Inhibition Of Arachidonic Acid Research Articles

Omega-3 and omega-6 polyunsaturated fatty acids and their potential therapeutic role in protozoan infections.

Protozoa exert a serious global threat of growing concern to human, and animal, and there is a need for the advancement of novel therapeutic strategies to effectively treat or mitigate the impact of associated diseases. Omega polyunsaturated fatty acids (ω-PUFAs), including Omega-3 (ω-3) and omega-6 (ω-6), are constituents derived from various natural sources, have gained significant attention for their therapeutic role in parasitic infections and a variety of essential structural and regulatory functions in animals and humans. Both ω-3 and ω-6 decrease the growth and survival rate of parasites through metabolized anti-inflammatory mediators, such as lipoxins, resolvins, and protectins, and have both in vivo and in vitro protective effects against various protozoan infections. The ω-PUFAs have been shown to modulate the host immune response by a commonly known mechanism such as (inhibition of arachidonic acid (AA) metabolic process, production of anti-inflammatory mediators, modification of intracellular lipids, and activation of the nuclear receptor), and promotion of a shift towards a more effective immune defense against parasitic invaders by regulation the inflammation like prostaglandins, leukotrienes, thromboxane, are involved in controlling the inflammatory reaction. The immune modulation may involve reducing inflammation, enhancing phagocytosis, and suppressing parasitic virulence factors. The unique properties of ω-PUFAs could prevent protozoan infections, representing an important area of study. This review explores the clinical impact of ω-PUFAs against some protozoan infections, elucidating possible mechanisms of action and supportive therapy for preventing various parasitic infections in humans and animals, such as toxoplasmosis, malaria, coccidiosis, and chagas disease. ω-PUFAs show promise as a therapeutic approach for parasitic infections due to their direct anti-parasitic effects and their ability to modulate the host immune response. Additionally, we discuss current treatment options and suggest perspectives for future studies. This could potentially provide an alternative or supplementary treatment option for these complex global health problems.

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-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.

Ginkgetin effectively mitigates collagen and AA-induced platelet activation via PLCγ2 but not cyclic nucleotide-dependent pathway in human.

Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 μM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca2+ ]i) levels and thromboxane A2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3β and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoproteinSer157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs.

Aktivitas Antiinflamasi In Vitro dan In Vivo Ekstrak Etanol Daun Mangga Arumanis (Mangifera indica L.)

Inflammation is a normal process designed to protect oneself and promote healing of body tissues. The mechanism of action of flavonoids as antiinflammatory is through the inhibition of arachidonic acid and the secretion of lysosomal enzymes. The ethanol extract of Mangifera indica L. leaves (EEML) contains flavonoids and is widely used as a medicine empirically. This study was conducted to evaluate the antiinflammatory effect of EEML in vitro and in vivo. In vitro study of the antiinflammatory activity of EEML based on its effect on the stability of blood membranes using UV-Vis spectrophotometry method. While the in vivo activity was tested using the carrageenan induction method on white rats of the Wistar strain. In vitro activity was expressed as EC50 and in vivo activity was expressed as % edema reduction. The results showed that ethanol extract was able to increase membrane stability with an EC50 value of 8.56 ?g/ml better than positive control aspirin with EC50 21.57 ?g/ml. In vivo test results showed that the administration of EEML doses of 200 mg/kg.bw, 400 mg/kg.bw, and 800 mg/kg.bw was not significantly different from the positive control in reducing edema. EEML has shown antiinflammatory effects in vitro and in vivo, so it has the potential to be developed as an antiinflammatory agent.

Structure-Activity Relationship Analysis of Analogs of Rhosin, a RhoA Inhibitor, Reveals a New Generation of Improved Antiplatelet Agents

