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Background Amyloid β oligomers (oAβ) are a key pathogenic driver in Alzheimer’s Disease (AD). Neuronal G protein-gated inwardly rectifying K + (GIRK/Kir3) channels are important regulators of neuronal excitability and prominent somatodendritic effectors for inhibitory G protein-coupled receptors, including the γ-aminobutyric acid type B receptor (GABA B R). We previously reported a male-specific suppression of GIRK channel activity in hippocampal (HPC) neurons evoked by oAβ in in vitro, ex vivo, and in vivo mouse models of AD, and showed that this adaptation correlated with synaptic and cognitive impairment. Using pharmacological approaches, we showed that this adaptation is mediated by co-activation of cellular prion protein (PrP C ) and metabotropic glutamate receptor 5 (mGluR5) and requires activation of cytosolic phospholipase A2 α (cPLA 2 α). However, the mechanisms underlying the sex specificity was unknown. Given the clinical context that females exhibit a 2-fold higher incidence of AD than males, and the loss of neuroprotective estrogen by menopause contributes to the sex differences in AD, we postulated that estrogen-associated resilience underlies this sex dimorphism of oAβ action. Methods To examine the strength of GIRK-dependent signaling in HPC neurons, we performed electrophysiology in primary HPC cultures from neonatal male and female mice and then measured whole-cell currents evoked by the direct-acting GIRK channel agonist ML297 and the GABA B R-selective agonist baclofen. We used an array of genetic and pharmacological approaches to investigate the molecular mechanism(s) underlying the vulnerability and resilience of GIRK channel activity to oAβ in male and female HPC neurons, respectively. Results We found that resilience to the oAβ-induced and PrP C /mGluR5-dependent suppression of GIRK channel activity in female HPC neurons is conferred by membrane-associated estrogen receptor α (mERα) and caveolin 1 (Cav1). When this resilience factor is blocked or absent, oAβ suppresses GIRK channel activity in female HPC neurons via the same PrP C -mGluR5-cPLA 2 α signaling pathway identified previously in male neurons. Conclusion As estrogen levels decline with aging and menopause, the protective influence of mERα/Cav1 may diminish, unmasking the oAβ-induced suppression of GIRK channel activity and exacerbating disease progression in females. Plain english summary While amyloid β plaques (Aβ) are notable hallmarks of Alzheimer’s Disease (AD), cognitive impairment in the early stages of the disease tracks more closely with the level of soluble Aβ oligomers (oAβ) in the brain. oAβ promotes cognitive deficits by disrupting the balance of excitatory and inhibitory influences on neurons in brain regions important for learning and memory such as the hippocampus, but the underlying molecular targets of oAβ and its pathogenic mechanisms are not fully understood. We recently demonstrated that oAβ weakens the activity of a prominent inhibitory influence on neuronal excitability (the GIRK channel) in the hippocampus of male but not female mice. This sexually dimorphic effect of oAβ was interesting and unexpected given that women are twice as likely to develop AD than men, and because disease progression is more aggressive in women. In this study, we investigated the mechanisms underlying the resilience of GIRK channels in female hippocampal neurons to oAβ. We found that resilience is conferred by estrogen and one of its receptors. When the influence of this receptor is diminished using pharmacological or genetic interventions, oAβ weakens GIRK channel activity in female and male neurons to a similar degree, and via the same mechanism. We speculate that with the onset of menopause, the protective influence of estrogen on GIRK channel activity in the hippocampus begins to wane. This, combined with other female-specific effects of oAβ on neuronal activity, contributes to the increased incidence and severity of AD in females. Highlights In hippocampal (HPC) neurons from female mice, local estrogen production protects GIRK channel activity from suppression by oligomeric amyloid β (oAβ). Inhibition of estrogen receptor α (ERα), but not estrogen receptor β (ERβ) or G protein-coupled estrogen receptor (GPER), unmasked the oAβ-induced suppression of GIRK channel activity in female HPC neurons. Without ERα protection, oAβ suppressed GIRK channel activity in female HPC neurons through the same mechanism identified previously in male HPC neurons involving cellular prion protein (PrP C ), metabotropic glutamate receptor 5 (mGluR5), and cytosolic phospholipase A 2 α (cPLA 2 α). mGluR5-associated regulation of GIRK channel plasticity is blocked in female HPC neurons independent of oAβ. Resilience of GIRK channel activity in female HPC neurons to mGluR5 agonist and oAβ requires membrane-associated ERα (mERα) and caveolin-1 (Cav1).

