Cytosolic Phospholipase A2 Activity Research Articles

Abstract 2097: Jak2 V617f Clonal Hematopoiesis Promotes Arterial Thrombosis Via Platelet Activation And Cross-talk

JAK2 V617F ( JAK2 VF ) clonal hematopoiesis (CH) is associated with a marked increase in atherothrombotic cardiovascular disease (CVD). The role of JAK2 VF CH in arterial thrombosis has not been assessed. Using mice simulating JAK2 VF CH, we showed accelerated carotid artery thrombosis, with increased platelet counts and activation and increased leukocytes and red blood cells. There was increased platelet activation in both Jak2 VF and wild type (WT) platelets in Jak2 VF CH suggesting cross-talk between mutant and WT platelets. To assess platelet-specific effects, we used Gp1ba-Cre (VFGp1ba) to selectively express Jak2 VF in platelets. VFGp1ba increased platelet counts to a similar level as in 20% Jak2 VF CH mice but had no effect on leukocyte counts. Like Jak2 VF CH, VFGp1ba increased platelet activation and accelerated arterial thrombosis. Mechanistically, Jak2 VF platelets showed 2.8-fold up-regulation of COX-1, increased activation of cytosolic phospholipase A2, an enzyme important for arachidonic acid generation, and increased thromboxane A2 production. Conditioned media from activated Jak2 VF platelets induced WT platelet activation that was reversed by a thromboxane receptor antagonist. Low dose aspirin ameliorated carotid thrombosis in VFGp1ba and Jak2 VF CH but not in WT control mice. This study shows increased arterial thrombosis in Jak2 VF CH related to Jak2 VF platelet activation and cross-talk with WT platelets via thromboxane, suggesting a potential beneficial role of aspirin treatment in JAK2 VF CH.

Gut microbial co-metabolite 2-methylbutyrylcarnitine exacerbates thrombosis via binding to and activating integrin α2β1

Thrombosis represents the leading cause of death and disability upon major adverse cardiovascular events (MACEs). Numerous pathological conditions such as COVID-19 and metabolic disorders can lead to a heightened thrombotic risk; however, the underlying mechanisms remain poorly understood. Our study illustrates that 2-methylbutyrylcarnitine (2MBC), a branched-chain acylcarnitine, is accumulated in patients with COVID-19 and in patients with MACEs. 2MBC enhances platelet hyperreactivity and thrombus formation in mice. Mechanistically, 2MBC binds to integrin α2β1 in platelets, potentiating cytosolic phospholipase A2 (cPLA2) activation and platelet hyperresponsiveness. Genetic depletion or pharmacological inhibition of integrin α2β1 largely reverses the pro-thrombotic effects of 2MBC. Notably, 2MBC can be generated in a gut-microbiota-dependent manner, whereas the accumulation of plasma 2MBC and its thrombosis-aggravating effect are largely ameliorated following antibiotic-induced microbial depletion. Our study implicates 2MBC as a metabolite that links gut microbiota dysbiosis to elevated thrombotic risk, providing mechanistic insight and a potential therapeutic strategy for thrombosis.

Neuroprotective Potential of Polydatin in Combating Parkinson’s Disease through the Inhibition of Monoamine Oxidase-B and Catechol-o-Methyl Transferase

