This study aims to explore the effects of arachidonic acid lipoxygenase metabolism in vascular calcification. We used 5/6 nephrectomy and high-phosphorus feeding to establish a model of vascular calcification in mice. Six weeks after nephrectomy surgery, vascular calcium content was measured, and Alizarin Red S and Von Kossa staining were applied to detect calcium deposition in aortic arch. Control aortas and calcified aortas were collected for mass spectrometry detection of arachidonic acid metabolites, and active molecules in lipoxygenase pathway were analyzed. Real-time quantitative PCR was used to detect changes in the expression of lipoxygenase in calcified aortas. Lipoxygenase inhibitor was used to clarify the effect of lipoxygenase metabolic pathways on vascular calcification. The results showed that 6 weeks after nephrectomy surgery, the aortic calcium content of the surgery group was significantly higher than that of the sham group (P < 0.05). Alizarin Red S staining and Von Kossa staining showed obvious calcium deposition in aortic arch from surgery group, indicating formation of vascular calcification. Nine arachidonic acid lipoxygenase metabolites were quantitated using liquid chromatography/mass spectrometry (LC-MS) analysis. The content of multiple metabolites (12-HETE, 11-HETE, 15-HETE, etc.) was significantly increased in calcified aortas, and the most abundant and up-regulated metabolite was 12-HETE. Furthermore, we examined the mRNA levels of metabolic enzymes that produce 12-HETE in calcified blood vessels and found the expression of arachidonate lipoxygenase-15 (Alox15) was increased. Blocking Alox15/12-HETE by Alox15 specific inhibitor PD146176 significantly decreased the plasma 12-HETE content, promoted calcium deposition in aortic arch and increased vascular calcium content. These results suggest that the metabolism of arachidonic acid lipoxygenase is activated in calcified aorta, and the Alox15/12-HETE signaling pathway may play a protective role in vascular calcification.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid ; Animals ; Arachidonate 12-Lipoxygenase ; Arachidonate 15-Lipoxygenase/metabolism* ; Arachidonic Acid ; Hydroxyeicosatetraenoic Acids ; Lipoxygenase/metabolism* ; Mice ; Signal Transduction ; Vascular Calcification

With the acceleration of aging society, delaying aging or promoting healthy aging has become a major demand for human health. 5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid into leukotrienes (LTs), which is a potent mediator of the inflammatory response. Previous studies showed that abnormal activation of 5-LOX and overproduction of LTs are closely related to the occurrence and development of aging-related inflammatory diseases. Therefore, inhibiting 5-LOX activation is a possibly potential strategy for treating age-related diseases. In this paper, the latest research progress in 5-LOX activation, 5-LOX in mediating aging-related diseases and its small molecule inhibitors is briefly reviewed to provide scientific theoretical basis and new ideas for the prevention and treatment of aging-related inflammatory diseases.
Humans ; Arachidonate 5-Lipoxygenase ; Leukotrienes ; Arachidonic Acid ; Aging ; Lipoxygenase Inhibitors/pharmacology*

OBJECTIVETo detect the expression of the plateau adaptablity gene(EPAS1, EGLN1 and PPARα) and proteins(HIF-2, PHD2 and PPARα) in rats blood, heart, liver, lung and kidney tissue after the rats exposed to high altitude.

METHODSThe Wistar rats were randomly divided into plain group(Shanghai, 55 m), acute exposure to high altitude 3400 m group, acute exposure to high altitude 4300 m group. Blood and organs of rats were collected in 1, 3, 5 days after arrival. Real time PCR and ELISA were used to compare the expression of plateau adaptablity gene and related protein between plain group and high altitude exposure groups.

RESULTSThe count of red blood cells, hemoglobin and HCT in high altitude 4300 m were higher than those in plain group. Compared with plain group, the expression of EPAS1 gene in blood, heart, liver and kidney tissue of rats at high altitude increased obviously(all P<0.05); the expression of EGLN1 in the heart, liver, brain and kidney increased, and PPARα gene in the heart, liver and kidney increased(all P<0.05). Compared with plain group, the expression of HIF-2 protein increased significantly at high altitudes in the liver, brain and kidney tissues. PHD2 and PPARα increased in the heart, liver and kidney.

