修回日期: 2005-09-01
接受日期: 2005-09-06
在线出版日期: 2005-10-28
目的: 探讨潘托拉唑对大鼠胃黏膜损伤保护的作用及其机制.
方法: 在乙醇诱导大鼠胃黏膜损伤前, 预先给予(iv)潘托拉唑(20 mg/kg)、L-硝基-精氨酸甲酯(L-NAME, 4 mg/kg)及L-精氨酸(250 mg/kg). 采用激光多普勒血流计(LDF)测定胃黏膜血流量(GMBF), 采用镉粒还原和比色法测定胃黏膜和血浆NO-2/NO-3含量, 并观察了胃黏膜损伤指数(ulcer index, UI)、溃疡坏死组织和中性粒细胞浸润严重程度的变化.
结果: 与模型损伤组比, 潘托拉唑组大鼠UI明显降低(5.7±2.1 vs 25.4±2.5, P<0.01), 溃疡坏死组织和中性粒细胞浸润程度明显减轻. 预先用L-NAME处理后, 潘托拉唑保护胃黏膜损伤作用明显减弱; L-NAME抑制作用可被L-精氨酸拮抗. iv潘托拉唑, 可增加GMBF、胃黏膜和血浆NO-2/NO-3 , L-NAME可逆转这种作用, 但对潘托拉唑抑制酸分泌作用无明显影响.
结论: 潘托拉唑通过一氧化氮介导对大鼠胃黏膜损伤有重要的保护作用, 而与潘托拉唑抑制酸分泌作用无关.
引文著录: 乐桥良, 张志坚, 林克荣, 王雯. 潘托拉唑对胃黏膜损伤保护作用及其机制. 世界华人消化杂志 2005; 13(20): 2480-2484
Revised: September 1, 2005
Accepted: September 6, 2005
Published online: October 28, 2005
AIM: To explore the protective effect of pantoprazole on the gastric mucosal lesions in rats and its related mechanism.
METHODS: Before the gastric mucosal lesion was in-duced by pure alcohol, the rats were intravenously injected with pantoprazole (20 mg/kg), Nω-nitro-L-arginine methyl ester (L-NAME, 4 mg/kg), and L-arginine (250 mg/kg). The gastric mucosal blood flow (GMBF) was detected by laser Doppler flowmetry (LDF), and the level of NO-2/NO-3 in the gastric mucosa and serum was measured by cadmium granulation reduction and colorimetric method. The ulcer index (UI), the severity of tissue necrosis, and neutrophils infiltration were observed.
RESULTS: Compared with that in the control group, the UI was significantly decreased (5.7±2.1 vs 25.4±2.5, P <0.01), and the degrees of tissue necrosis and neutrophils infiltration were markedly lightened. The protective effect of pantoprazole was significantly decreased by L-NAME. The inhibitory effect of L-NAME was antagonized by prior administration of L-arginine. The GMBF and the content of NO-2/NO-3 in gastric mucosa and serum were increased by the intravenous administration of pantoprazole. This effect was prevented by the pretreatment of L-NAME, but the antisecretion effect of pantoprazole was not affected by L-NAME.
CONCLUSION: Pantoprazole can protect gastric muc-osa against the lesions in rats by the mediation of nitric oxide (NO), and the protective effect has no relation with its antisecretory action of gastric acid.
- Citation: Le QL, Zhang ZJ, Lin KR, Wang W. Protective effect of pantoprazole on gastric mucosal lesions in rats and its mechanism. Shijie Huaren Xiaohua Zazhi 2005; 13(20): 2480-2484
- URL: https://www.wjgnet.com/1009-3079/full/v13/i20/2480.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v13.i20.2480
潘托拉唑(pantoprazole)显著抑制酸分泌作用的机制已基本阐明[1-3]; 但它对胃黏膜具有保护作用[4-13]的机制尚未完全阐明. 一氧化氮(nitric oxide, NO)是扩血管物质, 可增加胃黏膜血流量, 对胃黏膜具有保护作用[14-23]. 有关NO是否介导潘托拉唑对胃黏膜保护作用尚未见报道. 我们研究了潘托拉唑对大鼠胃黏膜保护作用, 并探讨NO是否参与了这一过程.
1.1.1 主要药品和试剂: 潘托拉唑是杨子江药业公司提供的产品, L-硝基-精氨酸甲酯(Nω-nitro-L-arginine methyl ester, L-NAME)为Sigma公司产品, L-精氨酸为上海华美工程公司产品. 镉粒为上海亭新工厂产品, 甘氨酸、亚硝酸盐为上海试剂一厂产品, 硫酸铜、N-1-奈乙胺盐酸盐为上海试剂三厂产品, 对氨基苯磺酸为上海山海科技研究所产品, 硫酸、硫酸锌、氢氧化钠、酚红为上海试剂二厂产品.
