Hawthorn leaf flavonoids alleviate the deterioration of atherosclerosis by inhibiting SCAP-SREBP2-LDLR pathway through sPLA2-â ¡A signaling in macrophages in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Hawthorn leaves are a combination of the dried leaves of the Rosaceae plants, i.e., Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge. var. major N. E. Br., is primarily cultivated in East Asia, North America, and Europe. hawthorn leaf flavonoids (HLF) are the main part of extraction. The HLF have demonstrated potential in preventing hypertension, inflammation, hyperlipidemia, and atherosclerosis. However, the potential pharmacological mechanism behind its anti-atherosclerotic effect has yet to be explored. AIM OF THE STUDY: The in vivo and in vitro effects of HLF on lipid-mediated foam cell formation were investigated, with a specific focus on the levels of secreted phospholipase A2 type IIA (sPLA2-II A) in macrophage cells. MATERIALS AND METHODS: The primary constituents of HLF were analyzed using ultra-high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. In vivo, HLF, at concentrations of 5 mg/kg, 20 mg/kg, and 40 mg/kg, were administered to apolipoprotein E knockout mice (ApoE-/-) fed by high-fat diet (HFD) for 16 weeks. Aorta and serum samples were collected to identify lesion areas and lipids through mass spectrometry analysis to dissect the pathological process. RAW264.7 cells were incubated with oxidized low-density lipoprotein (ox-LDL) alone, or ox-LDL combined with different doses of HLF (100, 50, and 25 µg/ml), or ox-LDL plus 24-h sPLA2-IIA inhibitors, for cell biology analysis. Lipids and inflammatory cytokines were detected using biochemical analyzers and ELISA, while plaque size and collagen content of plaque were assessed by HE and the Masson staining of the aorta. The lipid deposition in macrophages was observed by Oil Red O staining. The expression of sPLA2-IIA and SCAP-SREBP2-LDLR was determined by RT-qPCR and Western blot analysis. RESULTS: The chemical profile of HLF was studied using UPLC-Q-TOF-MS/MS, allowing the tentative identification of 20 compounds, comprising 1 phenolic acid, 9 flavonols and 10 flavones, including isovitexin, vitexin-4″-O-glucoside, quercetin-3-O-robibioside, rutin, vitexin-2″-O-rhamnoside, quercetin, etc. HLF decreased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) levels in ApoE-/- mice (P < 0.05), reduced ox-LDL uptake, inhibited level of inflammatory factors, such as IL-6, IL-8, TNF-α, and IL-1ꞵ (P < 0.001), and alleviated aortic plaques with a thicker fibrous cap. HLF effectively attenuated foam cell formation in ox-LDL-treated RAW264.7 macrophages, and reduced levels of intracellular TC, free cholesterol (FC), cholesteryl ester (CE), IL-6, TNF-α, and IL-1ß (P < 0.001). In both in vivo and in vitro experiments, HLF significantly downregulated the expression of sPLA2-IIA, SCAP, SREBP2, LDLR, HMGCR, and LOX-1 (P < 0.05). Furthermore, sPLA2-IIA inhibitor effectively mitigated inflammatory release in RAW264.7 macrophages and regulated SCAP-SREBP2-LDLR signaling pathway by inhibiting sPLA2-IIA secretion (P < 0.05). CONCLUSION: HLF exerted a protective effect against atherosclerosis through inhibiting sPLA2-IIA to diminish SCAP-SREBP2-LDLR signaling pathway, to reduce LDL uptake caused foam cell formation, and to slow down the progression of atherosclerosis in mice.

Syringol isolated from Eleusine coracana (L.) Gaertn bran suppresses inflammatory response through the down-regulation of cPLA2, COX-2, IκBα, p38 and MPO signaling in sPLA2 induced mice paw oedema.

