Phospholipase Cγ Research Articles

Aberrant B Cell Receptor Signaling in Naïve B Cells from Patients with Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease in which an impaired healing response to recurrent micro-injuries is thought to lead to fibrosis. Recent findings hint at a role for B cells and autoimmunity in IPF pathogenesis. We previously reported that circulating B cells from a fraction of patients, compared with healthy controls, express increased levels of the signaling molecule Bruton’s tyrosine kinase (BTK). However, it remains unclear whether B cell receptor (BCR) signaling is altered in IPF. Here, we show that the response to BCR stimulation is enhanced in peripheral blood B cells from treatment-naïve IPF patients. We observed increased anti-immunoglobulin-induced phosphorylation of BTK and its substrate phospholipase Cγ2 (PLCγ2) in naïve but not in memory B cells of patients with IPF. In naïve B cells of IPF patients enhanced BCR signaling correlated with surface expression of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) but not B cell activating factor receptor (BAFFR), both of which provide pro-survival signals. Interestingly, treatment of IPF patients with nintedanib, a tyrosine kinase inhibitor with anti-fibrotic and anti-inflammatory activity, induced substantial changes in BCR signaling. These findings support the involvement of B cells in IPF pathogenesis and suggest that targeting BCR signaling has potential value as a treatment option.

Fast-Trk(B)ing the mechanism of antidepressants

The mechanism by which antidepressants elicit clinical improvements has proven elusive. In a recent publication in Cell, Casarotto etal. (2021) reveal a surprising direct interaction between antidepressants and TrkB. This link provides an important mechanistic insight into synaptic remodeling that may assist in the design of improved antidepressant therapeutics.

PLCγ1 promotes phase separation of T cell signaling components.

The T cell receptor (TCR) pathway receives, processes, and amplifies the signal from pathogenic antigens to the activation of T cells. Although major components in this pathway have been identified, the knowledge on how individual components cooperate to effectively transduce signals remains limited. Phase separation emerges as a biophysical principle in organizing signaling molecules into liquid-like condensates. Here, we report that phospholipase Cγ1 (PLCγ1) promotes phase separation of LAT, a key adaptor protein in the TCR pathway. PLCγ1 directly cross-links LAT through its two SH2 domains. PLCγ1 also protects LAT from dephosphorylation by the phosphatase CD45 and promotes LAT-dependent ERK activation and SLP76 phosphorylation. Intriguingly, a nonmonotonic effect of PLCγ1 on LAT clustering was discovered. Computer simulations, based on patchy particles, revealed how the cluster size is regulated by protein compositions. Together, these results define a critical function of PLCγ1 in promoting phase separation of the LAT complex and TCR signal transduction.

Enriched Environment Promotes Adult Hippocampal Neurogenesis through FGFRs.

The addition of new neurons to existing neural circuits in the adult brain remains of great interest to neurobiology because of its therapeutic implications. The premier model for studying this process has been the hippocampal dentate gyrus in mice, where new neurons are added to mature circuits during adulthood. Notably, external factors such as an enriched environment (EE) and exercise markedly increase hippocampal neurogenesis. Here, we demonstrate that EE acts by increasing fibroblast growth factor receptor (FGFR) function autonomously within neurogenic cells to expand their numbers in adult male and female mice. FGFRs activated by EE signal through their mediators, FGFR substrate (FRS), to induce stem cell proliferation, and through FRS and phospholipase Cγ to increase the number of adult-born neurons, providing a mechanism for how EE promotes adult neurogenesis.SIGNIFICANCE STATEMENT How the environment we live in affects cognition remains poorly understood. In the current study, we explore the mechanism underlying the effects of an enriched environment on the production of new neurons in the adult hippocampal dentate gyrus, a brain area integral in forming new memories. A mechanism is provided for how neural precursor cells in the adult mammalian dentate gyrus respond to an enriched environment to increase their neurogenic output. Namely, an enriched environment acts on stem and progenitor cells by activating fibroblast growth factor receptor signaling through phospholipase Cγ and FGF receptor substrate proteins to expand the pool of precursor cells.

Compound Cocktail Inhibits Influenza Viral Pneumonia via Phospholipase Cγ1 Phosphorylation-Related Necroptosis and Partial Autophagy in Natural Killer Cells.