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA₂) binds via its calciumdependent C2 domain (cPLA₂-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA₂ and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA₂ at cytoplasmic resting-Ca2+ concentrations. cPLA₂-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA₂-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

Background Apolipoprotein 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 A 2 (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. Methods Mouse 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. Results Greater 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. Conclusions Our 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.

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.

ObjectiveTumour necrosis factor alpha (TNF-α) signalling plays a central role in the pathogenesis of various autoimmune diseases, particularly inflammatory arthritis. This study aimed to repurpose clinically approved drugs as potential inhibitors of TNF-α signalling in treatment of inflammatory arthritis.MethodsIn vitro and in vivo screening of an Food and Drug Administration (FDA)-approved drug library; in vitro and in vivo assays for examining the blockade of TNF actions by fexofenadine: assays for defining the anti-inflammatory activity of fexofenadine using TNF-α transgenic (TNF-tg) mice and collagen-induced arthritis in DBA/1 mice. Identification and characterisation of the binding of fexofenadine to cytosolic phospholipase A2 (cPLA2) using drug affinity responsive target stability assay, proteomics, cellular thermal shift assay, information field dynamics and molecular dynamics; various assays for examining fexofenadine inhibition of cPLA2 as well as the dependence of fexofenadine’s anti-TNF activity on cPLA2.ResultsSerial screenings of a library composed of FDA-approved drugs led to the identification of fexofenadine as an inhibitor of TNF-α signalling. Fexofenadine potently inhibited TNF/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) signalling in vitro and in vivo, and ameliorated disease symptoms in inflammatory arthritis models. cPLA2 was isolated as a novel target of fexofenadine. Fexofenadine blocked TNF-stimulated cPLA2 activity and arachidonic acid production through binding to catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505. Further, deletion of cPLA2 abolished fexofenadine’s anti-TNF activity.ConclusionCollectively, these findings not only provide new insights into the understanding of fexofenadine action and underlying mechanisms but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions, particularly inflammatory rheumatic diseases.

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-3β, 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.

Background Scutellaria baicalensis Georgi, a traditional Chinese herb, is known for its various biological effects, including antibacterial, anti-inflammatory, antioxidative, and antitumor properties. However, the function and mechanisms of methanol extract of Scutellaria baicalensis Georgi (MESB) in treating hepatic fibrosis remain unclear. Methods This study utilized a CCl4-induced mouse model of hepatic fibrosis to assess the effects of MESB through histopathological analysis and serum tests. The anti-fibrosis mechanism of MESB was investigated using qPCR, Western blotting, RNA interference, proteomics, and metabolomics. Spatial metabolomics identified key components of MESB in liver tissue, while molecular docking determined their targets. Results Treatment with MESB alleviated hepatic pathological changes and reversed hepatic fibrosis in the CCl4-induced models, as evidenced by decreased collagen fibers deposition, reduced expression of hepatic fibrosis markers COL1A1, FN, and PAI-1, and lowered serum levels of AST and ALT. In vitro, MESB inhibited the proliferation of LX-2 cells and the expression of hepatic fibrosis markers. Furthermore, MESB intervention modulated various pathways, particularly those involved in metabolic pathways. Subsequent metabolomics analysis demonstrated that MESB disrupted glycerophospholipid metabolism and suppressed arachidonic acid metabolism. MESB downregulated the expression of cPLA2 in LX-2 cells, leading to decreased production of arachidonic acid and its downstream inflammatory mediators. Meanwhile, MESB inhibited the expression of cPLA2 and its downstream NF-κB pathway in the liver tissues of models induced by CCl4. Additionally, silencing cPLA2 markedly reduced the expressions of COL1A1, FN, and PAI-1. Spatial metabolomics analysis confirmed the penetration of baicalein, wogonin and wogonoside into liver tissue. Further results indicated that baicalein and wogonin inhibited the expression of cPLA2, while baicalin and wogonoside do not exhibit this effect. Moreover, molecular docking suggested that baicalein and wogonin possess the potential to directly interact with cPLA2. Conclusion This study reveals that MESB is crucial in preventing hepatic fibrosis via the cPLA2-mediated arachidonic acid metabolic pathway, highlighting its key active components as potential drugs for fibrosis treatment.