Eleusine coracana (L.) Gaertn (E. coracana) is one of the highest consuming food crops in Asia and Africa. E. coracana is a plant with several medicinal values including anti-ulcerative, anti-diabetic, anti-viral and anti-cancer properties. However, the anti-inflammatory property of E. coracana remains to be elucidated. Therefore, the objective of present study was to investigate the potential in isolated molecule from E. coracana via a combination of in vitro, in vivo and in silico methods. In this study, we have isolated, purified and characterized an anti-inflammatory molecule from E. coracana bran extract known as syringol. Purification of syringol was accomplished by combination of GC-MS and RP-HPLC techniques. Syringol significantly inhibited the enzymes activity of sPLA2 (IC50 = 3.00 µg) and 5-LOX (IC50 = 0.325 µg) in vitro. The inhibition is independent of substrate concentration, calcium ion concentration and was irreversible. Syringol interacts with purified sPLA2 enzymes as evidenced by fluorescence and molecular docking studies. Further, the syringol molecule dose dependently inhibited the development of sPLA2 and λ-carrageenan induced edema. Furthermore, syringol decreases the expression of cPLA2, COX-2, IκBα, p38 and MPO in edematous tissues as demonstrated by western blots. These studies revealed that syringol isolated from E. coracana bran may develop as a potent anti-inflammatory molecule.

Oleanolic acid protects against cognitive decline and neuroinflammation-mediated neurotoxicity by blocking secretory phospholipase A2 IIA-activated calcium signals.

Neuroinflammation causes neurotoxic injury and underlies the pathogenesis of neurodegenerative disorders including Alzheimer's disease (AD). Astrocytes are the predominant immunoregulatory cells in AD. Oleanolic acid (OA) is a promising anti-inflammatory therapeutic agent that can ameliorate cerebral damage in ischemic environments, but its role in AD remains poorly elucidated. Here, preconditioning with OA inhibited the transcription and secretion of inflammatory cytokines IL-6, TNF-α, and IL-1ß in amyloid-beta peptide (Aß)-activated astrocytes. Moreover, OA ameliorated primary neuron death triggered by incubation in conditioned medium from Aß-treated astrocytes. Furthermore, OA also suppressed Aß-induced expression and production of group IIA secretory phospholipase A2 (sPLA2-IIA) in astrocytes. Supernatants supplemented with exogenous sPLA2-IIA reversed the protective role of OA against astrocyte activation-mediated neurotoxicity by suppressing cell viability and increasing LDH release, apoptosis, the contents of neurotoxic mediator arachidonic acid, and prostaglandin D2. Simultaneously, treatment with sPLA2 inhibitor aristolochic acid also counteracted neurotoxicity induced by Aß-activated astrocytes through increasing cell viability, inhibiting cell apoptosis, and reducing the releases of arachidonic acid and prostaglandin D2. Additionally, OA restrained Ca2+ influx in neurons after incubation with supernatants from Aß-activated astrocytes, which was abrogated by adding sPLA2-IIA. Activating Ca2+ signaling with BayK, an L-type Ca2 + channel agonist, reversed the beneficial role of OA against neurotoxicity induced by astrocyte activation-mediated inflammatory response. OA also ameliorated cognitive deficits in an adolescent rat model of Aß-evoked AD. These findings confirm that OA abrogates neuroinflammation and subsequent neurotoxicity induced by conditioned media from Aß-activated astrocytes in sPLA2-IIA mediated-calcium signals. Therefore, OA may protect neurons from injury caused by neighboring astrocyte activation in AD, indicating a promising therapeutic strategy against AD.

Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress.

