蛋白酶抑制剂ONO-3403对胰泌素刺激的大鼠胰腺外分泌的影响
陈少夫,中国医科大学第二临床学院消化内科 辽宁省沈阳市110004
山本光勝,大槻眞.日本产业医科大学第三内科 日本北九州市807
陈少夫，男，1962-11-2生，江苏省沭阳县人，汉族，1985年毕业于中国医科大学医学系，医学博士，现为消化内科副教授，主要从事胃肠激素与胃肠疾病的研究，发表文章26篇.
项目负责人 陈少夫,110004,中国医科大学第二临床学院消化内科,沈阳市和平区三好街36号. csf196211@yahoo.com.cn
收稿日期 2002-04-05 接受日期 2002-05-12
The effects of protease inhibitor ONO-3403 on pancreatic exocrine response to secretin in fed rats
Shao-Fu Chen, Mitsuyoshi Yamamoto, Otsuki Makoto
Shao-Fu Chen, Mitsuyoshi Yamamoto,Otsuki Makoto,Department of Gastroenterology,Second clinical college of China Medical University,Shenyang 110004,China;and Third Department of Internal Medicine,School of Medicine,University of Occupational and Environmental Health,Japan,Kitakyushu 807,Japan
Correspondence to: Dr.Shao-Fu Chen,Department of Gastroenterology,Second clinical college of China Medical University,Shenyang 110004, Liaoning Province,China. csf 196211@yahoo.com.cn
Received 2002-04-05 Accepted 2002-05-12
AbstractsAIM: To examine the pancreatic exocrine response to secretin and to clarify the mechanism of the pancreatic exocrine hypersecretion after oral administration of synthetic protease inhibitor ONO-3403 in rats.
METHODS: A single oral dose of synthetic protease inhibitor ONO-3403 was given to rats by orogastric tube 12h before experiment. The pancreatic juice was collected before and after stimulation of stepwise increasing doses of secretin. The output of protein, amylase and bicarbonate in pancreatic juice or pancreatic tissue were determined by Lowry method, chromogenic method with blue-dyed starch polymer, and the DST 800 multititration system, respectively.
RESULTS: Oral administration of ONO-3403 caused a significant increase in pancreatic juice flow (peak level 226±10 ul per 30min vs 44±5ul per 30 min, P<0.01), protein (peak level 985±215ug per 30min vs 254±47ug per 30min, P<0.01) and bicarbonate output (peak level 18±0.7umol per 30min vs 5.9±0.8umol per 30min,P<0.01) before and after secretin stimulation at 12h with pretreatment of ONO-3403. The pancreatic weight, pancreatic contents of protein in ONO-3403 pretreated rats were similar to those in control rats, and pancreatic content of amylasein in ONO-3403 pretreated rats was significantly lower than that in control rats (3114±372 U 103/Pancreas vs 5746±261U 103,P<0.05).
CONCLUSIONS: ONO-3403 can increase pancreatic exocrine secretion and sensitivity to secretin stimulation. The mechanism of ONO3403 induced pancreatic exocrine hypersecretion may be feedback regulation of the pancreas by increasing CCK secretion.
Chen SF, Yamamoto M, Makoto O. Effects of protease inhibitor ONO-3403 on pancreatic exocrine response to secretin in rats. Shijie Huaren xiaohua Zazhi 2002;10(7):792-795
摘要
目的:观察新型蛋白酶抑制剂ONO-3403对进食大鼠基础的及胰泌素刺激的胰腺外分泌的影响，探讨ONO-3403增加胰腺外分泌的机制.
方法:对进食的Wistar大鼠，分别于实验前12h经胃管给与ONO-3403(20mg/kg)后收集基础的和静脉注射胰泌素后的胰液，并对胰腺组织匀浆处理.用Lowry法，产色素法分别测定胰液及胰腺组织中蛋白含量，淀粉酶含量.用DST800多项滴定系统测定胰液HCO-3含量.
