Phosphorylation Of Cytosolic Phospholipase A2 Research Articles

Bromocriptine protects perilesional spinal cord neurons from lipotoxicity after spinal cord injury.

Recent studies have revealed that lipid droplets accumulate in neurons after brain injury and evoke lipotoxicity, damaging the neurons. However, how lipids are metabolized by spinal cord neurons after spinal cord injury remains unclear. Herein, we investigated lipid metabolism by spinal cord neurons after spinal cord injury and identified lipid-lowering compounds to treat spinal cord injury. We found that lipid droplets accumulated in perilesional spinal cord neurons after spinal cord injury in mice. Lipid droplet accumulation could be induced by myelin debris in HT22 cells. Myelin debris degradation by phospholipase led to massive free fatty acid production, which increased lipid droplet synthesis, β-oxidation, and oxidative phosphorylation. Excessive oxidative phosphorylation increased reactive oxygen species generation, which led to increased lipid peroxidation and HT22 cell apoptosis. Bromocriptine was identified as a lipid-lowering compound that inhibited phosphorylation of cytosolic phospholipase A2 by reducing the phosphorylation of extracellular signal-regulated kinases 1/2 in the mitogen-activated protein kinase pathway, thereby inhibiting myelin debris degradation by cytosolic phospholipase A2 and alleviating lipid droplet accumulation in myelin debris-treated HT22 cells. Motor function, lipid droplet accumulation in spinal cord neurons and neuronal survival were all improved in bromocriptine-treated mice after spinal cord injury. The results suggest that bromocriptine can protect neurons from lipotoxic damage after spinal cord injury via the extracellular signal-regulated kinases 1/2-cytosolic phospholipase A2 pathway.

Artocarpesin acts on human platelet aggregation through inhibition of cyclic nucleotides and MAPKs

The cardiovascular diseases (CVDs) are becoming a critical threat to our lives in these years. It is now widely accepted that platelets play an important role in cardiovascular disease as they have a fundamental role in thrombosis. Therefore, many drugs or natural substances have been developed to treat CVDs. Cudrania tricuspidata is a regional plant containing various constituents, such as xanthones, flavonoids, organic acids, and polysaccharides. It has been widely used in East Asia as an important ethnomedicine for the treatment of many diseases such as eczema, mumps, tuberculosis and acute arthritis. Therefore, we evaluated antiplatelet effects using artocarpesin isolated from C. tricuspidata. Confirmation of the antiplatelet function of artocarpesin was made according to the following analyzes. Artocarpesin inhibited collagen-induced human platelet aggregation, calcium mobilization, glycoprotein IIb/IIIa activation and thrombin-induced clot retraction through the regulation of associated signaling molecules. Artocarpesin increased the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and inositol 1, 4, 5-triphosphate receptor I (IP3RI). On the other hand, the phosphorylation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinases p38, JNK and phosphoinositide 3-kinase (PI3K)/Akt decreased. Thus, the study highlights that artocarpesin has an inhibitory effect on platelet activity and thrombus formation, showing its potential value in preventing platelet-induced cardiovascular disease.

Piezo1 activation induces fibronectin reduction and PGF2α secretion via arachidonic acid cascade

Glaucoma is a neurodegenerative disease that leads to blindness, and lowering intraocular pressure (IOP) is very important in glaucoma treatment. The trabecular meshwork is responsible for aqueous humor outflow, and the accumulation of fibronectin in trabecular meshwork is known to cause ocular hypertension. We have already shown that Piezo1 activation has an IOP lowering effect in mice and suppresses fibronectin expression level in human trabecular meshwork cells (HTMC). In this study, we report the mechanism of the reduction of fibronectin caused by Piezo1 activation. Activation of Piezo1 in HTMC showed increased expression of matrix metalloproteinase-2 (MMP-2) and cyclooxygenase (COX)-2, and decreased fibronectin expression. In addition, Piezo1 activation enhanced phosphorylation of cytosolic phospholipase A2 (cPLA2), and inhibitors targeting cPLA2 and COX-2 suppressed Yoda 1, a Piezo1 agonist, induced fibronectin reduction. These results indicate that the arachidonic acid cascade underlies this reaction, and, in support of this hypothesis, activation of Piezo1 promoted secretion of prostaglandin F2α (PGF2α) in HTMC. These results indicate that the activation of Piezo1 in HTMC promotes the degrading of fibronectin by promoting the arachidonic acid cascade and increasing the expression of PGF2α and MMP-2.

