Knockout of the Trpc1 gene reveals that TRPC1 can promote recovery from anaphylaxis by negatively regulating mast cell TNF-α production

Antigen-mediated mast cell (MC) degranulation is the critical early event in the induction of allergic reactions. Transient receptor potential channels (TRPC), particularly TRPC1, are thought to contribute to such MC activation. To explore the contribution of TRPC1 in MC-driven allergic reactions, we examined antigen-mediated anaphylaxis in Trpc1^?/? and WT mice, and TRPC1 involvement in the activation of MCs derived from the bone marrow (BMMCs) of these mice. In vivo, we observed a similar induction of passive systemic anaphylaxis in the Trpc1^?/? mice compared to WT controls. Nevertheless, there was delayed recovery from this response in Trpc1^?/? mice. Furthermore, contrary to expectations, Trpc1^?/? BMMCs responded to antigen with enhanced calcium signaling but with little defect in degranulation or associated signaling. In contrast, antigen-mediated production of TNF-α, and other cytokines, was enhanced in the Trpc1^?/? BMMCs, as were calcium-dependent events required for these responses. Additionally, circulating levels of TNF-α in response to antigen were preferentially elevated in the Trpc1^?/? mice, and administration of an anti-TNF-α antibody blocked the delay in recovery from anaphylaxis in these mice. These data thus provide evidence that, in this model, TRPC1 promotes recovery from the anaphylactic response by repressing antigen-mediated TNF-α release from MCs.     

The Inhibitory Effects of Low-Dose Ionizing Radiation in IgE-Mediated Allergic Responses

Ionizing radiation has different biological effects according to dose and dose rate. In particular, the biological effect of low-dose radiation is unclear. Low-dose whole-body gamma irradiation activates immune responses in several ways. However, the effects and mechanism of low-dose radiation on allergic responses remain poorly understood. Previously, we reported that low-dose ionizing radiation inhibits mediator release in IgE-mediated RBL-2H3 mast cell activation. In this study, to have any physiological relevance, we investigated whether low-dose radiation inhibits allergic responses in activated human mast cells (HMC-1(5C6) and LAD2 cells), mouse models of passive cutaneous anaphylaxis and the late-phase cutaneous response. High-dose radiation induced cell death, but low-dose ionizing radiation of <0.5 Gy did not induce mast cell death. Low-dose ionizing radiation that did not induce cell death significantly suppressed mediator release from human mast cells (HMC-1(5C6) and LAD2 cells) that were activated by antigen-antibody reaction. To determine the inhibitory mechanism of mediator released by low-dose ionizing radiation, we examined the phosphorylation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, and protein kinase C, as well as the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). The phosphorylation of signaling molecules and [Ca²⁺]_i following stimulation of FcεRI receptors was inhibited by low dose ionizing radiation. In agreement with its in vitro effect, ionizing radiation also significantly inhibited inflammatory cells infiltration, cytokine mRNA expression (TNF-α, IL-4, IL-13), and symptoms of passive cutaneous anaphylaxis reaction and the late-phase cutaneous response in anti-dinitrophenyl IgE-sensitized mice. These results indicate that ionizing radiation inhibits both mast cell-mediated immediate- and delayed-type allergic reactions in vivo and in vitro.     

Inhibitory effect of Japanese black vinegar on IgE-mediated degranulation of RBL-2H3 cells and a murine model of Japanese cedar pollinosis

Japanese black vinegar (JBV) is a traditional vinegar manufactured with steamed unpolished rice. After screening, beneficial effects of JBV on IgE-mediated allergic responses were found. In this study, acetic acid-free JBV was used to evaluate its antiallergic effects. JBV suppressed degranulation of rat basophilic leukemia RBL-2H3 cells in a dose-dependent manner without cytotoxicity. The inhibitory effect of JBV on the degranulation seemed to be caused by the bioactive ingredients other than proteins, because the activity was not affected by heat treatment or protease digestion. JBV inhibited the elevation in the intracellular Ca²⁺ concentration induced by antigen. Immunoblot analysis revealed that JBV suppresses degranulation of RBL-2H3 cells by downregulated phosphorylation of PI3K, Akt, and PLCγ1. In addition, oral administration of JBV significantly suppressed passive cutaneous anaphylaxis reaction in mice and an allergic symptom in Cry j1-induced pollinosis model mice. Thus, JBV has a potential as a health-promoting food with the antiallergy effect.     

