<0.0 001; 9.4±1.1 vs 2.9±0.4, P<0.0 001), 而胃酸度也没有显著差别(P = 0.11); 腹腔内应用阿斯匹林后总胃酸分泌量, 胃酸度以及总酸度分别为1.5±0.3、16.1±1.4和8.7±2.7, 与基础水平相比均有显著差异(P = 0.03, 0.009, 0.03); 在Balb/c小鼠, 腹腔内应用消炎痛后总胃酸分泌量, 胃酸度以及总酸度分别为3.4±0.3、5.8±1.0和6.5±1.2, 前二者与基础水平相比有显著差异(P = 0.007, 0.01); 腹腔内应用阿斯匹林后总胃酸分泌量, 胃酸度以及总酸度与基础水平相比均没有显著差异(P = 0.15, 0.15, 0.7). 应用消炎痛后, C57BL/6和 Balb/c小鼠Lanza score分别为12.6±0.9 和16.4±1.1 (P = 0.03); 应用阿斯匹林后, C57BL/6和 Balb/c小鼠Lanza score 为10.4±1.0和11.0±1.1 (P = 0.91). 应用[14C]-氨基比林蓄积试验观察2种小鼠离体胃对组胺刺激的效应, 发现C57BL/6小鼠对不同浓度组胺刺激的效应显著低于Balb/c小鼠(10-5 mol/L: 30.8±8.5 vs120.8±23.0, P = 0.006; 10-4 mol/L: 45.1±7.1 vs 236.1±48.7, P = 0.005; 10-3 mol/L: 37.9±5.3 vs199.4±35.2, P = 0.002)

结论: C57BL/6和Balb/c小鼠的胃酸分泌生理功能存在显著差异. 消炎痛和阿斯匹林对C57BL/6和Balb/c小鼠的胃酸分泌功能和胃黏膜损害产生不同的效应.

关键词: 消炎痛; 阿斯匹林; C57BL/6; Balb/c; 胃酸
引文著录: 王昌成. 消炎痛和阿斯匹林对C57BL/6和Balb/c小鼠胃酸分泌的效应. 世界华人消化杂志 2005; 13(14): 1717-1720
Effects of indomethacin and aspirin on gastric acid secretion in C57BL/6 and Balb/c mice
Chang-Cheng Wang
Chang-Cheng Wang, Department of Gastroenterology, Huaian Second People's Hospital, Huaian 223002, Jiangsu Province, China
Correspondence to: Dr. Chang-Cheng Wang, Department of Gastroe-nterology, Huaian Second People's Hospital, Huaian 223002, Jiangsu Province, China. wangchangcheng8001@163.com
Received: April 26, 2005
Revised: May 5, 2005
Accepted: May 15, 2005
Published online: July 28, 2005

AIM: To investigate the effects of indomethacin and aspirin on the gastric acid secretion and gastric lesions in C57BL/6 and Balb/c mice.

METHODS: The total acid volume (mL), acidity ([H+] mmol/L) and total 3-hour acid output (mmol/h) were determined after intraperitoneal administration of indomethacin and aspirin in the two different strains of mice by pylorus ligation technique and the gastric lesions were evaluated by Lanza score. Histamine-stimulated [14C]-aminopyrine accumulation was used to determine the changes of gastric acid secretion in the two strains of mice in vitro.

RESULTS: In baseline, total acid volume and total 3-hour acid output were significantly different between C57BL/6 and Balb/c mice (0.82±0.06 vs 2.32±0.18, P <0.0 001; P = 0.3 772.9±0.4 vs 9.7±1.6, P = 0.0 005), while the acidity was not (P = 0.377). In C57BL/6 mice, after administration of indomethacin, the total acid volume and total 3-hour acid output were 2.08±0.16 and 2.9±0.4 respectively, which were significantly higher than those in baseline (both P<0.0 001), while the acidity wasn't (P = 0.11); After administration of aspirin, the total acid volume, acidity and total 3-hour acid output were 1.5±0.3, 16.1±1.4 and 8.7±2.7 respectively, which were all markedly increased as compared with those in baseline (P = 0.03, 0.009, 0.03). In Balb/c mice, after administration of indomethacin, the total acid volume and acidity were 3.4±0.3 and 5.8±1.0respectively, which were obviously higher than those in baseline (P = 0.007, 0.01), while the total 3-hour acid output wasn't (P= 0.16); After administration of aspirin, the total acid volume, acidity and total 3-hour acid output were all not significantly different from those in baseline (P = 0.15, 0.15, 0.7). After administration of indomethacin, the Lanza scores in C57BL/6 and Balb/c mice were 12.6±0.9 and 16.4±1.1 respectively (P = 0.03), and after administration of aspirin, those were 10.4±1.0 and 11.0±1.1 respectively (P = 0.91). Disintegrations per second (DPS) of gastric glandular cells were significantly lower in C57BL/6 mice than that in Balb/c ones when different concentrations of histamine were used (10-5 mol/L: 30.8±8.5 vs120.8±23.0, P = 0.006; 10-4 mol/L: 45.1±7.1 vs 236.1±48.7, P = 0.005; 10-3 mol/L: 37.9±5.3 vs 199.4±35.2, P = 0.002).