Platelet activation and aggregation play a key role in mediating hemostasis and thrombosis. The antiplatelet therapies currently available in the market are associated with a high risk of hemorrhage and are mostly irreversible in suppressing platelet activity; hence, there is a need to develop better therapeutic agents. Previous genetic and pharmacological studies have implicated the small GTPase RhoA in multiple platelet signaling pathways. We devised a lead RhoA activity-specific inhibitor, Rhosin/G04, based on the structure-function relationship of RhoA interaction with its activator, guanine nucleotide exchange factor (GEF) (Figure 1A). Rhosin/G04 binds to RhoA directly with micromolar affinity at a surface groove that is essential for GEF recognition and blocks GEF-mediated GTP loading to RhoA. Rhosin/G04 inhibits platelet spreading on fibrinogen and thrombin-induced platelet aggregation, mimicking effects of RhoA gene targeting. In the current work, we have utilized the inhibitory activity of G04 for platelet activation and its biochemical activity to define its structure-activity relationship (SAR) and to understand its mechanism of action in an effort to improve efficacy and druggability.The structure of G04 in a groove of RhoA interaction was hypothesized based on the docking studies using Molsoft ICM-Pro. Cincinnati Children's Hospital Medical Center's compound library of over 360,000 chemicals was scanned for G04 analogs by similarity and substructure searches. In the initial screen, a human platelet aggregation assay was performed at both a low concentration (1 µg/ml) and a high concentration (5 µg/ml) of collagen. The first round similarity search resulted in a set of 7 compounds (Set-1), from which, compound 177629 showed significantly enhanced potency relative to G04 (Figure 1B). The second round of similarity searches for compounds more closely related to 177629 (Set-2) identified 14 compounds. The third-round search for other related compounds (Set-3) led to 9 additional compounds that add to the understanding of the SAR. The compounds that showed enhanced antiplatelet activity were examined for their potency and selectivity in in vitro biochemical binding assays and in suppressing RhoA-GTP formation and downstream phosphorylation of myosin light chain (p-MLC) signaling in platelets. The active compounds were further examined for their anti-platelet activities under diverse stimuli including thrombin, ADP, U46619 (a stable thromboxane receptor agonist), and arachidonic acid.The most active compounds from Set-1, Set-2, and Set-3 inhibited platelet aggregation by at least 70% and showed IC 50 values below 6 µM. Of these compounds, 12 showed significantly greater potency than the initial compound, G04. The most active compounds were 177618, 177619, 177628, 177629, 177633, and 177634. These compounds specifically inhibited RhoA activity and blocked p-MLC. SAR analyses led us to believe that the quinoline is optimally attached to the hydrazine at the 4-position. The halogen (choloro- or trifluoromethyl-) substitution at the 7- or 8- position improved activity, and the 7- position may be slightly favored. The aryl group is considerably variable with similar potency between the indole, methylphenyl, and dichlorophenyl- groups.Rhosin/G04 is the R enantiomer (i.e. Rhosin is R-G04), so its S enantiomer, S-G04 was also evaluated (Figure 1C). S-G04 is significantly more potent than R-G04 in inhibiting collagen-stimulated RhoA-GTP formation and aggregation of platelets, and its effect is completely reversible by washing the platelets. Finally, R-G04 and S-G04 showed differential inhibition of arachidonic acid and U46619 stimulated primary and secondary aggregation, highlighting the potential utilities of the inhibitors in dissecting different platelet activation mechanisms. S-G04 is active in inhibiting thrombin, ADP, U46619, and arachidonic acid-mediated platelet activation at submicromolar concentration, suggesting a broad role of RhoA signaling in integrating platelet signal cross talk.In summary, evaluation of Rhosin/R-G04 analogs in a platelet activity screen identified a new generation of improved small-molecule RhoA inhibitors, including an enantiomer with significantly improved efficacy. These analog studies of novel anti-platelet agents provide a new approach to effectively and reversibly manipulate platelet activities. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New Delta‐5‐Desaturase Inhibitor Suppress Lung Cancer Progression: A Paradigm Shift on COX‐2 Biology in Lung Cancer Treatment

ObjectiveAs the first leading cause of cancer deaths in the United States, lung cancer still attracts much research attention. Cyclooxygenase‐2 (COX‐2) is considered a classic target due to overexpression in nearly 80% non‐small‐cell lung cancer (NSCLC, major type) patients. The traditional concept of the COX‐2 inhibitor was designed to limit the COX‐2‐catalyzed arachidonic acid (AA) peroxidation and the formation of the PGE2 (subsequent cancer promoter). However, many COX‐2 inhibitors have failed to show clinical efficacy. Instead of inhibiting COX‐2, our previous studies have demonstrated that the genetically knocking down the delta‐5‐desaturase (D5D) by siRNA in cancer cells can lead to the inhibition of AA and PGE2 formation. This phenomenon promotes the COX‐2 catalyzed DGLA peroxidation to form 8‐hydroxyoctanoic acid (8‐HOA), which could be served as an anti‐cancer free radical byproduct in cancer therapy. However, the application of siRNA is still hindered by its in vivo instability and off‐target effect. Therefore, continued research into a new D5D small molecule inhibitor for lung cancer is required to expand the clinical benefit to the patient and to improve outcomes in NSCLC. In this study, we have first proposed the 10,11‐Dihydro‐5H‐dibenz[b,f]azepine as a newly synthesized D5D inhibitor and evaluated its effect on lung cancer by both in vitro and in vivo model.MethodAfter the treatment of novel D5D inhibitor in vitro, the DGLA, AA, PGE2, and 8‐HOA profile was measured by LC/MS or GC/MS to assess the D5D activity in A549 NSCLC cells. Colony formation assay, wound‐healing assay, transwell assay, flow cytometry analysis, and western blot analysis was performed on A549 cells to evaluate the effect of D5D inhibitor on cancer cell proliferation, migration, and apoptosis. The in vivo effect of the D5D inhibitor was determined by xenograft mouse models in a 4 weeks treatment period along with supplementation of DGLA.ResultsThe fatty acid profile results indicated that the treatment of D5D inhibitor (10 μM) significantly suppressed the AA and PGE2 but promoted 8‐HOA formation in A549 cells. The cell survival, migration, and proliferation of A549 cells were improved by the DGLA (100 μM) and D5D inhibitor treatment. Consistent with our in vitro data, the in vivo results suggested that the D5D inhibitor (15 mg/kg) along with DGLA supplementation could promote 8‐HOA formation to a threshold level, resulting in significant tumor reduction in both A549 and LLC xenograft mouse (nu/nu or C57B6) models. D5D inhibitor also suppressed the metastasis of LLC cells from the site of injection to the lung. Additionally, western blotting and immunohistochemistry results indicated that the D5D inhibitor also promoted apoptosis in tumor tissues.ConclusionOur new concept makes active use of the commonly overexpressed COX‐2 in cancer cells (no longer to be considered a problem, but instead a benefit), and concurrently targets D5D to also elicit DGLA’s anti‐cancer effect; these two efforts will act in concert to result in more effective and safer therapeutic outcomes for lung cancer treatment.Support or Funding InformationNIHR15CA195499