The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.     

Inhibitory effects of flavonoids isolated from Fragaria ananassa Duch on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3

We isolated the 4 kinds of flavonoids from strawberry ‘Nohime’ and examined the effect of these flavonoids on the degranulation in RBL-2H3 cells. The flavonoids were found to suppress the degranulation from Ag-stimulated RBL-2H3 cells to different extents. To disclose the inhibitory mechanism of degranulation by flavonoids, we examined their effects on the intracellular free Ca²⁺ concentration ([Ca²⁺]i) and the intracellular signaling pathway such as Lyn, Syk, and PLCγs. The intracellular free Ca²⁺ concentration ([Ca²⁺]i) was elevated by FcεRI activation, but these flavonoid treatments reduced the elevation of [Ca²⁺]i by suppressing Ca²⁺ influx. Kaempferol strongly suppressed the activation of Syk and PLCγs. It was thus suggested that suppression of Ag-stimulated degranulation by the flavonoids is mainly due to suppression of [Ca²⁺]i elevation and Syk activation. These results suggested that strawberry would be of some ameliorative benefit for the allergic symptoms.     

Syntaxin Binding Protein 1 Is Not Required for Allergic Inflammation via IgE-Mediated Mast Cell Activation

Mast cells play a central role in both innate and acquired immunity. When activated by IgE-dependent FcεRI cross-linking, mast cells rapidly initiate a signaling cascade and undergo an extensive release of their granule contents, including inflammatory mediators. Some SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor) proteins and SM (Sec1/Munc18) family proteins are involved in mast cell degranulation. However, the function of syntaxin binding protein 1 (STXBP1), a member of SM family, in mast cell degranulation is currently unknown. In this study, we examined the role of STXBP1 in IgE-dependent mast cell activation. Liver-derived mast cells (LMCs) from wild-type and STXBP1-deficient mice were cultured in vitro for the study of mast cell maturation, degranulation, cytokine and chemokine production, as well as MAPK, IκB-NFκB, and NFAT signaling pathways. In addition, in vivo models of passive cutaneous anaphylaxis and late-phase IgE-dependent inflammation were conducted in mast cell deficient W^sh mice that had been reconstituted with wild-type or STXBP1-deficient mast cells. Our findings indicate that STXBP1 is not required for any of these important functional mechanisms in mast cells both in vitro and in vivo. Our results demonstrate that STXBP1 is dispensable during IgE-mediated mast cell activation and in IgE-dependent allergic inflammatory reactions.     

Expression and Function of Allergin-1 on Human Primary Mast Cells

Mast cells (MC) play an important role in allergic and non-allergic immune responses. Activation of human MC is modulated by several cell surface inhibitory receptors, including recently identified Allergin-1 expressed on both human and mouse MC. Although Allergin-1 suppresses IgE-mediated, mast cell–dependent anaphylaxis in mice, the expression profile and function of Allergin-1 on human primary MC remains undetermined. Here, we established a seven-color flow cytometry method for assessing expression and function of a very small number of human primary MC. We show that Allergin-1S1, a splicing isoform of Allergin-1, is predominantly expressed on human primary MC in both bronchoalveolar lavage (BAL) fluid and nasal scratching specimens. Moreover, Allergin-1S1 inhibits IgE-mediated activation from human primary MC in BAL fluid. These results indicate that Allergin-1 on human primary MC exhibits similar characteristics as mouse Allergin-1 in the expression profile and function.     