Aims: This study aims to deduce the plausible contribution of polydatin in dopamine replenishment and dopaminergic neuroprotection to unveil its potential as a drug candidate for PD. Background: Available therapies for the management of dopaminergic degradation in Parkinson’s disease (PD) provide only symptomatic relief and are associated with various adverse effects. Levodopa (L-DOPA) is an age-old therapy in the treatment paradigm of PD, either used as mono-therapy or in combination with the inhibitors of the dopamine catabolising enzymes monoamine oxidase-B (MAO-B) and catechol O-methyltransferase (COMT) for replenishing the levels of the neurotransmitter. The discovery of plant-based novel drug therapies would help to target multiple pathways underlying the disease pathogenesis and are associated with minimal side effects. Polydatin, the precursor of resveratrol, has been explored recently to possess neuroprotective efficacy, however, the molecular mechanisms that underlie the Parkinsonism-associated neurobehavioral recovery as well as the neurorescue potential of polydatin has not been illustrated yet. Objective: The present study aimed to unveil the role of polydatin in dopamine upregulation in PD, by determining its dual inhibitory potential on the enzymes responsible for its breakdown, MAO-B and COMT. The study further aimed to elucidate the role of this molecule in regulating the enzymatic activity of Cytosolic Phospholipase A2 (cPLA2), the crucial enzyme underlying several pathogenic pathways leading to neurodegeneration. Methods: Molecular docking simulation of polydatin with the dopamine catabolizing enzymes MAO-B and COMT, as well as cPLA2, along with their respective known inhibitors was performed using the Molegro Virtual Docker (MVD) 2.1 package. Results: In-silico analyses revealed that polydatin could significantly inhibit the activities of this dopamine catabolizing enzyme, MAO-B, and COMT with comparable docking scores and more numbers of hydrogen bonds, and weaker interactions as that of their respective available synthetic inhibitors. Moreover, it was found that polydatin could regulate the activity of cPLA2 comparable to its known inhibitors. Conclusion: Polydatin exhibited efficacy as a potent dopamine replenishing agent by inhibiting its metabolizing enzymes as well as found to have efficacy against neuroinflammation, thereby highlighting the significance of designing novel phyto drugs for combating dopamine deficiency in PD.

Protective effect of arctiin against Toxoplasma gondii HSP70-induced allergic acute liver injury by disrupting the TLR4-mediated activation of cytosolic phospholipase A2 and platelet-activating factor

Toxoplasma gondii (T. gondii)-derived heat shock protein 70 (T.g.HSP70) is a toxic protein that downregulates host defense responses against T. gondii infection. T.g.HSP70 was proven to induce fatal anaphylaxis in T. gondii infected mice through cytosolic phospholipase A2 (cPLA2) activated-platelet-activating factor (PAF) production via Toll-like receptor 4 (TLR4)-mediated signaling. In this study, we investigated the effect of arctiin (ARC; a major lignan compound of Fructus arctii) on allergic liver injury using T.g.HSP70-stimulated murine liver cell line (NCTC 1469) and a mouse model of T. gondii infection. Localized surface plasmon resonance, ELISA, western blotting, co-immunoprecipitation, and immunofluorescence were used to investigate the underlying mechanisms of action of ARC on T. gondii-induced allergic acute liver injury. The results showed that ARC suppressed the T.g.HSP70-induced allergic liver injury in a dose-dependent manner. ARC could directly bind to T.g.HSP70 or TLR4, interfering with the interaction between these two factors, and inhibiting activation of the TLR4/mitogen-activated protein kinase/nuclear factor-kappa B signaling, thereby inhibiting the overproduction of cPLA2, PAF, and interferon-γ. This result suggested that ARC ameliorates T.g.HSP70-induced allergic acute liver injury by disrupting the TLR4-mediated activation of inflammatory mediators, providing a theoretical basis for ARC therapy to improve T.g.HSP70-induced allergic liver injury.

Thrombin Induced Apoptosis through Calcium-Mediated Activation of Cytosolic Phospholipase A2 in Intestinal Myofibroblasts.

Thrombin is a serine protease that participates in a variety of biological signaling through protease-activated receptors. Intestinal myofibroblasts play central roles in maintaining intestinal homeostasis. In this study, we found that thrombin-induced apoptosis is mediated by the calcium-mediated activation of cytosolic phospholipase A2 in the CCD-18Co cell. Thrombin reduced cell viability by inducing apoptosis and proteinase-activated receptor-1 antagonist attenuated thrombin-induced cell death. Endogenous ceramide did not affect the cell viability itself, but a ceramide-mediated pathway was involved in thrombin-induced cell death. Thrombin increased intracellular calcium levels and cytosolic phospholipase A2 activity. The ceramide synthase inhibitor Fumonisin B1, intracellular calcium chelator BAPTA-AM, and cytosolic phospholipase A2 inhibitor AACOCF3 inhibited thrombin-induced cell death. Thrombin stimulated arachidonic acid release and reactive oxygen species generation, which was blocked by AACOCF3, BAPTA-AM, and the antioxidant reagent Trolox. Taken together, thrombin triggered apoptosis through calcium-mediated activation of cytosolic phospholipase A2 in intestinal myofibroblasts.