Cancer cells driven by the Ras oncogene scavenge unsaturated fatty acids (FAs) from their environment to counter nutrient stress. The human group X secreted phospholipase A2 (hGX sPLA2) releases FAs from membrane phospholipids, stimulates lipid droplet (LD) biogenesis in Ras-driven triple-negative breast cancer (TNBC) cells and enables their survival during starvation. Here we examined the role of LDs, induced by hGX sPLA2 and unsaturated FAs, in protection of TNBC cells against nutrient stress. We found that hGX sPLA2 releases a mixture of unsaturated FAs, including ω-3 and ω-6 polyunsaturated FAs (PUFAs), from TNBC cells. Starvation-induced breakdown of LDs induced by low micromolar concentrations of unsaturated FAs, including PUFAs, was associated with protection from cell death. Interestingly, adipose triglyceride lipase (ATGL) contributed to LD breakdown during starvation, but it was not required for the pro-survival effects of hGX sPLA2 and unsaturated FAs. High micromolar concentrations of PUFAs, but not OA, induced oxidative stress-dependent cell death in TNBC cells. Inhibition of triacylglycerol (TAG) synthesis suppressed LD biogenesis and potentiated PUFA-induced cell damage. On the contrary, stimulation of LD biogenesis by hGX sPLA2 and suppression of LD breakdown by ATGL depletion reduced PUFA-induced oxidative stress and cell death. Finally, lipidomic analyses revealed that sequestration of PUFAs in LDs by sPLA2-induced TAG remodelling and retention of PUFAs in LDs by inhibition of ATGL-mediated TAG lipolysis protect from PUFA lipotoxicity. LDs are thus antioxidant and pro-survival organelles that guard TNBC cells against nutrient and lipotoxic stress and emerge as attractive targets for novel therapeutic interventions.

Non-Clinical Studies for Evaluation of 8-C-Rhamnosyl Apigenin Purified from Peperomia obtusifolia against Acute Edema.

Compound 8-C-rhamnosyl apigenin (8CR) induced a moderate reduction in the enzymatic activity of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus and cytosolic phospholipase A2 (cPLA2), but the compound also significantly inhibited the enzymatic activity of the enzyme cyclooxygenase. In vitro assays showed that the compound induced a slight change in the secondary structure of sPLA2 from Crotalus durissus terrificus snake venom. In vivo assays were divided into two steps. In the first step, the 8CR compound was administered by intraperitoneal injections 30 min prior to administration of sPLA2. In this condition, 8CR inhibited edema and myonecrosis induced by the sPLA2 activity of Crotalus durissus terrificus in a dose-dependent manner by decreasing interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), prostaglandin E2 (PGE2), and lipid peroxidation. This has been demonstrated by monitoring the levels of malondialdehyde (MDA) in rat paws after the course of edema induced by sPLA2. These results, for the first time, show that sPLA2 of Crotalus durissus terrificus venom induces massive muscle damage, as well as significant edema by mobilization of cyclooxygenase enzymes. Additionally, its pharmacological activity involves increased lipid peroxidation as well as TNF-α and IL-1ß production. Previous administration by the peritoneal route has shown that dose-dependent 8CR significantly decreases the enzymatic activity of cyclooxygenase enzymes. This resulted in a decrease of the amount of bioactive lipids involved in inflammation; it also promoted a significant cellular protection against lipid peroxidation. In vivo experiments performed with 8CR at a concentration adjusted to 200 µg (8 mg/kg) of intraperitoneal injection 15 min after sPLA2 injection significantly reduced sPLA2 edema and the myotoxic effect induced by sPLA2 through the decrease in the enzymatic activity of cPLA2, cyclooxygenase, and a massive reduction of lipid peroxidation. These results clearly show that 8CR is a potent anti-inflammatory that inhibits cyclooxygenase-2 (COX-2), and it may modulate the enzymatic activity of sPLA2 and cPLA2. In addition, it was shown that Crotalus durissus terrificus sPLA2 increases cell oxidative stress during edema and myonecrosis, and the antioxidant properties of the polyphenolic compound may be significant in mitigating the pharmacological effect induced by sPLA2 and other snake venom toxins.

Effects of Statins and Xuezhikang on the Expression of Secretory Phospholipase A2, Group IIA in Rat Vascular Smooth Muscle Cells.

