Effects of melatonin on the expression of iNOS and COX－2 in rat models of colitis

AIM: To investigate the effects of melatonin (MT) on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in rat models of colitis. METHODS: Healthy adult Sprague-Dawlay (SD) rats of both sexes, weighing 280+/-30 g, were employed in the present study. The rat models of colitis were induced by either acetic acid or 2,4,6-trinitrobenzene sulfonic acid (TNBS) enemas. The experimental animals were randomly divided into melatonin treatment and model control group that were intracolicly treated daily with melatonin at doses of 2.5, 5.0, 10.0 mg/kg(-1) and equal amount of saline respectively from 24 h following induction of colitis in rats inflicted with acetic acid enema and the seventh day in rats with TNBS to the end of study. A normal control group of rats treated with neither acetic acid nor TNBS but saline enema was also included in the study. On the 28(th) day of the experiment, the rat colon mucosal damage index (CDMI) was calculated, and the colonic prostaglandin E(2) (PGE(2)), nitric oxide (NO), as well as the iNOS and COX-2 expression were also determined biochemically or immunohistochemically. RESULTS: CDMI increased to 2.87+/-0.64 and 3.12+/-1.12 respectively in rats treated with acetic acid and TNBS enema, which was in accordance with the significantly elevated colonic NO and PGE(2) contents, as well as the up-regulated colonic iNOS and COX-2 expression in both of the two rat models of colitis. With treatment by melatonin at the doses of 5.0 and 10.0 mg/kg(-1), CDMI in both models of rat colitis was significantly decreased (P<0.05-0.01), which accorded synchronously and unanimously with the reduced colonic NO and PGE(2) content, as well as the down-regulated expression of colonic iNOS and COX-2. CONCLUSION: Melatonin has a protective effect on colonic injury induced by both acetic acid and TNBS enemas, which is probably via a mechanism of local inhibition of iNOS and COX-2 expression in colonic mucosa.     

COX-2: an in vivo evidence of its participation in heat stress-induced myocardial preconditioning

OBJECTIVE: Heat stress (HS) is known to induce delayed protection against myocardial infarction. We have previously shown that inducible nitric oxide synthase (iNOS), was involved in mediating this form of preconditioning. Since iNOS and cyclooxygenase-2 (COX-2) are co-induced in various cell types, the goal of this study was to investigate whether COX-2 could also participate to the HS-induced cardioprotection. METHODS AND RESULTS: A total of 78 male Wistar rats, subjected to either heat stress (42 degrees C for 15 min) or sham anaesthesia were used for this study. Twenty-four hours later, they were treated or not with a selective COX-2 inhibitor, either celecoxib (3 mg kg(-1), i.p.) or NS-398 (5 mg kg(-1), i.p.), 30 min before being subjected to a 30-min occlusion of the left coronary artery followed by a 120-min reperfusion, in vivo. HS resulted in a marked increase in myocardial COX-2 protein expression at 24 h, associated with a significant protection against infarction (46.0+/-1.4% in sham vs. 26.8+/-3.8% in HS group) (P相似文献   

Cyclo-oxygenase-2 inhibitors ameliorate the severity of experimental colitis in rats