Calcium-dependent cytosolic phospholipase A2 activation is implicated in neuroinflammation and oxidative stress associated with ApoE4

BackgroundApolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer’s disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known.MethodsMouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress.ResultsGreater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of persons with AD dementia carrying APOE3/4 compared to APOE3/3. Higher phosphorylated p38 MAPK but not ERK1/2 was found in ApoE4 primary astrocytes and mouse brains than that in ApoE3. Greater cPLA2 translocation to cytosol was observed in human postmortem frontal cortical synaptosomes with recombinant ApoE4 than ApoE3 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition.ConclusionsOur findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.

Modified lipid metabolism and cytosolic phospholipase A2 activation in mesangial cells under pro-inflammatory conditions.

Diabetic kidney disease is a consequence of hyperglycemia and other complex events driven by early glomerular hemodynamic changes and a progressive expansion of the mesangium. The molecular mechanisms behind the pathophysiological alterations of the mesangium are yet to be elucidated. This study aimed at investigating whether lipid signaling might be the missing link. Stimulation of human mesangial cells with high glucose primed the inflammasome-driven interleukin 1 beta (IL-1β) secretion, which in turn stimulated platelet-derived growth factor (PDGF-BB) release. Finally, PDGF-BB increased IL-1β secretion synergistically. Both IL-1β and PDGF-BB stimulation triggered the formation of phosphorylated sphingoid bases, as shown by lipidomics, and activated cytosolic phospholipase cPLA2, sphingosine kinase 1, cyclooxygenase 2, and autotaxin. This led to the release of arachidonic acid and lysophosphatidylcholine, activating the secretion of vasodilatory prostaglandins and proliferative lysophosphatidic acids. Blocking cPLA2 release of arachidonic acid reduced mesangial cells proliferation and prostaglandin secretion. Validation was performed in silico using the Nephroseq database and a glomerular transcriptomic database. In conclusion, hyperglycemia primes glomerular inflammatory and proliferative stimuli triggering lipid metabolism modifications in human mesangial cells. The upregulation of cPLA2 was critical in this setting. Its inhibition reduced mesangial secretion of prostaglandins and proliferation, making it a potential therapeutical target.

MO614: Modified Lipid Metabolism and Cytosolic Phospholipase A2 Activation in Mesangial Cells Under Pro-Inflammatory Conditions

Abstract BACKGROUND AND AIMS Diabetic kidney disease (DKD) is thought to be a consequence of hyperglycemia and driven by early glomerular hemodynamic changes and progressive expansion of the mesangium. The molecular mechanism behind the pathophysiological alterations of the mesangium is yet to be elucidated. This study aimed at investigating whether lipid signaling might be the missing link. METHOD Human mesangial cells (HMCs) were stimulated with high glucose, IL-1β or PDGF-BB. Production of IL-1β and PDGF-BB, COX-2 induction and cPLA2 activation and prostaglandin secretion were investigated using a combination of ELISA, western blot, lipidomics, qPCR and activity assays. Inhibition of cPLA2 was evaluated using ELISA, migration and proliferation assays. Validation of data was performed in silico using the Nephroseq database [1] and a recently published glomerular transcriptomic DKD database. [2] RESULTS Stimulation of HMCs with high glucose primed IL-1β secretion through inflammasome NLRP3 signaling. In turn, IL-1β was able to stimulate PDGF-BB release. PDGF-BB increased IL-1β secretion synergistically. Both IL-1β and PDGF-BB stimuli caused lipid metabolism variations and in particular triggered the formation of phosphorylated sphingoid bases (PSBs) and the activation of the enzymes cPLA2 and COX-2. This led to the formation of prostanoids from arachidonic acid produced by cPLA2. As a result, prostaglandins were secreted. Another product of cPLA2 activation was lysophosphatidic acid (LPA), a proliferation effector. Blocking cPLA2-mediated release of arachidonic acid could in fact reduce HMCs’ proliferation and secretion of prostaglandins. CONCLUSION Hyperglycemia primes glomerular inflammatory and proliferative stimuli triggering lipid metabolism modifications in HMCs. The upregulation of cPLA2 was found to be critical in this setting. cPLA2 inhibition with AACOCF3 reduced mesangial secretion of prostaglandins and proliferation, making it a potential target for DKD therapy.