Turmeric/curcumin

Curcumin is the primary curcuminoid present in the turmeric plant, Curcuma longa, and gives turmeric its orange-yellow color. The names “turmeric” and “curcumin” are often used interchangeably, especially for the spice used in cooking. Turmeric extracts and curcumin are used primarily for osteoarthritis (OA) and inflammatory conditions. They also may have activity in diabetes, obesity, and cardiovascular diseases. Curcumin has anti-inflammatory activity via inhibition of arachidonic acid, lipoxygenase, COX, nuclear factor-kappa B, tumor necrosis factor-alpha, and interleukins. In a study of knee OA, 30 mg of curcumin extract three times daily decreased COX-2 secretion into synovial fluid, similar to the effect of 25 mg of diclofenac three times daily. In another study of knee OA, serum levels of Coll2-1, a cartilage-specific biomarker, were significantly reduced. Clinical trials demonstrating benefit have used 1,000–1,500 mg per day in divided doses. The oral bioavailability of turmeric/curcumin may be low, so products that contain black pepper extracts or other compounds as absorption enhancers are preferred. The most common adverse effects are GI discomfort and nausea. Human studies have shown good tolerance even with higher dosages. Curcumin inhibits platelet aggregation in vitro and in vivo, so patients taking antithrombotic agents such as warfarin or clopidogrel should use this botanical cautiously. Multiple small studies have evaluated curcumin and related compounds for treatment of OA and rheumatoid arthritis (RA), alone or in combination with other natural products. Although some studies were placebo controlled, others compared curcumin with NSAIDs. An Iranian study of 40 participants with knee OA compared treatment with 1,500 mg of curcuminoids per day in three divided doses with placebo for 6 weeks. Treatment with curcuminoids resulted in greater improvements on standard OA assessment tools, including the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Lequesne’s pain functional index, and visual analogue scale (VAS), with few adverse effects. Pain and physical function also improved, but not stiffness. An Indian trial1Amalraj A. et al.J Med Food. 2017; 20: 1022-1030Crossref PubMed Scopus (62) Google Scholar randomized 36 participants with RA into three groups to receive 250 mg or 500 mg of curcumin or placebo twice daily for 90 days. The active treatment groups had significant improvement in their clinical symptoms at study end. Favorable changes in disease markers also occurred, including the erythrocyte sedimentation rate, C-reactive protein levels, and rheumatoid factor values. A 2018 Indian study2Karlapudi V. et al.J Med Food. 2018; 21: 511-520Crossref PubMed Scopus (9) Google Scholar of 105 patients with OA evaluated a combination curcumin product versus placebo in a 90-day trial that demonstrated improvements in pain, physical function, and quality of life. A noninferiority trial of 367 participants with OA compared 1,500 mg of curcumin extract daily to 1,200 mg of ibuprofen daily for 4 weeks. The WOMAC total, pain, stiffness, and function scores were similar between the curcumin and ibuprofen groups, with about two-thirds of participants rating themselves as “improved” on the global assessment. Curcumin-treated patients had less abdominal pain. An OA trial of 120 participants evaluated several natural products. Patients were randomized to receive 500 mg of curcumin extract, 750 mg of glucosamine sulfate, curcumin extract/glucosamine sulfate, or placebo twice daily for 42 days. The VAS pain scores were reduced from baseline, with the greatest reduction in the curcumin extract group. The WOMAC scores were also improved in the curcumin extract group. Improvements from baseline were noted with glucosamine, but less than those with curcumin. Evidence for curcumin/turmeric is promising given its safety, and it may be an option for patients with mild OA symptoms. Inform patients that the curcumin formulations used in studies may not be widely available. Products containing compounds such as black pepper extract are recommended and should be taken with meals with fats or oils to enhance absorption.