BACKGROUND: Both in experimental colitis and in inflammatory bowel disease, colonic eicosanoid generation is enhanced and may contribute to the pathogenesis of the inflammatory response. AIMS: To evaluate the effect of selective cyclo-oxygenase-2 (COX-2) inhibitors on the extent and severity of two models of experimental colitis. METHODS: Colitis was induced by intra-caecal administration of 2 ml 5% acetic acid or intra-colonic administration of 0.1 ml 3% iodoacetamide. Rats were treated intra-gastrically with nimesulide 2 x 10 mg/kg/day, or once with SC-236 6 mg/kg, and killed 1 or 3 days after damage induction. The colon was isolated, weighed, macroscopic damage was measured, and mucosal samples were obtained for histology and for determination of myeloperoxidase (MPO) and nitric oxide synthase (NOS) activities and eicosanoid generation. The serum levels of thromboxane B2 (TXB2), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were determined. RESULTS: Nimesulide significantly decreased the extent of colitis induced by acetic acid. Both nimesulide and SC-236 significantly decreased the extent of iodoacetamide-induced colonic damage. The decrease in the extent of colitis induced by nimesulide was accompanied by a significant decrease in mucosal MPO and NOS activities. Nimesulide and SC-236 decreased the enhanced colonic eicosanoid generation in acetic acid and iodoacetamide-induced colitis, and, in iodoacetamide-treated rats, nimesulide also decreased the elevated serum TNF-alpha and IL-1beta levels. CONCLUSIONS: The effective nimesulide and SC-236-induced amelioration of the severity of the colitis in acetic acid and iodoacetamide-treated rats confirms the role of eicosanoids in their pathogenesis and suggests that COX-2 inhibitors may be of value in the treatment of inflammatory bowel disease.     

Protective effect of Radix Acanthopanacis Senticosi capsule on colon of rat depression model

AIM: To investigate the abnormity of rat colon caused by depression and the ameliorative effects of Radix Acanthopanacis Senticosi (RAS) capsule on colon and their mechanisms in rat depression model. METHODS: Chronic stress-induced model of depression of Wistar rats was produced. The experimental animals were randomly divided into model control, 5-aminosalicylic acid (5-ASA) therapy group and three RAS capsule therapy groups. These five groups were intracolonically treated daily (8:00 a.m.) for 2 wk with normal saline, 5-ASA (100 mg/kg) and RAS capsule at the doses of 300, 600 and 900 mg/kg, respectively. A normal control group of rats was also included in the study. Colonic activities of nitric oxide (NO) and superoxide dismutase (SOD), levels of malondialdehyde (MDA) and inducible nitric oxide synthase (iNOS) were determined by ultraviolet spectrophotometry. The expression of cyclooxygenase-2 (COX-2) in colonic tissue was detected by immunohistochemistry. RESULTS: Enhanced colon inflammatory response and oxidative stress were observed in the chronic stress-induced rat depression model, which manifested as the significant increase of MDA, iNOS and NO levels, as well as the expressions of COX-2 in the colon tissue, but the colonic SOD activity was significantly decreased compared with the normal control (MDA: 10.34±2.77 vs2.55±0.70; iNOS: 1.11±0.44 vs 0.25±0.16; COX-2: 53.26±8.16 vs 4.87±1.65; NO: 11.28±5.66 vs 4.76±1.55; SOD: 53.39±11.15 vs 84.45±22.31; P<0.01). However, these parameters were significantly ameliorated in rats treated locally with RAS capsule at the doses of 300, 600 and 900 mg/kg (iNOS: 0.65±0.31,0.58±0.22 and 0.64±0.33; NO: 5.99±2.73, 6.87±1.96 and 6.50±1.58; MDA: 2.92±0.75, 3.19±1.08 and 3.26±1.24; SOD: 70.81±12.36, 73.30±15.30 and 69.09±11.03, respectively). The expressions of COX-2 in the colon were significantly ameliorated (28.83±9.48 and 27.04±9.56, respectively) when RAS capsule was administered at the doses of 600 and 900 mg/kg. CONCLUSION: Administration of RAS capsule intracolonically may have significant therapeutic effects on the colon of rat depression model, which are probably due to its antioxidative action and inhibition of arachidonic acid metabolism.     