CONCLUSION: The gastric acid secretion is significantly different between C57BL/6 and Balb/c mice. Different effects of indomethacin and aspirin on gastric acid secretion exist between the two strains of mice.

Key Words: Indomethacin; Aspirin; C57BL/6; Balb/c; Gastric acid
0 引言

胃酸分泌是由涉及到内分泌, 神经分泌和旁分泌机制的中枢和外周通路调控的. 非甾体类抗炎药是临床上应用最广泛的药物之一, 可抑制胃黏膜中前列腺素的合成使胃酸分泌增加, 因此常常导致胃黏膜的损害. 我们应用幽门结扎术, 通过腹腔内应用消炎痛和阿斯匹林, 观察他们对C57BL/6和Balb/c小鼠离体胃酸分泌功能的影响. 同时应用[14C]-氨基比林蓄积试验观察小鼠离体胃对组胺刺激后胃酸分泌的生理学效应.

1 材料和方法
1.1 材料

雄性C57BL/6(31-50g, 52wk)和Balb/c(22-31 g, 52 wk)小鼠在加拿大McMaster大学动物实验中心喂养, 温度21-23 ℃, 湿度 40-50%. 所有动物实验前禁食24 h, 自由饮水. 实验中动物的应用经过McMaster大学动物研究伦理委员会批准.

1.2 方法

动物于禁食24 h后, 按照Shay et al[1]方法, 在轻度麻醉下打开腹腔, 找到幽门并在其远端结扎, 迅速关闭腹腔. 然后腹腔内给予消炎痛40 mg/kg或阿斯匹林250 mg/kg, 3 h后结扎食管下端, 收集胃内容物离心后记录胃液量, 同时用0.1 mol/L NaOH 滴定50 mL 样本至pH 7.0(Extech pH, Canada)测定胃酸度([H+] mmol/L)以及3 h总酸量(mmol/h). 打开胃腔, 按照Lanza et al[2]评分观察胃黏膜损害程度: (0没有可见的糜烂; 1＋1个出血或糜烂; 2＋2-10出血或糜烂; 3＋11-25出血或糜烂; 4＋>25出血或糜烂或一个任何大小的侵入性溃疡). 按照Berglindh[3]的方法准备胃腺体, 动物被杀死后, 迅速取出胃并沿胃小弯打开, 放在37 ℃ pH 7.3含纯氧的PBS中, 用钝刀轻轻刮取小鼠胃黏膜, 用含纯氧的PBS洗涤2次(1 400 r/min, 5 min), 将刮取的胃黏膜置于含有2 g/L葡萄糖, 1 g/L小牛血清白蛋白(BSA, Sigma A-7888, USA), 0.25 g/L Ⅱ-s型大豆胰酶抑制剂(Sigma T-9128)和0.23 g/L Ⅳ型胶原酶(Sigma C-5138)的PBS(应用前充入950 mL/L O2和50 mL/L CO2气体至少15 min)溶液中. 然后置小鼠胃黏膜于100 mL含有消化酶的PBS中37 ℃ 30 min(同时用磁棒约100 r/min均匀搅动). 酶消化结束后, 用尼龙网(孔径500 mm, Small Parts Inc, USA)过滤残渣以及未消化的胃黏膜, 然后用不含消化酶的PBS中洗涤3次(2 000 r/min, 5 min), 最后将胃腺体标本置于含有2 g/L葡萄糖, 2 g/L小牛血清白蛋白, 1.2 mmol/L MgSO4和1 mmol/L CaCl2的15 mL 纯氧PBS中准备使用. 应用[14C]-弱碱氨基比林蓄积试验测定胃酸, 将0.5 mL胃腺体加入含有25 mL不同浓度组胺(10-5mol/L, 10-4 mol/L, 10-3 mol/L)和20 mL(相当于0.25 mCi)氨基比林(Amersham Pharmacia Biotech, Canada;specific activity 4255GBq /mol)试管中孵育60 min并予以旋转, 应用25 mL蒸馏水为空白对照. 孵育结束后, 用含有2 g/L葡萄糖, 2 g/L小牛血清白蛋白, 1.2 mmol/L MgSO4和1 mmol/L CaCl2的PBS洗涤3次(2 600 r/min, 5 min), 然后样本被转至闪烁计数管中同时加入1 mL组织溶解剂(NCS-2, Amersham)过夜. 第2 d样本中加入10 mL冰醋酸和4 mL闪烁液(ACS, Amersham)后置于Bechman LS 5801(USA)闪烁计数器中测定胃酸含量, 结果用胃腺体细胞每秒钟衰变数(DPS)表示.