结果:给与ONO-3403的大鼠胰液容积，无论基础的及胰泌素刺激的，均较对照组明显增加，并在胰泌素每小时为125ng/kg时胰液容积达高峰(226±10ul per30min vs 44±5ulper 30min,P<0.01),提示ONO-3403能增加进食大鼠的胰液外分泌.给与ONO-3403的大鼠胰液蛋白含量与对照组相比，无论基础的和胰泌素刺激的均明显增加，并在胰泌素每小时为62.5ng/kg时胰液蛋白含量达高峰(985±215ug per 30min vs 254±47ug per30min, P<0.01)，提示ONO-3403能增加进食大鼠的胰液外分泌中的蛋白含量.给与ONO-3403的大鼠胰液中HCO3-含量与对照组相比,无论基础的和胰泌素刺激的均明显增加，并在胰泌素每小时为250ng/kg时HCO3-含量达高峰(18±0.7umol per 30min vs 5.9±0.8umolper 30min, P<0.01),提示ONO-3403能增加进食大鼠的胰液中HCO3-含量.给与ONO-3403的大鼠胰腺重量和胰腺蛋白含量与对照组相比，差异不显著(P>0.05).而胰腺淀粉酶含量与对照组相比明显降低(3114±372U×103 vs 5746±261U×103,P<0.05).
结论:ONO-3403能增加进食大鼠的胰腺外分泌及对胰泌素刺激的敏感性，其机制可能是通过CCK调节的胰腺反馈而起作用的.
陈少夫,山本光勝,大槻眞. 蛋白酶抑制剂ONO-3403对胰泌素刺激的大鼠胰腺外分泌的影响.世界华人消化杂志 2002;10(7):792-795
0 引言
胰腺外分泌的反馈调节是通过十二指肠内胰蛋白酶和糜蛋白酶而起作用的[1-5].向十二指肠内输入人工合成的蛋白酶抑制剂camostat能引起肠腔内蛋白酶的抑制，而使血浆胆囊收缩素(CCK)浓度升高，并能刺激胰腺的外分泌，因此推测CCK在胰蛋白酶对胰腺外分泌的反馈调节中起主导作用[6-8].我们选择一种新型的蛋白酶抑制剂ONO-3403，他在抑制蛋白酶活性方面比camostat强5-10倍，作用时间更长.现观察大鼠口服ONO-3403后，是否能促进胰腺外分泌及增加对胰泌素刺激的敏感性，以寻找ONO-3403对胰腺外分泌的作用机制及与胰腺外分泌相应的反馈机制.蛋白酶抑制剂在急,慢性胰腺炎的治疗中起重要的作用[9-16]，探讨新型蛋白酶抑制剂对胰腺的作用机制有较重要的意义[17].
1 材料和方法1.1 材料 ♂wistar大鼠，体重250-300g,大鼠饲养在23℃，12h明暗交替的环境中，可以自由的饮水和获得标准的饲料饮食.(饲料由日本东京东方发酵公司生产).ONO-3403(ethylN-allyl-N-|(E)-2-methyl-3-[4-amino-phenoxy(arbonyl)phenyl]propenoyl| amino acetate methansulfonate)是一种人工合成的蛋白酶抑制剂，由日本小野制药公司提供,实验前用蒸馏水溶解并调整pH至6.4备用.胰泌素(secretin)购自日本大阪肽蛋白研究所.