Atherosclerosis is a multifactorial vascular disease characterized by formation of inflammatory lesions. Secretory phospholipase A2, group IIA (sPLA2-IIA) is involved in this process and plays a critical role. However, the exact role of sPLA2-IIA in cardiovascular inflammation is more complicated and remains unclear. Furthermore, both statins and Xuezhikang (XZK) are widely used in the prevention and treatment of cardiovascular disease risk because of their pleiotropic effects on the cardiovascular system. However, their effects on sPLA2-IIA are still controversial. We investigated the regulation of sPLA2-IIA by rat thoracic aorta smooth muscle cells (VSMCs) in culture. Cells were first incubated with IL-1ß alone to induce expression of sPLA2-IIA and then treated with several concentrations of statins or XZK for different times in the absence or presence of IL-1ß. We tested the expression of sPLA2-IIA, including sPLA2-IIA mRNA, protein, as well as activity. We found that statins or IL-1ß increase the expression of sPLA2-IIA in VSMCs and the effect is based on a synergetic relationship between them. However, for the first time, we observed that XZK effectively reduces sPLA2-IIA expression in IL-1ß-treated VSMCs. Our findings may shine a new light on the clinical use of XZK and statins in the prevention and treatment of atherosclerosis-related thrombosis.

Evaluating the inhibitory potential of Withania somnifera on platelet aggregation and inflammation enzymes: An in vitro and in silico study.

Context Withania somnifera (L.) Dunal is traditionally used for treating various ailments, but lacks scientific evaluation. Objective This study evaluates Withania somnifera (WS) for its effect on platelet activity and inflammatory enzymes. Materials and methods Aqueous and ethanolic (1:1) leaf extracts were subjected to in vitro indirect haemolytic activity using Naja naja venom, human platelet aggregation was quantified for lipid peroxidation using arachidonic acid (AA) as agonist and 5-lipoxygenase (5-LOX) levels were determined using standard spectrometric assays. Further, molecular docking was performed by the ligand fit method using molegro software package (Molegro ApS, Aarhus, Denmark). Results The study found that aqueous and ethanol extracts have very negligible effect (15%) with an IC50 value of 13.8 mg/mL on PLA2 from Naja naja venom. Further, extracts of WS also had very little effect (18%) with an IC50 value of 16.6 mg/mL on malondialdehyde (MDA) formation. However, a 65% inhibition of 5-LOX with an IC50 value of 0.92 mg/mL was observed in 1:1 ethanol extracts. The same was evident from SAR model with the active ingredient withaferin A binding predominantly on Phe 77, Tyr 98, Arg 99, Asp 164, Leu 168, Ser 382, Arg 395, Tyr 396 and Tyr 614 with an atomic contact energy value of -128.96 compared to standard phenidone (-103.61). Thus, the current study validates the application of WS for inflammatory diseases. Conclusion This study reveals the inhibitory potential of W. somnifera on inflammatory enzymes and platelet aggregation. Thus, WS can serve as a newer, safer and affordable medicine for inflammatory diseases.

Therapeutic effect of Qingyi decoction in severe acute pancreatitis-induced intestinal barrier injury.