Phosphorylation of SNAP-23 by IkappaB kinase 2 regulates mast cell degranulation

Mast cells are known to play a pivotal role in allergic diseases. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) leads to degranulation and allergic inflammation; however, the regulatory mechanisms of IgE-dependent exocytosis remain unknown. We show here that IkappaB kinase (IKK) 2 in mast cells plays critical roles in IgE-mediated anaphylaxis in vivo, and IgE-mediated degranulation in vitro, in an NF-kB-independent manner. Upon FcvarepsilonRI stimulation, IKK2 phosphorylates SNAP-23, the target membrane soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor (SNARE), and ectopic expression of a phospho-mimetic mutant of SNAP-23 partially rescued the impaired IgE-mediated degranulation in IKK2-deficient mast cells. These results suggest that IKK2 phosphorylation of SNAP-23 leads to degranulation and anaphylactic reactions. While this reaction is NF-kB-independent, we additionally show that IKK2 also regulates late-phase allergic reactions promoted by the release of proinflammatory cytokines in an NF-kB-dependent manner. The findings suggest that IKK2 is a central player in allergic reactions.     

Inhibitory function of Shikonin on MRGPRX2-mediated pseudo-allergic reactions induced by the secretagogue

BackgroundMast cells (MCs) are crucial effectors in allergic disorders by secreting inflammatory mediators. The Mas-related G-protein-coupled receptor X2 (Mrgprx2) was shown to have a key role in IgE-independent allergic reactions. Therefore, potential drug candidates that directly target Mrgprx2 could be used to treat pseudo-allergic diseases. Shikonin, an active ingredient derived from Lithospermum erythrorhizon Sieb. et Zucc has been used for its anti-inflammatory properties since ancient China.PurposeTo investigate the inhibitory effects of Shikonin on IgE-independent allergy both in vitro and in vivo, as well as the mechanism underlying its effects.Methods/study designsThe anti-anaphylactoid activity of Shikonin was evaluated in PCA and systemic anaphylaxis models, Calcium imaging was used to assess intracellular Ca²⁺ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of PLCγ-PKC-IP3 signaling pathway. The analytical method of surface plasmon resonance was employed to study the interaction between Shikonin and potential target protein Mrgprx2.ResultsShikonin can suppress compound 48/80 (C48/80)-induced PCA, active systemic anaphylaxis, and MCs degranulation in mice in a dose-dependent manner. In addition, Shikonin reduced C48/80-induced calcium flux and suppressed LAD2 cell degranulation via PLCγ-PKC-IP3 signaling pathway. Moreover, Shikonin was found to inhibit C48/80-induced Mrgprx2 expression in HEK cells, displaying specific interactions with the Mrgprx2 protein.ConclusionShikonin could be a potential antagonist of Mrgprx2, thereby inhibiting pseudo-allergic reactions through Ca²⁺ mobilization.     

Inhibitory effects of sesquiterpene lactones isolated from Eupatorium chinense L. on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice

Sesquiterpene lactones (SQTLs) have been shown to suppress the degranulation as inferred by histamine release in rat basophilic leukemia RBL-2H3 cells. In this study, we isolated the 9 kinds of SQTLs from Eupatorium chinense L. and examined the effects of these SQTLs on the degranulation in RBL-2H3 cells. The chemical structures of two novel compounds (SQTL-3 and 8) were determined. All the SQTLs suppressed the degranulation from Ag-stimulated RBL-2H3 cells. To disclose the inhibitory mechanism of degranulation by SQTLs, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCγs and intracellular free Ca²⁺ concentration ([Ca²⁺]i). None of these SQTLs showed the activation of Syk and PLCγs. The intracellular free Ca²⁺ concentration ([Ca²⁺]i) was elevated by FcεRI activation, but SQTLs treatment reduced the elevation of [Ca²⁺]i by suppressing Ca²⁺ influx. Thus, it was suggested that the suppression of Ag-stimulated degranulation by these SQTLs is mainly due to the decreased Ca²⁺ influx.Furthermore, in order to clarify the in vivo effect of SQTL-rich extract, we administered SQTL-rich extract to the type I allergic model mice and measured the passive cutaneous anaphylaxis (PCA) reaction induced by IgE-antigen complex. The SQTLs remarkably suppressed PCA reaction in a dose-dependent manner. Thus, it was suggested that SQTLs would be a candidate as an anti-allergic agent.     