1.2 方法[18-20] 将大鼠20只随机分成2组，每组10只，即对照组可自由的饮食水，不给予ONO-3403;实验组于实验开始前12h，经胃管将ONO-3403 (20mg/kg)灌入大鼠胃内，亦可自由的饮食水.对两组大鼠在实验开始先用Urethare(1.6g/kg)腹腔注射麻醉后，剥离出颈内静脉，用聚乙烯导管插入颈内静脉，以备注射胰泌素.行腹正中切口，结扎幽门，行胆管和胰管插管，胆管在肝门的下方和胰腺的近端结扎，将插入胆管的聚乙烯导管引入十二指肠，保持在整个实验过程中胆汁流入十二指肠，在总胆胰管的远端近十二指肠入口处插入胰管插管，以获得纯净的胰液，每30min收集1次胰液.在1h的平衡期后，先收集30min的基础胰液，然后进行胰泌素的剂量反应的研究，每30min阶梯式增加胰泌素的剂量，每小时分别为31.25,62.5,125,250,500,1000,2000ng/kg-，以每小时2ml的速度经颈内静脉输入体内，收集每个剂量的胰泌素刺激下的胰液，以备测定胰液的容积，蛋白含量，HCO3-的含量.上述实验结束后，通过放血法处死大鼠，迅速切除胰腺，清除其中的淋巴结，脂肪组织后，称其量，切除约300g的胰腺组织，放入-70℃冰箱中保存，以备测定胰腺组织中蛋白和淀粉酶的含量.测定前，切取胰腺组织的一部分，用组织匀浆器，2000r/min,在4℃0.15mol/L的氯化钠溶液中进行胰腺组织匀浆.胰液及胰腺组织中蛋白含量用Lowry法测定，使用小牛血清蛋白为标准品.淀粉酶含量用产色素法测定，以Somogyi单位表示.HCO3-含量用DST800多项滴定系统(radiometer,copenhagen,denmark),在采集胰液后迅速进行检测[21-23].
统计学处理 数据均用x±s表示，用两个样本均数差异的t检验法，进行显著性检验.
2 结果2.1 胰液容积，12h前给与ONO-3403的大鼠胰液容积与对照组相比,无论基础的和胰泌素刺激的胰液容积均明显增加,(P<0.01表1)，并在胰泌素每小时125ng/kg时胰液容积30min达高峰为226±10ul，提示ONO-3403能增加进食大鼠的胰液外分泌.
2.2 胰液蛋白含量, 12h前给与ONO-3403的大鼠胰液蛋白含量与对照组相比，无论基础的和胰泌素刺激的均明显增加,(P<0.01表2)，并在胰泌素每小时62.5ng/kg时胰液蛋白含量30min达高峰为985±215ug，以后逐渐下降至与对照组相同水平，提示ONO-3403能增
进食大鼠的胰液外分泌中的蛋白含量，与胰液容积增加是同步的.
表1 ONO-3403对胰泌素刺激的胰液容积的影响(n=10, x±s ,ulper30min)
bP<0.01vs对照组.
表2 ONO-3403对胰泌素刺激的胰液蛋白含量的影响(n=10, x±s , ug per30min)
bP<0.01vs对照组。
表3 ONO-3403胰泌素刺激的胰液中HCO3-含量的影响(n=10, x±s , umol per30min)
aP<0.05，bP<0.01vs对照组。
2.3 胰液中HCO3-含量,12h前给与ONO-3403的大鼠胰液中HCO3-含量与对照组相比，无论基础的和胰泌素刺激的均明显增加，(P<0.05或P<0.01表3)，并在胰泌素每小时250ng/kg时HCO3-含量达高峰为18±0.7umol per 30min,提示ONO-3403能增加进食大鼠的胰液中HCO3-含量.
2.4 胰腺重量，胰腺组织中的蛋白含量和淀粉酶含量， 12h前给与ONO-3403的大鼠胰腺重量和胰腺蛋白含量与对照组相比，差异不显著，P>0.05.而胰腺淀粉酶含量实验组与对照组相比明显降低，(P <0.05,表4).