AIM: To investigate the effect of Qingyi decoction on the expression of secreted phospholipase A2 (sPLA2) in intestinal barrier injury. METHODS: Fifty healthy Sprague-Dawley rats were randomly divided into control, severe acute pancreatitis (SAP), Qingyi decoction-treated (QYT), dexamethasone-treated (DEX), and verapamil-treated (VER) groups. The SAP model was induced by retrograde infusion of 1.5% sodium deoxycholate into the biliopancreatic duct of the rats. All rats were sacrificed 24 h post-SAP induction. Arterial blood, intestine, and pancreas from each rat were harvested for investigations. The levels of serum amylase (AMY) and diamine oxidase (DAO) were determined using biochemical methods, and serum tumor necrosis factor (TNF)-α level was measured by an enzyme linked immunosorbent assay. Pathologic changes in the harvested tissues were investigated by microscopic examination of hematoxylin and eosin-stained tissue sections. The expressions of sPLA2 at mRNA and protein levels were detected by reverse transcriptase PCR and Western blot, respectively. A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was used to investigate apoptosis of epithelial cells in the intestinal tissues. RESULTS: Compared to the control group, the expression of sPLA2 at both the mRNA and protein levels increased significantly in the SAP group (0.36 ± 0.13 vs 0.90 ± 0.38, and 0.16 ± 0.05 vs 0.64 ± 0.05, respectively; Ps < 0.01). The levels of AMY, TNF-α and DAO in serum were also significantly increased (917 ± 62 U/L vs 6870 ± 810 U/L, 59.7 ± 14.3 ng/L vs 180.5 ± 20.1 ng/L, and 10.37 ± 2.44 U/L vs 37.89 ± 5.86 U/L, respectively; Ps < 0.01). The apoptosis index of intestinal epithelial cells also differed significantly between the SAP and control rats (0.05 ± 0.02 vs 0.26 ± 0.06; P < 0.01). The serum levels of DAO and TNF-α, and the intestinal apoptosis index significantly correlated with sPLA2 expression in the intestine (r = 0.895, 0.893 and 0.926, respectively; Ps < 0.05). The levels of sPLA2, AMY, TNF-α, and DAO in the QYT, VER, and DEX groups were all decreased compared with the SAP group, but not the control group. Qingyi decoction intervention, however, gave the most therapeutic effect against intestinal barrier damage, although the onset of its therapeutic effect was slower. CONCLUSION: Qingyi decoction ameliorates acute pancreatitis-induced intestinal barrier injury by inhibiting the overexpression of intestinal sPLA2. This mechanism may be similar to that of verapamil.

Long-chain fatty alcohols from evening primrose oil inhibit the inflammatory response in murine peritoneal macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Evening primrose (Oenothera biennis L., Onagraceae) is a wild medicinal plant of Central American origin that is now one of the most widely used herbal medicines in different parts of the world. Oil extracted from it seeds is traditionally used in the treatment of eczema, asthma, rheumatoid arthritis, breast problem, premenstrual and menopausal syndrome, all they have an inflammatory component. The present study demonstrates the in vitro anti-inflammatory effect of long-chain fatty alcohols, minor compounds isolated from Evening primrose oil (EPO). MATERIAL AND METHODS: A mixture of long chain fatty alcohols (LCFAs) was isolated from the non-triacylglycerol fraction of the EPO. Hexacosanol (C26OH: 38.65%), tetracosanol (C24OH: 31.59%), docosanol (C22OH: 11.36%) and octocosanol (C28OH: 7.64%), were the major constituents, identified and quantified by GC and GC-MS. LCFA was tested with LPS stimulated murine peritoneal macrophage. This fraction, significantly and dose-dependently decreased nitric oxide production induced by LPS (P<0.001) and the inhibitory effect seems to be consequence of an action at the level of the inducible nitric-oxide synthethase (iNOS) gene enzyme expression rather than to a direct inhibitory action on enzyme activity. The release of PLA2 and TXB2 also was significantly inhibited by LCFAs (P<0.001) although LCFAs did not affect to PGE2 generation, however the western blot assay showed that LCFAs reduced cyclooxygenase-2 enzyme gene expression at all doses assayed. In the same way, the secretion of inflammatory cytokines interleukin 1ß (IL-1ß) and tumour necrosis factor α (TNF-α) from LPS-stimulated murine macrophage, were also significantly reduced (P<0.001). CONCLUSION: These results demonstrates the anti-inflammatory activity of LCFAs, providing an additional value about the role of bioactive minor compounds in the beneficial effect of EPO and supports its traditional uses in inflammatory processes management.

A comparative study: In vitro effects of EPA and DHA on immune functions of head-kidney macrophages isolated from large yellow croaker (Larmichthys crocea).