Tumor suppressor p53 functions as a negative regulator in IgE-mediated mast cell activation

Mast cells are known to play a pivotal role in allergic diseases such as allergic rhinitis, asthma, and atopic dermatitis by releasing granules containing histamine, LTC4, and other preformed chemical mediators. Previous reports have demonstrated that IKK2 (also called IKKβ), a central intracellular component of NF-κB activation pathways, plays a critical role in IgE-mediated degranulation of mast cells and anaphylaxis in mice. In this study, we show that protein levels of tumor suppressor p53 are up-regulated upon IgE-mediated activation in mast cells and lack of p53 results in enhanced responses in both early and late phase anaphylaxis. p53 inhibits not only the catalytic activity of IKK2 presumably through the modulation of glycosylation but also p65 (RelA)-mediated transactivation. Our findings are the first to demonstrate that p53 functions as a negative regulator in mast cells.     

Tyrosol Suppresses Allergic Inflammation by Inhibiting the Activation of Phosphoinositide 3-Kinase in Mast Cells

Allergic diseases such as atopic dermatitis, rhinitis, asthma, and anaphylaxis are attractive research areas. Tyrosol (2-(4-hydroxyphenyl)ethanol) is a polyphenolic compound with diverse biological activities. In this study, we investigated whether tyrosol has anti-allergic inflammatory effects. Ovalbumin-induced active systemic anaphylaxis and immunoglobulin E-mediated passive cutaneous anaphylaxis models were used for the immediate-type allergic responses. Oral administration of tyrosol reduced the allergic symptoms of hypothermia and pigmentation in both animal models. Mast cells that secrete allergic mediators are key regulators on allergic inflammation. Tyrosol dose-dependently decreased mast cell degranulation and expression of inflammatory cytokines. Intracellular calcium levels and activation of inhibitor of κB kinase (IKK) regulate cytokine expression and degranulation. Tyrosol blocked calcium influx and phosphorylation of the IKK complex. To define the molecular target for tyrosol, various signaling proteins involved in mast cell activation such as Lyn, Syk, phosphoinositide 3-kinase (PI3K), and Akt were examined. Our results showed that PI3K could be a molecular target for tyrosol in mast cells. Taken together, these findings indicated that tyrosol has anti-allergic inflammatory effects by inhibiting the degranulation of mast cells and expression of inflammatory cytokines; these effects are mediated via PI3K. Therefore, we expect tyrosol become a potential therapeutic candidate for allergic inflammatory disorders.     

α-Linolenic acid attenuates pseudo-allergic reactions by inhibiting Lyn kinase activity

BackgroundPseudo-allergic reactions are potentially fatal hypersensitivity responses caused by mast cell activation. α-linolenic acid (ALA) is known for its anti-allergic properties. However, its potential anti-pseudo-allergic effects were not much investigated.PurposeTo investigate the inhibitory effects of ALA on IgE-independent allergy in vitro, and in vivo, as well as the mechanism underlying its effects.Methods/study designsThe anti-anaphylactoid activity of ALA was evaluated in passive cutaneous anaphylaxis reaction (PCA) and systemic anaphylaxis models. Calcium imaging was used to assess intracellular Ca²⁺ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of Lyn-PLCγ-IP3R-Ca²⁺ and Lyn-p38/NF-κB signaling pathway.ResultsALA (0, 1.0, 2.0, and 4.0 mg/kg) dose-dependently reduced serum histamine, chemokine release, vasodilation, eosinophil infiltration, and the percentage of degranulated mast cells in C57BL/6 mice. In addition, ALA (0, 50, 100, and 200 μM) reduced Compound 48/80 (C48/80) (30 μg/ml)-or Substance P (SP) (4 μg/ml)-induced calcium influx, mast cell degranulation and cytokines and chemokine release in Laboratory of Allergic Disease 2 (LAD2) cells via Lyn-PLCγ-IP3R-Ca²⁺ and Lyn-p38/NF-κB signaling pathway. Moreover, ALA (0, 50, 100, and 200 μM) inhibited C48/80 (30 μg/ml)- and SP (4 μg/ml)-induced calcium influx in Mas-related G-protein coupled receptor member X2 (MrgX2)-HEK293 cells and in vitro kinase assays confirmed that ALA inhibited the activity of Lyn kinase. In response to 200 μM of ALA, the activity of Lyn kinase by (7.296 ± 0.03751) × 10⁻⁵ units/μl and decreased compared with C48/80 (30 μg/ml) by (8.572 ± 0.1365) ×10⁻⁵ units/μl.ConclusionOur results demonstrate that ALA might be a potential Lyn kinase inhibitor, which could be used to treat pseudo-allergic reaction-related diseases such as urticaria.     

Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice

Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity.     

Identification of anti-allergic effect of Clonorchis sinensis-derived protein venom allergen-like proteins (CsVAL)

Previous studies demonstrated that Clonochis sinensis-derived crude antigens suppress development of allergic responses. We investigated the effects of C. sinensis venom allergen-like (CsVAL) proteins on immune-modulating activities in allergic inflammatory response. Using RBL-2H3 rat mast cells, we demonstrated that CsVAL inhibits antigen-induced β-hexosaminidase release from immunoglobulin E-sensitized RBL-2H3 cells, and this inhibitory activity occurs by suppressing Lyn phosphorylation and intracellular reactive oxygen species production. In addition, CsVAL peptide treatment inhibits activation of protein kinase C-α and extracellular signal-regulated kinase 1/2, which are involved in degranulation of immunoglobulin E-sensitized mast cells. Furthermore, immunization with CsVAL suppressed development of skin inflammation by assessing ear thickness and cutaneous infiltration by eosinophils and mast cells in oxazolone-induced contact hypersensitivity in vivo mouse model. These results suggest that CsVAL is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases.     

Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism

PGE(2) is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE(2) on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE(2) alone or in the context of IgE-mediated activation. PGE(2) treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-gamma, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE(2) stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE(2), in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP(1), EP(3), and EP(4) PGE receptor subtypes, including a novel splice variant of the EP(1) receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE(2) is mediated through EP(1) and/or EP(3) receptors. Our results suggest that PGE(2) may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP(1)/EP(3)-dependent mechanism.     

Bifidobacterium suppresses IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells

Sixteen heat-killed bifidobacteria isolated from human intestine and a probiotic strain Lactobacillus GG were tested for their ability to influence IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells in vitro . The bifidobacteria suppressed IgE-mediated degranulation of RBL-2H3 cells by 1.6–56.4% in a strain-dependent manner. Bifidobacteria from healthy infants expressed high inhibitory effects on IgE-mediated degranulation (41–55%), while those from allergic infants varied greatly in their effects against degranulation. Bifidobacteria taxonomically identified as Bifidobacterium bifidum exhibited much stronger inhibitory effects against IgE-mediated degranulation than those taxonomically identified as B. adolescentis ( P < 0.05).These results indicate that the intestinal bifidobacteria might be one of factors influencing IgE-mediated allergic responses.     

Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47^phox, a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47^phox and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-α_IIbβ₃ activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.     

Kaempferol is an estrogen-related receptor α and γ inverse agonist

Kaempferol is a dietary flavonoid that is thought to function as a selective estrogen receptor modulator. In this study, we established that kaempferol also functions as an inverse agonist for estrogen-related receptors alpha and gamma (ERRα and ERRγ). We demonstrated that kaempferol binds to ERRα and ERRγ and blocks their interaction with coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Kaempferol also suppressed the expressions of ERR-target genes pyruvate dehydrogenase kinase 2 and 4 (PDK2 and PDK4). This evidence suggests that kaempferol may exert some of its biological effect through both estrogen receptors and estrogen-related receptors.

Structured summary:

MINT-6824653:PGC-1 alpha (uniprotkb:Q9UBK2) and ERR gamma (uniprotkb: P62508) bind (MI:0407) by surface plasmon resonance (MI:0107)