表4 ONO-3403对胰腺质量、胰腺蛋白和淀粉酶含量的影响(n=10, x±s)
aP <0.05 vs对照组
3 讨论
胰液容积，胰液蛋白含量，淀粉酶含量，及HCO3-含量的变化能反映胰液外分泌的变化.蛋白，淀粉酶，是由胰腺腺胞细胞分泌的，水和HCO3-是由胰管上皮细胞分泌的.CCK既能作用于胰腺腺胞细胞，促进其分泌胰酶；也能作用于胰管上皮细胞，促进其分泌水和HCO3-[24,25]，胰泌素主要作用于胰管上皮细胞，促进其分泌水和HCO3-[26].我们的研究结果表明，ONO-3403能增加进食大鼠胰液流量，蛋白质及HCO3-的分泌，并能增强胰腺对胰泌素刺激的敏感性而使胰腺外分泌增加.其机制可能是通过CCK调节的胰腺反馈而起作用的.在进食大鼠的十二指肠内有较多的蛋白酶底物即食物蛋白，此状态下的胰腺外分泌较空 腹时强，分泌至十二指肠腔内的蛋白水解酶的活性较高，口服蛋白酶抑制剂ONO-3403后在十二指肠内抑制了蛋白水解酶的活性，而使肠腔内蛋白未能充分代谢，蛋白可刺激CCK的释放，CCK可促进胰腺外分泌增加，包括胰液流量，蛋白含量，淀粉酶含量，HCO3-含量.胰腺的这一负反馈机制可能是通过肠腔内的多种内源性的CCK释放肽而引起的，其中一种是称为监视肽monitor peptide(MP)[27],是胰腺分泌至胰液中的，其他的是小肠分泌至肠腔内的，如腔内CCK释放因子(LCRF)[28-30]和diazepam-binding inhibitor(DBI)[31].这些CCK释放肽都对胰蛋白酶敏感，可被其降解.当经胃管给与ONO-3403后，抑制了十二指肠内蛋白酶的活性，使这些CCK释放肽的降解减少，促进了CCK的释放，而使胰腺外分泌增强.
我们推测，在口服ONO-3403后，降低了十二指肠内蛋白酶的活力或直接作用于小肠的CCK产生细胞，使CCK的产生明显的增加.CCK刺激胰酶的分泌，包括MP和胰蛋白酶，糜蛋白酶同时进入十二指肠.在空腹状态下，这些蛋白水解酶使MP,LCRP和DBI水解失活，这样就抑制了CCK的释放，而在进食状态下，食物蛋白能与CCK释放肽竟争胰蛋白酶和糜蛋白酶，使CCK释放肽失活减少，能持续刺激CCK的释放.CCK是小肠膜中I细胞分泌的一种激素[32]，其重要的生理作用是刺激胰腺的腺胞细胞分泌胰酶等物质[33,34]，胰泌素是由小肠膜中S细胞分泌的，其主要的作用是促进胰管上皮细胞分泌水和HCO3-.在给与蛋白酶抑制剂ONO-3403后，胰腺外分泌对胰泌素的敏感性与对照组相比明显增强，说明ONO-3403能使内源性CCK分泌增强，使血中CCK浓度增加，加上外源性胰泌素，使胰腺外分泌明显增加.胰腺质量的增加是胰腺炎症水肿的标志，但我们的实验证明给与ONO-3403后大鼠的胰腺质量，胰腺蛋白含量与对照组相比无明显变化，说明经口给与ONO-3403后不能造成胰腺明显水肿，同时他可使胰腺淀粉酶含量降低，提示ONO-3403能使胰腺淀粉酶合成减少.
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山本光勝,大槻眞.日本产业医科大学第三内科 日本北九州市807
陈少夫，男，1962-11-2生，江苏省沭阳县人，汉族，1985年毕业于中国医科大学医学系，医学博士，现为消化内科副教授，主要从事胃肠激素与胃肠疾病的研究，发表文章26篇.
