Role of the route of leukotrienes in an experimental model of oral mucositis induced by 5-fluorouracil

Abstract Purpose: To investigate the participation of cysteinyl leukotrienes in the pathophysiology of oral mucositis. Methods: Oral mucositis was induced in hamsters using 5-fluorouracil (5-FU; 60 and 40 mg/kg; i.p., on days 1 and 2, respectively, and with excoriations in jugal mucosa on day 4). Montelukast (10, 20, or 40 mg/kg/d; gavage), MK886 (3 mg/kg/d, i.p.), or saline or celecoxib (7.5 mg/kg/d; i.p.) was administered 1 h prior to 5-FU and daily, until the fourth (MK886) or tenth day, when the animals were euthanized and their jugal mucosa was collected for macroscopic, histopathological, and immunohistochemical evaluation. Results: Neither montelukast nor MK-886 prevented the oral mucositis induced by 5-FU, as observed by histopathological evaluation. In addition, we did not find significant differences in the expression of inducible nitric oxide synthase-2, cyclooxygenase-2, or interleukin (IL)-1β between the experimental and control groups. However, we did observe a significant decrease in tumor necrosis factor (TNF)-α expression for all doses of montelukast; we also observed a significant decrease in IL-10 with 40 mg/kg/d and MK 886. Conclusions: Cysteinyl leukotrienes do not play an important role in experimental oral mucositis induced by 5-FU. There is a modulating action specifically on TNF-α.

Arachidonic acid metabolic pathway-related genes interactions increase the incidences of stroke and vulnerable plaques / 中华神经科杂志

Objective To investigate whether metabolic pathway-related gene polymorphisms are associated with arterial plaque stability and their gene-gene interactions increase the risk of cerebral infarctions.Methods Totally 294 patients with atherothrombosis stroke admitted to the Department of Neurology, the Third Affiliated Hospital of Wenzhou Medical University from September 2010 to December 2012 were divided into a carotid vulnerable plaque group ( n=69 ) and a stable plaque group ( n=225 ) according to the results of carotid B-mode ultrasonography.A total of 282 healthy volunteers excluded carotid plaque and stroke were enrolled as well.Genetic polymorphisms of ALOX5AP and CYP3A5, CYP2C9*2, CYP2C9*3 and EPHX2 were genotyped using polymerase chain reaction and mass spectrometry analysis.The SPSS16.0 software was used to compare genotype frequencies and the generalized multifactor dimensionality reduction ( GMDR ) method was applied for gene-gene interaction analyses.Results The results showed that EPHX2 GG genotype might protect against stroke ( OR =0.520, 95% CI 0.288 -0.940, P=0.030).The distribution of CYP3A5 genotypes showed statistically significant differences (χ2 =7.284, P=0.026) between the vulnerable plaque ( AA: 5 cases, AG: 36 cases, GG: 28 cases) and stable plaque ( AA: 26 cases, AG: 77 cases, GG: 122 cases ) groups.Multivariate Logistic regression analysis showed that the GG genotype of CYP3A5 was protective factor for unstable plaques ( OR=0.405, 95%CI 0.178 -0.920, P =0.031 ).Differences in other SNPs did not reach statistical significance between the two groups.The GMDR analysis showed a significant gene-gene interaction between SG13S114 and A6986G, with scores of 10 for cross-validation consistency and 9 for the sign test (P=0.011).The best model for ischemic stroke was found to be SG13S114 AA and A6986G AA.Adjusting for age, hypertension and diabetes, the certain gene-gene interaction predicted a significantly higher risk of cerebral infarction (OR=1.804, 95%CI 1.180-2.759, P=0.006).Conclusions The EPHX2 G860A gene might be linked with the incidence of cerebral infarctions.Only a CYP3A5 gene polymorphism might be associated with carotid plaque instability in patients with stroke.The gene-gene interaction predicts a significantly higher risk of cerebral infarction.There is a 1.804-fold increased risk for ischemic stroke in individuals with these combined genetic factors.

Gastroprotective Effect of Cochinchina momordica Seed Extract in Nonsteroidal Anti-Inflammatory Drug-Induced Acute Gastric Damage in a Rat Model

BACKGROUND/AIMS: The major compounds of Cochinchina momordica seed extract (SK-MS10) include momordica saponins. We report that the gastroprotective effect of SK-MS10 in an ethanol-induced gastric damage rat model is mediated by suppressing proinflammatory cytokines and downregulating cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LOX), and the activation of calcitonin gene-related peptide. In this study, we evaluated the gastroprotective effects of SK-MS10 in the nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage rat model. METHODS: The pretreatment effect of SK-MS10 was evaluated in the NSAID-induced gastric damage rat model using aspirin, indomethacin, and diclofenac in 7-week-old rats. Gastric damage was evaluated based on the gross ulcer index by gastroenterologists, and the damage area (%) was measured using the MetaMorph 7.0 video image analysis system. Myeloperoxidase (MPO) was measured by enzyme-linked immunosorbent assay, and Western blotting was used to analyze the levels of cyclooxygenase (COX)-1, COX-2, cPLA2, and 5-LOX. RESULTS: All NSAIDs induced gastric damage based on the gross ulcer index and damage area (p<0.05). Gastric damage was significantly attenuated by SK-MS10 pretreatment compared with NSAID treatment alone (p<0.05). The SK-MS10 pretreatment group exhibited lower MPO levels than the diclofenac group. The expression of cPLA2 and 5-LOX was decreased by SK-MS10 pretreatment in each of the three NSAID treatment groups. CONCLUSIONS: SK-MS10 exhibited a gastroprotective effect against NSAID-induced acute gastric damage in rats. However, its protective mechanism may be different across the three types of NSAID-induced gastric damage models in rats.

Expression of lipoxin A4, leukotriene C4 and lipoxygenase-5 in severe preeclampsia / 中华围产医学杂志

Objective To investigate the expression and clinical significance of lipoxin A4,leukotrienc C4,lipoxygenase-5 in peripheral blood of pregnant women with different types of severe preeclampsia.Methods Forty-five singleton pregnant women who accepted antenatal care and delivered in First Affiliated Hospital of Wenzhou Medical College were enrolled in this study from December 2010 to June 2011.All objects were divided into normal pregnancy group (n=20),early onset severe preeclampsia group (n=10) and late onset severe preeclampsia group (n=15).Enzymelinked immunosorbent assay was used to detect lipoxin A4 and leukotriene C4 levels in peripheral blood.The level of lipoxygenase-5 mRNA in white blood cells was detected by real time fluorescence quantitative reverse transcription-polymerase chain reaction.The differences of lipoxin A4,leukotriene C4 and lipoxygenase-5 mRNA among groups were compared by analysis of variance and LSD-t test;and correlations among their expressions were analyzed by linear regression.Results Lipoxin A4 level in early and late onset severe preeclampsia group was (355.3±116.0) pg/ml and (389.7±117.5) pg/ml,which were both significantly lower than that in normal pregnancy group [(555.0±139.8) pg/ml] (t=-4.03 and-3.77,P＜0.05 respectively).The leukotriene C4 level in early and lateonset severe preeclampsia and normal pregnaney group was (591.3±185.5) pg/ml,(510.3±197.1) pg/ml and (496.9 ± 158.8) pg/ml,no statistical difference were found (F=0.889,P＞0.05) ; neither did the expression of lipoxygenase 5 mRNA,which was 4.2± 1.9 in normal pregnancy group,4.8 ± 2.0 in early onset severe preeclampsia group and 4.4 ± 1.2 in late onset severe preeclampsia group (F=0.311,P＞0.05).There was no correlation among the levels of lipoxin A4,leukotriene C4 and lipoxygenase-5 mRNA in each group (P ＞ 0.05).Conclusions Early and remarkable decreasing of lipoxin A4 level might contribute to the development of early onset severe preeclampsia.

Effect of isoflurane preconditioning on expression of 5-1ipoxygenase during focal cerebral ischemia-reperfusion in rats / 中华麻醉学杂志

Objective To investigate the effect of isoflurane preconditioning on the expression of 5-lipoxy-genase (5-LOX) during focal cerebral ischemia-reperfusion (I/R) in rats.Methods Thirty-nine male adult Sprague-Dawley rats weighing 250-300 g were randomly divided into 3 groups (n =13 each):sham operation group (group S); focal cerebral I/R group (group I/R); isoflurane preconditioning group (group Ⅰ).Focal cerebral I/R was produced by mid-cerebral artery occlusion in anesthetized rats.The rats inhaled 2 h of 2％ isoflurane and focal cerebral I/R was produced 24 h later in group I.The neurological deficits were scored at 24 h of reperfusion.The animals were then sacrificed.The brains were immediately removed for determination of the infarct size.The expression of 5-LOX,myeloid differentiation factor88 (MyD88) and nuclear factor kappa B (NF-κB) protein and mRNA was detected using Western blot and RT-PCR respectively.Results Compared with group S,the neurological deficit score was significantly increased,the infarct size was enlarged in groups I/R and I,the expression of 5-LOX,MyD88 and NF-κB protein and mRNA was up-regulated in group I/R,and the expression of 5-LOX mRNA and MyD88 protein and mRNA was up-regulated in group I (P ＜ 0.05).Compared with group I/R,the neurological deficit score was significantly lower,the infarct size was smaller,and the expression of 5-LOX,MyD88 and NF-κB protein and mRNA was lower in group I (P ＜ 0.05).Conclusion Isoflurane preconditioning can reduce focal cerebral I/R injury by down-regulating the expression of 5-LOX and inhibiting MyD88/NF-κB signaling pathway in rats.

Expressions of cyclooxygenase-2 and 5-1ipoxygenase in colonic mucosa of ulcerative colitis and the relationship between them / 中华消化杂志

Objective To investigate the expressions of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) in colonic mucosa of patients with ulcerative colitis (UC)and to analyze the relationship between them. Methods The specimens of colonic mucosa from 32 UC patients were graded according to endoscopic and histological grading standards, and specimens of colonic mucosa from 26 healthy controls were also collected. The expressions of COX-2 and 5-LOX mRNA and protein in colonic mucosa were determined using real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemistry. The correlation between COX-2 and 5-LOX was analyzed. Results For UC patients,10 were class 1, 19 class 2 and 3 class 3 according to endoscopic grading, whereas 19 were class Ⅰ , 9 class Ⅱ and 4 class Ⅲ according to histological grading. The expressions of COX-2 and 5-LOX mRNA in active UC patients were 81. 25% and 53.13%, respectively, and were 11.54% and 19. 23% in healthy controls, respectively, with significant differences between the two groups (all P values＜0.01). The positive expressions of COX2 and 5-LOX increased in accordance with increasing of endoscopic grading and histological grading.The levels of COX-2 and 5-LOX were 20. 08±1.17 and 37.83 ±1.48 in colonic mucosa tissues of UC patients, respectively, and 48.42 ± 1.69 and 11.28 ± 1.62 in healthy controls, respectively. There was significant difference between the two groups (all P values＜0.05). A good positive correlation was found between COX-2 and 5-LOX. Conclusions The expressions of COX-2 is closely related to 5-LOX in UC patients. Both may play a pivotal role in inflammation of UC.

5-(4-Hydroxy-2,3,5-trimethylbenzylidene) thiazolidine-2,4-dione attenuates atherosclerosis possibly by reducing monocyte recruitment to the lesion

A variety of benzylidenethiazole analogs have been demonstrated to inhibit 5-lipoxygenase (5-LOX). Here we report the anti-atherogenic potential of 5-(4-hydroxy-2,3,5-trimethylbenzylidene) thiazolidin-2,4-dione (HMB-TZD), a benzylidenethiazole analog, and its potential mechanism of action in LDL receptor-deficient (Ldlr-/-) mice. HMB-TZD Treatment reduced leukotriene B4 (LTB4) production significantly in RAW264.7 macrophages and SVEC4-10 endothelial cells. Macrophages or endothelial cells pre-incubated with HMB-TZD for 2 h and then stimulated with lipopolysaccharide or tumor necrosis factor-alpha (TNF-alpha) displayed reduced cytokine production. Also, HMB-TZD reduced cell migration and adhesion in accordance with decreased proinflammatory molecule production in vitro and ex vivo. HMB-TZD treatment of 8-week-old male Ldlr-/- mice resulted in significantly reduced atherosclerotic lesions without a change to plasma lipid profiles. Moreover, aortic expression of pro-atherogenic molecules involved in the recruitment of monocytes to the aortic wall, including TNF-alpha , MCP-1, and VCAM-1, was downregulated. HMB-TZD also reduced macrophage infiltration into atherosclerotic lesions. In conclusion, HMB-TZD ameliorates atherosclerotic lesion formation possibly by reducing the expression of proinflammatory molecules and monocyte/macrophage recruitment to the lesion. These results suggest that HMB-TZD, and benzylidenethiazole analogs in general, may have therapeutic potential as treatments for atherosclerosis.