P666Anti-platelet therapy in the primary prevention of cardiovascular disease in patients with chronic obstructive pulmonary disease: randomised controlled proof of concept trial (APPLE COPD-ICON 2)

Abstract The APPLE COPD-ICON2 trial is a prospective 2x2 factorial, double blinded proof of concept randomised controlled trial targeting patients with chronic obstructive pulmonary disease (COPD) at high risk of cardiovascular disease. The primary goal was to investigate if antiplatelet therapy (APT) will produce the predefined cut-off of platelet inhibition measured using the Multiplate test. We also assessed inflammatory biomarkers in serum. Patients were randomised to Aspirin plus placebo, ticagrelor plus placebo, Aspirin plus ticagrelor or placebo only for 6 months. The primary outcome is inhibition of arachidonic acid (ASPI-test, cut-off <40) and adenosine diphosphate (ADP-test, cut-off <46) induced platelet aggregation at 6 months based on intention to treat (ITT) and sensitivity per protocol (PP) analyses. Safety outcomes included rates of major/minor bleeding. Of 543 patients screened, 120 were recruited (mean age of 67.5 years). The ITT response rate to Aspirin was 48.3% (95% confidence interval [CI] 35.8–61.0%) according to ASPI-test and the response rate to ticagrelor was 41.4% (95% CI 29.3–54.6%) according to ADP-test. The PP ASPI-test response rate to Aspirin was 68.3% (95% CI 52.3–80.9%) and the PP ADP-test response rate to ticagrelor was 68.8% (95% CI 50.4–82.6%). There were no differences between the groups in the changes in Quality of Life using questionnaires (EQ5D 5L, St. George's COPD-C), inflammatory markers, carotid intima media thickness and vascular stiffness from baseline to 6-months. There were 5 type 1 bleeds according to the BARC criteria recorded in this study; 2 in the placebo arm, 2 in the Aspirin arm, and 1 in the ticagrelor arm. The MRC Dyspnoea score, FEV1 and FVC was similar across the groups. Primary outcome measures Aspirin No Aspirin Ticagrelor No Ticagrelor ITT analysis set* n 60 60 58 62 Baseline No. of responders 1 6 4 1 % (95% CI) 1.7 (0.2, 11.3) 10 (4.5, 20.8) 6.9 (2.6, 17.3) 1.6 (0.2, 10.9) 6 months No. of responders 29 7 24 2 % (95% CI) 48.3 (35.8, 61) 11.7 (5.6, 22.8) 41.4 (29.3, 54.6) 3.2 (0.8, 12.3) PP analysis set** n 41 45 32 54 6 months No. of responders 28 7 22 2 % (95% CI) 68.3 (52.3, 80.9) 15.6 (7.5, 29.6) 68.8 (50.4, 82.6) 3.7 (0.9, 14.0) *Descriptive statistics for the primary outcome of response for the comparative groups at baseline and 6 months for the ITT analysis set and **PP analysis set. Note that response is ASPI response in the Aspirin and No Aspirin columns and ADP response in the ticagrelor and No ticagrelor columns. Primary outcome measure Nearly one third of COPD patients did not have a platelet response to antiplatelet therapy with Aspirin and ticagrelor. These findings support the high pro-thrombotic milieu and the need for further research in COPD patients. Acknowledgement/Funding Astra Zeneca (Funder reference number ISSBRIL0303)

Antiplatelet therapy in the primary prevention of cardiovascular disease inpatients with chronic obstructive pulmonary disease: a randomised controlled proof-of-concept trial.

The APPLE COPD-ICON2 trial is a prospective 2×2 factorial, double-blinded proof-of-concept randomised controlled trial targeting patients with chronic obstructive pulmonary disease (COPD) without prior history of cardiovascular disease. The primary goal of this trial is to investigate if treatment with antiplatelet therapy will produce the predefined cut-off of platelet inhibition measured using the Multiplate test in COPD patients.Eligible patients were randomised to aspirin plus placebo, ticagrelor plus placebo, aspirin plus ticagrelor or placebo only for 6 months. The primary outcome comprises inhibition (binary response) of arachidonic acid- (ASPI test, cut-off <40) and adenosine diphosphate- (ADP test, cut-off <46) induced platelet aggregation at 6 months.543 patients were screened and 120 patients were recruited with mean age of 67.5 years; 47.5% patients were male. The per-protocol ASPI test response rate to aspirin was 68.3% (95% CI 52.3–80.9%). The per-protocol ADP test response rate to ticagrelaor was 68.8% (95% CI 50.4–82.6%).Platelet response to antiplatelet therapy with aspirin and ticagrelor was not observed in nearly one-third of COPD patients without prior history of cardiovascular disease. These findings support the high pro-thrombotic milieu and the need for further research to determine the effect of antiplatelet/antithrombotic therapy on cardiovascular morbidity and mortality in COPD patients.