项目负责人 陈少夫,110004,中国医科大学第二临床学院消化内科,沈阳市和平区三好街36号. csf196211@yahoo.com.cn
收稿日期 2002-04-05 接受日期 2002-05-12
The effects of protease inhibitor ONO-3403 on pancreatic exocrine response to secretin in fed rats
Shao-Fu Chen, Mitsuyoshi Yamamoto, Otsuki Makoto
Shao-Fu Chen, Mitsuyoshi Yamamoto,Otsuki Makoto,Department of Gastroenterology,Second clinical college of China Medical University,Shenyang 110004,China;and Third Department of Internal Medicine,School of Medicine,University of Occupational and Environmental Health,Japan,Kitakyushu 807,Japan
Correspondence to: Dr.Shao-Fu Chen,Department of Gastroenterology,Second clinical college of China Medical University,Shenyang 110004, Liaoning Province,China. csf 196211@yahoo.com.cn
Received 2002-04-05 Accepted 2002-05-12
AbstractsAIM: To examine the pancreatic exocrine response to secretin and to clarify the mechanism of the pancreatic exocrine hypersecretion after oral administration of synthetic protease inhibitor ONO-3403 in rats.
METHODS: A single oral dose of synthetic protease inhibitor ONO-3403 was given to rats by orogastric tube 12h before experiment. The pancreatic juice was collected before and after stimulation of stepwise increasing doses of secretin. The output of protein, amylase and bicarbonate in pancreatic juice or pancreatic tissue were determined by Lowry method, chromogenic method with blue-dyed starch polymer, and the DST 800 multititration system, respectively.
RESULTS: Oral administration of ONO-3403 caused a significant increase in pancreatic juice flow (peak level 226±10 ul per 30min vs 44±5ul per 30 min, P<0.01), protein (peak level 985±215ug per 30min vs 254±47ug per 30min, P<0.01) and bicarbonate output (peak level 18±0.7umol per 30min vs 5.9±0.8umol per 30min,P<0.01) before and after secretin stimulation at 12h with pretreatment of ONO-3403. The pancreatic weight, pancreatic contents of protein in ONO-3403 pretreated rats were similar to those in control rats, and pancreatic content of amylasein in ONO-3403 pretreated rats was significantly lower than that in control rats (3114±372 U 103/Pancreas vs 5746±261U 103,P<0.05).
CONCLUSIONS: ONO-3403 can increase pancreatic exocrine secretion and sensitivity to secretin stimulation. The mechanism of ONO3403 induced pancreatic exocrine hypersecretion may be feedback regulation of the pancreas by increasing CCK secretion.
Chen SF, Yamamoto M, Makoto O. Effects of protease inhibitor ONO-3403 on pancreatic exocrine response to secretin in rats. Shijie Huaren xiaohua Zazhi 2002;10(7):792-795
摘要
目的:观察新型蛋白酶抑制剂ONO-3403对进食大鼠基础的及胰泌素刺激的胰腺外分泌的影响，探讨ONO-3403增加胰腺外分泌的机制.
方法:对进食的Wistar大鼠，分别于实验前12h经胃管给与ONO-3403(20mg/kg)后收集基础的和静脉注射胰泌素后的胰液，并对胰腺组织匀浆处理.用Lowry法，产色素法分别测定胰液及胰腺组织中蛋白含量，淀粉酶含量.用DST800多项滴定系统测定胰液HCO-3含量.
结果:给与ONO-3403的大鼠胰液容积，无论基础的及胰泌素刺激的，均较对照组明显增加，并在胰泌素每小时为125ng/kg时胰液容积达高峰(226±10ul per30min vs 44±5ulper 30min,P<0.01),提示ONO-3403能增加进食大鼠的胰液外分泌.给与ONO-3403的大鼠胰液蛋白含量与对照组相比，无论基础的和胰泌素刺激的均明显增加，并在胰泌素每小时为62.5ng/kg时胰液蛋白含量达高峰(985±215ug per 30min vs 254±47ug per30min, P<0.01)，提示ONO-3403能增加进食大鼠的胰液外分泌中的蛋白含量.给与ONO-3403的大鼠胰液中HCO3-含量与对照组相比,无论基础的和胰泌素刺激的均明显增加，并在胰泌素每小时为250ng/kg时HCO3-含量达高峰(18±0.7umol per 30min vs 5.9±0.8umolper 30min, P<0.01),提示ONO-3403能增加进食大鼠的胰液中HCO3-含量.给与ONO-3403的大鼠胰腺重量和胰腺蛋白含量与对照组相比，差异不显著(P>0.05).而胰腺淀粉酶含量与对照组相比明显降低(3114±372U×103 vs 5746±261U×103,P<0.05).
