Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

Cdk5-mediated oligodendrocyte myelin breakdown and neuroinflammation: Implications for the link between Type 2 Diabetes and Alzheimer's disease.

Oligodendrocytes, crucial myelinating glia in the central nervous system, play a vital role in maintaining axonal integrity and facilitating efficient nerve impulse conduction. The degradation of myelin in oligodendrocytes has been implicated in Alzheimer's disease (AD) and cognitive dysfunction. Interestingly, individuals with Type 2 Diabetes (T2D) have a significantly higher likelihood of developing cognitive impairment, possibly due to insulin resistance and glucose toxicity within the central nervous system (CNS). However, the precise relationship between these two disorders remains elusive. Our study proposes a potential link between T2D and AD, involving Cdk5-mediated breakdown of oligodendrocyte myelin and neuroinflammation. In the context of T2D, glucose toxicity in oligodendrocytes leads to heightened Cdk5 kinase activity and cPLA2 hyperactivation, resulting in chronic inflammation and myelin deterioration. This myelin breakdown in oligodendrocytes is thought to contribute to the development of AD and cognitive dysfunction. Notably, the administration of a Cdk5 inhibitor (TFP5) effectively alleviates neuroinflammation and myelin degradation. Moreover, our findings demonstrate heightened activity of Cdk5, cPLA2, and phospho-cPLA2 levels in the brain of a mouse model with Type 2 Diabetes (T2D). Hence, our findings suggest that targeting Cdk5 could be a promising therapeutic strategy to counteract AD pathogenesis in T2D-related conditions.

Alveolar macrophage-derived gVPLA2 promotes ventilator-induced lung injury via the cPLA2/PGE2 pathway.

BACKGROUND: Ventilator-induced lung injury (VILI) is a clinical complication of mechanical ventilation observed in patients with acute respiratory distress syndrome. It is characterized by inflammation mediated by inflammatory cells and their secreted mediators. METHODS: To investigate the mechanisms underlying VILI, a C57BL/6J mouse model was induced using high tidal volume (HTV) mechanical ventilation. Mice were pretreated with Clodronate liposomes to deplete alveolar macrophages or administered normal bone marrow-derived macrophages or Group V phospholipase A2 (gVPLA2) intratracheally to inhibit bone marrow-derived macrophages. Lung tissue and bronchoalveolar lavage fluid (BALF) were collected to assess lung injury and measure Ca2 + concentration, gVPLA2, downstream phosphorylated cytoplasmic phospholipase A2 (p-cPLA2), prostaglandin E2 (PGE2), protein expression related to mitochondrial dynamics and mitochondrial damage. Cellular experiments were performed to complement the animal studies. RESULTS: Depletion of alveolar macrophages attenuated HTV-induced lung injury and reduced gVPLA2 levels in alveolar lavage fluid. Similarly, inhibition of alveolar macrophage-derived gVPLA2 had a similar effect. Activation of the cPLA2/PGE2/Ca2 + pathway in alveolar epithelial cells by gVPLA2 derived from alveolar macrophages led to disturbances in mitochondrial dynamics and mitochondrial dysfunction. The findings from cellular experiments were consistent with those of animal experiments. CONCLUSIONS: HTV mechanical ventilation induces the secretion of gVPLA2 by alveolar macrophages, which activates the cPLA2/PGE2/Ca2 + pathway, resulting in mitochondrial dysfunction. These findings provide insights into the pathogenesis of VILI and may contribute to the development of therapeutic strategies for preventing or treating VILI.

Apolipoprotein C-III itself stimulates the Syk/cPLA2-induced inflammasome activation of macrophage to boost anti-tumor activity of CD8+ T cell.