Retraction Note: Calcium-dependent cytosolic phospholipase A2 activation is implicated in neuroinflammation and oxidative stress associated with ApoE4

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s13024-021-00438-3.

TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

The role of the Ca2+-permeable ion channel TRPC5 in regulating vasocontraction in obesity is poorly understood. Here, we investigated whether TRPC5 contributes to vascular dysfunction in obesity by promoting endothelium-dependent contraction via activation of cytosolic phospholipase A2 (cPLA2) in the aortic endothelial cells of obese mice. Acetylcholine-induced endothelium-dependent relaxation and contraction in the aorta were measured using wire myography. PLA2 activity was measured by the fluorogenic PLA2 substrate Bis-BODIPY™ FL C11-PC. The intracellular Ca2+ level in response to acetylcholine was measured by Fluo-4 fluorescence. Endothelium-derived contracting factors were assessed by enzyme immunoassay. Diet-induced obesity (DIO) attenuated endothelium-dependent vasodilation, enhanced endothelium-dependent contraction (EDC), and increased the expression of TRPC5 in the mouse aorta. Activation of TRPC5 promoted EDC in the wild-type mouse aorta, whereas pharmacological inhibition and genetic knockout of TRPC5 decreased EDC in the DIO mouse aorta. Moreover, cPLA2 phosphorylation and activity were higher in aortic endothelial cells from DIO mice, and this was attenuated by inhibition and knockout of TRPC5. Cyclooxygenase 2 (COX-2) expression was increased in DIO mouse endothelium and was decreased by a TRPC5 inhibitor and knockout of TRPC5. Release of prostaglandins F2α (PGF2α) and E2 (PGE2) was involved in TRPC5-regulated EDC in DIO mice. This study demonstrated that TRPC5 contributes to endothelial and vascular dysfunction and is involved in EDC through activation of cPLA2 and enhanced COX-2-PGF2α/PGE2 levels in DIO mice.

Membrane phosphatidylserine allosterically regulates the cytosolic phospholipase A2 activity via an electrostatic-switch mechanism.

Phospholipase A2 (PLA2) is a peripheral membrane protein that plays an essential role in many inflammatory responses. However, the activation mechanisms of PLA2 on the membrane surface have not been fully understood. Herein, we have combined experimental techniques and theoretical approaches to investigate the activation and association of the PLA2 protein on an artificial phospholipid membrane. Using a phosphatidylserine (PS) nanodomain containing membrane to mimic the inflammatory conditions, we found that the activity of cytosolic PLA2s (cPLA2s) increases with higher ratios of PS in the membrane. Molecular dynamics simulations reveal that significant changes in the protein structure are related to negatively charged membranes. In particular, the alteration of negatively charged residues in the C2 domain brings about an opened binding pocket and the catalytic site access to the substrate phospholipid. Meanwhile, the negative residues in the loop 650-665 facilitate the optimal interfacial orientation of the protein with a closed binding pocket on the membrane surface. These results lead us to suggest an electrostatic-switch allosteric mechanism for cPLA2 activation on the cell membrane surface under the inflammatory state.