统计学处理 按每100 g体重计算, 用均数±标准误表示, 各组之间总胃酸分泌量(mL), 胃酸度([H+] mmol/L), 测3 h酸度(mmol/h)以及DPS用GraphPad InStat 3统计软件进行非配对t检验(双尾法);Lanza score评分用Mann-Whitney U检验. P<0.05认为具有统计学差异.

2 结果
2.1 胃酸分泌

在基础状态下, C57BL/6(n = 10)和Balb/c(n = 9)小鼠的总胃酸分泌量(mL), 胃酸度([H+]mmol/L)和酸度(mmol/h)分别为0.82±0.06 vs 2.32±0.18(t = 8.174, P<0.0001), 10.4±1.3 vs 12.3±1.7(t = 0.907, P = 0.377)和2.9±0.4 vs 9.7±1.6(t = 4.275, P = 0.0 005).

在C57BL/6小鼠, 腹腔内应用(n = 7)消炎痛后总胃酸分泌量(mL), 胃酸度([H+]mmol/L)和酸度(mmol/h)分别为2.08±0.16 vs 0.82±0.06(t = 8.281, P<0.0 001), 13.4±1.0 vs 10.4±1.3(t = 1.689, P = 0.11)和 9.4±1.1vs 2.9±0.4(t = 6.222, P<0.0 001); 腹腔内应用(n = 8)阿斯匹林后总胃酸分泌量(mL), 胃酸度([H+]mmol/L)以及酸度(mmol/h)分别为1.52±0.32 vs 0.82±0.06(t = 2.348, P = 0.03), 16.1±1.4 vs 10.4±1.3(t = 2.993, P = 0.009)和8.7±2.7 vs 2.9±0.4(t = 2.366, P = 0.03). 在Balb/c小鼠, 腹腔内应用(n = 7)消炎痛后总胃酸分泌量(mL), 胃酸度([H+]mmol/L)和酸度(mmol/h)分别为3.4±0.31 vs 2.32±0.18(t = 3.173, P = 0.007), 5.8±1.0 vs 12.3±1.7(t = 3.005, P = 0.01)和6.5±1.2 vs 9.7±1.6(t = 1.493, P = 0.16); 腹腔内应用(n = 7)阿斯匹林后总胃酸分泌量(mL), 胃酸度([H+]mmol/L)和酸度(mmol/h)分别为3.1±0.5 vs 2.32±0.18(t = 1.514, P = 0.15), 9.1±0.8 vs 12.3±1.7(t = 1.506, P = 0.15)和8.9±1.1 vs 9.7±1.6(t = 0.389, P = 0.7).

2.2 小鼠胃Lanza score评分

应用消炎痛后, C57BL/6(n = 7)和 Balb/c(n = 7)小鼠Lanza score分别为12.6±0.9 vs16.4±1.1(P = 0.03); 应用阿斯匹林后, C57BL/6(n = 8)和Balb/c(n = 7)小鼠Lanza score分别为10.4±1.0 vs11.0±1.1(P = 0.91).

2.3 小鼠对组胺刺激的效应

C57BL/6(n = 15)小鼠对组胺刺激的效应显著低于Balb/c小鼠(n = 14)[组胺浓度10-5 mol/L, 10-4 mol/L和10-3 mol/L时胃腺体细胞每秒钟衰变数(DPS)分别为30.8±8.5 vs 120.8±23.0(t = 3.674, P = 0.006), 45.1±7.1 vs 236.1±48.7(t = 3.88, P = 0.005)和37.9±5.3 vs 199.4±35.2(t = 4.542, P = 0.002); 空白对照组C57BL/6和Balb/c的DPM分别为17.1±6.2和20.3±4.1(t = 0.423, P = 0.68).