Ligation of CD40 receptor in human B lymphocytes triggers the 5-lipoxygenase pathway to produce reactive oxygen species and activate p38 MAPK

Previously, we reported that CD40-induced production of reactive oxygen species (ROS) by NADPH oxidase requires the TNF receptor-associated factor (TRAF) 3, as well as the activities of phosphatidylinositol 3-kinase (PI3K) and Rac1. Here we investigated the possible mechanisms of the production of ROS after CD40 ligation in B cells. We describe an alternative ROS production pathway that is triggered by CD40 ligation, involves 5-lipoxygenase (5-LO), and results in activation of p38 MAPK. Our studies in Raji human B lymphomas revealed that CD40-induced ROS production by 5-LO also requires the activities of PI3K and Rac1. In contrast to the NADPH oxidase pathway, however, TRAF molecules are not required for the CD40-induced ROS production by 5-LO. The association of CD40 with 5-LO is dependent on CD40 ligation in Raji B cells, and co-immunoprecipitation experiments using epitope-tagged proteins transiently expressed in human embryonic kidney 293T cells revealed the role of the regulatory subunit of PI3K, p85, in this association. Collectively, these data suggest a separate pathway for the CD40-induced ROS production in B cells and demonstrate that this pathway requires 5-LO via direct association of p85 with both CD40 and 5-LO.

The expression of 5-LOX in endometriosis and the relationship with angionesis / 中国医师杂志

Objective To investigate the expression of 5-lipoxygenase (5-LOX) in endometriosis and analyze the relationship of 5-LOX and angionesis in endometriosis. Methods Immunochemical method was used to determine the expression of 5-LOX, VEGF, and the value of MVD in ectopic endometrium (ec-topic group) and eutopic endometrium (eutopic group) of 32 patients with EMs and in eutopic endometrium ( control group) of 25 patients without EMs. Result The expressions of 5-LOX and VECF and the value of MVD in ectopic groups were 0. 43 ± 0.09,0. 39 ±0.07 and 0.40 ± 0.09, respectively, which were higher than that of eutopic group and control group. The expressions of 5-LOX and VEGF and the value of MVD in eutopic group were 0. 35 ± 0. 08, 0.30 ± 0. 06 and 0. 33 ± 0. 08, respectively, which were higher than that of control group ( P <0.05). In ectopic group, the expressions of 5-LOX and VEGF, and the value of MVD in stage III/IV, were much higher than that of stage I/II ( P <0. 05); the expressions of 5-LOX and VEGF and the value of MVD were related to the severe of endometriosis through Spearman correlation analysis. The expressions of 5-LOX were shown to be significantly related to the expressions of VEGF and the value of MVD in ectopic and eutopic groups ( P <0. 05). Conclusion There is a high expression of 5-LOX in endometriosis, suggesting that 5-LOX may be participate in the pathogenesis of endometriosis. 5-LOX may promote angiogenesis of EMs through up - regulating the expression of VEGF.

Quantitative analysis of the 5-lipoxygenase mRNA in the rat peritoneal macrophages cultured in vitro by capillary electrophoresis / 中华检验医学杂志

Objective To quantify the mRNA of 5-lipoxygenase (5-LO) in the rat peritoneal macrophages cultured in vitro by capillary electrophoresis (CE). Methods The rat peritoneal macrophages were isolated and cultured for indicated time. The mRNA of 5-LO was detected by RT-PCR, and the products of RT-PCR were quantified by CE. Results The DNA fragments in the 100 bp DNA marker and the products of the RT-PCR were separated successfully by CE with the sieving buffer containing 1.8% hydroxypropylmethylcellulose (HPMC). It proved that there were no significant changes on the expressions of 5-LO mRNA when the cells were cultured for 72 h quantified by CE. The mRNA of 5-LO significantly decreased by almost 80% by CE with the cells cultured for 120 h in vitro.Conclusions The products of RT-PCR could be separated and quantified by CE directly.The 5-LO mRNA could express normally in the rat peritoneal macrophages for 72 h in vitro.