Early upregulation of cytosolic phospholipase A2\u03b1 in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by the selective death of motor neurons. Cytosolic phospholipase A2 alpha (cPLA2α) upregulation and activation in the spinal cord of ALS patients has been reported. We have previously shown that cPLA2α upregulation in the spinal cord of mutant SOD1 transgenic mice (SOD1G93A) was detected long before the development of the disease, and inhibition of cPLA2α upregulation delayed the disease’s onset. The aim of the present study was to determine the mechanism for cPLA2α upregulation.MethodsImmunofluorescence analysis and western blot analysis of misfolded SOD1, cPLA2α and inflammatory markers were performed in the spinal cord sections of SOD1G93A transgenic mice and in primary motor neurons. Over expression of mutant SOD1 was performed by induction or transfection in primary motor neurons and in differentiated NSC34 motor neuron like cells.ResultsMisfolded SOD1 was detected in the spinal cord of 3 weeks old mutant SOD1G93A mice before cPLA2α upregulation. Elevated expression of both misfolded SOD1 and cPLA2α was specifically detected in the motor neurons at 6 weeks with a high correlation between them. Elevated TNFα levels were detected in the spinal cord lysates of 6 weeks old mutant SOD1G93A mice. Elevated TNFα was specifically detected in the motor neurons and its expression was highly correlated with cPLA2α expression at 6 weeks. Induction of mutant SOD1 in primary motor neurons induced cPLA2α and TNFα upregulation. Over expression of mutant SOD1 in NSC34 cells caused cPLA2α upregulation which was prevented by antibodies against TNFα. The addition of TNFα to NSC34 cells caused cPLA2α upregulation in a dose dependent manner.ConclusionsMotor neurons expressing elevated cPLA2α and TNFα are in an inflammatory state as early as at 6 weeks old mutant SOD1G93A mice long before the development of the disease. Accumulated misfolded SOD1 in the motor neurons induced cPLA2α upregulation via induction of TNFα.

The binding of autotaxin to integrins mediates hyperhomocysteinemia-potentiated platelet activation and thrombosis in mice and humans

AbstractHyperhomocysteinemia (HHcy) is associated with an exaggerated platelet thrombotic response at sites of vascular injury. In this study, human medical examination showed that elevated human plasma Hcy levels correlated positively with enhanced blood coagulation and platelet activity, suggesting that humans with HHcy are more prone to thrombus formation at the sites of vascular injury. Accordingly, we observed accelerated platelet activation, primary hemostasis, and thrombus formation in apolipoprotein E-deficient (ApoE−/−) mice with acute or chronic HHcy. Upon homocysteine (Hcy) administration in C57BL/6J mice, platelet aggregation, spreading and clot retraction were markedly induced. More important, Hcy increased the affinity of platelet integrin αIIbβ3 with ligands and enhanced integrin outside-in signaling by promoting membrane phosphatidylserine exposure in vitro. Mechanistically, lipidomics analysis showed that lysophosphatidylcholines were the primary metabolites leading to clustering of HHcy-stimulated platelets. Cytosolic phospholipase A2 (cPLA2) activity and autotaxin (ATX, a secreted lysophospholipase D) secretion were upregulated by Hcy, leading to membrane phospholipid hydrolysis and PS exposure. Moreover, secreted ATX directly interacted with integrin β3. Inhibitors of cPLA2 and ATX activity blocked integrin αIIbβ3 outside-in signaling and thrombosis in HHcy ApoE−/− mice. In this study, we identified a novel mechanism by which HHcy promotes platelet membrane phospholipid catabolism and extracellular ATX secretion to activate integrin outside-in signaling, consequently exacerbating thrombosis and the results revealed an innovative approach to treating HHcy-related thrombotic diseases.

Tyrosine phosphatase STEP is a key regulator of glutamate-induced prostaglandin E2 release from neurons