Influenza viral infections are prone to global outbreaks and cause pneumonia in affected populations. High morbidity and mortality caused by pneumonia occur during an influenza pandemic. Antivirals or control of inflammation is the primary means of influenza treatment. A compound cocktail composed of arctiin, daidzein, glycyrrhizic acid, and liquiritin inhibited mouse pneumonia resulting from a PR8 viral infection and caused a weight gain after oral administration. Natural killer cell activating receptors, both Ly49D and Ly49H in the lungs, were increased in the treatment in mice. In H3N2 virus-infected natural killer-92MI cells, the cocktail treatment had different effects on phosphorylation sites of phospholipase Cγ1 (PLCγ1) and killed infected cells through necroptosis or late apoptosis, in which RIP3 was increased and both caspase-3 and phosphorylated-JNK in the cells were downregulated. Acid phosphatase activity in viral-infected natural killer-92MI cells was induced by the compound cocktail treatment, which could be related to the p62 decrease in natural killer-92MI cells. In addition, an autophagic flux induction was observed in alveolar basal epithelial cells (A549). Protein p65, but not phosphorylated-p65, was significantly decreased by the treatment. Our results indicate that the compound cocktail strengthened the phosphorylation of PLCγ1-related necroptosis and partial autophagy in natural killer cells, which could yield an inhibitory effect on viral pneumonia in influenza.

Tryptophan improves porcine intestinal epithelial cell restitution through the CaSR/Rac1/PLC-γ1 signaling pathway.

This study aimed to investigate the effect of tryptophan on cell migration and its underlying mechanism in porcine intestine epithelial cells (IPEC-J2). This study shows that tryptophan can modulate IPEC-J2 cell proliferation, enhance cell migration and the protein concentration of calcium-sensing receptors (CaSR), total ras-related C3 botulinum toxin substrate 1 (total Rac1), Rho family member 1 of GTP-binding protein (GTP-rac1), and phosphorylated phospholipase Cγ1 (p-PLC-γ1). Moreover, Rac1, phospholipase C-γ1 (PLC-γ1) silencing or CaSR inhibitor (NPS2143) inhibited tryptophan-induced upregulation of cell migration. In contrast, tryptophan enhanced the cell migration area and protein concentration of total Rac1, GTP-rac1, and phosphorylated PLCγ1 in cells transfected with wild type CaSR. The overexpression of CaSR increased cell migration, which was reduced by Rac1 or PLC-γ1 silencing. Collectively, our results suggested that tryptophan can improve IPEC-J2 cell migration through the CaSR/Rac1/PLC-γ1 signaling pathway.

PLCγ1 inhibition combined with inhibition of apoptosis and necroptosis increases cartilage matrix synthesis in IL-1β-treated rat chondrocytes.

Osteoarthritis (OA) is an age-related, chronic degenerative disease. With the increasing median age of the population, this disease has become an important public health problem. New, disease-modifying therapies are needed. A potential novel molecular target is phospholipase Cγ1 (PLCγ1), a critical enzyme with important functions including calcium signaling regulation and cell proliferation. In rat chondrocytes treated with IL-1β (20ng·mL-1 for 36h), inhibition of PLCγ1 with U73122 (2μm for 12h) increased levels and expression of the cartilage matrix components Collagen2 and Aggrecan. This beneficial effect of PLCγ1 inhibition was counteracted by increased chondrocyte apoptosis and necroptosis, increased cell death, and increase levels of ROS, all potentially negative for OA. Combined treatment of IL-1β+U73122-treated chondrocytes with inhibitors of apoptosis (Z-VAD, 10μm) and necroptosis (Nec-1, 30μm) enhanced the increases in levels and expression of Collagen2 and Aggrecan, and prevented the increases in cell death and ROS levels. These results suggest that PLCγ1 inhibition may be a viable approach for an OA therapy, if combined with targeted inhibition of chondrocyte apoptosis and necroptosis.

Eudebeiolide B Inhibits Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Regulating RANKL-Induced NF-κB, c-Fos and Calcium Signaling

Eudebeiolide B is a eudesmane-type sesquiterpenoid compound isolated from Salvia plebeia R. Br., and little is known about its biological activity. In this study, we investigated the effects of eudebeiolide B on osteoblast differentiation, receptor activator nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vitro and ovariectomy-induced bone loss in vivo. Eudebeiolide B induced the expression of alkaline phosphatase (ALP) and calcium accumulation during MC3T3-E1 osteoblast differentiation. In mouse bone marrow macrophages (BMMs), eudebeiolide B suppressed RANKL-induced osteoclast differentiation of BMMs and bone resorption. Eudebeiolide B downregulated the expression of nuclear factor of activated T-cells 1 (NFATc1) and c-fos, transcription factors induced by RANKL. Moreover, eudebeiolide B attenuated the RANKL-induced expression of osteoclastogenesis-related genes, including cathepsin K (Ctsk), matrix metalloproteinase 9 (MMP9) and dendrocyte expressed seven transmembrane protein (DC-STAMP). Regarding the molecular mechanism, eudebeiolide B inhibited the phosphorylation of Akt and NF-κB p65. In addition, it downregulated the expression of cAMP response element-binding protein (CREB), Bruton’s tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2) in RANKL-induced calcium signaling. In an ovariectomized (OVX) mouse model, intragastric injection of eudebeiolide B prevented OVX-induced bone loss, as shown by bone mineral density and contents, microarchitecture parameters and serum levels of bone turnover markers. Eudebeiolide B not only promoted osteoblast differentiation but inhibited RANKL-induced osteoclastogenesis through calcium signaling and prevented OVX-induced bone loss. Therefore, eudebeiolide B may be a new therapeutic agent for osteoclast-related diseases, including osteoporosis, rheumatoid arthritis and periodontitis.