结论:ONO-3403能增加进食大鼠的胰腺外分泌及对胰泌素刺激的敏感性，其机制可能是通过CCK调节的胰腺反馈而起作用的.
陈少夫,山本光勝,大槻眞. 蛋白酶抑制剂ONO-3403对胰泌素刺激的大鼠胰腺外分泌的影响.世界华人消化杂志 2002;10(7):792-795
0 引言
胰腺外分泌的反馈调节是通过十二指肠内胰蛋白酶和糜蛋白酶而起作用的[1-5].向十二指肠内输入人工合成的蛋白酶抑制剂camostat能引起肠腔内蛋白酶的抑制，而使血浆胆囊收缩素(CCK)浓度升高，并能刺激胰腺的外分泌，因此推测CCK在胰蛋白酶对胰腺外分泌的反馈调节中起主导作用[6-8].我们选择一种新型的蛋白酶抑制剂ONO-3403，他在抑制蛋白酶活性方面比camostat强5-10倍，作用时间更长.现观察大鼠口服ONO-3403后，是否能促进胰腺外分泌及增加对胰泌素刺激的敏感性，以寻找ONO-3403对胰腺外分泌的作用机制及与胰腺外分泌相应的反馈机制.蛋白酶抑制剂在急,慢性胰腺炎的治疗中起重要的作用[9-16]，探讨新型蛋白酶抑制剂对胰腺的作用机制有较重要的意义[17].
1 材料和方法1.1 材料 ♂wistar大鼠，体重250-300g,大鼠饲养在23℃，12h明暗交替的环境中，可以自由的饮水和获得标准的饲料饮食.(饲料由日本东京东方发酵公司生产).ONO-3403(ethylN-allyl-N-|(E)-2-methyl-3-[4-amino-phenoxy(arbonyl)phenyl]propenoyl| amino acetate methansulfonate)是一种人工合成的蛋白酶抑制剂，由日本小野制药公司提供,实验前用蒸馏水溶解并调整pH至6.4备用.胰泌素(secretin)购自日本大阪肽蛋白研究所.
1.2 方法[18-20] 将大鼠20只随机分成2组，每组10只，即对照组可自由的饮食水，不给予ONO-3403;实验组于实验开始前12h，经胃管将ONO-3403 (20mg/kg)灌入大鼠胃内，亦可自由的饮食水.对两组大鼠在实验开始先用Urethare(1.6g/kg)腹腔注射麻醉后，剥离出颈内静脉，用聚乙烯导管插入颈内静脉，以备注射胰泌素.行腹正中切口，结扎幽门，行胆管和胰管插管，胆管在肝门的下方和胰腺的近端结扎，将插入胆管的聚乙烯导管引入十二指肠，保持在整个实验过程中胆汁流入十二指肠，在总胆胰管的远端近十二指肠入口处插入胰管插管，以获得纯净的胰液，每30min收集1次胰液.在1h的平衡期后，先收集30min的基础胰液，然后进行胰泌素的剂量反应的研究，每30min阶梯式增加胰泌素的剂量，每小时分别为31.25,62.5,125,250,500,1000,2000ng/kg-，以每小时2ml的速度经颈内静脉输入体内，收集每个剂量的胰泌素刺激下的胰液，以备测定胰液的容积，蛋白含量，HCO3-的含量.上述实验结束后，通过放血法处死大鼠，迅速切除胰腺，清除其中的淋巴结，脂肪组织后，称其量，切除约300g的胰腺组织，放入-70℃冰箱中保存，以备测定胰腺组织中蛋白和淀粉酶的含量.测定前，切取胰腺组织的一部分，用组织匀浆器，2000r/min,在4℃0.15mol/L的氯化钠溶液中进行胰腺组织匀浆.胰液及胰腺组织中蛋白含量用Lowry法测定，使用小牛血清蛋白为标准品.淀粉酶含量用产色素法测定，以Somogyi单位表示.HCO3-含量用DST800多项滴定系统(radiometer,copenhagen,denmark),在采集胰液后迅速进行检测[21-23].