CONCLUSIONPlateau adaptive genes(EPAS1, EGLN1 and PPARα) and protein(HIF-2, PHD2 and PPARα) differed in different altitude and different organizations. They might be used as target markers of plateau hypoxia.

Adaptation, Physiological ; Altitude ; Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Brain ; China ; Heart ; Hypoxia ; Hypoxia-Inducible Factor-Proline Dioxygenases ; metabolism ; Kidney ; Liver ; Lung ; PPAR alpha ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Rats ; Rats, Wistar

PURPOSE: The aim of this work was to investigate the beneficial effects of rhamnazin against inflammation, reactive oxygen species (ROS)/reactive nitrogen species (RNS), and anti-oxidative activity in murine macrophage RAW264.7 cells. METHODS: To examine the beneficial properties of rhamnazin on inflammation, ROS/ RNS, and anti-oxidative activity in the murine macrophage RAW264.7 cell model, several key markers, including COX and 5-LO activities, NO•, ONOO-, total reactive species formation, lipid peroxidation, •O₂ levels, and catalase activity were estimated. RESULTS: Results show that rhamnazin was protective against LPS-induced cytotoxicity in macrophage cells. The underlying action of rhamnazin might be through modulation of ROS/RNS and anti-oxidative activity through regulation of total reactive species production, lipid peroxidation, catalase activity, and •O₂, NO•, and ONOO• levels. In addition, rhamnazin down-regulated the activities of pro-inflammatory COX and 5-LO. CONCLUSION: The plausible action by which rhamnazin renders its protective effects in macrophage cells is likely due to its capability to regulate LPS-induced inflammation, ROS/ RNS, and anti-oxidative activity.
Arachidonate 5-Lipoxygenase ; Catalase ; Inflammation* ; Lipid Peroxidation ; Macrophages* ; Nitrogen ; Reactive Nitrogen Species ; Reactive Oxygen Species

Cysteinyl leukotrienes (cys-LTs) are potent mediators of inflammation derived from arachidonic acid through the 5-lipoxygenase/leukotriene C4 synthase pathway. The derivation of their chemical structures and identification of their pharmacologic properties predated the cloning of their classical receptors and the development of drugs that modify their synthesis and actions. Recent studies have revealed unanticipated insights into the regulation of cys-LT synthesis, the function of the cys-LTs in innate and adaptive immunity and human disease, and the identification of a new receptor for the cys-LTs. This review highlights these studies and summarizes their potential pathobiologic and therapeutic implications.
Adaptive Immunity ; Arachidonate 5-Lipoxygenase ; Arachidonic Acid ; Asthma ; Clone Cells ; Cloning, Organism ; Humans ; Inflammation Mediators ; Leukotrienes*

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 microg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-kappaB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-kappaB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-kappaB pathways in monocytes.
Arachidonate 5-Lipoxygenase ; Atherosclerosis ; Monocytes* ; NF-kappa B ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Transcription Factors

5-Lipoxygenase, one of lipoxygenase isozymes, is a well-studied oxidative metabolism enzyme. It widely exists in various human tissues and cells, participates in the oxidative metabolism of endogenous and exogenous chemicals, and produces a variety of metabolites, all of which contribute to the occurrence of human diseases, such as inflammation, asthma, atherosclerosis, and tumor and so on. The expression of 5-lipoxygenase is at low level in normal human tissues while at high level in abnormal tissues. 5-Lipoxygenase is closely related to many kinds of diseases in human ovary, brain, cardiovascular system, lung, liver, pancreas and other tissues. The abnormal expression of 5-lipoxygenase tends to promote the development of the disease.
Arachidonate 5-Lipoxygenase ; physiology ; Atherosclerosis ; enzymology ; Humans ; Inflammation ; enzymology ; Neoplasms ; enzymology

OBJECTIVETo determine whether 5-lipoxygenase (5-LOX) is involved in rotenone-induced injury in PC12 cells, which is a cell model of Parkinson disease.

METHODSAfter rotenone treatment for various durations, cell viability was determined by colorimetric MTT reduction assay, and 5-LOX translocation was detected by immunocytochemistry. The effect of 5-LOX inhibitor zileuton was also investigated.

