一氧化氮对日本血吸虫童虫细胞毒作用的研究

目的 研究一氧化氮(NO)对日本血吸虫童虫的杀伤作用。 方法 用LPS或LPS+IFN-γ诱导离体的小鼠腹腔巨噬细胞,使其产生NO;加入机械断尾的日本血吸虫童虫,测定48 h内童虫的死亡率。为进一步证实NO对童虫的杀伤作用,用诱导型一氧化氮合酶(iNOS)的抑制剂L-NNA(Nω-nitro-L-arginine)抑制NO的产生,与未加抑制剂组比较,观察童虫死亡率的变化。 结果 LPS或LPS+IFN-γ均能有效诱导巨噬细胞产生NO,2.0×10~5细胞产生的NO浓度分别为(109.96±3.70)μmol/L和(113.50±7.38)μmol/L,其相应的杀虫率分别达91.07％±2.92％和96.86％±2.36％。加入2 mmol/L的L-NNA后,巨噬细胞的NO产量明显降低,其杀虫率也相应减低。 结论 巨噬细胞诱生的NO对日本血吸虫童虫具杀伤作用。     

Low Direct Cytotoxicity of Nabumetone on Gastric Mucosal Cells

Prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for clinical purposes because they are not harmful to the gastrointestinal mucosa. We recently showed that NSAIDs have direct cytotoxicity in NSAID-induced gastric lesions. We show here that under conditions where the NSAIDs indomethacin and celecoxib clearly induce cell death, an NSAID prodrug, nabumetone, and its active metabolite 6-methoxy-2-naphthylacetic acid (6MNA), did not have such effects. Moreover, nabumetone and 6MNA exhibited much lower membrane permeabilizing activities than did indomethacin and celecoxib. We recently reported that when an orally administered NSAID was used in combination with a low dose of intravenously administered indomethacin, the severity of gastric lesions produced in rats depended on the cytotoxicity of the orally administered NSAID. Using a similar protocol, we show here that gastric lesions were produced when the orally administered NSAID was celecoxib, but not when nabumetone was used. We thus propose that the low direct cytotoxicity of nabumetone observed in vitro is maintained in vivo, and that the use of nabumetone does not harm the gastric mucosa. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labour, and Welfare of Japan, as well as by the Suzuken Memorial Foundation, and the Japan Research Foundation for Clinical Pharmacology.     

Cyclooxygenase-2 Inhibitor Nimesulide Suppresses Telomerase Activity by Blocking Akt/PKB Activation in Gastric Cancer Cell Line

Nonsteroidal antiinflammatory drugs (NSAIDs) have been reported to have antiproliferative effects in neoplastic cells of different origin during the past few decades. We aimed to study the effects of the selective COX-2 inhibitor, nimesulide, on cell viability and telomerase and Akt/PKB activity in the human gastric cancer cell line MKN-45 and to explore the molecular mechanism for the antitumor activity of the selective COX-2 inhibitor. We tested the influence of nimesulide on the gastric cancer cell line MKN-45 in vitro. Trypan blue exclusion was used to determine the cell viability after incubation for 0, 12, 24, and 48 hr in different concentrations of nimesulide 0, 25, 50, 100, 200 microM). After treatment or no treatment with 100 microM nimesulide for 0, 12, 24, or 48 hr in the presence or absence of 300 nM okadaic acid for 2 hr, telomerase and Akt/PKB activity was measured using TRAP PCR-ELISA and nonradioactive IP kinase assays, respectively. In the gastric cancer cell line MKN-45 nimesulide caused a time- and dose-dependent reduction in cell numbers and significantly inhibited telomerase and Akt/PKB activity; the inhibition of telomerase activity was partly associated with the attenuation of Akt/PKB activity. These results demonstrate that the selective COX-2 inhibitor suppresses the telomerase activity of gastric cancer cells, in part by blocking the activation of protein kinase B, which provides a new signaling mechanism responsible for the anticancer effects of the selective COX-2 inhibitor.     

Functional Mechanism Underlying COX-2 Expression Following Administration of Indomethacin in Rat Stomachs: Importance of Gastric Hypermotility

The expression of COX-2 is up-regulated in the rat stomach after administration of indomethacin, and the inhibition of this enzyme may be a key to NSAID-induced gastric damage. The present study investigated the mechanism for COX-2 expression induced in the rat stomach by indomethacin, in relation with the ulcerogenic processes. The animals were given indomethacin or SC-560 p.o., and the gastric mucosa was examined 8 hr later. Indomethacin decreased the mucosal PGE2 content and produced gross damage with gastric hypermotility and the expression of COX-2 mRNA in the mucosa. Although SC-560 did not produce damage, this agent caused a decrease in the PGE2 content and an increase in gastric motility as well as the up-regulation of COX-2 expression, and provoked damage in the presence of rofecoxib. Gastric lesions induced by indomethacin were prevented by both atropine (even in the presence of exogenous HCl) and omeprazole, although the hypermotility response was inhibited only by atropine. The COX-2 expression induced by indomethacin or SC-560 was inhibited by atropine, even in the presence of exogenous HCl, while omeprazole had no effect. The mucosal PGE2 content was decreased by SC-560 at 2 hr but recovered 8 hr later, and this recovery of PGE2 was attenuated by both atropine and rofecoxib but not omeprazole. These results suggested that the COX-2 expression in the stomach following treatment with indomethacin is functionally associated with gastric hypermotility response induced by COX-1 inhibition. Luminal acid does not play a role in the up-regulation of COX-2 expression in the stomach following administration of indomethacin.     

尼美舒利对人胃癌SGC-7901细胞PGE2合成及COX-2、VEGF表达的影响

目的观察尼美舒利对人胃癌SGC-7901细胞前列腺素E2（PGE2）合成及环氧合酶（COX）-2、血管内皮生长因子（VEGF）表达的影响，探讨其抑制胃癌血管生成的机制。方法对人胃癌SGC-7901细胞进行培养．免疫组织化学、放射免疫法检测不同浓度尼美舒利作用后细胞COX-2、PGE2、VEGF的表达。结果与阴性对照组相比。尼美舒利100、200μmol／L作用48h后SGC-7901细胞的COX-2、VEGF表达明显降低（P〈0．05），COX-2与VEGF的表达呈正相关（r=0．8080，P〈0．05）；随着尼美舒利浓度的升高，PGE2分泌逐渐降低。结论抑制COX-2的活性与表达、减少PGE。的合成及由此引起的VEGF表达降低，在抑制胃癌血管生长中可能具有一定作用。     

冠状动脉粥样硬化斑块组成成分与斑块破裂的关系

为观察氧化型低密度脂蛋白对内皮细胞一氧化氮生成的影响,采用体外细胞培养方法将含不同浓度氧化型低密度脂蛋白的ＤＭＥＭ 培养液（ 氧化型低密度脂蛋白终浓度分别为０ ｍｇＬ、５０ ｍｇＬ 、１００ ｍｇＬ 、２００ ｍｇＬ）,与内皮细胞共同孵育（３７ ℃、５％ ＣＯ２）２４ ｈ 后,分别测上清液中乳酸脱氢酶活性和一氧化氮含量及细胞中一氧化氮合酶活性,并用细胞化学染色法记数各组细胞一氧化氮合酶阳性染色细胞数。结果发现,随着培养液中氧化型低密度脂蛋白浓度升高,上清液中乳酸脱氢酶活性增加和一氧化氮含量下降,细胞中一氧化氮合酶活性和一氧化氮合酶阳性染色细胞数下降,可见,氧化型低密度脂蛋白可损伤血管内皮细胞,降低细胞中一氧化氮合酶活性,抑制一氧化氮产生,这可能是氧化型低密度脂蛋白致动脉粥样硬化的原因之一。     