Protective effect of angelica sinensis polysaccharide on experimental immunological colon injury in rats

AIM: To study the effect of angelica sinensis polysaccharide (ASP) on immunological colon injury and its mechanisms in rats.METHODS: Immunological colitis model of rats was induced by intracolon enema with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) and ethanol. The experimental animals were randomly divided into normal control, model control, 5-aminosalicylic acid therapy groups and three doses of ASP therapy groups. The 6 groups were treated intracolonically with normal saline, normal saline, 5-aminosalicylic acid (100 mg.kg-1), and ASP daily (8:00 am) at the doses of 200, 400 and 800 mg.kg-1 respectively for 21 days 7 d following induction of colitis. The rat colon mucosa damage index (CMDI), the histopathological score (HS), the score of occult blood test (OBT), and the colonic MPO activity were evaluated. The levels of SOD, MDA, NO, TNF-α, IL-2 and IL-10 in colonic tissues were detected biochemically and immunoradiometrically. The expressions of TGF-β and EGF in colonic tissues were also determined immunochemically.RESULTS: Enhanced colonic mucosal injury, inflammatory response and oxidative stress were observed in colitis rats,which manifested as significant increases of CMDI, HS, OBT,MPO activity, MDA and NO contents, as well as the levels of TNF-α and IL-2 in colonic tissues, although colonic TGF-β protein expression, SOD activity and TL-10 content were significantly decreased compared with the normal control (P＜0.01). However, these parameters were found to be significantly ameliorated in colitis rats treated intracolicly with ASP at the doses of 400 and 800 mg@kg-1 (P＜0.05-0.01).Meantime, colonic EGF protein expression in colitis rats was remarkably up-regulated.CONCLUSION: ASP has a protective effect on immunological colon injury induced by TNBS and ethanol enema in rats,which was propably due to the mechanism of antioxidation,immunomodulation and promotion of wound repair.     

Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A(2). We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34(+) hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34(+) cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34(+) cells synthesized more PGE(2) than TXB(2) (214 +/- 50 vs. 30 +/- 10 pg/10(6) cells), whereas the reverse was true in mature megakaryocytes (TXB(2) 8,440 +/- 2,500 vs. PGE(2) 906 +/- 161 pg/10(6) cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE(2) and TXB(2) to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE(2) and TXA(2) may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.     

Acetic acid-derived prostaglandin-dependent colonic adaptive cytoprotection is preserved in chronic colitis: role of cyclo-oxygenase

BACKGROUND AND AIMS: We recently reported the phenomenon of prostaglandin-dependent colonic adaptive cytoprotection (CAP) against acute colonic injury induced by acetic acid (AA) in the normal colon. This study investigated whether the CAP is preserved in the chronic inflamed colon. MATERIALS AND METHODS: Normal rats and a chronic colitis model, induced by trinitrobenzene sulfonic acid, received an intracolonal administration (0.5 ml) of saline or AA at low concentration (1%) followed by high concentration (8%) 30 min later. The distal colon was removed 48 h after 8% AA administration, and colitis was assessed by macroscopic scoring and measurement of the myeloperoxidase (MPO) activity. Indomethacin (5 mg/kg), a nonselective cyclo-oxygenase (COX) inhibitor, or N-[2-cyclohexyloxy-4-nitrophenyl] methane-sulfonamide (NS398, 1 mg/kg), a COX type 2 selective inhibitor, was injected intraperitoneally 1 h before pretreatment with 1% AA. RESULTS: Intracolonal administration of 8% AA induced colonic mucosal damage (macroscopic score 10.0+/-0.9) and elevated MPO activity (2.8+/-0.2 U/g), which were significantly reduced to 3.3+/-0.8 and 1.8+/-0.2 U/g by 1% AA pretreatment, respectively. Indomethacin abolished the gross mucosal protective effect by 1% AA pretreatment in 8% AA-derived colitis in normal rats while the NS398 had no effect. Both indomethacin and NS398 reversed the MPO activity reduction induced by 1% AA pretreatment. In chronic inflamed colon 8% AA treatment resulted in an increase in the macroscopic score to 11.5+/-0.4 from 4.7+/-0.4, but not the MPO activity, which was significantly reduced to 5.7+/-0.9 by 1% AA pretreatment. This gross mucosal protective effect by 1% AA pretreatment in chronic inflamed colon was reversed by indomethacin while the NS398 had no effect. CONCLUSION: These data show that COX-1 and COX-2 derived prostaglandins induced by low concentration AA pretreatment reduce the colonic mucosal injury and the increase in the MPO activity in colitis, respectively. The protective effect of COX-1 is preserved in chronic inflamed colon. These findings support the existence of a low concentration of AA-derived prostaglandin-dependent CAP and suggest that colonic AA, which is derived from bacterial breakdown of carbohydrate and protein in the colon, plays a crucial role in the endogenous defense mechanisms.     