Comparative effects of different concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on immune responses of head-kidney macrophages isolated from large yellow croaker were studied in vitro. After exposing to serum-free medium for 1 day, cultured cells were incubated in medium supplemented with graded levels of EPA or DHA (0, 5, 25, 100, 200 and 1000 µM, respectively) in the form of fatty acid bovine serum albumin (FA-BSA) complex for 12 h, 24 h and 36 h, respectively. Control samples were incubated in the absence of EPA or DHA (2% bovine serum albumin, BSA). Following stimulation, cell viability, lipid peroxidation, secretary phopholipase A2 (sPLA2) and prostaglandin E2 (PGE2) production as well as some immune parameters including phagocytosis, respiratory burst activity and interleukin 1ß (IL-1ß) production were determined. Results showed that EPA and DHA affected cell viability in dose-dependent and time-dependent manners. In particular, cell viability was significantly decreased after 24 h and 36 h incubation with 1000 µM EPA or DHA (P < 0.05). Higher levels of EPA (200 and 1000 µM) caused a significant increase in the production of malondialdehyde (MDA) (P < 0.05), while DHA did not significantly affect the MDA production. EPA significantly increased the intracellular superoxide anion synthesis which, on the contrary, was significantly reduced by DHA. Phagocytosis percentage (PP) values were significantly higher in treatments with 5 µM DHA (P < 0.05), but significantly decreased by 200 and 1000 µM EPA and DHA compared to the control group (P < 0.05). Decreased PGE2 production was produced by cells treated with relatively low doses of EPA or DHA. When high levels of stimulants (1000 µM EPA or DHA) were used, PGE2 levels were elevated and reached a significant level (P < 0.05). Both EPA and DHA significantly inhibited the production of sPLA2, where DHA exerted the more potent inhibitory effects than EPA. No pronounced effect was observed on IL-1ß production among all the treatments, and IL-1ß level in cell culture supernatant was fairly low (only approximately 6 pg/ml). Those findings suggested that EPA and DHA could influence the immunity and physiological conditions of macrophages from head kidney of large yellow croaker in vitro.

Inhibition of cPLA2 and sPLA2 activities in primary cultures of rat cortical neurons by Centella asiatica water extract.

Leaf extract of Centella asiatica has been used as an alternative medicine for memory improvement in the Indian Ayurvedic system of medicine for a long time. Although several studies have revealed its effect in ameliorating the cognitive impairment in rat models of Alzheimer's disease, the molecular mechanism of C. asiatica on neuroprotection still remains unexplained. In this study, we investigated the effects of C. asiatica water extract on activity of subtypes of phospholipase A2 (PLA2) in primary cultures of rat cortical neurons and quantified by HPLC a possible molecule responsible for the activity. The cPLA2 and sPLA2 activities were inhibited in vitro by asiaticoside present in the water extract of C. asiatica. This extract may be a candidate for the treatment of neurodegenerative processes because of its pharmacological activity in the brain and its low toxicity, as attested by its long popular use as a natural product.

Gabapentin's minimal action on markers of rat brain arachidonic acid metabolism agrees with its inefficacy against bipolar disorder.

In rats, FDA-approved mood stabilizers used for treating bipolar disorder (BD) selectively downregulate brain markers of the arachidonic acid (AA) cascade, which are upregulated in postmortem BD brain. Phase III clinical trials show that the anticonvulsant gabapentin (GBP) is ineffective in treating BD. We hypothesized that GBP would not alter the rat brain AA cascade. Chronic GBP (10 mg/kg body weight, injected i.p. for 30 days) compared to saline vehicle did not significantly alter brain expression or activity of AA-selective cytosolic phospholipase A(2) (cPLA(2)) IVA or secretory (s)PLA(2) IIA, activity of cyclooxygenase-2, or prostaglandin E(2) or thromboxane B(2) concentrations. Plasma esterified and unesterified AA concentration was unaffected. These results, taken with evidence of an upregulated AA cascade in the BD brain and that approved mood stabilizers downregulate the rat brain AA cascade, support the hypothesis that effective anti-BD drugs act by targeting the brain AA cascade whereas ineffective drugs (such as GBP) do not target this pathway, and suggest that the rat model might be used for screening new anti-BD drugs.