3 讨论

酸分泌在许多食管, 胃和十二指肠疾病的发病机制中起至关重要的作用. 在动物模型和人类如非甾体类抗炎药诱发的糜烂和溃疡中, 胃黏膜的分泌状态可能与他们不同的形态学变化密切相关. 非甾体类抗炎药如消炎痛和阿斯匹林等常可引起胃黏膜的损害, 他是导致消化道溃疡和出血的主要原因之一. 氨基比林蓄积试验是一种研究人类酸分泌[4]和动物壁细胞或胃腺体[3]的可靠方法. 应用H.felis感染的Balb/c小鼠发现局部酸产生对胃内Helicobacter种类的分布至关重要[5-6]. 在这些小鼠, H.felis主要局限于幽门和贲门, 仅在伴随药物性酸抑制的时候才侵犯胃体部. 与此相反, H.felis感染的C57BL/6小鼠则趋向于定植在分泌胃酸的胃体黏膜, 导致壁细胞数量的减少和随之发生的胃萎缩[7-9]. 这种小鼠对H. pylori感染反应的种系特异性差异主要由于参与主导免疫反应类型的不同, Balb/c小鼠主要与TH2有关, 而C57BL/6主要与TH1有关[10-11].Kuroda et al[12]研究表明, 用LPS刺激后, Balb/c小鼠腹膜和脾脏中巨噬细胞产生的PEG2显著高于C57BL/6小鼠, 同时Balb/c和C57BL/6小鼠巨噬细胞中COX-2蛋白的表达明显不同. 他们认为, Balb/c小鼠巨噬细胞产生大量PGE2是由微粒体PEG合成酶和磷脂酶A2而不是由COX-2调控的, 与Th1细胞受抑制有关. 本研究表明, 在基础状态下, Balb/c小鼠的胃酸量和3 h总酸量显著高于C57BL/6小鼠, 胃酸度稍高于C57BL/6小鼠; 同时应用[14C]-氨基比林蓄积试验观察两种离体小鼠胃对组胺刺激的效应, 发现C57BL/6小鼠对组胺刺激的效应显著低于Balb/c小鼠, 这些结果表明在基础状态下两种小鼠的胃酸分泌功能存在显著差异. 在C57BL/6小鼠, 应用消炎痛和阿斯匹林后胃酸产生显著增多; 在Balb/c小鼠, 应用消炎痛后胃酸产生显著增加, 但应用阿斯匹林后除了胃酸容量稍有增加外, 胃酸度和3 h总酸度却有减少, 提示两种不同基因特性的小鼠对非甾体抗炎药呈现不同的胃酸分泌效应. Kuroda et al[12]研究发现, Balb/c小鼠巨噬细胞产生的PEG2显著高于C57BL/6小鼠, 而PEG2可抑制胃酸的分泌. Takeuchi et al[13]应用消炎痛等药物对SD大鼠进行研究, 发现口服30 mg/kg消炎痛在8 h内可引起明显的胃黏膜损害, 而同样剂量的选择性COX-1抑制剂(SC-560)和COX-2抑制剂(rofecoxib)却不引起明显的胃黏膜损害; 在给予消炎痛和SC-560后2 h内胃黏膜PEG2含量较正常下降20%, 而rofecoxib却未产生任何影响; 在幽门结扎术前1 h给予口服消炎痛30 mg/kg没有发现对大鼠的胃酸分泌产生效应[对照组和消炎痛组的容量(mL)和酸度(mmol/h)分别为7.1±0.8 vs 6.7±1.1和178.8±12.4 vs 162.4±8.4]. 同时我们应用Lanza score法对消炎痛和阿斯匹林引起的胃黏膜损害进行评价, 发现应用消炎痛后, C57BL/6小鼠的Lanza score显著低于Balb/c小鼠(P = 0.03); 但应用阿斯匹林后, C57BL/6和Balb/c小鼠的Lanza score却没有显著统计学差异. 我们认为这主要与基础状态下Balb/c小鼠胃酸分泌显著高于C57BL/6小鼠有关. 消炎痛和阿斯匹林同属于非选择性非甾体类抗炎药物, 其COX-1/COX-2比率有所不同, 这可能是导致他们对不同种系特异性小鼠产生不同效应的原因之一, 其确切机制有待于进一步的研究.

我们的研究结果表明, C57BL/6和Balb/c小鼠胃酸分泌功能存在显著差异. 消炎痛和阿斯匹林对C57BL/6和Balb/c小鼠的胃酸分泌功能和胃黏膜损害产生不同的影响效应.

致谢

感谢加拿大McMaster大学Ireneusz T Padol和Richard H.Hunt教授.

编辑:潘伯荣 审读:张海宁

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