Functional phenotype of phosphoinositide 3-kinase p85α-null platelets characterized by an impaired response to GP VI stimulation

AbstractPhosphoinositide 3-kinases (PI3Ks), a family of lipid kinases comprising 3 classes with multiple isoforms, have been shown to participate in different phases of platelet signaling. To investigate the roles that enzymes play in platelet function in vivo and determine which isoforms are important for particular signaling events, we analyzed platelet function of gene knockout mice deficient in the p85α regulatory subunit of heterodimeric class IA PI3K. The kinase activity of p85α–/– platelets was only 5% of the activity of platelets from wild-type littermates. Platelet aggregation induced by adenosine diphosphate (ADP), thrombin, U46619, phorbol 12-myristate 13-acetate (PMA), or botrocetin was not defective in p85α–/– mice, compared with wild-type animals. In contrast, aggregation induced by collagen and collagen-related peptide (CRP) was partially but readily impaired in p85α–/– mice. Both P-selectin expression and fibrinogen binding in response to CRP were also decreased to a similar extent in p85α–/– platelets. Platelets from p85α–/– mice appeared to spread poorly over a CRP-coated surface with intact filopodial protrusions. Significant attenuation of CRP-induced tyrosine phosphorylation in known PI3K effectors such as Btk, Tec, PKB/Akt, and phospholipase Cγ2 were observed with p85α–/– platelets, whereas no alteration was noted in upstream molecules of Syk, LAT, and SLP-76. Considered as a whole, these results provide the first genetic evidence that PI3K p85α plays a significant role in platelet function, almost exclusively in the glycoprotein (GP) VI/Fc receptor γ chain complex-mediated signaling pathway.

Physiological Roles of Phospholipase Cγ and Its Mutations in Human Disease

Physiological Roles of Phospholipase Cγ and Its Mutations in Human Disease

Identification and Differential Expression of microRNA in Response to Elevated Phospholipase Cγ Expression in Liver RH 35 Carcinoma Cells

Our study has primarily shown the positive effect of PLCγ2 on liver tumor cell proliferation, but the molecular basis for its function remains elusive. miRNAs have been widely accepted as important modulators of various cellular activities. This study attempts to characterize the global influence of PLCγ2 on miRNA expressions in liver cancer RH35 cells. Firstly, the recombinant adenovirus AdPLCγ2 was infected into the cells. High-throughput sequencing technology was applied to measure miRNA expressions in PLCγ2-overexpressing cells. Moreover, the target genes and signaling pathways modulated by PLCγ2-specifc miRNAs were identified using target prediction program, GO annotation and KEGG analysis. As a result, totally 246 known and 1075 novel candidate miRNAs were identified, among which 34 known and 191 novel miRNAs exhibited ≥2-fold changes in the AdPLCγ2-infected cells. Correspondingly, 6985 target genes of above 225 differently-expressed miRNAs were predicted, mainly involved in Hippo signaling, Wnt signaling etc., and responsible for tumor development, cell proliferation, apoptosis, migration, lipid metabolism and so on. In aggregate, PLCγ2 induces the significant alterations in miRNA expression, thus providing mechanistic insights into tumorgenesis mediated by PLCγ2, and maybe offers some clues on identifying potential candidates for controlling liver cancer cell growth.

PhospholipaseCγ1/calcium-dependent membranous localization of Gsdmd-N drives endothelial pyroptosis, contributing to lipopolysaccharide-induced fatal outcome.