Vicagrel enhances aspirin-induced inhibition of both platelet aggregation and thrombus formation in rodents due to its decreased metabolic inactivation

Both aspirin and vicagrel are effective antiplatelet drugs, with the potential for concomitant use as another dual-antiplatelet therapy for the prevention of recurrent thrombotic or ischemic events. Because they both are the substrates of carboxylesterase 2 (CES2), aspirin attenuated the metabolic activation of and platelet response to vicagrel in mice treated with the two drugs concomitantly. In this study, we sought to clarify whether vicagrel could affect platelet responses to aspirin and their underlying mechanisms. Plasma levels of aspirin and salicylic acid were determined by liquid chromatography-tandem mass spectrometry, inhibition of arachidonic acid (AA)-induced whole-blood platelet aggregation by aspirin was assessed with an aggregometer, and their antithrombotic effects were evaluated by arteriovenous shunt thrombosis model. The results showed that concomitant use of vicagrel (5, 10, or 20 mg/kg) led to an average of 55% and 77% increases in systemic exposure of aspirin (Cmax and AUC0-t) and 2.8-fold increase in suppression of AA-induced platelet aggregation in mice when compared with use of aspirin alone. In the rat thrombus formation model, vicagrel (1 mg/kg) enhanced inhibition of thrombosis formation by aspirin (5 mg/kg), but not vice versa. We conclude that vicagrel increases platelet responses to aspirin and also enhances inhibition of thrombus formation of aspirin due to decreased CES2-catalyzed aspirin inactivation in rodents, and that an integrated net effect on thrombus formation in vivo is superior to inhibition of AA- or ADP-induced platelet aggregation ex vivo by either of the two drugs if taken concomitantly.

Development of an original phospholipase A2-targeted peptide able to reduce amyloid pathology in a mouse model of Alzheimer’s disease

Event Abstract Back to Event Development of an original phospholipase A2-targeted peptide able to reduce amyloid pathology in a mouse model of Alzheimer’s disease Séverine André1, 2, Jean M. Atakana Lukulu1, 2, Marine Lecomte1, 2, Robert N. Muller1, 2, 3, Luce Vander Elst1, 2, Sophie Laurent1, 2, 3 and Carmen Burtea1, 2* 1 University of Mons, Belgium 2 Laboratory of Nuclear Magnetic Resonance and Molecular Imaging, University of Mons, Belgium 3 Centre for Microscopy and Molecular Imaging (CMMI), Belgium Introduction Alzheimer’s disease (AD) is the most common dementia worldwide and, since 1993, no new drug was approved by the FDA for its treatment because more than 95% of them have failed during clinical trials [1]. Moreover, actual therapies are only symptomatic and do not slow the progression of the disease. In this context, identification of new targets is crucial and phospholipase A2 (PLA2) signaling pathway has been proposed to be involved in this pathology [2], its inhibition showing neuron protection against apoptosis induced by amyloid beta (Aβ) [3]. Using the phage-display technology, we have identified a PLA2-targeted peptide (PLP25) aiming to reversibly and specifically inhibit this enzyme, contrariwise to existing inhibitory molecules. In order to improve its brain availability, we combined this peptide to a vector peptide (LRP2) targeting the LDL receptor (LDLR) and able to cross the blood-brain barrier. This complex (PLP25-LRP2), evaluated in vitro and in vivo, is expected to be beneficial for AD therapy. Methods The in vitro evaluation of PLP25-LRP2 aimed to determine: (i) the inhibition of arachidonic acid (AA) release by activated PLA2 in H2O2, glutamate or A stimulated cells; (ii) the effect on cellular localization of PLA2 and of AA-depending enzymes such as COX2 and ALOX5; and (iii) the actin cytoskeleton reorganization in cells treated with this complex. For in vivo validation, the PLP25-LRP2 complex was injected to APP/PS1 mice. Its therapeutic potential was first evaluated by assessing the spatial memory of APP/PS1 mice by the Barnes maze and detecting amyloid burden by Magnetic Resonance Imaging (MRI) using USPIO-PHO (peptide PHO identified by phage display and specific to Aβ, grafted to Ultrasmall Superparamagnetic Particles of Iron Oxide) [4,5]. Secondly, different biomarkers were evaluated ex vivo by immunohistochemistry, such as the PLA2 itself, amyloid plaques (AP), phosphorylated Tau protein (p-Tau) and NMDA receptors (NMDAR). Results H2O2, glutamate and A stimulate PLA2 activity and AA released by the induced cells, whereas our PLP25-LRP2 therapeutic complex reduces this release by 20% to 30%. In neuronal cells, glutamate and Aβ stimulate PLA2 translocation from cytosol to organelle membranes and plasma membrane processes, whereas PLP25-LRP2 is able to prevent this translocation, leading to PLA2 inhibition. Using AA released by PLA2, COX2 and ALOX5 relocated like PLA2 upon activation, while the reduced AA restricted their relocation. Finally, PLA2 inhibition by PLP25-LRP2 has also restrained the filopodial dynamics and reorganization of actin cytoskeleton, phenomena associated to neuronal excitotoxicity, characteristic to AD. Injected to APP/PS1 mice, our therapeutic complex seems to improve the spatial memory as shown by a lower number of errors and a higher percentage of time spent in the correct quadrant of the Barnes maze, compared to the mice injected with a non-specific peptide (NSP). Moreover, the PLP25-LRP2-treated mice exhibit a lower number of AP than mice injected with NSP, shown by both MRI and IHC. They also present a cellular localization of p-Tau similar to healthy mice, revealing the key role of Aβ in Tau pathology; the expression levels of PLA2 and NMDAR tend to be restored to the healthy control levels. Acknowledgements This work is supported by the Rotary Foundation – Hope in Head campaign 2015, the FRMH (Fond pour la Recherche Médicale en Hainaut) and the grant “Actions de Recherche Concertée” financed by the Fédération Wallonie-Bruxelles, the Waloon Region, Feder, Interreg and COST actions.

Investigation of the inhibition effect of arachidonic acid on the core structure of the HIV-1 gp41

The gp41 transmembrane domain of the envelope glycoprotein of the human immunodeficiency virus (HIV) modulates the conformation of the viral envelope spike. During the HIV fusion process, C-terminal heptad repeat (CHR, C34) wrap antiparallel to the N-terminal heptad repeat (NHR, N36) helices to form a stable six-helix bundle (6-HB) core structure, which brings the viral and cell membranes into close proximity for fusion. Therefore, inhibiting the formation of 6-HB is considered to be a key activity of an effective HIV-1 fusion inhibitor. The level of arachidonic acid (AA) is increased in HIV infected patients. Our study provides a new insight into the functional role of AA during the formation of HIV-1 gp41 6-HB. Native polyacrylamide gel electrophoresis (N-PAGE), enzyme-linked-immunosorbent serologic assay (ELISA) and circular dichroism (CD) spectroscopy were used to investigate the inhibition of AA for the formation of 6-HB. Molecular docking technique was adopted to explore the underlying mechanism. HIV-1 JR-FL (R5 strain) Envelope was adopted to determine the inhibition effect of AA. AA is shown to interfere with the formation of α-helical complexes of N36 and C34 by N-PAGE, ELISA and CD spectroscopy. The isotherm titration microcalorimetry (ITC) results indicate there is a single class of binding site on N36. ΔH and ΔS are −12.43 kJ mol−1 and 70.07 J mol−1 K−1, respectively, indicating hydrophobic interaction and electrostatic forces are the main acting forces. The molecular docking results manifest that AA interacts with the hydrophobic residues (Trp-571, Leu-568, Val-570 and Leu-576) and ionic interactions occur between Arg-579 and the -COOH of AA. The inhibitory activity of AA on HIV-1 JR-FL is quantified by 50% effective concentration (EC50) and 90% effective concentration (EC90), which are 31.42 ± 1.08 and 133.47 ± 18.10 μg mL−1, respectively. All the results indicate that AA is able to inhibit the formation of 6-HB but cannot disrupt the preformed 6-HB. Therefore, AA is a potential inhibitor for the viral fusion/entry.

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm2 for 12hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease.

Effects of phenidone (DuCLOX-2/5 inhibitor) against N-methyl-N-nitrosourea induced mammary gland carcinoma in albino rats

The present study was designed to evaluate the effects of phenidone (Dual inhibitor of COX-2 and 5-LOX, DuCLOX-2/5 inhibitor) on various aspects of cancer chemoprevention. Treatment with the phenidone was inquested to validate the implications of dual inhibition of arachidonic acid (AA) metabolism against MNU induced mammary gland carcinogenesis. MNU treated rat showed altered hemodynamic profile, distorted cellular architecture, upregulated inflammatory enzyme markers (COX, LOX, Nitric oxide and hydrogen sulfide) and distorted oxidative stress markers (thiobarbituric acid reactive substances, protein carbonyl, superoxide dismutase, catalase and glutathione). Phenidone treatment regulated histological architecture in the experimental animals similar to control. The treatment with phenidone favorably regulated the levels of inflammatory markers, and oxidative stress markers against toxic treatment. Our findings emphasize the potential role of phenidone in suppression of mammary gland carcinoma against the deleterious effects of MNU.