Varespladib methyl in cardiovascular disease.

IMPORTANCE OF THE FIELD: The high risk of recurrent cardiovascular events amongst patients with cardiovascular disease receiving evidence-based therapies has prompted investigations into complimentary treatments that may reduce residual risk. Analyses of clinical trials in statin-treated patients demonstrate that elevated lipid levels and an activated systemic inflammatory state are associated with a higher risk of recurrent cardiovascular events. AREAS COVERED IN THIS REVIEW: This article reviews evidence supporting the causal role for secretory phospholipase A(2) (sPLA(2)) in experimental atherosclerosis, the involvement of various sPLA(2) isozymes as mediators of pro-atherogenic lipoprotein remodeling and participants in vascular and systemic inflammatory responses, and the evidence that sPLA(2) inhibition reduces atherosclerosis in experimental models and biomarkers associated with cardiovascular events in coronary heart disease (CHD) patients. WHAT THE READER WILL GAIN: The experimental basis for sPLA(2) inhibition with varespladib methyl as a potential candidate for lowering recurrent cardiovascular events particularly in acute coronary syndrome patients is discussed. TAKE HOME MESSAGE: Varespladib methyl therapy reduces atherogenic lipoprotein concentrations and systemic inflammatory markers in CHD patients. The future role of varespladib methyl in CHD patients awaits the results of ongoing clinical trials.

Genistein, a potent inhibitor of secretory phospholipase A2: a new insight in down regulation of inflammation.

OBJECTIVES: Some of the legumes, spices and medicinal herbs rich in genistein are known for their anti-inflammatory properties. Anti-inflammatory property of these herbs is determined by subjecting secretory phospholipase A(2) (sPLA(2)) inhibition, a key enzyme in the inflammatory reactions by genistein. MATERIALS AND METHODS: Genistein was assessed for inhibition of sPLA(2) activity using (14)C-oleate radiolabelled Escherichia coli membrane as substrate. The enzyme-inhibitor interaction was established by intrinsic fluorescence and circular dichroism studies. The in vivo anti-inflammatory activity was tested by injecting sPLA(2), Vipera russelli venom phospholipase-V (VRV-PL-V) with different concentrations of genistein in the range of 3-21 muM into intra plantar surface of right hind footpad of mice. Systemic effect was tested by administering the genistein (21 muM) i.p. 30 min before and immediately after sPLA(2) injection. RESULT: Genistein inhibited sPLA(2) enzymes of inflammatory exudates (human synovial fluid and human pleural fluid) and snake venoms (VRV-PL-V and Naja naja phospholipase-I) in a concentration dependent manner with IC(50) values ranging from 5.75 to 11.75 muM. Increasing the calcium (Ca(2+)) concentration from 2.5 to 15 mM and substrate concentration up to 120 nM did not alter the level of inhibition. Genistein alters the intrinsic fluorescence intensity and shown apparent shift in far ultra violet-circular dichroism spectra of VRV-PL-V, indicating the direct interaction with enzyme. Genistein also inhibited the VRV-PL-V induced mouse paw oedema in a concentration dependent manner. The genistein at 21 muM concentration administered immediately after the VRV-PL-V injection, effectively neutralized the oedema inducing activity. CONCLUSION: Genistein inhibited sPLA(2) activity of both inflammatory exudates and snake venoms in a concentration dependent manner and sPLA(2) induced mouse paw oedema. The study partially explains the observed anti-inflammatory property of several medicinal herbs which containing genistein.