The neuron-specific tyrosine phosphatase striatal-enriched phosphatase (STEP) is emerging as a key regulator of excitotoxicity, which is involved in the pathogenesis of both acute and chronic neurological diseases. However, the intracellular mechanisms that are regulated by STEP to confer neuroprotection against excitotoxic insults are not well understood. The present study investigates the role of STEP in regulating neuronal release of the proinflammatory prostanoid prostaglandin E2 (PGE2), which is associated with a wide range of pathological conditions. The findings show that glutamate-mediated activation of the N-methyl-D-aspartic acid receptor in STEP-deficient neurons leads to rapid and sustained increase in the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), a signaling molecule involved in the production of inflammatory mediators. Such sustained p38 MAPK activation increases the activity of cytosolic phospholipase A2, which catalyzes the release of arachidonic acid, the initial substrate for PGE2 biosynthesis. Sustained p38 MAPK activation also induces nuclear factor-κB–mediated increase in expression of cyclooxygenase-2 that is involved in the conversion of arachidonic acid to prostanoids, resulting in enhanced biosynthesis and release of PGE2 from neurons. Restoration of STEP function with a STEP mimetic (TAT-STEP-myc peptide) significantly decreases the activation of p38 MAPK–mediated cytosolic phospholipase A2/cyclooxygenase-2/PGE2 signaling cascade. This study identifies an important mechanism involved in the neuronal release of the proinflammatory mediator PGE2 after excitotoxic insult and highlights for the first time the immunomodulatory ability of a neuronal tyrosine phosphatase.

RETRACTED ARTICLE: Calcium-dependent cytosolic phospholipase A2 activation is implicated in neuroinflammation and oxidative stress associated with ApoE4

BackgroundApolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer’s disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known.MethodsMouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress.ResultsGreater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of patients with AD carrying APOE3/E4 compared to APOE3/E3. Greater cPLA2 phosphorylation was also observed in human postmortem frontal cortical synaptosomes and primary astrocytes after treatment with recombinant ApoE4 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition.ConclusionsOur findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.

Berberine Suppresses Colonic Inflammation in Dextran Sulfate Sodium-Induced Murine Colitis Through Inhibition of Cytosolic Phospholipase A2 Activity.

Ulcerative colitis (UC) causes chronic inflammation and damage to the colonic mucosal layer. Recent studies have reported significant changes in phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in UC patients and oral administration of PC has considerable therapeutic effects against UC, suggesting the metabolism of phosphatidylcholine may be involved in the UC development. Our previous work has demonstrated that berberine effectively suppresses inflammation and protects colonic mucosa injury in DSS-induced colitic mice. However, whether the therapeutic effects of berberine are attributed to its action on the PC metabolism remains unknown. In the present study, we have shown that berberine significantly reduces the lysophosphatidylcholine (LPC) levels in the sera of DSS-induced experimental colitis mice and LPS-stimulated macrophage RAW 264.7 cells. The cytosolic phospholipase A2a (PLA2G4A), an enzyme for hydrolyzing PC to LPC, was found to be up-regulated in the colon tissue of experimental colitis mice and inflamed macrophage RAW 264.7 cells. We then demonstrated berberine inhibits the phosphorylation of cytosolic phospholipase A2a (PLA2G4A) in the colon tissue of experimental colitis mice and inflamed macrophage RAW 264.7 cells. Subsequently, we revealed berberine suppressed the expression of pro-inflammatory factors including TNF-alpha and IL-6 through regulating PLA2G4A dysfunction in macrophage RAW 264.7 cells. Mechanistically, we found that berberine directly binds to PLA2G4A and inhibits MAPK/JNK signaling pathway to inhibit PLA2G4A activity in inflammatory status. Therefore, we concluded that berberine inhibits colonic PLA2G4A activity to ameliorate colonic inflammation in experimental colitic mice, suggesting modulation of the PC metabolism via PLA2G4A might be beneficial for establishing new therapies strategy for UC.

P.441 Impact of CYP2C19 and CYP2D6 polymorphism on antidepressants and antipsychotics exposure; a meta-analysis

Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2−) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2− release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2−. However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs’ function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell.