统计学处理 数据均用x±s表示，用两个样本均数差异的t检验法，进行显著性检验.
2 结果2.1 胰液容积，12h前给与ONO-3403的大鼠胰液容积与对照组相比,无论基础的和胰泌素刺激的胰液容积均明显增加,(P<0.01表1)，并在胰泌素每小时125ng/kg时胰液容积30min达高峰为226±10ul，提示ONO-3403能增加进食大鼠的胰液外分泌.
2.2 胰液蛋白含量, 12h前给与ONO-3403的大鼠胰液蛋白含量与对照组相比，无论基础的和胰泌素刺激的均明显增加,(P<0.01表2)，并在胰泌素每小时62.5ng/kg时胰液蛋白含量30min达高峰为985±215ug，以后逐渐下降至与对照组相同水平，提示ONO-3403能增
进食大鼠的胰液外分泌中的蛋白含量，与胰液容积增加是同步的.
表1 ONO-3403对胰泌素刺激的胰液容积的影响(n=10, x±s ,ulper30min)
| 分组 | 胰泌素每小时剂量( ng/kg) | |||||||
| 0 | 31.25 | 62.5 | 125 | 250 | 500 | 1000 | 2000 | |
| 对照组 | 31±2 | 36±6 | 46±3 | 44±5 | 54±5 | 57 ±4 | 58±3 | 71±5 |
| 实验组 | 172±10b | 191±8 b | 221±7 b | 226±10 b | 224±11 b | 215±9 b | 208±12b | 202±16 b |
bP<0.01vs对照组.
表2 ONO-3403对胰泌素刺激的胰液蛋白含量的影响(n=10, x±s , ug per30min)
| 分组 | 胰泌素每小时剂量(ng/kg) | |||||||
| 0 | 32.25 | 62.5 | 125 | 250 | 500 | 1000 | 2000 | |
| 对照组 | 323±86 | 295±64 | 254±47 | 171±50 | 171±21 | 138±19 | 229±71 | 172±37 |
| 实验组 | 636±1606 | 637±190b | 985±215b | 930±241b | 575±140b | 434±104b | 300±65.8 | 246±62.6 |
bP<0.01vs对照组。
表3 ONO-3403胰泌素刺激的胰液中HCO3-含量的影响(n=10, x±s , umol per30min)
| 分组 | 胰泌素每小时剂量(ng/kg) | |||||||
| 0 | 31.25 | 62.5 | 125 | 250 | 500 | 1000 | 2000 | |
| 对照组 | 3±0.2 | 3.7±0.7 | 4.9±0.6 | 4.7±0.8 | 5.9±0.8 | 6.1±0.7 | 6±0.6 | 7±0.6 |
| 实验组 | 8±1.3a | 11±1.2b | 14.8±1.2b | 16.5±0.7b | 18±0.7b | 17.1±0.3b | 16.1±1.1b | 14.9±1.1a |
aP<0.05，bP<0.01vs对照组。
2.3 胰液中HCO3-含量,12h前给与ONO-3403的大鼠胰液中HCO3-含量与对照组相比，无论基础的和胰泌素刺激的均明显增加，(P<0.05或P<0.01表3)，并在胰泌素每小时250ng/kg时HCO3-含量达高峰为18±0.7umol per 30min,提示ONO-3403能增加进食大鼠的胰液中HCO3-含量.
2.4 胰腺重量，胰腺组织中的蛋白含量和淀粉酶含量， 12h前给与ONO-3403的大鼠胰腺重量和胰腺蛋白含量与对照组相比，差异不显著，P>0.05.而胰腺淀粉酶含量实验组与对照组相比明显降低，(P <0.05,表4).