Although impaired insulin signaling in the brain has been recognized as a key factor in the development and progression of Alzheimer’s disease (AD), the underlying mechanisms remain incompletely understood. Given that overactivation of cytosolic phospholipase A2 (cPLA2) has been implicated in AD, we tested the hypothesis that oligomeric tau (oTau) activates cPLA2, which in turn negatively affects Caveolin-1 (Cav-1) and insulin signaling. In the cerebral cortex and hippocampus of 12-months-old 3xTg-AD mice, we observed an upregulation of phosphorylated cPLA2 (p-cPLA2), accompanied by downregulation of Cav-1 and impaired insulin signaling. Specifically, we found significant decreases in insulin receptor-α (IR-α) and insulin receptor-β (IR-β) expression, along with increased levels of phospho-insulin receptor substrate 1 at Ser307 [p-IRS-1 (Ser307)] and decreased levels of p-IRS-1 (Tyr895), compared to wild-type (WT) mice. To further investigate the role of cPLA2 in insulin signaling impairment in AD, we demonstrated that oTau activated cPLA2 in primary mouse cerebral endothelial cells (CECs), leading to Cav-1 downregulation and disrupted insulin signaling. Notably, these detrimental effects of oTau on Cav-1 and insulin signaling were abolished when cPLA2 expression was depleted using small interfering RNA (siRNA). In conclusion, our study highlights the pivotal role of cPLA2 in regulating Cav-1 function and insulin signaling in AD, offering insights into potential therapeutic targets for mitigating insulin resistance associated with the disease.

Obesity is a worldwide epidemic problem and correlates to varieties of acute or chronic lung diseases such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, and pulmonary fibrosis. An increase of leptin, a kind of adipokine, in lean mice plasma has been found to impair immune responses and facilitate the infection of Klebsiella pneumoniae, resulting in increased pneumonia severity. Also, a higher leptin level is found in exhaled breath condensates of obese or asthmatic subjects, compared to healthy ones, suggesting that leptin is involved in the occurrence or exacerbation of lung injury. In previous studies, we showed that leptin stimulated cytosolic phospholipase A2-α (cPLA2α) gene expression in lung alveolar type II cells via mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB)-activated coactivator p300. Herein, we show that the in vivo application of leptin in the respiratory system upregulated the expression of inflammatory proteins cPLA2α and cyclooxygenase-2 (COX-2) together with leukocyte infiltration. Treatment with an ROS scavenger (N-acetylcysteine, NAC), an NADPH oxidase inhibitor (apocynin), or an activating protein (AP)-1 inhibitor (tanshinone IIA) attenuated leptin-mediated cPLA2α/COX-2 expression and leukocyte recruitment in the lung. Leptin increased intracellular oxidative stress in a leptin receptor (OB-R) and NADPH oxidase-dependent manner, leading to the phosphorylation of the AP-1 subunit c-Jun. In summation, leptin increased lung cPLA2α/COX-2 expression and leukocyte recruitment via the NADPH oxidase/ROS/AP-1 pathway. Understanding the inflammatory effects of leptin on the pulmonary system provides opportunities to develop strategies against lung injury related to metabolic syndrome or obesity.