Increased prevalence of cancer in obese individuals is involved with dyslipidemia- induced chronic inflammation and immune suppression. Although apolipoprotein C-III (ApoC3)-transgenic mice (ApoC3TG mice) or poloxamer 407 (P407)-treated mice had hyperlipidemia, CD8+ T cells with upregulated antitumor activities were observed in ApoC3TG mice, and decreased CD8+ T cell activities were observed in P407-treated mice. Increased ApoC3 expression in hepatocellular carcinoma was associated with increased infiltration of CD8+ T cells and predicted survival. Recombinant ApoC3 had no direct effects on CD8+ T cells. The upregulation of CD8+ T cells in ApoC3TG mice was due to cross-talk with context cells, as indicated by metabolic changes and RNA sequencing results. In contrast to dendritic cells, the macrophages of ApoC3TG mice (macrophagesTG) displayed an activated phenotype and increased IL-1ß, TNF-α, and IL-6 production. Coculture with macrophagesTG increased CD8+ T cell function, and the adoptive transfer of macrophagesTG suppressed tumor progression in vivo. Furthermore, spleen tyrosine kinase (Syk) activation induced by TLR2/TLR4 cross-linking after ApoC3 ligation promoted cellular phospholipase A2 (cPLA2) activation, which in turn activated NADPH oxidase 2 (NOX2) to promote an alternative mode of inflammasome activation. Meanwhile, mitochondrial ROS produced by increased oxidative phosphorylation of free fatty acids facilitated the classical inflammasome activation, which exerted an auxiliary effect on inflammasome activation of macrophagesTG. Collectively, the increased antitumor activity of CD8+ T cells was mediated by the ApoC3-stimulated inflammasome activation of macrophages, and the mimetic ApoC3 peptides that can bind TLR2/4 could be a future strategy to target liver cancer.

[Electroacupuncture improves limb motor function by down-regulating cytosolic phospholi-pase A2 and reducing apoptosis of nerve cells in rats with spinal cord injury].

OBJECTIVE: To observe the effect of electroacupuncture（EA） on the expression of cytosolic phospholipase A2 （cPLA2） and apoptosis of nerve cells in rats with spinal cord injury （SCI）, so as to explore its mechanisms underlying improvement of SCI. METHODS: Seventy-two female SD rats were randomly divided into model, EA, antagonist and EA+antagonist groups, with 18 rats in each group and other 18 rats were used as the sham operation （sham） group. The SCI model was established by referring to modified Allen's method with a weight impactor. The hindlimb motor function was assessed by using Basso-Beattie-Bresnahan （BBB） score. Rats of the EA group were subjected to EA stimulation at "Dazhui"（GV14）, "Yaoyangguan"（GV3）, bilateral "Ciliao"（BL32） and "Zusanli"（ST36） for 20 min, once a day for 14 days. Rats of the antagonist group received intravenous injection followed by intraperitoneal injection of arachidonyl trifluoromethyl ketone （AACOCF3, antagonist of cPLA2）, once every other day. Rats of the EA+antagonist group received EA treatment combined with antagonist injection. After the treatment, the rats were sacrificed and the spinal cord tissue was collected for detecting the protein expression of cPLA2, p-cPLA2, Bcl-2, Bax and Caspase-3 by Western blot, and the mRNA expression of cPLA2, Bcl-2, Bax and Caspase-3 using qRT-PCR. The morphological changes of the spinal cord were detected by Nissl staining. RESULTS: In comparison with the sham group, the BBB score, expression of Bcl-2 protein and mRNA were significantly down-regulated （P<0.01）, whereas the expression levels of Bax, Caspase-3 and p-cPLA2 proteins and mRNAs were considerably up-regulated in the model group （P<0.01）. Compared with the model group, the BBB score, expression levels of Bcl-2 protein and mRNA were significantly up-regulated （P<0.01, P<0.05）, while the expression levels of Bax, Caspase-3 and p-cPLA2 proteins in the EA, antagonist and EA+antagonist groups, Bax and cPLA2 mRNAs in both antagonist and EA+antagonist groups, and Caspase-3 mRNA in the EA+antagonist group were obviously down-regulated （P<0.01, P<0.05）. The effect of EA+antagonist was significantly superior to EA in increasing BBB score and in lowering expression of Bax and cPLA2 mRNAs （P<0.01, P<0.05）. Nissl staining showed reduced number of nerve cells and Nissl bodies, and striped dark blue cells in the model group, which was milder in the EA and antagonist groups, particularly in the EA+antagonist group. CONCLUSION: EA may improve the limb motor function of SCI rats, which may be related to its functions in down-regulating the expression of p-cPLA2, Bax and Caspase-3 and up-regulating Bcl-2 to reduce the apoptosis of nerve cells in the regional spinal cord.

Structure Activity Relationship and Molecular Docking of Some Quinazolines Bearing Sulfamerazine Moiety as New 3CLpro, cPLA2, sPLA2 Inhibitors.

The current work was conducted to synthesize several novel anti-inflammatory quinazolines having sulfamerazine moieties as new 3CLpro, cPLA2, and sPLA2 inhibitors. The thioureido derivative 3 was formed when compound 2 was treated with sulfamerazine. Also, compound 3 was reacted with NH2-NH2 in ethanol to produce the N-aminoquinazoline derivative. Additionally, derivative 4 was reacted with 4-hydroxy-3-methoxybenzaldehyde, ethyl chloroacetate, and/or diethyl oxalate to produce quinazoline derivatives 5, 6, and 12, respectively. The results of the pharmacological study indicated that the synthesized 4-6 and 12 derivatives showed good 3CLpro, cPLA2, and sPLA2 inhibitory activity. The IC50 values of the target compounds 4-6, and 12 against the SARS-CoV-2 main protease were 2.012, 3.68, 1.18, and 5.47 µM, respectively, whereas those of baicalein and ivermectin were 1.72 and 42.39 µM, respectively. The IC50 values of the target compounds 4-6, and 12 against sPLA2 were 2.84, 2.73, 1.016, and 4.45 µM, respectively, whereas those of baicalein and ivermectin were 0.89 and 109.6 µM, respectively. The IC50 values of the target compounds 4-6, and 12 against cPLA2 were 1.44, 2.08, 0.5, and 2.39 µM, respectively, whereas those of baicalein and ivermectin were 3.88 and 138.0 µM, respectively. Also, incubation of lung cells with LPS plus derivatives 4-6, and 12 caused a significant decrease in levels of sPLA2, cPLA2, IL-8, TNF-α, and NO. The inhibitory activity of the synthesized compounds was more pronounced compared to baicalein and ivermectin. In contrast to ivermectin and baicalein, bioinformatics investigations were carried out to establish the possible binding interactions between the newly synthesized compounds 2-6 and 12 and the active site of 3CLpro. Docking simulations were utilized to identify the binding affinity and binding mode of compounds 2-6 and 12 with the active sites of 3CLpro, sPLA2, and cPLA2 enzymes. Our findings demonstrated that all compounds had outstanding binding affinities, especially with the key amino acids of the target enzymes. These findings imply that compound 6 is a potential lead for the development of more effective SARS-CoV-2 Mpro inhibitors and anti-COVID-19 quinazoline derivative-based drugs. Compound 6 was shown to have more antiviral activity than baicalein and against 3CLpro. Furthermore, the IC50 value of ivermectin against the SARS-CoV-2 main protease was revealed to be 42.39 µM, indicating that it has low effectiveness.

Co-exposure to Fe, Zn, and Cu induced neuronal ferroptosis with associated lipid metabolism disorder via the ERK/cPLA2/AA pathway.

Excessive amounts of iron (Fe), zinc (Zn), and copper (Cu) can be toxic to neuronal cells, even though these are essential trace elements for animals and humans. However, the precise mechanisms underlying the neurotoxicity of exposure to mixtures of Fe, Zn, and Cu are still mostly unclear. The research aimed to investigate the influence of co-exposure to iron, zinc and copper and the related mechanisms in HT22 murine hippocampal neuronal cells. Intracellular metal content, markers of oxidative damage, and biomarkers of ferroptosis were respectively detected. Afterward, metabolomic analyses were performed to obtain a comprehensive understanding of the metal mixtures on metabolism, and the functions of key enzymes on metabolic pathways were validated. The results showed that metal co-exposure resulted in cellular iron overload and increased lipid peroxidation, accompanied by significant pathological damage and mitochondrial abnormalities in HT22 cells. Meanwhile, it was found that GSH depletion, decreased GPX4, and increased expression of the lipid metabolism gene ACSL4 play important roles in ferroptosis induced by metal mixture. Further, metabolomic analysis revealed metal co-exposure induced significant alterations in metabolite levels, especially in the glycerophospholipid metabolism pathway and the arachidonic acid metabolism pathway. The levels of cPLA2 and its metabolite, arachidonic acid, were significantly increased after metal co-exposure. Then, inhibition of cPLA2 decreased the level of arachidonic acid and attenuated ferroptosis in neuronal cells. Collectively, our findings unveiled ferroptosis induced by metal co-exposure associated with crucial molecular changes in neuronal cells, providing a novel perspective on the comprehensive toxicity risk assessment of metal mixtures.

Mume Fructus (Prunus mume Sieb. et Zucc.) extract accelerates colonic mucosal healing of mice with colitis induced by dextran sulfate sodium through potentiation of cPLA2-mediated lysophosphatidylcholine synthesis.

BACKGROUND: Mume Fructus (MF) is the fruit of Prunus mume Sieb. et Zucc, a plant of Rosaceae family. Previous studies demonstrated that MF was capable of ameliorating ulcerative colitis (UC) in mice, its action mechanism needs to be clarified. PURPOSE: This study deciphered whether and how MF extract accelerates colonic mucosal healing, the therapeutic endpoint of UC. METHODS: Biochemical, histopathological and qRT-PCR analyses were utilized to define the therapeutic efficacy of MF on dextran sulfate sodium (DSS)-induced colitis in mice. UHPLC-QTOF-MS/MS-based metabolomics technique was adopted to explore the changes of endogenous metabolites associated with UC and responses to MF intervention. qRT-PCR analysis was performed to confirm the molecular pathway in vivo. The effects of MF and lysophosphatidylcholine (LPC) on cell viability, wound healing, proliferation, and migration were examined through a series of in vitro experiments. Moreover, the effects of different subtypes of phospholipase A2 (PLA2) inhibitors on MF-treated colonic epithelial cells were detected by wound healing test and transwell assay. RESULTS: Orally administered MF could alleviate colitis in mice mainly by accelerating the healing of colonic mucosa. Guided by an unbiased metabolomics screen, we identified LPC synthesis as a major modifying pathway in colitis mice after MF treatment. Notably, MF facilitated the synthesis of LPC by enhancing the expression of PLA2 in colitis mice. Mechanistically, MF and LPC accelerated wound closure by promoting cell migration. Moreover, the promotion of MF on wound healing and migration of colonic epithelial cells was blunted by a cytosolic phospholipase A2 (cPLA2) inhibitor. CONCLUSION: MF can facilitate colonic mucosal healing of mice with colitis through cPLA2-mediated intestinal LPC synthesis, which may become a novel therapeutic agent of UC.

Identification and functional analysis of cytosolic phospholipase A2 (cPLA2) from the red swamp crayfish Procambarus clarkii: The first evidence of cPLA2 involved in immunity in invertebrates.

Cytosolic phospholipase A2 (cPLA2) specifically liberates the arachidonic acids from the phospholipid substrates. In mammals, cPLA2 serves as a key control point in inflammatory responses due to its diverse downstream products. However, the role of cPLA2 in animals lower than mammals largely remains unknown. In the current research, a homolog of cPLA2 was first identified and characterized in the red swamp crayfish Procambarus clarkii. The full-length cDNA of PccPLA2 was 4432 bp in length with a 3036 bp-long open reading frame, encoding a putative protein of 1011 amino acids that contained a protein kinase C conserved region 2 and a catalytic subunit of cPLA2. PccPLA2 was ubiquitously expressed in all examined tissues with the highest expression in the hepatopancreas, and the expression in hemocytes as well as hepatopancreas was induced upon the immune challenges of WSSV and Aeromonas hydrophila. After the co-treatment of RNA interference and bacterial infection, the decline of bacteria clearance capability was observed in the hemolymph, and the expression of some antimicrobial peptides (AMPs) was significantly suppressed. Additionally, the phagocytosis of A. hydrophila by primary hemocytes decreased when treated with the specific inhibitor CAY10650 of cPLA2. These results indicated the participation of PccPLA2 in both cellular and humoral immune responses in the crayfish, which provided an insight into the role that cPLA2 played in the innate immunity of crustaceans, and even in invertebrates.

Antiplatelet Effect of Daphnetin Is Regulated by cPLA2-Mediated Thromboxane A2 Generation in Mice.

Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.

Co-exposure to particulate matter and humidity increases blood pressure in hypertensive mice via the TRPV4-cPLA2-COX2 pathway.

Epidemiological studies have demonstrated that particulate matter (PM) can induce or exacerbate hypertension. High relative humidity has been associated with elevated blood pressure in certain regions. However, the coupling effect of humidity and PM on elevated blood pressure and the underlying mechanisms remain unknown. Herein, we aimed to explore the effects of exposure to PM and/or high relative humidity on hypertension, as well as elucidate underlying mechanisms. Male C57/BL6 mice were intraperitoneally administered NG-nitro-L-arginine methyl ester (L-NAME) to establish a hypertensive mouse model. The hypertensive mice were exposed to PM (0.15 mg/kg/day) and/or different relative humidities (45/90%) for eight weeks. Histopathological changes, systolic blood pressure (SBP), endothelial-derived contracting factors (thromboxane B2 [TXB2], Prostaglandin F2α [PGF2α], endothelin-1 [ET-1], and angiotensin II [Ang II]), and relaxing factors (prostaglandin I2 [PGI2] and nitric oxide [NO]) were measured to assess the effects of PM exposure and humidity on hypertension in mice. Levels of transient receptor potential vanilloid 4 (TRPV4), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase 2 (COX2) were measured to explore their potential mechanisms. Herein, exposure to 90% relative humidity or PM alone had a slight but insignificant effect on hypertension. However, pathological changes and elevated blood pressure were markedly exacerbated following exposure to PM and 90% relative humidity. Levels of PGF2α, TXB2, and ET-1 were significantly increased, whereas the PGI2 level was substantially decreased. HC-067047-mediated blockade of TRPV4 suppressed TRPV4, cPLA2, and COX2 expression and effectively alleviated the increased blood pressure induced by exposure to PM and 90% relative humidity. These results indicate that 90% relative humidity and PM can activate the TRPV4-cPLA2-COX2 ion channel in the aorta, altering the endothelial-derived contracting and relaxing factors and enhancing blood pressure in hypertensive mice.

Involvement of p38 MAPK/cPLA2 and arachidonic acid metabolic pathway in Shengmai injection-induced pseudo-allergic reactions.

ETHNOPHARMACOLOGICAL RELEVANCE: Adverse reactions to traditional Chinese medicine injections involve pseudo-allergic reactions (PARs). However, in clinical practice, "immediate allergic reactions" and PARs in response to these injections are not often differentiated. AIM OF THE STUDY: This study aimed to clarify the type of reactions produced by Shengmai injections (SMI) and elucidate the possible mechanism. MATERIALS AND METHODS: A mouse model was used to evaluate vascular permeability. Metabolomic and arachidonic acid metabolite (AAM) analyses were performed using UPLC-MS/MS, and the p38 MAPK/cPLA2 pathway was detected by western blotting. RESULTS: The first exposure to intravenous SMI rapidly and dose-dependently induced edema and exudative reactions in the ears and lungs. These reactions were not IgE-dependent and were likely to be PARs. Metabolomic analysis showed that endogenous substances were perturbed in SMI-treated mice, in which the arachidonic acid (AA) metabolic pathway was the most affected. SMI substantially increased the levels of AAMs in lung, including prostaglandins (PGs), leukotrienes (LTs), and hydroxy-eicosatetraenoic acids (HETEs). The p38 MAPK/cPLA2 signaling pathway was activated after a single SMI dose. Inhibitors of cyclooxygenase-2 and 5-lipoxygenase enzymes reduced exudation and inflammation in the ears and lungs of mice. CONCLUSION: Production of inflammatory factors that increase vascular permeability may result in SMI-induced PARs, and p38 MAPK/cPLA2 signaling pathway and downstream AA metabolic pathway are involved in the reactions.

Elamipretide alleviates pyroptosis in traumatically injured spinal cord by inhibiting cPLA2-induced lysosomal membrane permeabilization.

