5-LOX mRNA Expression Research Articles

Dietary chitosan affects metabolism of arachidonic acid in weaned piglets

The effects of chitosan on immune function via arachidonic acid (AA) pathway in weaned piglets were investigated. A total of 180 piglets (Duroc × Yorkshire × Landrace) were randomly assigned to 5 dietary treatments and fed a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. Results showed that serum AA, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) contents in piglets were increased in a linear or quadratic dose-dependent manner with increasing chitosan on day 28 (P < 0.05). Chitosan increased serum cytosolic-phospholipase A2 (cPLA2) activity in a linear or quadratic dose-dependent manner on day 14 or 28, and improved 5-lipoxygenase (5-LOX) activity in a linear manner and cyclooxygenase-2 (COX-2) activity quadratically on day 28 (P < 0.05). Moreover, chitosan elevated gene expression of cPLA2 mRNA quadratically in the small intestine on days 14 and 28, increased the COX-2 mRNA expression in the duodenum or jejunum in a linear or quadratic manner on day 28, and improved the 5-LOX mRNA expression quadratically in the small intestine (P < 0.05). These results implied that the metabolism of AA was regulated by chitosan in a dose-dependent relationship, which may be one reason why chitosan affected immune function via AA pathway in weaned piglets.

5-Lipoxygenase contributes to the progression of hepatocellular carcinoma

5-Lipoxygenase (5-LOX) has been implicated in the development and progression of lung, pancreatic and esophageal cancers. However, its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore the role of 5-LOX in the pathogenesis of HCC. The expression of 5-LOX was detected in human HCC, HepG2 cells and diethylnitrosamine (DEN)-induced rat HCC using immunohistochemistry (IHC) staining or reverse transcriptase-polymerase chain reaction. Apoptosis in rat HCC was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Cell viability and apoptosis were determined in HepG2 cells by MTT assay and flow cytometry, respectively. IHC staining showed that the 5-LOX protein was highly expressed in human HCC, HepG2 cells and rat HCC, but not in the normal liver tissues. 5-LOX mRNA expression in human and rat HCC was also significantly increased compared to normal liver tissues. Zileuton, a 5-LOX inhibitor, reduced the nodule incidence and the mean number of nodules per nodule-bearing liver in DEN-induced rats. Further study using TUNEL assay showed that zileuton treatment induced apoptosis in the liver as the result of inhibition on 5-LOX levels. This result is consistent with our observation of significantly higher apoptotic indices in rats treated with DEN/zileuton, which were significantly higher compared to those from the control groups. In addition, zileuton reduced cell viability and induced apoptosis in a concentration- and time-dependent manner as detected using HepG2 cells in our in vitro analysis. In conclusion, 5-LOX is expressed in HCC, and the inhibition of 5-LOX blocks the development of HCC via the induction of apoptosis in tumor cells.

5-lipoxygenase inhibitor zileuton attenuates ischemic brain damage: involvement of matrix metalloproteinase 9

Objectives: Cerebral ischemia causes an increased expression and activation of 5-lipoxygenase (5-LOX) and matrix metalloproteinase 9 (MMP-9). Recent works demonstrated that the selective 5-LOX inhibitor zileuton down-regulates MMP-9 expression in vascular diseases and cancer. In the present work, we first studied the neuroprotective effect of zileuton on focal cerebral ischemia in rats and further investigated the effect of zileuton on the expression of 5-LOX and MMP-9 in ischemic brain.Methods: Adult Sprague–Dawley rats underwent permanent middle cerebral artery occlusion (pMCAO). The rats then received treatment with zileuton 5, 10 or 50 mg/kg or vehicle. Cerebral water content and infarct volume were measured 24 hours after pMCAO. Expression of 5-LOX messenger RNA (mRNA) and MMP-9 mRNA were determined by reverse transcription polymerase chain reaction. The levels of the proform and the active form of MMP-9 were detected by gelatin zymography.Results: Oral treatment of zileuton at 10 or 50 mg/kg significantly reduced cerebral water content and infarct volume. It also down-regulated the expression of 5-LOX mRNA and MMP-9 mRNA. Zileuton at 50 mg/kg could inhibit the active form of MMP-9.Conclusion: These results suggest that selective 5-LOX inhibitor zileuton attenuates ischemic brain damage in rats, which may be partly associated with MMP-9 inhibition.

The anti-inflammatory pharmacology of Pycnogenol ® in humans involves COX-2 and 5-LOX mRNA expression in leukocytes

We investigated the effects of Pycnogenol ® supplementation on the arachidonic acid pathway in human polymorphonuclear leukocytes (PMNL) in response to an inflammatory stimulus. Pycnogenol is a standardised extract of French maritime pine bark consisting of procyanidins and polyphenolic monomers. Healthy volunteers aged 35 to 50 years were supplemented with 150 mg Pycnogenol a day for five days. Before and after the final day of supplementation, blood was drawn and PMNL were isolated. PMNL were primed with lipopolysaccharide (LPS) and stimulated with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic acid pathway and the biosynthesis of leukotrienes, thromboxane and prostaglandins. Pycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Interestingly, Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1. Here we show for the first time that Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.