RESULTRotenone (0.3-30 μmol/L) induced PC12 cell injury, and zileuton (3-100 μmol/L) attenuated this injury. Rotenone also time-and concentration-dependently induced 5-LOX translocation into the nuclear envelope, and zileuton (1-30 μmo/L) significantly inhibited rotenone-induced 5-LOX translocation.

CONCLUSION5-LOX is involved in rotenone-induced injury in PC12 cells, and 5-LOX inhibitor zileuton can reduce rotenone-induced 5-LOX activation and cell injury.

Animals ; Arachidonate 5-Lipoxygenase ; metabolism ; physiology ; Cell Survival ; drug effects ; Hydroxyurea ; analogs & derivatives ; pharmacology ; Lipoxygenase Inhibitors ; pharmacology ; PC12 Cells ; Rats ; Rotenone ; pharmacology

OBJECTIVETo investigate the effect of intracellular 5-lipoxygenase on oxidation of benzidine in HBE cells and to provide further evidence that lipoxygenase is an alternative pathway for the oxidation of xenobiotics mediated by cytochrome P450.

METHODSEnzyme system test: Soybean lipoxygenase (SLO), substrate (benzidine) and other components reacted in the enzyme system, followed by detection of the reaction products by spectrophotometry. In vitro test: After HBE cells were exposed to benzidine, the protein levels of 5-lipoxygenase in HBE cells were assessed by Western-blot, and the DNA damage by the single cell gel electrophoresis. At last, the effect of the specific inhibitor of 5-lipoxygenase (AA861) on 5-lipoxygenase protein expression and DNA damage in HBE cells were detected.

RESULTSSLO could catalyze the co-oxidation of benzidine to generate benzidine diimine in the presence of hydrogen peroxide. Under optimal condition, numax value of the oxidation of benzidine catalyzed by SLO was 1.42 nmol*min(-1) SLO, and the Km value of benzidine was 1.48 mmol/L. EGCG could inhibit the oxidation of benzidine by SLO. Benzidine could induce 5-lipoxygenase protein expression in HBE cells, but AA861 was invalid. Benzidine caused DNA damage in HBE cells, which could be significantly inhibited by AA861.

CONCLUSION5-LOX protein expression in HBE cells can be regulated by benzidine, which suggests that the co-oxidation of benzidine by 5-LOX could produce into electrophile that could covalently bind to DNA and induce DNA damage, which could be one of the mechanisms for carcinogenesis of BZD. 5-LOX inhibitor AA861 can inhibit this effect.

Arachidonate 5-Lipoxygenase ; metabolism ; Benzidines ; pharmacokinetics ; toxicity ; Cells, Cultured ; DNA Damage ; drug effects ; Epithelial Cells ; drug effects ; enzymology ; metabolism ; Humans

OBJECTIVETo investigate the protective effect of Crocin against hypoxia damage of cardiac myocytes of neonatal rats and the regulation of HIF-1 and prolyhydroxylase (PHDs).

METHODSA model of CoCl2 simulated hypoxia damage was established in primary cultural myocardial cell. Expression levels of HIF-1alpha, VEGF, iNOS, as well as PHD1, 2, 3 protein in myocardial cells were detected by Western blot.

RESULTSCompared with CoCl2 group, the viability of myocardial cell was significantly increased after treated 24 h at 10(-5)mol/L Crocin (P < 0.01), HIF-1alpha, VEGF and iNOS were expressed higher than those in Crocin + CoCl2 group (P < 0.01), the expression of PHD2 was significantly increased (P < 0.01), while the expression of PHD3 was remarkably reduced in Crocin + CoCl2 Group (P < 0.01).

CONCLUSIONCrocin has better protective effect on hypoxic damage of myocardial cell. The mechanisms of protective effect of Crocin may be related to the activation of HIF-1-mediated pathway of the hypoxia response. PHDs may be involved in the pathophysiology regulated process of myocardial cells.

Animals ; Carotenoids ; pharmacology ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Homeodomain Proteins ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Hypoxia-Inducible Factor-Proline Dioxygenases ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism