Biosynthesis of leukotriene B4.

Leukotriene B4 (LTB4) is a lipid mediator derived from arachidonic acid (AA) by the sequential action of 5-lipoxygenase (5-LOX), 5-lipoxygenase-activating protein (FLAP) and LTA4 hydrolase (LTA4H). It was initially recognized for its involvement in the recruitment of neutrophils and is one of the most potent chemotactic agents known to date. A large body of data has indicated that LTB4 plays a significant role in many chronic inflammatory diseases, such as arthritis, chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer and more recently, metabolic disorder. In this review, we focus on the biosynthesis of LTB4 and its biological effects. In particular, we will describe a basic biochemical understanding integrated with recent developments in the field of structural biology of the three key enzymes (5-LOX, FLAP and LTA4H) in LTB4 biosynthesis, and also summarize the most outstanding work on in vivo biological and pathogenic roles of these enzymes and the development of enzyme inhibitors.

Singapore Grouper Iridovirus Disturbed Glycerophospholipids Homeostasis: Cytosolic Phospholipase A2 Was Essential for Virus Replication.

Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, causes great economic losses in the aquaculture industry. Previous studies demonstrated the lipid composition of intracellular unenveloped viruses, but the changes in host-cell glyceophospholipids components and the roles of key enzymes during SGIV infection still remain largely unknown. Here, the whole cell lipidomic profiling during SGIV infection was analyzed using UPLC-Q-TOF-MS/MS. The lipidomic data showed that glycerophospholipids (GPs), including phosphatidylcholine (PC), phosphatidylserine (PS), glycerophosphoinositols (PI) and fatty acids (FAs) were significantly elevated in SGIV-infected cells, indicating that SGIV infection disturbed GPs homeostasis, and then affected the metabolism of FAs, especially arachidonic acid (AA). The roles of key enzymes, such as cytosolic phospholipase A2 (cPLA2), 5-Lipoxygenase (5-LOX), and cyclooxygenase (COX) in SGIV infection were further investigated using the corresponding specific inhibitors. The inhibition of cPLA2 by AACOCF3 decreased SGIV replication, suggesting that cPLA2 might play important roles in the process of SGIV infection. Consistent with this result, the ectopic expression of EccPLA2α or knockdown significantly enhanced or suppressed viral replication in vitro, respectively. In addition, the inhibition of both 5-LOX and COX significantly suppressed SGIV replication, indicating that AA metabolism was essential for SGIV infection. Taken together, our results demonstrated for the first time that SGIV infection in vitro disturbed GPs homeostasis and cPLA2 exerted crucial roles in SGIV replication.

Zinc and iron complexes of oleanolic acid, (OA) attenuate allergic airway inflammation in rats.

Oleanolic acid (OA) is a hydroxyl pentacyclic triterpene acid (HTAs) used in various ailments. Inflammatory diseases may be profoundly influenced by iron (Fe) and zinc (Zn) status. We studied the anti-asthmatic effects of two metal complexes (Fe and Zn) of OA in the ovalbumin (OVA)-induced rat model. Delayed type hypersensitivity (DTH) was measured. Total and differential leucocyte count was done in blood as well as bronchoalveolar lavage fluid (BALF). The mRNA expression levels of pro-inflammatory cytokines were measured in lung tissue by reverse transcription polymerase chain reaction. The levels of cyclooxygenase-2 (COX-2), immunoglobulin E (IgE) and 5-lipoxygenase (5-LOX) were estimated by enzyme linked immunosorbent assay. Splenocyte proliferation was performed through BrdU uptake method and nitric oxide levels were measured by colorimetric assay kit. The acute toxicity study was also done for the complexes. The asthmatic group developed allergic airway inflammation shown by increased DTH and inflammatory markers in blood and BALF. OA + Fe and OA + Zn displayed significant decrease in DTH, NO, expression of IL-4, 5, 13, 17, toll-like receptor-2, nuclear factor-kappa B and tumor necrosis factor-α; serum IgE, COX-2, and 5-LOX. The metal complexes also attenuated OVA-stimulated splenocyte proliferation. While no hepatotoxic or nephrotoxic potential was shown by OA + Fe and OA + Zn. Our findings indicate that both OA + Fe and OA + Zn possess significant anti-asthmatic effect which may be ascribed to its immunomodulatory and anti-inflammatory features.

Computer modeling in predicting the bioactivity of human 5-lipoxygenase inhibitors.

5-Lipoxygenase (5-LOX) is a key enzyme in the inflammatory path. Inhibitors of 5-LOX are useful for the treatment of diseases like arthritis, cancer, and asthma. We have collected a dataset including 220 human 5-LOX inhibitors for classification. A self-organizing map (SOM), a support vector machine (SVM), and a multilayer perceptron (MLP) algorithm were used to build models with selected descriptors for classifying 5-LOX inhibitors into active and weakly active ones. MACCS fingerprints were used in this model building process. The accuracy (Q) and Matthews correlation coefficient (MCC) of the best SOM model (Model 1A) were 86.49% and 0.73 on the test set, respectively. The Q and MCC of the best SVM model (Model 2A) were 82.67% and 0.64 on the test set, respectively. The Q and MCC of the best MLP model (Model 3B) were 84.00% and 0.67 on the test set, respectively. In addition, 180 inhibitors with bioactivities measured by fluorescence method were further used for a quantitative prediction. Multiple linear regression (MLR) and SVM algorithms were used to build models to predict the [Formula: see text] values. The correlation coefficients (R) of the MLR model (Model Q1) and the SVM model (Model Q2) were 0.72 and 0.74 on the test set, respectively.

ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, possesses characteristic alterations to multiple metabolic pathways, including the accumulation of cytosolic lipid droplets. However, the pathways that drive lipid droplet accumulation in ccRCC cells and their importance to cancer biology remain poorly understood. METHODS: We sought to identify the carbon sources necessary for lipid droplet accumulation using Oil red O staining and isotope-tracing lipidomics. The role of the acyl-CoA synthetase (ACSL) family members, an important group of lipid metabolic enzymes, was investigated using siRNA and drug mediated inhibition. CTB and XTT assays were performed to determine the effect of ACSL3 knockdown and lipid starvation on ccRCC cell viability and shRNA was used to study the effect of ACSL3 in an orthotopic mouse model. The relationship between ferroptosis susceptibility of ccRCC and ACSL3 controlled lipid metabolism was examined using CTB and FACS-based assays. The importance of 5-LOX in ferroptosis susceptibility in ccRCC was shown with XTT survival assays, and the expression level and predictive value of 5-LOX in TCGA ccRCC data was assessed. RESULTS: We found that ccRCC cells obtain the necessary substrates for lipid droplet accumulation by metabolizing exogenous serum derived lipids and not through de novo lipogenesis. We show that this metabolism of exogenous fatty acids into lipid droplets requires the enzyme acyl-CoA synthetase 3 (ACSL3) and not other ACSL family proteins. Importantly, genetic or pharmacologic suppression of ACSL3 is cytotoxic to ccRCC cells in vitro and causes a reduction of tumor weight in an orthotopic mouse model. Conversely, ACSL3 inhibition decreases the susceptibility of ccRCC cells to ferroptosis, a non-apoptotic form of cell death involving lipid peroxidation. The sensitivity of ccRCC to ferroptosis is also highly dependent on the composition of exogenous fatty acids and on 5-lipoxygenase (5-LOX), a leukotriene producing enzyme which produces lipid peroxides that have been implicated in other cancers but not in ccRCC. CONCLUSIONS: ACSL3 regulates the accumulation of lipid droplets in ccRCC and is essential for tumor growth. In addition, ACSL3 also modulates ferroptosis sensitivity in a manner dependent on the composition of exogenous fatty acids. Both functions of ACSL3 could be exploited for ccRCC therapy.

Edaravone, a free radical scavenger, protects neuronal cells' mitochondria from ischemia by inactivating another new critical factor of the 5-lipoxygenase pathway affecting the arachidonic acid metabolism.

To investigate the neuroprotective effect of edaravone was dependent on 5-lipoxygenase (5-LOX) signalling pathway or not. Middle cerebral artery occlusion (MCAO) and oxygen glucose deprivation (OGD) were established in SD rats and PC12 cells to mimic ischemic injury. In vivo, edaravone can significantly reduce neurological deficit scores, infarct volume, ROS level and expression of 5-LOX. For in vitro experiment, reduced viability, cell death which occurred via necrosis and apoptosis were shown after OGD and even severer in OGD-reperfusion (OGD-R). Interestingly, edaravone (0.01, 0.1, 1 µmol/L) and caffeic acid (5-LOX inhibitor) can dramatically attenuate OGD/OGD-R injuries. Profoundly, mitochondrial transmembrane potential was ameliorated and cristae membranes (detected by electron microscope) were swollen in OGD/OGD-R cells; however, edaravone preserved the normal ultrastructure of mitochondria and reduced ROS. Astonishingly, immunohistochemistry analyses showed that 5-LOX was first located in the cytosol, dendrites and nuclei of control cells and then translocated to the nuclear membrane after OGD/OGD-R, which indicated the activation of 5-LOX pathway. Edaravone and caffeic acid can inhibit 5-LOX translocation to the nuclear membrane after OGD/OGD-R and reduce cysteinyl leukotrienes (CysLTs), which are metabolites of 5-LOX. Our results are the first to indicate that the protective action of edaravone may function, at least in part, by inhibiting 5-LOX activation, maintaining the ultrastructure and integrated function of mitochondria, thus protecting neuronal cells from ischemia. Furthermore, the instability of mitochondria may be another critical factor in 5-LOX activation.

Blockage of cytosolic phospholipase A2 alpha by monoclonal antibody attenuates focal ischemic brain damage in mice.

The purposes of the current study were to investigate the effects of a monoclonal antibody (mAb) on cytosolic phospholipase A2 alpha (cPLA2α) in mice with cerebral ischemia-reperfusion (IR) injury and to ascertain the potential mechanisms of those effects. This study evaluated whether the use of anti-cPLA2α mAb could reduce stroke injury in a mouse model of cerebral IR injury. The expression/activity of cPLA2α and cPLA2α- derived proinflammatory lipid mediators such as prostaglandin E2 (PGE2), leukotriene B4 (LTB4), lysophosphatidylcholine (LPC), and free fatty acids (FFA) was assessed. This study also evaluated neurological deficits, motor function, pathological changes, apoptosis, and the area of infarction in the injured mice. Mice treated with anti-cPLA2α mAb recovered neurological function and their condition improved, apoptosis in the brain decreased and infarct volume decreased, and expression of cPLA2α, 5-LOX, COX-2, FFA, LPC, PGE2, and LTB4 was attenuated. Our findings indicate that cPLA2α plays a key role in cerebral IR injury and that treatment with anti-cPLA2α mAb after cerebral IR injury helps to reduce levels of proinflammatory cytokines, alleviate tissue damage, and reduce levels of deleterious lipid mediators. Thus, anti-cPLA2α mAb treatment has the potential to treat ischemic brain damage.