开口箭提取物对大鼠实验性结肠炎的治疗机制研究

目的研究中药开口箭有效成分对大鼠实验性结肠炎的治疗作用及其机制。方法建立三硝基苯磺酸大鼠结肠炎模型，用开口箭醇提物（主要成分为甾体皂苷）灌肠治疗2周，计算治疗前后结肠黏膜损伤指数（CMDI）和组织学评分（HS），测定其结肠组织超氧化物歧化酶（SOD）活性、中性粒细胞髓过氧化物酶（MPO）、丙二醛（MDA）、血小板表面P-选择素（P-选择素）以及血小板最大聚集率（PagT）改变。结果治疗后大鼠CMDI及HS降低，MDA降低，MPO活性下降，PagT和P-选择素计数下降，SOD活性增加。结论开口箭醇提物对大鼠实验性结肠炎有治疗作用，其机制可能为清除氧自由基、减轻脂质过氧化物对组织的损伤以及抑制血小板的聚集与活化，减少炎性因子的释放。     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups of eight each: normal, NS, model Ⅰ, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks, the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO)activities assayed. NF-~B p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-α (and ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κB p65 and other parameters were analyzed. RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25&#177;1.39 and 6.24&#177;1.04, respectively), which were in accordance with the significantly elevated MPO activity (1.69+0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model Ⅱ group were higher than that of normal control(9.7&#177;1.96 vs 1.7&#177;0.15, 84.09&#177;14.52 vs 16.03&#177;6.21,77.69&#177;8.09 vs 13.41&#177;4.91 P&lt;0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities, macroscopical and histological indices in TNBS-induced colitis, respectively (r = 0.8235, 0.8780, 0.8572, 0.9152,0.8247; P&lt;0.05). CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

Eradication of Helicobacter pylori significantly reduced gastric damage in nonsteroidal anti-inflammatory drug-treated Mongolian gerbils

AIM: To examine the effect of eradication of Hellcobacter pylori prior to usage of NSAIDs, by investigating gastric inflammatory activity, myeloperoxidase (MPO) activity, prostaglandin (PG) E_2 synthesis in H Pylori-infected, and H pylori-eradicated gerbils followed by administration of indomethacin and rofecoxib. METHODS: Six-week-old male gerbils were orally inoculated with H pylori. Seven weeks later, anti-H pylori triple therapy and vehicle were given to gerbils respectively and followed by oral indomethacin (2 mg/kg·d) or rofecoxib (10 mg/kg·d) for 2 wk. We examined the area of lesions, gastric inflammatory activity, PGE_2 synthesis and MPO activity in the stomach. RESULTS: In indomethacin and rofecoxib-treated gerbils, the following results were obtained in H pylori-infected group vs H pylori-eradicated group respectively: hyperplasia area of the stomach (mm~2): 82.4±9.2 vs 13.9±3.5 (P＜0.05), 30.5±5.1 vs 1.3±0.6 (P＜0.05); erosion and ulcer area (mm~2): 14.4±4.9 vs 0.86±0.5 (P＜0.05), 1.3±0.6 vs0.4±0.3 (P＜0.05); score of gastritis: 7.0±0.0 vs3.6±0.5 (P＜0.05), 7.0±0.0 vs 2.7±0.5 (P＜0.05); MPO activity (μmol H_2O_2/min/g tissue): 104.7±9.2 vs9.0±2.3(P＜0.05), 133.5±15.0 vs2.9±0.7 (P＜0.05); PGE_2 synthesis (pg/mg wet weight/min): 299.2±81.5 vs102.8±26.2 (P＜0.05), 321.4±30.3 vs 11.9±4.8(P＜0.05). CONCLUSION: Eradication of H pylori reduced gastric damage of NSAID-treated Mongolian gerbils. Rofecoxib caused less severe gastric damage than indomethacin in H pylori-eradicated gerbils.     