Effect of Ibuprofen on Cyclooxygenase and Nitric Oxide Synthase of Gastric Mucosa: Correlation with Endoscopic Lesions and Adverse Reactions

The aim of this study was to evaluate the effect of ibuprofen on gastric mucosa and enzymes involved in gastroprotection in healthy volunteers. Twenty-four Helicobacter pylori-negative subjects were randomized to treatment with ibuprofen or ibuprofen-arginate (each 600 mg/6 hr during 3 days). Endoscopies were performed 1 week before and after treatment. Biopsies were taken from the gastric antrum and corpus for determination of prostaglandin E2 (PGE2) by ELISA and cyclooxygenase (COX-1 and COX-2) and nitric oxide synthase (eNOS and iNOS) by western blot. All subjects had at least one gastric lesion except for two individuals taking ibuprofen-arginate. Ibuprofen-arginate caused a lower rate of clinical adverse reactions than ibuprofen. Subjects with gastric lesions or adverse reactions had lower PGE2 levels. COX-1, COX-2, eNOS, and iNOS were detectable in all subjects. The constitutive enzymes (COX-1 and eNOS) did not change after treatment. COX-2 was higher in corpus than antrum and it increased after ibuprofen treatment. iNOS tended to increase mildly in the corpus in subjects with adverse reactions or endoscopic lesions. There were no significant differences between ibuprofen and ibuprofen-arginate in PGE2, or enzymes.     

Effects of Indomethacin on Resting Cerebral Hemodynamic and During Suctioning in Preterm Neonates

The effect of therapeutic doses of indomethacin versus a placebo on cerebral hemodynamics was studied in nine preterm infants using Doppler ultrasound. Three doses of indomethacin (0.2 mg/kg) induced a significant decrease in the mean frequency compared to the placebo. Heart rate, blood pressure, transcutaneous carbon dioxide tension, and transcutaneous oxygen tension remained stable throughout the study. The changes in mean frequency occurred rapidly and were sustained at significantly diminished levels for at least 1 hour. All infants were mechanically ventilated, and the increase of mean frequency secondary to suctioning was significantly attenuated after each dose of indomethacin as compared to the placebo. The results confirmed that infusion of indomethacin caused a reduction in cerebral blood flow, probably by vasoconstriction of cerebral vessels. Indomethacin also seemed to attenuate the cerebrovascular response to hypercapnia induced by endotracheal suctioning. Even so, and because of the alteration of resting cerebral hemodynamics, we do not support the recommendation that indomethacin should be used prophylactically to prevent patent ductus arterious or periventricular intraventricular hemorrhage.     

一氧化氮在低密度脂蛋白氧化中的双向作用

为观察一氧化氮与低密度脂蛋白氧化的关系,揭示一氧化氮在动脉粥样硬化发病和防治中的作用,给小鼠腹腔注射云芝多糖,再用干扰素γ、脂多糖和Ｌ－精氨酸刺激小鼠腹腔巨噬细胞,然后检测一氧化氮释放量与脂氢过氧化物浓度。结果发现,一氧化氮与低密度脂蛋白氧化间有一个量的相关关系（ｒ＝０．７５３,Ｐ〈０．０５）,一定范围内（≤５０μｍｏｌ／Ｌ）的一氧化氮释放量与脂氢过氧化的量呈负相关,即抑制细胞对低密度脂蛋白的氧化;当一氧化氮产生过多时（≥９０μｍｏｌ／Ｌ）,脂氢过氧化物的量随之增加,则促进低密度脂蛋白的氧化。结果提示,一氧化氮在低密度脂蛋白氧化中发挥双重作用,它对低密度脂蛋白的氧化起抑制或促进作用取决于它的释放量。     

一氧化氮在低密度脂蛋白氧化中的作用及调控研究

通过给小鼠腹腔注射云芝多糖，并用干扰素γ，脂多糖及佛波酯醇刺激细胞，观察细胞一氧化氮的产量，测定脂氢过氧化物和硫代巴比妥酸反应物质，以反应低密度脂蛋白的氧化程度，从而部分揭示巨噬细胞氧化低密度脂蛋白的机制，探讨一氧化氮在低密度脂蛋白氧化中的作用和云芝多糖的抑制效果。     

选择性环氧合酶-2抑制剂塞来昔布在实验性结肠炎中的作用机制

目的　明确选择性环氧合酶 (COX) 2抑制剂—塞来昔布对 2 ,4 ,6 三硝基苯磺酸 (TNBS)诱导的大鼠结肠炎的作用 ,并初步探讨其作用机制。方法　将大鼠分为四组 :第 1组和第 2组为造模组 ,第 3组和第 4组为对照组。用 0 .2 5mlTNBS乙醇溶液 (TNBS浓度 2 5mg/ml,乙醇浓度 5 0 % )灌肠 ,诱导大鼠慢性实验性结肠炎模型。造模组大鼠于造模前 3h开始 ,分别给予塞来昔布 (1.2 5mg/kg ,第 1组 )和蒸馏水 (第 2组 ) ,每天 2次 ,共 7d。第 3组大鼠为正常对照组。第 4组大鼠以塞来昔布 (1.2 5mg/kg)灌胃 ,每天 2次 ,共 7d。于第 7天实验结束时处死所有存活动物 ,观察结肠黏膜的损伤程度 ,同时用放射免疫分析法测定结肠黏膜前列腺素E2 (PGE2 )水平。结果　造模组大鼠结肠损伤积分分别为11.15± 3.30 (第 1组 )和 8.5 0± 2 .82 (第 2组 ) ,均显著高于正常对照组 0 .6 2± 0 .0 9(P值均 <0 .0 1)。第 1组的结肠损伤积分显著高于第 2组 (P <0 .0 5 )。第 3组和第 4组的积分差异无显著性。造模组大鼠结肠黏膜PGE2浓度分别为 (12 .0 0± 4 .33)pg/ μg(第 1组 )和 (17.2 0± 9.6 2 ) pg/ μg(第 2组 ) ,均显著高于正常对照组的 (6 .0 2± 3.39) pg/ μg ,(P值均 <0 .0 5 )。第 1组的PGE2浓度显著低于第 2组 (P <0 .0 5 )     

Effects of leminoprazole, omeprazole and sucralfate on indomethacininduced delayed healing of kissing gastric ulcers in rats

We have examined whether or not repeated treatment with indomethacin delays the healing of kissing gastric ulcers induced in rats. The effects of leminoprazole, omeprazole and sucralfate on any delay in ulcer healing caused by indomethacin were also determined in relation to myeloperoxidase activity. Kissing gastric ulcers were induced by luminal application of an acetic acid solution. Indomethacin significantly delayed ulcer healing in a dose-dependent manner. Leminoprazole and omeprazole decreased the size and depth of ulcers, the healing of which was delayed by indomethacin, while sucralfate only decreased the ulcer depth. Histological studies showed that indomethacin inhibited tissue contraction and regeneration of the ulcerated mucosa. Leminoprazole and omeprazole prevented the inhibition of these parameters. The myeloperoxidase (MPO) activity of the ulcer portion in animals treated with indomethacin was markedly higher than in the control group. Both leminoprazole and omeprazole, but not sucralfate, significantly reduced MPO activity in contrast to the control value (in the presence of indomethacin). There was a significant relationship between the ulcerated area and myeloperoxidase activity. These results suggested that: (i) leminoprazole and omeprazole prevent the indomethacin-induced delay in ulcer healing by promoting tissue contraction and regeneration of the ulcerated mucosa; (ii) sucralfate prevents the indomethacin-induced delay in ulcer healing via the promotion of the formation of granulation tissue; and (iii) MPO activity will be useful to biochemically ensure the healing state of ulcers.     

The Effects of Chronic Administration of Indomethacin and Misoprostol on Renal Function in Cirrhotic Patients with and without Ascites

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) often cause renal dysfunction in cirrhotic patients with ascites through inhibition of prostaglandin synthesis. However, their renal effects in cirrhotic patients without ascites are controversial. In addition, the role of prostaglandins in cirrhotic patients with ascites and in non-ascitic cirrhotic patients receiving NSAIDs also remains elusive. Thus we evaluated the chronic renal effects of indomethacin and misoprostol in 9 cirrhotic patients with ascites (protocol 1) and 21 cirrhotic patients without ascites (protocol 2).

Methods: The patients of protocol I received 200 μg of misoprostol every 6 h for 7 consecutive days. In protocol 2, 11 patients received 25 mg indomethacin three times a day for 7 consecutive days. The other 10 patients received 25 mg indomethacin three times a day plus 200 μg misoprostol every 6 h for 7 consecutive days. Renal function tests, plasma renin activity, and plasma aldosterone concentration were measured before and after treatment.

Results: In protocol 1, misoprostol tended to reduce the urinary sodium excretion (p = 0.08). In protocol 2, indomethacin alone greatly impaired renal plasma flow (p < 0.05), creatinine clearance (p < 0.05), blood urea nitrogen (p < 0.05), and serum creatinine (p = 0.06) in 11 patients. Similar magnitudes of renal dysfunction were observed in the other 10 patients despite the concomitant misoprostol treatment.

Conclusion: Chronic administration of misoprostol may have caused a negative natriuretic effect in cirrhotic patients with ascites. In cirrhotic patients without ascites chronic administration of indomethacin may induce a renal dysfunction that cannot be reversed by misoprostol.     

氧化型低密度脂蛋白和维生素E对人脐静脉内皮细胞产生一氧化氮及合酶的影响

为研究氧化型低密度脂蛋白和维生素Ｅ对人脐静脉内皮细胞产生一氧化氮及合酶的影响,应用一氧化氮及一氧化氮合酶试剂盒测定培养的人脐静脉内皮细胞一氧化氮含量及一氧化氮合酶活性。结果发现,氧化型低密度脂蛋白能显著抑制培养的内皮细胞一氧化氮的产生,从对照组的１２６±２４ｍｍｏｌ／ｇ下降为４１±８ｍｍｏｌ／ｇ;并能明显降低一氧化氮合酶的活性,具有浓度和时间效应。抗氧化剂维生素Ｅ显著增加氧化型低密度脂蛋白刺激内皮细胞产生一氧化氮含量以及一氧化氮合酶活性。提示氧化型低密度脂蛋白可以通过抑制内皮细胞一氧化氮合酶的活性而抑制其产生一氧化氮。维生素Ｅ通过增加一氧化氮合酶活性而增加内皮细胞产生一氧化氮。     

胃乐散对大鼠溃疡组织TXA2、PGI2 及COX-2含量的影响及意义

目的 观察胃乐散对大鼠实验性溃疡血栓素A2(TXA2)和前列腺素I2( PGI2)及环氧合酶2(COX-2)的影响,并探讨其作用机理.方法 SD大鼠48只,随机分为正常对照组、模型组、胃乐散低剂量组、胃乐散中剂量组、胃乐散高剂量组和雷尼替丁组.采用冰醋酸烧灼法制备大鼠胃溃疡模型,连续用药后第14天处死大鼠,取溃疡部位组织提取蛋白.用ELISA法检测组织中血栓素B2(TXB2)和6-酮-前列腺素F1α(6-Keto-PGF1α)的水平变化,并观察TXB2/6-Keto-PGF1α的比值变化.Western blot检测组织中COX-2的含量.结果 与正常对照组比较,模型组6-Keto-PGF1α、COX-2水平降低(P＜0.05),TXB2及TXB2/6-Keto-PGF1α升高(P＜0.05);胃乐散高剂量组6-Keto-PGF1α的含量高于正常对照组(P＜0.05).与模型组比较,胃乐散中、高剂量组及雷尼替丁组6-Keto-PGF1α、COX-2的含量增加,特别是胃乐散高剂量组增加更为明显(P＜0.01),胃乐散高剂量组及雷尼替丁组TXB2低于模型组(P＜0.05),特别是TXB2/6-Keto-PGF1α降低更为显著(P＜0.01).结论 胃乐散治疗胃溃疡可能是通过促进PGI2分泌,纠正TXA2/PGI2的失衡来实现的.     