Assessment of three point-of-care platelet function assays in adult trauma patients

BackgroundAntiplatelet (AP) medication use is common among trauma patients and is associated with poor outcomes. Management options for platelet dysfunction in trauma patients are controversial, expensive, and potentially harmful. Although light transmission platelet aggregometry is considered the standard test to assess platelet function, it is cumbersome and not generally available. Currently, there are no widely accepted platelet function point-of-care tests for acute trauma. Study designProspective observational study from 2014 to 2015. Baseline Multiplate aggregometry aspirin area under the platelet aggregation curve (ASPI AUC), Thrombelastography Platelet Mapping percent inhibition of arachidonic acid (TEG-PM AA), and VerifyNow Aspirin Test (ARU) were compared for ability to detect any AP medication use (aspirin or clopidogrel), platelet dysfunction, and identify patients at risk for intracranial hemorrhage (ICH) progression by calculating the area under receiver operating characteristic curves (AUC), sensitivity, specificity, and positive and negative predictive values. Adenosine diphosphate assays were similarly evaluated. ResultsSixty-four patients were enrolled, 25 were taking AP medications. AP patients were older (71.6 versus 35.0 y, P < 0.001) and received more platelet transfusions, but other baseline characteristics were similar. Median ASPI AUC (22.0 versus 53.5 P < 0.001) and VerifyNow ARU (503.5 versus 629.0, P < 0.001) were lower, whereas TEG-PM AA (51.8% versus 18.3%, P < 0.001) was higher in AP patients. Multiplate ASPI AUC, TEG-PM AA percent inhibition, and VerifyNow ARU could identify AP medication use (AUC: 0.90, 0.77, and 0.90, respectively). Adenosine diphosphate assays did not correlate with AP medication use in this population. TEG-PM AA percent inhibition and VerifyNow ARU correlated well with Multiplate ASPI AUC to identify platelet dysfunction (AUC: 0.78, 0.89, respectively). ICH occurred in 29 patients; 12 of which had progression of their injury. ASPI AUC (AUC: 0.50) and VerifyNow ARU (AUC: 0.59) did not correlate, and TEG-PM AA percent inhibition (AUC: 0.66) minimally correlated with progression. ConclusionsMultiplate, TEG-PM, and VerifyNow are useful point-of-care tests which identify AP medication use and platelet dysfunction in trauma patients. Initial TEG-PM AA percent inhibition may be associated with risk for ICH progression. However, additional large, prospective studies are needed.

Asymmetric Binding and Metabolism of Polyunsaturated Fatty Acids (PUFAs) by CYP2J2 Epoxygenase.

Cytochrome P450 (CYP) 2J2 is the primary epoxygenase in the heart and is responsible for the epoxidation of arachidonic acid (AA), an ω-6 polyunsaturated fatty acid (PUFA), into anti-inflammatory epoxide metabolites. It also epoxidizes other PUFAs such as docosahexaenoic acid (DHA), linoleic acid (LA), and eicosapentaenoic acid (EPA). Herein, we have performed detailed thermodynamic and kinetic analyses to determine how DHA, LA, and EPA modulate the metabolism of AA by CYP2J2. We use the Nanodisc system to stabilize CYP2J2 and its redox partner, CYP reductase (CPR). We observe that DHA strongly inhibits CYP2J2-mediated AA metabolism, LA only moderately inhibits AA metabolism, and EPA exhibits insignificant inhibition. We also characterized the binding of these molecules using ebastine competitive binding assays and show that DHA binds significantly tighter to CYP2J2 than AA, EPA, or LA. Furthermore, we utilize a combined approach of molecular dynamics (MD) simulations and docking to predict key residues mediating the tight binding of DHA. We show that although all the tested fatty acids form similar contacts to the active site residues, the affinity of DHA for CYP2J2 is tighter because of the interaction of DHA with residues Arg-321, Thr-318, and Ser-493. To demonstrate the importance of these residues in binding, we mutated these residues to make two mutant variants, CYP2J2-T318A and CYP2J2-T318V/S493A. Both mutant variants showed weaker binding than the wild type (WT) to DHA and AA; DHA inhibition of AA was also mitigated in the mutants compared to the WT. Therefore, using a combined experimental and MD simulation approach, we establish that CYP2J2 inhibition of AA metabolism by DHA, EPA, and LA is asymmetric because of tighter binding of DHA to select residues in the active site.

Pharmacological basis of the use of the root bark of Zizyphus nummularia Aubrev. (Rhamnaceae) as anti-inflammatory agent.