The suppressive effect of tamarixetin, isolated from Inula japonica, on degranulation and eicosanoid production in bone marrow-derived mast cells.

The study aimed to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in immunoglobulin E /antigen-induced mouse bone marrow-derived mast cells (BMMCs). Tamarixetin isolated from Inula japonica was infected into BMMCs. The inhibitory effect of tamarixetin were analyzed by quantifying β-hexosaminidase, eicosanoid generation, intracellular calcium measurement, and Western blot analysis. Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of protein kinase B (Akt) and its downstream signal molecules including IκB kinase and nuclear factor-κB. Besides, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 and p38 mitogen-activated protein kinase.Conclusion: In summary, tamarixetin inhibits degranulation and eicosanoid generation through the phospholipase Cγ1 as well as Akt pathways. It could be potential for the prevention of allergic inflammatory diseases in BMMCs.

Suppressive effect of tamarixetin, isolated from Inula japonica, on degranulation and eicosanoid production in bone marrow-derived mast cells.

The aim of this study was to evaluate the inhibitory effect of tamarixetin on the production of inflammatory mediators in IgE/antigen-induced mouse bone marrow-derived mast cells (BMMCs). The effects of tamarixetin on mast cell activation were investigated with regard to degranulation, eicosanoid generation, Ca2+ influx, and immunoblotting of various signaling molecules. Tamarixetin effectively decreased degranulation and the eicosanoid generation such as leukotriene C4 and prostaglandin D2 in BMMCs. To elucidate the mechanism involved, we investigated the effect of tamarixetin on the phosphorylation of signal molecules. Tamarixetin inhibited the phosphorylation of Akt and its downstream signal molecules including IKK and nuclear factor κB. In addition, tamarixetin downregulated the phosphorylation of cytosolic phospholipase A2 (cPLA2) and p38 mitogen-activated protein kinase. Taken together, this study suggests that tamarixetin inhibits degranulation and eicosanoid generation through the PLCγ1 as well as Akt pathways in BMMCs, which would be potential for the prevention of allergic inflammatory diseases.

Tricin Isolated from Enzyme-Treated Zizania latifolia Extract Inhibits IgE-Mediated Allergic Reactions in RBL-2H3 Cells by Targeting the Lyn/Syk Pathway.

Tricin, a flavone present in rice bran, is confirmed as the major efficacious compound present in the enzyme-treated Zizania latifolia extract (ETZL), which protects against UVB-induced skin-aging. However, the suppressive mechanism of tricin on allergic responses remains unknown. The present study, therefore, aimed to determine the mechanisms of tricin and ETZL on mast cell degranulation in IgE-activated rat basophilic leukemia cell line (RBL-2H3) cells. We investigated the regulatory effects of tricin and ETZL on degranulation, production of cytokines and lipid mediators, and signaling proteins involved in the IgE-bound high-affinity IgE receptor activation, mitogen-activated protein kinase, arachidonic acid and Syk. The production of β–hexosaminidase, tumor necrosis factor-α, interleukin-4, leukotrienes (LT) B4, LTC4 and prostaglandin E2 in IgE-stimulated RBL-2H3 cells were significantly inhibited by exposure to tricin or ETZL. Moreover, tricin and ETZL inhibit the phosphorylation of cytosolic phospholipase A2, 5-lipoxygenase and cyclooxygenase-2. Furthermore, the phosphorylation of Akt, ERK, p38, JNK, protein kinase Cδ and phospholipase Cγ1 were effectively suppressed by both samples. Exposure to tricin or ETZL also significantly decreases the phosphorylation of Lyn and Syk, but has minimal effect on Fyn. Taken together, our data indicate that tricin and ETZL are potential anti-allergic materials that could be applied for the prevention of allergy-related diseases.