Rofecoxib, a COX-2 inhibitor, does not inhibit human gastric mucosal prostaglandin production

BACKGROUND & AIMS: Rofecoxib, an inhibitor of the inducible cyclooxygenase (COX)-2 enzyme, appears not to cause acute gastroduodenal injury or chronic ulceration. To attribute this to COX-2 selectivity with sparing of gastric mucosal prostaglandin synthesis requires direct proof. METHODS: Twenty-four healthy, nonsmoking Helicobacter pylori-negative volunteers were randomized to 1 of 2 separate concurrent blinded crossover studies. Sixteen volunteers received rofecoxib, 50 mg once daily, for 5 days in one treatment period and placebo in the other. Eight volunteers similarly received naproxen, 500 mg twice daily, and placebo. On day 5 of each period, antral mucosal prostaglandin E2 (PGE2) synthesis was measured by radioimmunoassay after vortexing for 3 minutes. Whole blood COX-1 activity was measured as serum thromboxane (TXB)2- and COX-2 activity as lipopolysaccharide (LPS)-induced PGE2. RESULTS: Naproxen decreased gastric mucosal PGE2 synthesis by 65% (90% confidence interval [CI], 53%-74%; P = 0.001 vs. placebo) in contrast to an 18% increase after rofecoxib (90% CI, -11% to 57%; P = 0.313 vs. placebo). Naproxen also significantly inhibited both serum TXB2 by 94% and LPS-induced PGE2 production by 77% (both P < or = 0.002 vs. placebo), but rofecoxib only inhibited COX-2-dependent LPS-induced PGE(2) (by 79%; P < 0.001 vs. placebo). CONCLUSIONS: Rofecoxib (50 mg) lacked naproxen's ability to reduce the availability of gastroprotective prostaglandins.     

Therapeutic effect of phenantroline in two rat models of inflammatory bowel disease.

BACKGROUND: Phenantroline is a zinc-chelator that inhibits biological activities of matrix metalloproteinases (MMPs). Over-expression of MMPs can accelerate tissue destruction and disrupt subsequent tissue repair. The effects of phenantroline in two rat models of inflammatory bowel disease (IBD) are evaluated: transmural colitis induced by trinitrobenzensulphonic acid (TNBS) and distal colitis caused by dextran sulphate sodium (DSS). METHODS: Transmural colitis was induced by TNBS in two groups of 15 rats each, and distal colitis was induced by DSS in two other groups of 15 rats each. Phenantroline was administered by oral gavage at 20 mg kg(-1) day(-1) to the test groups, whereas matched control groups received oral vehicle. On the last day of dosing, rats were subjected to intracolonic dialysis under anaesthesia for assessment of luminal eicosanoid release (PGE2, TXB2 and LTB4) and euthanized. Colons were removed and lesions were blindly scored according to macroscopic and histological scales. Myeloperoxidase (MPO) activity was measured in homogenates of colonic tissue. RESULTS: In the TNBS model, phenantroline treatment significantly reduced colonic strictures; in the DSS model, phenantroline significantly decreased scores of epithelial injury. In both models, the levels of PGE2, TXB2 and LTB4 and tissue MPO were not significantly altered. CONCLUSIONS: Although phenantroline did not modify the activity of inflammatory mediators, this compound substantially reduced intestinal injury associated with tissue remodelling.     

Regulatory role of phosphatidylinositol 3-kinase on TNF-alpha-induced cyclooxygenase 2 expression in colonic epithelial cells

BACKGROUND AND AIMS: Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells. METHODS: In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography. RESULTS: The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly. CONCLUSIONS: COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.     