BackgroundThe root bark of Zizyphus nummularia (Rhamnaceae) is traditionally used as an anti-inflammatory agent. The current study aimed to explore the anti-inflammatory activity (in vivo) of a crude ethanolic extract (EE) and the pure identified octadecahydro-picene-2,3,14,15-tetranone (IC) in the root bark of Z. nummularia. IC was further subjected to suitable in vitro and in silico studies to find out the mechanistic pharmacology.MethodsEE (100 and 200 mg/kg, p.o.) and (IC) (400 and 600 μg/kg, p.o.) were subjected to in vivo anti-inflammatory assays to evaluate the anti-inflammatory activity and predict the probable mechanism(s) of action. Suitable acute (carrageenan-induced paw edema, arachidonic acid-induced ear edema, xylene-induced ear edema) and chronic (cotton pellet granuloma) models were employed to investigate in vivo the anti-inflammatory activity. Based on in vivo observation, IC was further subjected to in vitro assays to estimate the inhibition of nitric oxide (NO), prostaglandin-E2 (PGE-2) and tumor necrosis factor-α (TNF-α) production in PBS stimulated RAW 264.7 cells. Based on the observation of in vitro studies, finally, ADME prediction and molecular docking studies of IC were performed for better understanding of interaction of IC with TNF-α.ResultsOral administration of EE (100 and 200 mg/kg) exhibited significant inhibition of carrageenan (p < 0.05) and arachidonic acid (p < 0.05) induced oedema, and the reduced the granuloma tissue formation (p < 0.05) in experimental mice. IC (400 and 600 μg/kg, p.o.) exhibited significant (p < 0.01) inhibition of carrageenan, xylene and arachidonic acid-induced edema, and reduced the granuloma tissue formation. In in vitro assays, IC caused a concentration-dependent inhibition of LPS stimulated NO (up to ~ 67.4 % at 50 μM) and TNF-α (~84.5 % at 50 μM) production. However, the PGE-2 inhibition did not follow dose dependent pattern. Based on in vitro observations, the molecular docking has been performed on the basis of interaction with TNF-α. In in silico studies, it was observed that IC showed hydrogen bonding with GLN 47 amino acid residue of TNF-α protein.ConclusionsIC possibly produces anti-inflammatory activity through inhibition of TNF-α and NO production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0942-7) contains supplementary material, which is available to authorized users.

ANTI-INFLAMMATORY HERBS AND THEIR MOLECULAR MECHANISMS OF ACTION

Background: Medicinal plant and plant products have shown tremendous potentials and are used beneficially in the treatment of inflammation and in the management of diseases with significant inflammatory components. Many medicinal plants employed as anti-inflammatory and anti-phlogistic remedies lack the gastro-erosive side effects of non-steroidal anti-inflammatory drugs (NSAID) or the plethora of unwanted side effects associated with steroidal anti-inflammatory drugs. In order to harness and optimise the applications of these herbs in inflammatory diseases, there is a need to understand how these herbs produce their anti-inflammatory actions.&#x0D; Materials and Methods: This paper is a review of some anti-inflammatory herbs and their molecular mechanisms of action. A literature search and analysis of published manuscript was employed to x-ray research findings that show how medicinal plants produce anti-inflammatory activities.&#x0D; Results: Many studies have shown that anti-inflammatory activities of herbal extracts and herb-derived compounds are mainly due to their inhibition of arachidonic acid (AA) metabolism, cyclo-oxygenase (COX), lipo-oxygenase (LOX), pro-inflammatory cytokines, inducible nitric oxide, and transcription activation factor (NF-κB). Some anti-inflammatory medicinal herbs are reported to stabilize lysosomal membrane and some cause the uncoupling of oxidative phosphorylation of intracellular signalling molecules. Many have also been shown to possess strong oxygen radical scavenging activities. &#x0D; Conclusion: Most of the mechanisms by which anti-inflammatory medicinal plants act are related and many herbal products have been shown to act through a combination of these molecular pathways.

Antiplatelet Effects of Flavonoids Mediated by Inhibition of Arachidonic Acid Based Pathway.

Flavonoids, important components of human diet, have been claimed to possess a significant antiplatelet potential, in particular due to their effects on the arachidonic acid cascade. Due to variable and incomplete results, this study was aimed at delivering a detailed analysis of the effects of 29 structurally relevant, mainly natural flavonoids on three consecutive steps of the arachidonic acid cascade.Only the isoflavonoids genistein and daidzein were shown to possess a marked cyclooxygenase-1 inhibitory activity, which was higher than that of acetylsalicylic acid using the isolated ovine enzyme, and physiologically relevant, although lower than acetylsalicylic acid in human platelets. None of the tested flavonoids possesses an effect on thromboxane synthase in a clinically achievable concentration. Contrarily, many flavonoids, particularly those possessing an isolated 7-hydroxyl group and/or a 4'-hydroxyl group, acted as antagonists on thromboxane receptors. Interestingly, the substitution of the free 7-hydroxyl group by glucose might not abolish the activity.In conclusion, the consumption of few flavonoids in a diet, particularly of the isoflavonoids genistein and daidzein, may positively influence platelet aggregation.

13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.