Activation of metabolite receptor GPR91 promotes platelet aggregation and transcellular biosynthesis of leukotriene C4

BackgroundSuccinate is a Krebs cycle intermediate whose formation is enhanced under metabolic stress, and for which a selective sensor GPR91 has been identified on various cell types including platelets. Platelet‐derived eicosanoids play pivotal roles in platelet activation/aggregation, which is key to thrombus formation and progression of atherothrombosis. ObjectivesThis study aims to decipher the molecular mechanism(s) and potential involvement of eicosanoids in succinate enhanced platelet activation/aggregation. MethodsWe used liquid chromatography‐mass spectrometry (LC‐MS)/MS‐based lipid mediator profiling to identify eicosanoids regulated by succinate. We ran light transmittance aggregometry and flow cytometry to assess platelet aggregation, P‐selectin expression, and platelet‐polymorphonuclear leukocyte (PMN) adherence. Various pharmacological tools were used to assess the contributions of GPR91 signalling and eicosanoids in platelet aggregation. ResultsSuccinate and two types of synthetic non‐metabolite GPR91 agonists—cis‐epoxysuccinate (cES) and Cmpd131—potentiated platelet aggregation, which was partially blocked by a selective GPR91 antagonist XT1. GPR91 activation increased production of 12‐hydroxy‐eicosatetraenoic acid (12‐HETE), thromboxane (TX) A2, and 12‐hydroxy‐heptadecatrienoic acid (12‐HHT) in human platelets, associated with phosphorylation of cytosolic phospholipase A2 (cPLA2), suggesting increased availability of free arachidonic acid. Blocking 12‐HETE and TXA2 synthesis, or antagonism of the TXA2 receptor, significantly reduced platelet aggregation enhanced by GPR91 signalling. Moreover, platelet‐PMN suspensions challenged with succinate exhibited enhanced transcellular biosynthesis of leukotriene C4 (LTC4), a powerful proinflammatory vascular spasmogen. ConclusionSuccinate signals through GPR91 to promote biosynthesis of eicosanoids, which contribute to platelet aggregation/activation and potentially vascular inflammation. Hence, GPR91 may be a suitable target for pharmacological intervention in atherothrombotic conditions.

Inhibitory effects of thromboxane A2 generation by ginsenoside Ro due to attenuation of cytosolic phospholipase A2 phosphorylation and arachidonic acid release

BackgroundThromboxane A2 (TXA2) induces platelet aggregation and promotes thrombus formation. Although ginsenoside Ro (G-Ro) from Panax ginseng is known to exhibit a Ca2+-antagonistic antiplatelet effect, whether it inhibits Ca2+-dependent cytosolic phospholipase A2 (cPLA2α) activity to prevent the release of arachidonic acid (AA), a TXA2 precursor, is unknown. In this study, we attempted to identify the mechanism underlying G-Ro-mediated TXA2 inhibition. MethodsWe investigated whether G-Ro attenuates TXA2 production and its associated molecules, such as cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS), cPLA2α, mitogen-activated protein kinases, and AA. To assay COX-1 and TXAS, we used microsomal fraction of platelets. ResultsG-Ro reduced TXA2 production by inhibiting AA release. It acted by decreasing the phosphorylation of cPLA2α, p38-mitogen-activated protein kinase, and c-Jun N-terminal kinase1, rather than by inhibiting COX-1 and TXAS in thrombin-activated human platelets. ConclusionG-Ro inhibits AA release to attenuate TXA2 production, which may counteract TXA2-associated thrombosis.