Neutral Lipid Cacostasis Contributes to Disease Pathogenesis in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by motor neuron (MN) death. Lipid dysregulation manifests during disease; however, it is unclear whether lipid homeostasis is adversely affected in the in the spinal cord gray matter (GM), and if so, whether it is because of an aberrant increase in lipid synthesis. Moreover, it is unknown whether lipid dysregulation contributes to MN death. Here, we show that cholesterol ester (CE) and triacylglycerol levels are elevated several-fold in the spinal cord GM of male sporadic ALS patients. Interestingly, HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, was reduced in the spinal cord GM of ALS patients. Increased cytosolic phospholipase A2 activity and lyso-phosphatidylcholine (Lyso-PC) levels in ALS patients suggest that CE accumulation was driven by acyl group transfer from PC to cholesterol. Notably, Lyso-PC, a byproduct of CE synthesis, was toxic to human MNs in vitro Elevations in CE, triacylglycerol, and Lyso-PC were also found in the spinal cord of SOD1G93A mice, a model of ALS. Similar to ALS patients, a compensatory downregulation of cholesterol synthesis occurred in the spinal cord of SOD1G93A mice; levels of sterol regulatory element binding protein 2, a transcriptional regulator of cholesterol synthesis, progressively declined. Remarkably, overexpressing sterol regulatory element binding protein 2 in the spinal cord of normal mice to model CE accumulation led to ALS-like lipid pathology, MN death, astrogliosis, paralysis, and reduced survival. Thus, spinal cord lipid dysregulation in ALS likely contributes to neurodegeneration and developing therapies to restore lipid homeostasis may lead to a treatment for ALS.SIGNIFICANCE STATEMENT Neurons that control muscular function progressively degenerate in patients with amyotrophic lateral sclerosis (ALS). Lipid dysregulation is a feature of ALS; however, it is unclear whether disrupted lipid homeostasis (i.e., lipid cacostasis) occurs proximal to degenerating neurons in the spinal cord, what causes it, and whether it contributes to neurodegeneration. Here we show that lipid cacostasis occurs in the spinal cord gray matter of ALS patients. Lipid accumulation was not associated with an aberrant increase in synthesis or reduced hydrolysis, as enzymatic and transcriptional regulators of lipid synthesis were downregulated during disease. Last, we demonstrated that genetic induction of lipid cacostasis in the CNS of normal mice was associated with ALS-like lipid pathology, astrogliosis, neurodegeneration, and clinical features of ALS.

Role of GluN2A NMDA receptor in homocysteine-induced prostaglandin E2 release from neurons.

Hyperhomocysteinemia or systemic elevation of homocysteine is a metabolic condition that has been linked to multiple neurological disorders where inflammation plays an important role in the progression of the disease. However, it is unclear whether hyperhomocysteinemia contributes to disease pathology by inducing an inflammatory response. The current study investigates whether exposure of primary cultures from rat and mice cortical neurons to high levels of homocysteine induces the expression and release of the proinflammatory prostanoid, Prostaglandin E2 (PGE2). Using enzymatic assays and immunoblot analysis we show concurrent increase in the activity of cytosolic phospholipase A2 (cPLA2) and level of cyclooxygenase-2 (COX2), two enzymes involved in PGE2 biosynthesis. The findings also show an increase in PGE2 release from neurons. Pharmacological inhibition of GluN2A-containing NMDAR (GluN2A-NMDAR) with NVP-AAM077 significantly reduces homocysteine-induced cPLA2 activity, COX2 expression, and subsequent PGE2 release. Whereas, inhibition of GluN2B-containing NMDAR (GluN2A-NMDAR) with Ro 25-6981 has no effect. Complementary studies in neuron cultures obtained from wild type and GluN2A knockout mice show that genetic deletion of GluN2A subunit of NMDAR attenuates homocysteine-induced neuronal increase in cPLA2 activity, COX2 expression, and PGE2 release. Pharmacological studies further establish the role of both extracellular-regulated kinase/mitogen-activated protein kinase and p38 MAPK in homocysteine-GluN2A NMDAR-dependent activation of cPLA2-COX2-PGE2 pathway. Collectively, these findings reveal a novel role of GluN2A-NMDAR in facilitating homocysteine-induced proinflammatory response in neurons.

Su1806 – Berberine Regulates Lysophosphatidylcholine to Protect Dss-Induced Murine Colitis Through Inhibition of Cytosolic Phospholipase A2 Activities

Su1806 – Berberine Regulates Lysophosphatidylcholine to Protect Dss-Induced Murine Colitis Through Inhibition of Cytosolic Phospholipase A2 Activities