Inhibition of pro-inflammatory secreted phospholipase A2 by extracts from Cynara cardunculus L.

The aim of the present work was to evaluate the anti-inflammatory properties of Cynara cardunculus L. (Asteraceae) during its growth using various solvents such as n-hexane, dichloromethane, acetone, and methanol for air-dried leaves and stems. The anti-inflammatory activities of crude extracts were evaluated by measuring the inhibition potency of mammalian non-pancreatic phospholipases A2 (hG-IIA). The methanol and acetone extracts of leaves harvested in February exhibit potent inhibition of hG-IIA (IC(50) = 50 and 70 microg/ml, respectively). However, the acetone extract of stems harvested in December inhibits the hG-IIA with a lower IC(50) around 130 microg/ml. Fractionation on silica gel and hydrophobic gel of the methanol extract of leaves harvested in February increases the inhibitory effect, and the IC(50) reached 10 microg/ml.

Docosahexaenoic acid down-regulates endothelial Nox 4 through a sPLA2 signalling pathway.

We investigated the anti-inflammatory and antioxidant activities of docosahexaenoic acid (DHA) by evaluating its modulation of the two enzymes most involved in vascular inflammation, i.e. endothelial secreted phospholipase A(2) (sPLA(2)) and NADPH oxidase 4 (Nox) 4. Exposure of human aortic endothelial cells (HAECs) to DHA led to its preferential incorporation into outer leaflet phospholipids. Pre-treatment with DHA abolished HAECs stimulation induced by A23187 and Ang II, whereas the effects on IL-1beta treatment were less pronounced. Group V sPLA(2) RNA was similarly modulated by DHA supplementation. In addition, DHA decreased Nox 4 expression and activity; this effect was associated with reduced production of reactive oxygen species. Further, the use of specific inhibitors allowed demonstrating that group V sPLA(2) is involved in the down-regulation of Nox 4 expression and activity by DHA. This interplay is mediated by ERK and PKC.

Inhibition of cyclooxygenase-2 aggravates secretory phospholipase A2-mediated progression of acute liver injury.

Our previous study [Bhave, V. S., Donthamsetty, S., Latendresse, J. R., Muskhelishvili, L., and Mehendale, H. M. 2008-this issue. Secretory phospholipase A(2) mediates progression of acute liver injury in the absence of sufficient COX-2. Toxicol Appl Pharmacol] showed that in the absence of sufficient induction and co-presence of cyclooxygenase-2 (COX-2), secretory phospholipase A(2) (sPLA(2)) appearing in the intercellular spaces for cleanup of post-necrotic debris seems to contribute to the progression of toxicant-initiated liver injury, possibly by hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas. To further test our hypothesis on the protective role of COX-2, male Fisher-344 rats were administered a selective COX-2 inhibitor, NS-398, and then challenged with a moderately toxic dose of CCl(4). This led to a 5-fold increase in the susceptibility of the COX-2 inhibited rats to CCl(4) hepatotoxicity and mortality. The CCl(4) bioactivating enzyme CYP2E1 protein, CYP2E1 enzyme activity, and the (14)CCl(4)-derived radiolabel covalently bound to the liver proteins were unaffected by the COX-2 inhibitor suggesting that the increased hepatotoxic sensitivity of the COX-2 inhibited rats was not due to higher bioactivation of CCl(4). Further investigation showed that this increased mortality was due to higher plasma and hepatic sPLA(2) activities, inhibited PGE(2) production, and progression of liver injury as compared to the non-intervened rats(.) In conclusion, inhibition of COX-2 mitigates the tissue protective mechanisms associated with COX-2 induction, which promotes sPLA(2)-mediated progression of liver injury in an acute liver toxicity model. Because increased sPLA(2) activity in the intercellular space is associated with increased progression of injury, and induced COX-2 is associated with hepatoprotection, ratios of hepatic COX-2 and sPLA(2) activities may turn out to be a useful tool in predicting the extent of hepatotoxicities.