罗格列酮对大鼠溃疡性结肠炎的疗效及其机制

目的观察罗格列酮对大鼠溃疡性结肠炎的疗效并探讨其可能存在的机制。方法应用三硝基苯磺酸(TNB)/乙醇灌肠制备大鼠溃疡性结肠炎模型。实验设正常对照组、模型对照组、阳性药物组(柳氮磺胺吡啶组,100 mg/kg)、罗格列酮给药组(2 mg、4mg、8 mg/kg),每天灌胃给药1次,给药时间从造模后第2天开始至实验结束共8 d,观察大鼠疾病活动指数(DAI)、结肠黏膜损伤指数(CMDI)及组织学评分(HS)。生化法检测大鼠结肠组织髓过氧化物酶(MPO)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)的含量。结果与正常组相比,模型组大鼠DAI、CMDI、HS明显增加(P<0.01),结肠组织MPO活性、MDA水平显著升高(P<0.01),SOD活性下降(P<0.01)。罗格列酮4 mg、8 mg/kg和SASP可不同程度改善DAI、CMDI和HS(P<0.05,P<0.01),降低MPO活性和MDA水平(P<0.01),增加SOD活性(P<0.01)。结论罗格列酮对大鼠溃疡性结肠炎有保护作用,其机制可能与减少脂质氧化,增加清除氧自由基的能力有关。     

Effects of advanced glycation end products on the expression of COX-2, PGE2 and NO in human osteoarthritic chondrocytes

OBJECTIVE: Advanced glycation end products (AGE) accumulate in articular cartilage with age. We investigated the effects of AGE in primary-cultured human OA chondrocytes. METHODS: Chondrocytes were cultured with/or without AGE-bovine serum albumin (AGE-BSA) and the expression levels of inducible nitric oxide (iNOS), cyclooxygenase (COX)-2 microsomal prostaglandin E synthase-1 (mPGES-1) were evaluated using RT-PCR and western blot analysis. Prostaglandin E(2) (PGE(2)) was analysed by ELISA and nitric oxide (NO) was analysed by Griess reaction assay. Pharmacological studies to elucidate the involved pathway were executed using specific inhibitors of MAPK and receptor for AGE (RAGE). RESULTS: We found that treatment of OA chondrocytes with AGE-BSA increased COX-2, mPGES-1 and iNOS mRNA and protein, as well as elevating production of PGE(2) and NO. Pre-treatment with the MAPK inhibitors SP600125 (JNK inhibitor), SB202190 (p38 inhibitor) or PD98059 (ERK inhibitor) significantly inhibited AGE-BSA induction of COX-2 expression and production of PGE(2). In contrast, SN50, a nuclear factor-kappaB (NF-kappaB) inhibitor, had no effect on levels of COX-2 and PGE(2). SB202190 and SN50, but not SP600125 and PD98059, decreased AGE-BSA-induced production of NO. Pre-treatment with soluble receptor for AGE (sRAGE) also reduced AGE-stimulated COX-2, iNOS and PGE(2), implicating the involvement of RAGE. CONCLUSIONS: These results show that AGE may augment inflammatory responses in OA chondrocytes by increasing PGE(2) and NO levels, possibly via the MAPK pathway for PGE(2) and the NF-kappaB pathway for NO.     

Recovery of ischaemic injured porcine ileum: evidence for a contributory role of COX-1 and COX-2