Effects of indomethacin on prostanoid synthesis and vasomotor responsiveness in endothelium-denuded abdominal and thoracic aortas of Wistar rats

In endothelium-denuded abdominal (but not thoracic) aortas of rats, the nonselective cyclooxygenase (COX) inhibitor, indomethacin, suppressed contractions evoked by α-adrenergic agonists hypothetically mediated by prostanoids. We aimed to identify these non-endothelial-derived contractile prostanoids released by α-adrenergic receptors activation. Endothelium-denuded abdominal and thoracic aortas of Wistar rats were used for biochemical and functional analyses. Western blot analysis showed that COX-1 and COX-2 protein levels were respectively equivalent in endothelium-denuded abdominal and thoracic aortas. Enzyme immunoassay data supported direct evidence of phenylephrine-stimulated release of prostanoids (PGI₂, PGE₂, and PGF_2α) by thoracic and abdominal aortas without endothelium, and their almost complete inhibition by 1 μM indomethacin. Isometric force measurements established that 10 μM indomethacin—but no lower concentrations—inhibited the contractions evoked by phenylephrine in endothelium-denuded abdominal aorta. In this preparation, 10 μM indomethacin also depressed the contractions provoked by angiotensin II and high K⁺ (80 mM). In fact, indomethacin (up to 1 mM) caused concentration-dependent reductions in all abovementioned contractile responses. In endothelium-denuded thoracic aortas, however, only 1 mM indomethacin significantly depressed the contractile activity stimulated by either phenylephrine, angiotensin II, or high K⁺. Hence, there was a clear quantitative difference in response to indomethacin between abdominal and thoracic aortas without endothelium. Altogether, the results indicate that prostanoids induced by phenylephrine in abdominal and thoracic aortas were derived from non-endothelial COX-mediated metabolism; notably, the decrease in prostanoid synthesis could not account for the inhibition of vasoconstrictor responses by indomethacin: Through COX-independent actions, indomethacin inhibited aortic smooth muscle contractility.     

Indomethacin treatment during initial period of acetic acid-induced rat gastric ulcer healing promotes persistent polymorphonuclear cell-infiltration and increases future ulcer recurrence

The study was performed to examine whether indomethacin administered during the initial period of acetic acid-induced gastric ulcer healing affects future ulcer recurrence. Gastric ulcers were produced in rats by subserosal injection of acetic acid. Indomethacin (1 mg/kg/day, orally) administered either alone or concomitant with ornoprostil (50µg/kg/day, orally) was started on the fourth day and continued for 56 days. In rats whose ulcer healed at the 90th day after production of ulcer, endoscopy was done every 30 days to examine recurrence of ulcer. Gastric specimens were obtained 10, 30, 60, 90, and 240 days after ulcer production for histology, to quantitate the height of regenerated mucosa, thickness of fibrous tissue, degree of polymorphonuclear cell infiltration, and PAS-positive cells. Cumulative ulcer recurrence rate was significantly higher in rats initially treated with indomethacin than in controls. Increased polymorphonuclear cell infiltration was the major histologic abnormality persisting after cessation of indomethacin. Ornoprostil reversed these abnormalities caused by indomethacin. In conclusion, the administration of indomethacin during the initial period of the ulcer healing promoted persistent polymorphonuclear cell infiltration and increased ulcer recurrence rates, possibly via a prostaglandin-dependent mechanism.The work was supported by The Ministry of Education, Science and Culture of Grant-in-Aid for General Scientific Research 02454236 and by the DVA Medical Research Service.