Protection of mouse brain from aluminum-induced damage by caffeic acid.

The natural product caffeic acid is a specific inhibitor of 5-lipoxygenase (5-LOX); it also possesses antioxidant and antiinflammatory properties. The current study was designed to determine whether the neuroprotective properties of caffeic acid are due to inhibition of 5-LOX. Cerebral damage was induced in mice by intracerebroventricular microinjection of aluminum (5.0 microg aluminum in 2.0 microL, once a day, for 5 days). Caffeic acid was administered intragastrically at 30 min prior to aluminum and repeated daily for an additional 10 days. The brain injury was determined by observation of behavioral changes in mice, as well as by measuring biochemical and pathological changes in the cerebral tissue. The levels of 5-LOX proteins and 5-LOX mRNA expression were measured in brain tissue. Aluminum impaired learning and memory in mice produced neuronal death in hippocampi, elevated brain malondialdehyde levels, increased protein expression of amyloid precursor protein (APP), amyloid beta, and 5-LOX. It also increased 5-LOX mRNA expression and decreased choline acetyl transferase (ChAT) protein expression in the brain tissue of mice. Caffeic acid prevented brain damage as well as behavioral and biochemical changes caused by aluminum overload. The results of this study suggest that overexpression of 5-LOX accompanies the cerebral injury induced by aluminum overload in mice, and that selective inhibitors of 5-LOX may have potential value in the treatment of aluminum neurotoxicity and conceivably of diseases associated with neuronal injury.

Minocycline inhibits 5-lipoxygenase activation and brain inflammation after focal cerebral ischemia in rats

To determine whether the anti-inflammatory effect of minocycline on postischemic brain injury is mediated by the inhibition of 5-lipoxygenase (5-LOX) expression and enzymatic activation in rats. Focal cerebral ischemia was induced for 30 min with middle cerebral artery occlusion, followed by reperfusion. The ischemic injuries, endogenous IgG exudation, the accumulation of neutrophils and macrophage/microglia, and 5-LOX mRNA expression were determined 72 h after reperfusion. 5-LOX metabolites (leukotriene B4 and cysteinyl leukotrienes) were measured 3 h after reperfusion. Minocycline (22.5 and 45 mg/kg, ip, for 3 d) attenuated ischemic injuries, IgG exudation, and the accumulation of neutrophils and macrophage/microglia 72 h after reperfusion. It also inhibited 5-LOX expression 72 h after reperfusion and the production of leukotrienes 3 h after reperfusion. Minocycline inhibited postischemic brain inflammation, which might be partly mediated by the inhibition of 5-LOX expression and enzymatic activation.

Inhibitory Effects of Mizolastine on Ultraviolet B‐Induced Leukotriene B4 Production and 5‐Lipoxygenase Expression in Normal Human Dermal Fibroblasts In Vitro

Ultraviolet B (UVB) irradiation exerts hazardous effects such as acute photodamage, skin cancer and photoaging. In this study we evaluated the protective effects of a nonsedative histamine H1-receptor antagonist, mizolastine, on UVB-exposed skin dermal fibroblasts. Therefore, primary human skin fibroblasts were incubated with mizolastine or dexamethasone after 100 mJ/cm(2) UVB irradiation. Leukotriene B4 (LTB4) in fibroblast supernatants was detected with enzyme immunoassays, expression of 5-lipoxygenase (5-LOX) messenger RNA (mRNA) in skin fibroblasts was examined by reverse transcriptase-polymerase chain reaction and expression of 5-LOX protein was measured by immune blotting and immunofluorescent staining with rabbit anti-human 5-LOX antibody. It was found that 0.01 mM mizolastine inhibited UVB-induced LTB4 production from skin fibroblasts at 12, 24 and 36 h. Meanwhile, mizolastine down-regulated 5-LOX mRNA expression and inhibited 5-LOX translocation from nucleus to cytoplasm in fibroblasts. On the basis of these findings, we propose that mizolastine might play a protective role in the pathogenesis of UV radiation-induced acute photodamage of the skin.

5-Lipoxygenase and Leukotriene B 4 Receptor Are Expressed in Human Pancreatic Cancers But Not in Pancreatic Ducts in Normal Tissue

The 5-lipoxygenase (5-LOX) pathway is critical for pancreatic cancer cell growth and escape from apoptosis. Inhibition of 5-LOX blocks proliferation and induces apoptosis in human pancreatic cancer cells. However, the expression of 5-LOX and its downstream signaling pathway have not been investigated in human pancreatic adenocarcinoma. Reverse transcriptase-polymerase chain reaction revealed expression of 5-LOX mRNA in all pancreatic cancer cell lines tested including, PANC-1, AsPC-1, and MiaPaCa2 cells, but not in normal pancreatic ductal cells. The expression of 5-LOX protein in pancreatic cancer cell lines was demonstrated by Western blotting. Finally, 5-LOX up-regulation in human pancreatic cancer tissues was verified by intense positive staining in cancer cells by immunohistochemistry. Staining for the 5-LOX protein was particularly evident in the ductal components of the more differentiated tumors but not in ductal cells in normal pancreatic tissues from cadaver donors. Immunohistochemistry also revealed strong staining of cancer tissues with an antibody to the receptor of the downstream 5-LOX metabolite, leukotriene B(4). The current study demonstrated marked expression of 5-LOX and the leukotriene B(4) receptor in human pancreatic cancer tissues. These findings provide further evidence of up-regulation of this pathway in pancreatic cancer and that LOX inhibitors are likely to be valuable in the treatment of this dreadful disease.