BACKGROUND: We have previously shown that the non-selective cyclooxygenase (COX) inhibitor indomethacin retards recovery of intestinal barrier function in ischaemic injured porcine ileum. However, the relative role of COX-1 and COX-2 elaborated prostaglandins in this process is unclear. AIMS: To assess the role of COX-1 and COX-2 elaborated prostaglandins in the recovery of intestinal barrier function by evaluating the effects of selective COX-1 and COX-2 inhibitors on mucosal recovery and eicosanoid production. METHODS: Porcine ileal mucosa subjected to 45 minutes of ischaemia was mounted in Ussing chambers, and transepithelial electrical resistance was used as an indicator of mucosal recovery. Prostaglandins E1 and E2 (PGE) and 6-keto-PGF1alpha (the stable metabolite of prostaglandin I2 (PGI2)) were measured using ELISA. Thromboxane B2 (TXB2, the stable metabolite of TXA2) was measured as a likely indicator of COX-1 activity. RESULTS: Ischaemic injured tissues recovered to control levels of resistance within three hours whereas tissues treated with indomethacin (5x10(-6) M) failed to fully recover, associated with inhibition of eicosanoid production. Injured tissues treated with the selective COX-1 inhibitor SC-560 (5x10(-6) M) or the COX-2 inhibitor NS-398 (5x10(-6) M) recovered to control levels of resistance within three hours, associated with significant elevations of PGE and 6-keto-PGF1alpha compared with untreated tissues. However, SC-560 significantly inhibited TXB2 production whereas NS-398 had no effect on this eicosanoid, indicating differential actions of these inhibitors related to their COX selectivity. CONCLUSIONS: The results suggest that recovery of resistance is triggered by PGE and PGI2, which may be elaborated by either COX-1 or COX-2.     

The effects of colloidal bismuth tartrate on colitis induced by immune-complex in rabbits

AIM To observe the therapeutic effect of colloidal bismuth tartrate in an animal colitis model.METHODS Immune-complex colitis was induced in groups of rabbits by formalin, and two hours later0.85 mL heat-aggregated rabbit IgG was given intravenously through the ear cannula. Animals wereintracolonically treated with colloidal bismuth tartrate (BITNAL), and its effect was compared withsulfasalazine (SASP), indomethacin (IND) and bifidobiogen (BIFG). Animals were killed, the mucosalappearance was scored (0-4), and tissue saved for histological studies, the number of neutrophils present ininflamed colonic tissue was quantitated by the myeloperoxidase (MPO) activity assay, the production oflipoxygenase and cyclo-oxygenase products was monitored and eicosanoid production were assayed byincubation colonic specimens and the media for prostaglandin E2(PGE2), leukotriene (LTB4), thromboxaneB2(TXPe) were examined by radiommunoassay.RESULTS Immune-complex colitis was induced by formalin and IgG, colonic damage persisted for at least1 wk by macrography. Histologically, the inflammatory response included mucosal and submucosalinfiltration by polymorphonuclear leukocytes, macrophages, lymphocytes and fibroblasts, the macroscopic,persent 2 wk after IgG, was correlated with greatly increased PGE2, LTB4 and TXB2 compared with levels incontrols. Treatment with BITNAL (500 mg/kg) resulted in a lowered inflammation index, lowered MPOactivity and inhibited the increased formation of PGF-2, LTB4 and TXB2 by the inflamed colon, and IND(500 mg/kg) markedly inhibited prostanoid formation in both inflamed and control colon but did not reducetissue damage, SASP (500 mg/kg) also inhibited the formation of PGE2, LTB4 and TXB2 but the effectswere less marked. BIFG (400 mg/kg) did not significantly reduce the colonic injury and the media sythesizedby the rabbit colon.CONCLUSION BITAL provides better therapeutic effects in experimental colitis than anti-inflammatorydrug IND or SASP.     

Mitochondrial dysfunction activates cyclooxygenase 2 expression in cultured normal human chondrocytes