Spinal cord injury (SCI) is a devastating injury that may result in permanent motor impairment. The active ingredients of medications are unable to reach the affected area due to the blood‒brain barrier. Elamipretide (SS-31) is a new and innovative aromatic cationic peptide. Because of its alternating aromatic and cationic groups, it freely crosses the blood‒brain barrier. It is also believed to decrease inflammation and protect against a variety of neurological illnesses. This study explored the therapeutic value of SS-31 in functional recovery after SCI and its possible underlying mechanism. A spinal cord contusion injury model as well as the Basso Mouse Scale, footprint assessment, and inclined plane test were employed to assess how well individuals could function following SCI. The area of glial scarring, the number of dendrites, and the number of synapses after SCI were confirmed by HE, Masson, MAP2, and Syn staining. Western blotting, immunofluorescence, and enzyme-linked immunosorbent assays were employed to examine the expression levels of pyroptosis-, autophagy-, lysosomal membrane permeabilization (LMP)- and MAPK signalling-related proteins. The outcomes showed that SS-31 inhibited pyroptosis, enhanced autophagy and attenuated LMP in SCI. Mechanistically, we applied AAV vectors to upregulate Pla2g4A in vivo and found that SS-31 enhanced autophagy and attenuated pyroptosis and LMP by inhibiting phosphorylation of cPLA2. Ultimately, we applied asiatic acid (a p38-MAPK agonist) to test whether SS-31 regulated cPLA2 partially through the MAPK-P38 signalling pathway. Our group is the first to suggest that SS-31 promotes functional recovery partially by inhibiting cPLA2-mediated autophagy impairment and preventing LMP and pyroptosis after SCI, which may have potential clinical application value.

Effect of atorvastatin on lipoxygenase pathway-related gene expression in an in vitro model of lipid accumulation in hepatocytes.

Lipid accumulation in hepatocytes can result from an imbalance between lipid acquisition and lipid catabolism. In recent years, it has been discovered that eicosanoids derived from arachidonic acid (AA) have the potential to create specialized pro-resolving lipid mediators to actively resolve inflammation, but it is not clear whether AA and lipoxygenases exert effects on hepatic inflammation. Here, the effects of atorvastatin on the expression of cytoplasmic phospholipase A2 (cPLA2) and lipoxygenase pathway genes (ALOX5, ALOX12, ALOX15, and ALOX15B) were evaluated in an in vitro model of palmitic acid (PA)-induced hepatocyte lipid accumulation in McA-RH7777 (McA) cells. Palmitic acid increased cPLA2 expression, intracellular AA levels, and ALOX12 expression (P < 0.05). Atorvastatin at various concentrations had no significant effects on AA levels or on cPLA2, ALOX15, and ALOX15B expressions. ALOX5 was not detected, despite multiple measurements. Pro-inflammatory IL-1ß expression levels were upregulated by PA (P < 0.01) and attenuated by atorvastatin (P < 0.001). TNFα did not differ among groups. The expression levels of anti-inflammatory IL-10 decreased in response to PA (P < 0.05), but were not affected by atorvastatin. In conclusion, in an in vitro model of lipid accumulation in McA cells, atorvastatin reduced IL-1ß; however, its effect was not mediated by AA and the lipoxygenase pathway at the established doses and treatment duration. Further research is required to investigate time-response data, as well as other drugs and integrated cell systems that could influence the lipoxygenase pathway and modulate inflammation in liver diseases.

Theoretical evaluation of the malathion and its chemical derivatives interaction with cytosolic phospholipase A2 from zebrafish.

Cytosolic phospholipase A2 (cPLA2) belongs to a large family of proteins and plays a crucial role in the regulation of arachidonic acid metabolism and inflammation cascade in zebrafish (Danio rerio). This enzyme with a molecular weight of 85 kDa, has two distinct domains. One is the regulatory and calcium-dependent (Ca2+) domain called C2, the other is the catalytic α/ß hydrolase Ca2+-independent domain, where serine and aspartic acid catalytic dyad residues are present. We investigated the interaction of malathion and their organophosphate metabolites in the cPLA2 using in silico tools. Molecular docking results showed hydrophobic interactions with the paraoxon and catalytic site residue (Ser 223). Malathion increases intracellular Ca2+ due to endoplasmic reticulum influx which in turn activities phospholipase A2 and arachidonic acid release. Molecular docking and homology modelling of proteins and ligands could be a complementary tool for ecotoxicology and environment pollution assessment.