1.1.2 实验动物: 纯种、♂、健康SD大鼠(上海西普尔-必凯实验动物有限公司提供), 体质量200-260 g, 实验前禁食24 h, 但不禁水.
1.1.3 主要仪器: XHF-1高速分解器为上海金山生化仪器厂产品, TG328 B型分析天平为上海天平仪器厂产品, 801型离心机为上海手术器械厂产品, 724微机型可见分光光度计为上海光学仪器厂产品, 眼科手术器械一套. 激光多普勒血流计(LDF), LDF-Ⅲ型, 南开大学制造, 技术参数如下: He-Ne光源, 功率2 mW, 输出功率>1 mW, 光纤长2.5 m, 测定范围为1 mm半球区域, 量程选×1档, 时间常数1 s档, 频率为4 kHz.
1.2.1 胃黏膜损伤模型制备: 大鼠禁食24 h后, 30 g/L戊巴比钠(4 mg/kg)ip麻醉, 用1 mL无水乙醇灌胃, 1 h后腹正中切口进入腹腔, 沿胃大弯处剪开胃, 并进行各项实验指标测定.
1.2.2 给药和分组: 取SD大鼠99只, 随机分为9组, 每组11只. Ⅰ组: 假手术组, 作为正常对照; Ⅱ组: 损伤模型组, 作为损伤对照; Ⅲ组: 潘托拉唑组, 在用无水乙醇灌胃30 min前, iv潘托拉唑(20 mg/kg). Ⅳ组: L-NAME组, 在iv潘托拉唑20 min前, iv L-NAME(4 mg/kg). Ⅴ组: L-精氨酸(L-arginine)组, 在iv L-NAME 15 min前, iv L-精氨酸(250 mg/kg). Ⅰ-Ⅳ组又分成2组, 其中1组用于胃酸、胃黏膜血流量、胃黏膜和血浆NO-2/NO-3测定, 另1组用于胃黏膜损伤指数判定和组织学观察. 对照组给予等量的生理盐水.
1.2.3 胃液pH测定: 取胃液2 mL, 加酚红指示剂2滴, 黄色表示有胃酸存在. 用0.1 mol/L氢氧化钠滴定至粉红色. 所耗氢氧化钠mL数×50, 即为胃酸浓度(mmol/L), 并换算成pH.
1.2.4 胃黏膜血流量(GMBF)测定: 采用LDF测定, 开机预热30 min调出LDF-Ⅲ GMBF测定程序, 从胃大弯处作一小切口, 用LDF的探头轻轻垂直接触胃黏膜表面, 待血流稳定后, 在胃体大小弯、胃窦大小弯取4点测定GMBF, 取其平均值, 以激光多普勒信号电压值(mV)表示血流量的相对数值.
1.2.5 胃黏膜和血浆NO-2/NO-3含量测定 取胃, 用钝性刀片刮取胃黏膜, 组织匀浆, 离心后, 取上清液, -20℃贮存待测; 取外周血2 mL, EDTA抗凝, 离心后, 取上清液, -20℃贮存待测; 采用镉粒还原和比色法测定[24].
1.2.6 胃黏膜损伤指数(ulcer index, UI)判定: 按Guth标准计算溃疡指数, 处死动物, 取出胃及十二肠, 沿大弯侧剪开后, 展平, 按溃疡或糜烂面积大小给予计分: 斑点状糜烂为1分, 糜烂<1 mm为2分, 糜烂介于1-2 mm之间为3分, 糜烂为2-4 mm为4分, 糜烂>4 mm为5分.
1.2.7 组织学观察: 将全胃固定于甲醛液, 制成石腊切片, HE染色, 光镜下观察, 评定标准如下: (1)坏死物质: 未见为0, 仅在溃疡底面边缘有少许为(+), 薄层覆盖整个溃疡底为(++), 厚层覆盖为(+++). (2)中性粒细胞浸润: 未见为0, 偶见(+), 明显(+++), 介于后两者之间为(++).
统计学处理 多组计量资料之间比较采用方差分析, 组间两两比较用q检验, 实验数据均以mean±SD表示; 计数资料之间采用χ2检验.
损伤模型组大鼠UI(25.4±2.5)比正常对照组(0)明显升高(P<0.01); 潘托拉唑组与损伤模型组比明显降低(P<0.01); L-NAME组与损伤模型组比无明显差异(P>0.05), 而比潘托拉唑组明显升高(P<0.01); L-精氨酸组与损伤模型组和L-NAME组比均明显降低(P<0.01)(表1).
损伤模型组大鼠溃疡底部可见大量坏死物质, 周围组织中中性粒细胞浸润明显; 潘托拉唑组溃疡底部坏死物质和周围组织中中性粒细胞浸润均比损伤模型组明显减轻(P<0.01); L-NAME组溃疡底部坏死物质和周围组织中中性粒细胞浸润与损伤模型组比均无明显差异(P>0.05), 而比潘托拉唑组严重(P<0.01); L-精氨酸组与损伤模型组和L-NAME组比二者均明显减轻(P<0.01), 与潘托拉唑组比无明显差异(P>0.05)(表1).