OBJECTIVE: Mitochondrial alterations play a key role in the pathogenesis of osteoarthritis (OA). This study evaluated a potential role of mitochondrial respiratory chain (MRC) dysfunction in the inflammatory response of normal human chondrocytes. METHODS: Commonly used inhibitors of the MRC were utilized to induce mitochondrial dysfunction in normal human chondrocytes. Levels of prostaglandin E(2) (PGE(2)) protein and expression of cyclooxygenase 2 (COX-2) and COX-1 messenger RNA (mRNA) and protein were analyzed. To identify the underlying mechanisms responsible for PGE(2) liberation, reactive oxygen species (ROS) were measured. Inhibitors of ROS, including vitamin E, and inhibitors of mitochondrial Ca(2+) and NF-kappaB were used to test their effects on the MRC. RESULTS: Antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) significantly increased the levels of PGE(2) (mean +/- SEM 505 +/- 132 pg/50,000 cells and 288 +/- 104 pg/50,000 cells, respectively, at 24 hours versus a basal level of 29 +/- 9 pg/50,000 cells; P < 0.05) and increased the expression of COX-2 at both the mRNA and protein levels. Expression of COX-1 did not show any modulation with either inhibitor. Further experiments revealed that antimycin A and oligomycin induced a marked increase in the levels of ROS. Production of PGE(2) and expression of COX-2 protein were inhibited by antioxidants, vitamin E, and mitochondrial Ca(2+) and NF-kappaB inhibitors. The response to blockers of mitochondrial Ca(2+) movement showed that ROS production was dependent on mitochondrial Ca(2+) accumulation. CONCLUSION: These results strongly suggest that, in human chondrocytes, the inhibition of complexes III and V of the MRC induces an inflammatory response, which could be especially relevant in relation to PGE(2) production via mitochondrial Ca(2+) exchange, ROS production, and NF-kappaB activation. These data may prove valuable for a better understanding of the participation of mitochondria in the pathogenesis of OA.     

IL-18BP和IL-4共表达腺病毒基因对关节炎小鼠滑膜组织COX-2和iNOS表达的作用

目的 本研究构建了共表达小鼠白细胞介素-18结合蛋白(IL-18BP)和IL-4的重组腺病毒载体(Ad mIL-18BP/mIL-4),将其用于胶原诱导型关节炎(CIA)小鼠局部关节基因治疗,以探讨对小鼠滑膜组织环氧化酶-2(COX-2)和诱导型一氧化氮合酶(iNOS)及其诱导产物前列腺素E2(PGE2)、一氧化氮(NO)表达水平的影响.方法 采用雄性的DBA-1/BOM小鼠,建立CIA的小鼠模型,经共表达小鼠IL-18BP和IL-4的重组腺病毒载体局部关节基因治疗后,分别采用半定量反转录一聚合酶链反应(RT-PCR)法和Westem印迹法检测滑膜组织COX-2、iNOS mRNA的表达水平和蛋白表达水平,用竞争ELISA和硝酸酶还原法检测关节滑膜液PGE2和NO的含量,并设AdLacZ组和磷酸盐缓冲液(PBS)组分别为病毒对照组和组织对照组.结果 AdmIL-18BP/mIL-4治疗组滑膜组织COX-2、iNOS mRNA表达水平明显低于AdLacZ对照组(0.15 vs 0.42,P<0.01;0.05 vs 0.77,P<0.01)和PBS对照组(0.15 vs 0.65,P<0.01;0.05 vs 0.64,P<0.01);AdmIL-18BP/mIL-4治疗组滑膜组织COX-2、iNOS的蛋白表达水平明显低于AdLacZ对照组(0.08 VS 0.92,P<0.01;0.11 vs 1.00,P<0.01)及PBS对照组(0.08 vs 0.77,P<0.01;0.11 vs 0.84,P<0.01);AdmIL-18BP/mIL-4治疗组关节滑膜液中PGE2及NO水平明显低于AdLacZ对照组[(0.68±0.06)vs(2.58±0.21)ng/ml,P<0.01;(23.4±2.5)vs(60.0±11.3) μmol/L,P<0.01]和PBS对照组[(0.68±0.06)vs(2.57±0.20)ng/mL,P<0.01;(23.4±2.5)vs(60.3±13.4)μmol/L,P<0.01].结论 共表达小鼠IL-18BP和IL-4的重组腺病毒基因治疗能明显下调COX-2和iNOS的表达水平,减少其诱导产物PGE2、NO的合成,这将为RA的基因治疗提供新的治疗策略.     

Nuclear factor-kappaB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). CONCLUSION: The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.