IRGM/Irgm1 deficiency inhibits neutrophil-platelet interactions and thrombosis in experimental atherosclerosis and arterial injury.

BACKGROUND: Neutrophil extracellular traps (NETs) closely link inflammation and thrombosis. The immune-related GTPase family M protein (IRGM) and its ortholog of mouse IRGM1 are positively correlated with plaque rupture during atherosclerosis process. However, whether and how IRGM/IRGM1 affects NETs formation and atherosclerotic thrombosis remains unknown, which will further promote the development of antithrombotic treatment tools. METHODS: The thrombi images, platelet activation makers and NETs makers were detected in the serum of STEMI patients and controls. To futher investigate IRGM/IRGM1 affects NETs formation and atherothrombosis in vivo, ApoE-/-Irgm1+/- and ApoE-/- mice received diets rich in fat and 2.5% FeCl3 was then used to induce experimental arterial thrombosis in an atherosclerosis background. In vitro, PMA and thrombin were used to stimulate neutrophils and platelets, respectively, and the expression of IRGM/IRGM1 were modified. To reveal the molecular mechanisms, MAPK-cPLA2 signals inhibitors were used. RESULTS: Serum IRGM was positively correlated with PF4 and neutrophil elastase. Subsequently, Irgm1 deficient mice have a longer occlusion time and lower growth rate. In vitro, as expected, IRGM/Irgm1 deficiency inhibits platelet activation and platelet-neutrophil interaction. More importantly, IRGM promoted NETs production through activating MAPK-cPLA2 signals in PMA stimulated neuropils, whereas inhibiting the production of NETs eliminated the difference in platelet activation and thrombosis caused by IRGM/Irgm1 modification in vivo and vitro. Similarly, inhibition of platelet activation also eliminated the influence of IRGM/Irgm1 modification on NETs production. CONCLUSIONS: Overall, our data indicate that IRGM/Irgm1 deficiency in neuropils inhibits the intense interaction between neutrophils and platelets, and ultimately inhibits thrombosis.

Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson's Disease.

Hyperactivation of cyclin-dependent kinase 5 (Cdk5) by p25, contributes to neuroinflammation causing neurodegeneration in Parkinson's disease (PD) and Alzheimer's disease. However, the mechanism by which Cdk5 induces neuroinflammation in the PD brain is largely unexplored. Here, we show that Cdk5 phosphorylates cytosolic phospholipase A2 (cPLA2) at Thr-268 and Ser-505 sites lead to its activation and generation of eicosanoid products. Mutational studies using site-directed mutagenesis and molecular simulations show that the architecture of the protein changes on each single-point mutation. Interestingly, double mutations also led to a severe decline in the activity of cPLA2 and to the disruption of its translocation to the plasma membrane. Further, the brain lysates of transgenic PD mouse models show hyperactivation of Cdk5, resulting in enhanced phosphorylation of Thr-268 and Ser-505 of cPLA2 and its heightened activity, confirming the findings observed in the cell culture model of PD. These phosphorylation sites of cPLA2 and Cdk5 could be explored as the future therapeutic targets against neuroinflammation in PD. Further, conjoint transcriptomic analysis of the publicly available human PD datasets strengthens the hypothesis that genes of the arachidonic acid, prostaglandin synthesis, and inflammatory pathways are significantly upregulated in the case of PD patients compared with that of healthy control subjects.

TCA-phospholipid-glycolysis targeted triple therapy effectively suppresses ATP production and tumor growth in glioblastoma.