WWL70 protects against chronic constriction injury-induced neuropathic pain in mice by cannabinoid receptor-independent mechanisms

BackgroundTargeting the endocannabinoid system has emerged as an effective strategy for the treatment of inflammatory and neurological diseases. Unlike the inhibition of the principal 2-arachidonyl glycerol (2-AG) hydrolytic enzyme monoacylglycerol lipase (MAGL), which leads to 2-AG overload and cannabinoid receptor desensitization, selective inhibition of the minor 2-AG hydrolytic enzyme alpha, beta-hydrolase domain 6 (ABHD6) can provide therapeutic benefits without producing cannabimimetic side effects. We have shown that inhibition of ABHD6 significantly reduces neuroinflammation and exerts neuroprotection in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 inhibition on neuropathic pain has not been explored.MethodsNeuropathic pain was induced by chronic constriction injury (CCI) of the mouse sciatic nerve and examined by Hargreaves and Von Frey tests. Activation of inflammatory cells and the production of cytokines and chemokines in the spinal cord dorsal horn, dorsal root ganglion (DRG), and sciatic nerve were assessed by qRT-PCR, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. The levels of 2-AG and arachidonic acid (AA) in sciatic nerve were quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).ResultsTreatment with the selective ABHD6 inhibitor WWL70 significantly alleviated CCI-induced thermal hyperalgesia and mechanical allodynia. Microglia activation, macrophage infiltration, and the production of nociceptive mediators were reduced in the ipsilateral lumbar spinal cord dorsal horn, DRG, and sciatic nerve of WWL70-treated animals. The diminished cytokine and chemokine production is likely due to the inhibitory effect of WWL70 on NF-κB phosphorylation. Surprisingly, the anti-nociceptive and anti-inflammatory effects of WWL70 were not reversed by addition of the cannabinoid receptor antagonists. Treatment with WWL70 did not alter the levels of 2-AG, AA, and the phosphorylation of cytosolic phospholipase A2 (cPLA2), but significantly reduced the production of prostaglandin E2 (PGE2) and the expression of cyclooxygenase-2 (COX-2) and prostaglandin E synthase-2 (PGES2) in the injured sciatic nerve.ConclusionsThis study reveals a novel mechanism for the antinociceptive effect of the 2-AG catabolic enzyme ABHD6 inhibitor WWL70. Understanding the interaction between endocannabinoid and eicosanoid pathways might provide a new avenue for the treatment of inflammatory and neuropathic pain.

1,6-O,O-Diacetylbritannilactone suppresses activation of mast cell and airway hyper-responsiveness

Mast cells play critical roles in allergic disorders such as atopic dermatitis and allergic asthma. The aim of this study was to investigate the anti-inflammatory and anti-asthmatic activities of 1,6-O,O-diacetylbritannilactone (OODBL) isolated from Inula japonica Thunb. (I. japonica) in a murine asthma model and bone marrow-derived mast cells (BMMCs). In an ovalbumin-induced asthma model, OODBL administration attenuated the airway hyper-responsiveness induced by aerosolized methacholine and serum IgE level in asthmatic mice. In vitro system, we found that OODBL reduced leukotriene C4 production and degranulation through the suppression of cytosolic phospholipase A2 phosphorylation and phospholipase Cγ-mediated Ca2+ influx in IgE/antigen-stimulated BMMCs. Taken together, OODBL may have therapeutic potential in the treatment of allergic diseases such as asthma.

Inhibitory Effect of Three Diketopiperazines from Marine-derived Bacteria on Secretory Group IIA Phospholipase A2

Diketopiperazines, natural products found in bacteria, fungi, marine sponges, gorgonian and red algae, are cyclic dipeptides possessing relatively-simple and rigid structures with chiral nature and various side chains. The compounds in this structure class have been known to possess diverse bioactivities including antibiotic activity, anti-cancer activity, neuroprotective activity, and anti-inflammatory activity. The expression of secretory group IIA phospholipase A2 (sPLA2-IIA) is enhanced by development of inflammatory disorders. Aim of this study is to determine the effects of diketopiperazines on the secretion and activity of sPLA2-IIA by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVECs). To do this, sPLA2-IIA expression was induced in the LPS-stimulated HUVECs and mice to evaluate the effect of diketopiperazines. Results showed that diketopiperazines remarkably suppressed the LPS-mediated protein expression and activity of sPLA2-IIA via inhibition of phosphorylation of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK) 1/2. These results demonstrated that diketopiperazines might play an important role in the modulation of sPLA2-IIA expression and activity in response to the inflammatory diseases.