表4 ONO-3403对胰腺质量、胰腺蛋白和淀粉酶含量的影响(n=10, x±s)
| 分组 | 胰腺质量(mg) | 蛋白含量(mg/pancreas) | 淀粉酶含量(103×U/pancreas) |
| 对照组 | 1016±21 | 144±7 | 5746±261 |
| 12 h组 | 1155±72 | 138±8 | 3114±372a |
aP <0.05 vs对照组
3 讨论
胰液容积，胰液蛋白含量，淀粉酶含量，及HCO3-含量的变化能反映胰液外分泌的变化.蛋白，淀粉酶，是由胰腺腺胞细胞分泌的，水和HCO3-是由胰管上皮细胞分泌的.CCK既能作用于胰腺腺胞细胞，促进其分泌胰酶；也能作用于胰管上皮细胞，促进其分泌水和HCO3-[24,25]，胰泌素主要作用于胰管上皮细胞，促进其分泌水和HCO3-[26].我们的研究结果表明，ONO-3403能增加进食大鼠胰液流量，蛋白质及HCO3-的分泌，并能增强胰腺对胰泌素刺激的敏感性而使胰腺外分泌增加.其机制可能是通过CCK调节的胰腺反馈而起作用的.在进食大鼠的十二指肠内有较多的蛋白酶底物即食物蛋白，此状态下的胰腺外分泌较空 腹时强，分泌至十二指肠腔内的蛋白水解酶的活性较高，口服蛋白酶抑制剂ONO-3403后在十二指肠内抑制了蛋白水解酶的活性，而使肠腔内蛋白未能充分代谢，蛋白可刺激CCK的释放，CCK可促进胰腺外分泌增加，包括胰液流量，蛋白含量，淀粉酶含量，HCO3-含量.胰腺的这一负反馈机制可能是通过肠腔内的多种内源性的CCK释放肽而引起的，其中一种是称为监视肽monitor peptide(MP)[27],是胰腺分泌至胰液中的，其他的是小肠分泌至肠腔内的，如腔内CCK释放因子(LCRF)[28-30]和diazepam-binding inhibitor(DBI)[31].这些CCK释放肽都对胰蛋白酶敏感，可被其降解.当经胃管给与ONO-3403后，抑制了十二指肠内蛋白酶的活性，使这些CCK释放肽的降解减少，促进了CCK的释放，而使胰腺外分泌增强.
我们推测，在口服ONO-3403后，降低了十二指肠内蛋白酶的活力或直接作用于小肠的CCK产生细胞，使CCK的产生明显的增加.CCK刺激胰酶的分泌，包括MP和胰蛋白酶，糜蛋白酶同时进入十二指肠.在空腹状态下，这些蛋白水解酶使MP,LCRP和DBI水解失活，这样就抑制了CCK的释放，而在进食状态下，食物蛋白能与CCK释放肽竟争胰蛋白酶和糜蛋白酶，使CCK释放肽失活减少，能持续刺激CCK的释放.CCK是小肠膜中I细胞分泌的一种激素[32]，其重要的生理作用是刺激胰腺的腺胞细胞分泌胰酶等物质[33,34]，胰泌素是由小肠膜中S细胞分泌的，其主要的作用是促进胰管上皮细胞分泌水和HCO3-.在给与蛋白酶抑制剂ONO-3403后，胰腺外分泌对胰泌素的敏感性与对照组相比明显增强，说明ONO-3403能使内源性CCK分泌增强，使血中CCK浓度增加，加上外源性胰泌素，使胰腺外分泌明显增加.胰腺质量的增加是胰腺炎症水肿的标志，但我们的实验证明给与ONO-3403后大鼠的胰腺质量，胰腺蛋白含量与对照组相比无明显变化，说明经口给与ONO-3403后不能造成胰腺明显水肿，同时他可使胰腺淀粉酶含量降低，提示ONO-3403能使胰腺淀粉酶合成减少.
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