Rationale: Glioblastoma (GBM) displays a complex metabolic reprogramming in cancer cells. Adenosine triphosphate (ATP) is one of the central mediators of cell metabolism and signaling. GBM cells generate ATP by glycolysis and the tricarboxylic acid (TCA) cycle associated with oxidative phosphorylation (OXPHOS) through the breaking-down of pyruvate or fatty acids to meet the growing energy demand of cancer cells. Therefore, it's urgent to develop novel treatments targeting energy metabolism to hinder tumor cell proliferation in GBM. Methods: Non-targeted metabolomic profiling analysis was utilized to evaluate cell metabolic reprogramming using a small molecule inhibitor (SMI) EPIC-0412 treatment. Cellular oxygen consumption rate (OCR) and the total proton efflux rate (PER), as well as ATP concentration, were tracked to study metabolic responses to specifically targeted inhibitors, including EPIC-0412, arachidonyl trifluoromethyl ketone (AACOCF3), and 2 deoxy-D-glucose (2-DG). Cancer cell proliferation was assessed by CCK-8 measurements and colony formation assay. Additionally, flow cytometry, immunoblotting (IB), and immunofluorescence (IF) analyses were performed with GBM cells to understand their tumorigenic properties under treatments. Finally, the anticancer effects of this combination therapy were evaluated in the GBM mouse model by convection-enhanced delivery (CED). Results: We found that SMI EPIC-0412 could effectively perturb the TCA cycle, which participated in the combination therapy of cytosolic phospholipase A2 (cPLA2)-inhibitor AACOCF3, and hexokinase II (HK2)-inhibitor 2-DG to disrupt the GBM energy metabolism for targeted metabolic treatments. ATP production was significantly declined in glioma cells when treated with monotherapy (EPIC-0412 or AACOCF3), dual therapy (EPIC-0412 + AACOCF3), or triple therapy (EPIC-0412 + AACOCF3 +2-DG) regimen. Our experiments revealed that these therapies hindered glioma cell proliferation and growth, leading to the reduction in ATP production and G0/G1 cell cycle arrest. We demonstrated that the combination therapy effectively extended the survival of cerebral tumor-bearing mice. Conclusion: Our findings indicate that the TCA-phospholipid-glycolysis metabolism axis can be blocked by specific inhibitors that significantly disrupt the tumor energy metabolism and suppress tumor proliferation in vitro and in vivo, suggesting that targeting ATP synthesis inhibition in cancer cells might be an attractive therapeutic avenue in GBM management.

Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation.

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger cytoskeletal reorganization and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in cell-cycle regulation. However, the exact link between mechanical forces and the biochemical pathways that control mitotic progression remains unknown. Here, we identify a tension-dependent signal on the nucleus that sets the time for nuclear envelope permeabilization (NEP) and mitotic entry. This signal relies on actomyosin contractility, which unfolds the nucleus during the G2-M transition, activating the stretch-sensitive cPLA2 on the nuclear envelope and regulating the nuclear translocation of cyclin B1. Our data demonstrate how nuclear tension during the G2-M transition contributes to timely and efficient mitotic spindle assembly and prevents chromosomal instability.

Eicosanoid lipidome activation in post-mortem brain tissues of individuals with APOE4 and Alzheimer's dementia.

BACKGROUND: Chronic neuroinflammation is one of the hallmarks of late-onset Alzheimer's disease (AD) dementia pathogenesis. Carrying the apolipoprotein ε4 (APOE4) allele has been associated with an accentuated response to brain inflammation and increases the risk of AD dementia progression. Among inflammation signaling pathways, aberrant eicosanoid activation plays a prominent role in neurodegeneration. METHODS: Using brains from the Religious Order Study (ROS), this study compared measures of brain eicosanoid lipidome in older persons with AD dementia to age-matched controls with no cognitive impairment (NCI), stratified by APOE genotype. RESULTS: Lipidomic analysis of the dorsolateral prefrontal cortex demonstrated lower levels of omega-3 fatty acids eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and DHA-derived neuroprotectin D1 (NPD-1) in persons with AD dementia, all of which associated with lower measures of cognitive function. A significant interaction was observed between carrying the APOE4 allele and higher levels of both pro-inflammatory lipids and pro-resolving eicosanoid lipids on measures of cognitive performance and on neuritic plaque burden. Furthermore, analysis of lipid metabolism pathways implicated activation of calcium-dependent phospholipase A2 (cPLA2), 5-lipoxygenase (5-LOX), and soluble epoxide hydrolase (sEH) enzymes. CONCLUSION: These findings implicate activation of the eicosanoid lipidome in the chronic unresolved state of inflammation in AD dementia, which is increased in carriers of the APOE4 allele, and identify potential therapeutic targets for resolving this chronic inflammatory state.