损伤模型组大鼠GMBF与正常对照组比明显降低(P<0.01), 潘托拉唑组与损伤模型组比明显升高(P<0.05), L-NAME组与损伤模型组比无明显差异(P>0.05), 而比潘托拉唑组明显降低(P<0.05)(表2).
损伤模型组大鼠胃黏膜和血浆NO-2/NO-3比正常对照组均非常显著增加(P<0.01); 潘托拉唑组比正常对照组非常显著增加(P<0.01), 亦比损伤模型组明显增加(P<0.05); L-NAME组与潘托拉唑组比显著减少(P<0.01), 而与正常对照组比无明显差异(P>0.05)(表2).
损伤模型组大鼠胃液pH与正常对照组比明显降低(P<0.05), 潘托拉唑组与损伤模型组比明显升高(P<0.01), L-NAME组与潘托拉唑组比无明显差异(P>0.05)(表2).
潘托拉唑是人工合成的质子泵抑制剂(proton pump inhibitor, PPI), 通过抑制胃壁细胞分泌小管的H+/K+-ATP酶, 使胃酸分泌的最终步骤阻断, 其抑制作用强而持久, 从而可以治疗消化性溃疡[1-3]. 有研究表明, 潘托拉唑对胃黏膜损伤具有保护作用[4-13]; Blandizzi et al[4]报道潘托拉唑能有效预防消炎痛引起大鼠胃体和胃窦的胃黏膜损伤, 在消炎痛引起的溃疡模型中, 也能明显改善其UI; Shah et al[5]报道潘托拉唑对乙醇引起的胃黏膜损伤有细胞保护作用的同时能加强细胞保护适应, 预先给阿司匹林、氯化氨或应激造成的实验性胃黏膜损伤的大鼠iv潘托拉唑, 能明显减轻胃黏膜损伤[6,7]; 潘托拉唑对非甾体抗炎药(NSAIDs)引起的胃黏膜损伤有很好的保护作用[8,9], Bianchi et al[8]对需要连续长期应用NSAIDs类药治疗的风湿性疾病患者同时给予潘托拉唑口服, 与对照组比, 潘托拉唑能非常有效的预防消化性溃疡发生; 潘托拉唑对危重患者应激性胃黏膜损害及胃黏膜出血有很好的预防作用[10-12]; 对于志愿者口服阿司匹林引起的胃黏膜损伤潘托拉唑也有保护作用[13]. 本实验结果显示, 预先用潘托拉唑给由乙醇诱导胃黏膜损伤大鼠iv, 与损伤模型组比, 胃黏膜损伤指数明显降低, 病理组织观察, 其溃疡坏死物质和周围中性粒细胞浸润亦明显减少, 表明潘托拉唑对大鼠胃黏膜损伤具有保护作用.
一氧化氮(NO)是由血管内皮细胞产生血管舒张因子[14,15], 近年来有报道, NO对胃黏膜损伤有保护作用[14-23]; Konturek et al[16]发现NO介导了vit C保护健康志愿者口服阿斯匹林诱导的胃黏膜损伤; NO抑制剂L-NAME能增加水应激的胃黏膜损伤, 而NO前体L-精氨酸能明显减轻这种胃黏膜损伤[17-19]; NO可减轻由乙醇诱导的实验性胃黏膜损伤的严重程度[20-23], Kalia et al[20]预先用L-NAME(NOS抑制剂)处理大鼠, 发现会加重由乙醇诱导的胃黏膜损伤, 并且这种胃黏膜损伤能被L-精氨酸逆转, 另外, L-NAME能明显降低大鼠GMBF和胃黏膜血红蛋含量[21], 表明NO对胃黏膜损伤有保护作用. 我们用L-NAME预先处理大鼠, 能明显升高潘托拉唑降低的胃黏膜损伤指数, 溃疡底部坏死物质增多, 周围中性粒细胞浸润也非常明显, 在L-NAME给药15 min前给予L-精氨酸则能抑制这种损伤作用, 表明NO介导了潘托拉唑对胃黏膜的保护作用.
本实验结果显示, iv潘托拉唑, 能明显增加胃黏膜和血浆NO-2/NO-3含量, 同时GMBF亦升高, 而在潘托拉唑iv 20 min前给予L-NAME则能逆转这种作用. 本实验结果还显示, 潘托拉唑能著显提高胃液pH, 抑制胃酸分泌, 而L-NAME则对潘托拉唑抑制胃酸分泌的作用无明显影响.
总之, 我们认为, 潘托拉唑对大鼠胃黏膜损伤具有保护作用, NO通过增加GMBF介导了这种保护作用, 而与潘托拉唑抑制胃酸分泌作用无关.
电编: 张敏 编辑: 菅鑫妍 审读: 张海宁
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