Lipoxygenase gene expression in baboon intrauterine tissues in late pregnancy and parturition.

Myometrium (upper and lower segment), cervix, chorion and decidua were obtained at the time of Caesarean hysterectomy in 15 baboons, 10 at varying gestational ages prior to the onset of labour in the last trimester of pregnancy, and five in spontaneous labour at term. Expression of currently recognized human lipoxygenase (LOX) genes was determined using Northern blot analysis relative to each of three housekeeping genes. Signals of similar size to human 5-LOX and human platelet 12-LOX genes were detected in all tissues. Expression of 5-LOX mRNA in the cervix decreased with advancing gestational age. In decidua, expression of 5-LOX mRNA was higher in tissues from animals in labour (compared to those not in labour), whereas in chorion, its expression was lower in tissues from animals in labour. Expression of the platelet 12-LOX gene decreased in chorion with advancing gestational age, and in cervix was lower in tissues from animals in labour. We postulate that the variation in expression of LOX genes may play a role in the onset or promotion of parturition in the baboon.

Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human fetal membranes throughout pregnancy and at term.

Lipoxygenase metabolites may be involved in human parturition. 5-lipoxygenase (5-LOX) catalyses the first steps in the synthesis of leukotrienes from arachidonic acid, and its activity is dependent on 5-LOX activating protein (FLAP). The expression of 5-LOX and FLAP were investigated in fetal membranes to determine whether there are changes with gestational age or at term with the onset of labour. No significant differences were found in the expression of 5-LOX or FLAP mRNA in the amnion at different gestational ages or at term. In the chorion-decidua, 5-LOX mRNA expression was significantly higher in the first trimester of pregnancy than in the second and third trimesters. At term, there was a significant increase in both 5-LOX mRNA and protein expression in the chorion-decidua in the time after labour, compared with the time before labour. The expression of FLAP mRNA was also significantly higher in the chorion-decidua in the first trimester of pregnancy compared with the third trimester, and at term in the time after labour compared with the time before labour. Expression of FLAP protein was not studied, as an antibody is not currently available. These results are consistent with a role for 5-LOX and FLAP in the control of parturition at term, and also suggest an involvement earlier in pregnancy.

Granulocyte-Macrophage Colony-Stimulating Factor Increases 5-Lipoxygenase Gene Transcription and Protein Expression in Human Neutrophils

The complex process of neutrophil activation and accumulation is orchestrated by a cascade of cytokines and bioactive lipids produced at the site of inflammation. Neutrophils are a rich source of the potent inflammatory lipid leukotriene B4(LTB4). Granulocyte-macrophage colony-stimulating factor (GM-CSF) can activate neutrophils for an exponential increase in LTB4 production in response to a number of subsequent stimuli. In this report, we examined the temporal regulation, by GM-CSF, of the gene and protein expression of 5-lipoxygenase (5-LOX), a key enzyme of the LTB4 production pathway. Human neutrophils were exposed to 10 ng/mL of GM-CSF for various periods of time and 5-LOX mRNA was measured by Northern blot analysis. We observed no change in 5-LOX mRNA at early time points (0.25 to 3 hours); however, by 18 hours we observed a significant augmentation of 5-LOX-specific message (4.3 +/- 1.7-fold increase; n = 6). Nuclear transcription assays indicated that the rate of 5-LOX gene transcription was augmented threefold in neutrophils incubated with GM-CSF, whereas the half-life of the message was not markedly changed. Parallel experiments indicated that the levels of 5-LOX protein were also increased by GM-CSF. The augmentation was observed within 30 minutes after stimulation and was maximal (5.23 +/- 2.6; n = 4) at 18 hours. Incubation of GM-CSF-stimulated neutrophils with protein synthesis inhibitors resulted in a time-dependent impairment of their ability to produce LTB4, with no inhibition seen during the first hours, a 75% decrease seen by 12 hours, and greater than 95% inhibition seen at 18 hours. Collectively, our data imply that GM-CSF can regulate LTB4 production by two distinct mechanisms: a short-term increase that is not related to increased 5-LOX mRNA expression and is independent of protein synthesis, and a sustained increase in LTB4 production that is associated with the transcriptional activation of the 5-LOX gene, increase in 5-LOX mRNA levels, and dependence on protein synthesis. Such transcriptional modulation of 5-LOX enzyme expression may provide new approaches for therapeutic intervention in protracted inflammatory conditions.