Leptin suppresses adenosine triphosphate-induced impairment of spinal cord astrocytes.

Spinal cord injury (SCI) causes long-term disability and has no clinically effective treatment. After SCI, adenosine triphosphate (ATP) may be released from neuronal cells and astrocytes in large amounts. Our previous studies have shown that the extracellular release of ATP increases the phosphorylation of cytosolic phospholipase A2 (cPLA2 ) and triggers the rapid release of arachidonic acid (AA) and prostaglandin E2 (PGE2) via the stimulation of epidermal growth factor receptor (EGFR) and the downstream phosphorylation of extracellular-regulated protein kinases 1 and 2. Leptin, a glycoprotein, induces the activation of the Janus kinase (JAK2)/signal transducers and activators of transcription-3 (Stat3) pathway via the leptin receptor. In this study, we found that 1) prolonged leptin treatment suppressed the ATP-stimulated release of AA and PGE2 from cultured spinal cord astrocytes; 2) leptin elevated the expression of caveolin-1 (Cav-1) via the JAK2/Stat3 signaling pathway; 3) Cav-1 blocked the interaction between Src and EGFR, thereby inhibiting the phosphorylation of EGFR and cPLA2 and attenuating the release of AA or PGE2; 4) pretreatment with leptin decreased ;he level of apoptosis and the release of interleukin-6 from cocultured neurons and astrocytes; and 5) leptin improved the recovery of locomotion in mice after SCI. Our results highlight leptin as a promising therapeutic agent for SCI. © 2016 Wiley Periodicals, Inc.

Inhibitory effect of fermented Arctium lappa fruit extract on the IgE-mediated allergic response in RBL‑2H3 cells.

Arctium lappa fruit has been used in traditional medicine, and it is known to exert beneficial effects, such as antioxidant, anti-inflammatory and anticancer effects. However, the effects of the Arctium lappa fruit on the allergic response remain unknown. In this study, we evaluated the anti-allergic effects of Arctium lappa fruit extract (AFE) and its fermented form (F-AFE) using immunoglobulin E (IgE)-activated RBL‑2H3 cells. To investigate the anti-allergic effects of AFE or F-AFE, we examined the release of β-hexosaminidase, a key biomarker of degranulation during an allergic reaction, and the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) in the cells treated with or without the above-mentioned extracts. AFE weakly inhibited the release of β-hexosaminidase, whereas F-AFE significantly suppressed the release of β-hexosaminidase in a dose-dependent manner. Consistently, F-AFE suppressed the production of TNF-α and PGE2 in a dose-dependent manner. F-AFE exerted an inhibitory effect on the production of β-hexosaminidase, TNF-α and PGE2 with an IC50 value of 30.73, 46.96 and 36.27 µg/ml, respectively. Furthermore, F-AFE inhibited the phosphorylation of Lyn, Fyn and Syk, which are involved in the FcεRI signaling pathway, that of phosphoinositide phospholipase C (PLC)γ1/2 and protein kinase C (PKC)δ, which are associated with the degranulation process, as well as that of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK), p38 and Akt, which are associated with cytokine expression. In the late phase, F-AFE partially suppressed the phosphorylation of cytosolic phospholipase A2 (cPLA2), but not the expression of cyclooxygenase (COX)-2. To compare and identify the major components of the two extracts, we used high-performance liquid chromatography. The levels of arctigenin, one of the major compounds, were elevated 6-fold in F-AFE compared with AFE, whereas the levels of arctiin, an arctigenin glycoside, were decreased in F-AFE by approximately 57.40%. These results suggest that arctigenin plays an important role in the anti-allergic effects of F-AFE. Taken together, F-AFE containing anti-allergic phytochemicals, including arctigenin, inhibited the activation of the FcεRI receptor induced by the antigen‑IgE complex. Such effects may provide further information for the development of a phytomedicine for allergic diseases.

Inhibitory effect of polyozellin on secretory group IIA phospholipase A2.

The expression of secretory group IIA phospholipase A2 (sPLA2-IIA) is enhanced by development of inflammatory disorders. In this study, sPLA2-IIA expression was induced in the lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells and mice to evaluate the effect of polyozellin. Polyozellin, a major constituent of a Korea edible mushroom Polyozellus multiplex, has been known to exhibit the biological activities such as anti-oxidative and anti-inflammatory effects. Polyozellin remarkably suppressed the LPS-mediated protein expression and activity of sPLA2-IIA via inhibition of phosphorylation of cytosolic phospholipase A2 and extracellular signal-regulated kinase 1/2. These results demonstrated that polyozellin might play an important role in the modulation of sPLA2-IIA expression and activity in response to the inflammatory diseases.

Ruscogenin suppresses mouse neutrophil activation: Involvement of protein kinase A pathway

Ruscogenin, a natural steroidal sapogenin, presents in both food and medicinal plants. It has been found to exert significant anti-inflammatory activities. Considering that activation of neutrophil is a key feature of inflammatory diseases, this study was performed to investigate the inhibitory effect of ruscogenin and its underlying mechanisms responsible for neutrophil activation. Ruscogenin displayed potent antioxidative effects against Formyl-Met-Leu-Phe (FMLP)-induced extra- and intracellular superoxide generation in mouse bone marrow neutrophils, with IC50 values of 1.07±0.32μM and 1.77±0.46μM, respectively. Phorbol myristate acetate (PMA)-elicited extra- and intracellular superoxide generation were also suppressed by ruscogenin, with IC50 values of 1.56±0.46μM and 1.29±0.49μM, respectively. However, ruscogenin showed weak inhibition in NaF-induced response. Inhibition of superoxide generation was mediated neither by a superoxide-scavenging ability nor by a cytotoxic effect. Furthermore, ruscogenin inhibited the membrane translocation of p47phox and p67phox. It reduced FMLP-induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and p21-activated kinase (PAK). The cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were increased by ruscogenin. Moreover, ruscogenin inhibited phosphorylation of protein kinase B (Akt), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). In addition, the inhibitory effects of ruscogenin on superoxide production and the phosphorylation of Akt, p38MAPK, and ERK1/2 were reversed by PKA inhibitor (H89), suggesting a PKA-dependent mechanism. In summary, our data suggest that ruscogenin inhibits activation of neutrophil through cPLA2, PAK, Akt, MAPKs, cAMP, and PKA signaling pathways. Increased PKA activity is associated with suppression of the phosphorylation of Akt, p38MAPK, and ERK1/2 pathways.

AS252424, a PI3Kγ Inhibitor, Downregulates Inflammatory Responsiveness in Mouse Bone Marrow-Derived Mast Cells

PI3Kγ is known to play an important role in inflammation and immune responses. Mast cells are closely involved in the initiation and regulation of immune responses and allergic inflammatory reactions. AS252424 is a specific PI3Kγ inhibitor. Until now, the anti-inflammatory effect of AS252424 on mast cells has not been reported. In this study, we investigated the anti-inflammatory effects of AS252424 on the inflammatory mediators in activated bone marrow-derived mast cells (BMMCs). AS252424 dramatically attenuated c-Kit ligand (KL)-induced leukotriene C4 (LTC4) generation and degranulation in BMMCs. Downregulating phosphorylation of cytosolic phospholipase A2 (cPLA2) and mitogen-activated protein kinase (MAPK) and inhibition of Ca(2+) liberation in BMMCs might be involved in the anti-inflammatory effects. These results suggested that AS252424 might be considered as a chemical tool or a drug candidate for prevention and therapy of inflammatory disease.

Anti-Allergic Action of Aged Black Garlic Extract in RBL-2H3 Cells and Passive Cutaneous Anaphylaxis Reaction in Mice

Garlic (Allium sativum) has been used as a food as well as a component of traditional medicine. Aged black garlic (ABG) is known to have various bioactivities. However, the effect of ABG on allergic response is almost unknown. In the present study, we investigated whether ABG can inhibit immunoglobulin E-mediated allergic response in RBL-2H3 cells as well as in vivo passive cutaneous anaphylaxis (PCA). In in vitro tests, ethyl acetate extract (EBG) of ABG significantly inhibited the release of β-hexosaminidase (IC₅₀, 1.53 mg/mL) and TNF-α (IC₅₀, 0.98 mg/mL). Moreover, BG10, an active fraction of EBG, dramatically suppressed the release of β-hexosaminidase (IC₅₀, 53.60 μg/mL) and TNF-α (IC₅₀, 27.80 μg/mL). In addition, BG10 completely blocked the formation of prostaglandin E₂ and leukotriene B₄ at ≥25 μg/mL. When the effect of BG10 on FcɛRI receptor cascade was investigated, BG10 significantly inhibited the phosphorylation of Syk, but not Lyn. Furthermore, BG10 dose dependently decreased the phosphorylation of cytosolic phospholipase A₂ (cPLA₂) and 5-lipoxygenase (5-LO) as well as the expression of cyclooxygenase-2 (COX-2). Consistent with what has been mentioned earlier, BG10 also significantly inhibited the PCA reaction in mice. In conclusion, these results indicate that ABG suppresses the allergic response, and the mechanism for its anti-allergic action may involve suppressions of Syk, cPLA₂, 5-LO, and COX-2. The anti-allergic actions of ABG, EBG, or BG10 suggest that they may be useful as functional foods for allergic diseases.

Dual role of the p38 MAPK/cPLA 2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A2 (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.

LIM kinase-1 selectively promotes glycoprotein Ib-IX–mediated TXA2 synthesis, platelet activation, and thrombosis

AbstractCurrent antithrombotic drugs have an adverse effect on bleeding, highlighting the need for new molecular targets for developing antithrombotic drugs that minimally affect hemostasis. Here we show that LIMK1−/− mice have defective arterial thrombosis in vivo but do not differ from wild-type mice with respect to bleeding time. LIMK1−/− mice show a selective defect in platelet activation induced through the von Willebrand Factor (VWF) receptor, the glycoprotein Ib-IX-V complex (GPIb-IX), but not by GPIb-IX–independent platelet agonists. In fact, LIMK1−/− platelets show an enhanced reaction to certain GPIb-IX–independent agonists. The defect of LIMK1−/− platelets in GPIb-IX–mediated platelet activation is attributed to a selective inhibition in VWF/GPIb-IX–induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and consequent thromboxane A2 (TXA2) production. Supplementing a TXA2 analog, U46619, corrected the defect of LIMK1−/− platelets in VWF-induced stable platelet adhesion. Although LIMK1−/− platelets also showed reduced actin polymerization after GPIb-IX–mediated platelet aggregation, actin polymerization inhibitors did not reduce TXA2 generation, but rather accelerated platelet aggregation, suggesting that the role of LIMK1 in GPIb-mediated platelet activation is independent of actin polymerization. Thus, LIMK1 plays a novel role in selectively mediating GPIb-IX–dependent TXA2 synthesis and thrombosis and represents a potential target for developing antithrombotic drugs with minimal bleeding side effect.

Regulation of Cytosolic Phospholipase A2 Phosphorylation by Proteolytic Cleavage of Annexin A1 in Activated Mast Cells

Annexin A1 (ANXA1) is cleaved at the N terminal in some activated cells, such as macrophages, neutrophils, and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced asthma model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrow-derived mast cells. This cleavage event was inhibited when intracellular Ca(2+) signaling was blocked. ANXA1-knockdown RBL-2H3 cells produced a greater amount of eicosanoids with simultaneous upregulation of cytosolic phospholipase A(2) (cPLA(2)) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found that cPLA(2) interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA(2) mainly interacted with full-length ANXA1 in the cytosol and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA(2) and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA(2) and production of eicosanoids during mast-cell activation.