Multiple organ perfusion is impaired in sepsis. Clinical studies suggest that persistent perfusion disturbances are prognostic of fatal outcome in sepsis. Pyroptosis occurs upon activation of caspases and their subsequent cleavage of gasdermin D (Gsdmd), resulting in Gsdmd-N (activated NH2-terminal fragment of Gsdmd) that form membrane pores to induce cell death in sepsis. In addition, Gsdmd-/- mice are protected from a lethal dose of lipopolysaccharide (LPS). However, how Gsdmd-mediated pyroptosis occurs in endothelial cells and leads to impaired perfusion remain unexplored in endotoxemia. We used transgenic mice with ablation of Gsdmd and determined that mice lacking Gsdmd exhibited reduced breakdown of endothelial barrier, improved organ perfusion, as well as increased survival in endotoxemia. Phospholipase Cγ1 (PLCγ1) contributed to Gsdmd-mediated endothelial pyroptosis in a calcium-dependent fashion, without affecting Gsdmd-N production. Cytosolic calcium signaling promoted Gsdmd-N translocation to the plasma membrane, enhancing endothelial pyroptosis induced by LPS. We used adeno-associated virus (AAV9) vectors carrying a short hairpin RNA (shRNA) against murine PLCγ1 mRNA under control of the tie1 core promoter (AAV-tie1-sh-PLCγ1) to uniquely downregulate PLCγ1 expression in the endothelial cells. Here, we showed that unique inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, and improved perfusion disturbances. Moreover, unique downregulate endothelial PLCγ1 expression markedly decreased mortality of mice in endotoxemia. Thus, we establish that endothelial injury as an important trigger of fatal outcome in endotoxemia. Additionally, these findings suggest that interfering with Gsdmd and PLCγ1-calcium pathway may represent a new treatment strategy for critically ill patients sustaining endotoxemia.NEW & NOTEWORTHY Our study newly reveals that Phospholipase Cγ1 (PLCγ1) contributes to gasdermin D (Gsdmd)-mediated endothelial pyroptosis in a calcium-dependent fashion. Cytosolic calcium signaling promotes activated NH2-terminal fragment of Gsdmd (Gsdmd-N) to translocate to the plasma membrane, enhancing endothelial pyroptosis induced by cytoplasmic LPS. Genetic or pharmacologic inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage, improve perfusion disturbances, and decrease mortality of mice in endotoxemia.

Phospholipase Cγ Signaling in Bone Marrow Stem Cell and Relevant Natural Compounds Therapy

Excessive bone resorption has been recognized play a major role in the development of bone-related diseases such as osteoporosis, rheumatoid arthritis, Paget's disease of bone, and cancer. Phospholipase Cγ (PLCγ) family members PLCγ1 and PLCγ2 are critical regulators of signaling pathways downstream of growth factor receptors, integrins, and immune complexes and play a crucial role in osteoclast. Ca2+ signaling has been recognized as an essential pathway to the differentiation of osteoclasts. With growing attention and research about natural occurring compounds, the therapeutic use of natural active plant-derived products has been widely recognized in recent years. In this review, we summarized the recent research on PLCγ signaling in bone marrow stem cells and the use of several natural compounds that were proven to inhibit RANKL-mediated osteoclastogenesis via modulating PLCγ signaling pathways.

Suppressing PLCγ1 enhances osteogenic and chondrogenic potential of BMSCs

Phosphatidylcholine-specific phospholipase Cγ1 (PLCγ1) is involved in regulating cell metabolism. However, little is known how PLCγ1 directs BMSC differentiation. Here, we investigated the role of PLCγ1 in rat BMSC differentiation into osteoblasts and chondrocytes. The results of Alizarin red and Alcian blue staining showed that PLCγ1 inhibitor U73122 significantly enhanced the mineralization capacity and proteoglycan deposition of BMSCs. The results of qPCR technique and Western blot analysis showed that long-term treatment of U73122 enhanced COL1A1 and OPG mRNA levels and Collagen 1A1, BMP2, and p-Smad1/5/9 protein levels and that short-term treatment of U73122 enhanced COL2A1 and SOX9 mRNA levels and Collagen 2, SOX9, Aggrecan, TGF-β3, and p-Smad2/3 protein levels. Decreased p-mTOR and p-P38 contributed to enhanced osteogenic potentials of BMSCs and increased p-P38 contributed to enhanced chondrogenic potentials of BMSCs. The scaffold transplantation with U73122+BMSC was more efficacious than BMSC alone for osteochondral defect repair in a rat model. Therefore, suppressing PLCγ1 could improve the capacity to effectively use BMSCs for cell therapy of osteochondral defect.

Alzheimer’s disease and the genetics of neuroprotection

As the world’s leading form of age-related dementia, Alzheimer’s disease (AD) is a devastating neurological disorder associated with severe emotional and economic burdens. Despite years of research, the underlying mechanism behind this disease remains unclear. The current prevailing theory relies on the deposition of toxic amyloid beta (Aβ), which promotes the formation of phosphorylated tau tangles, and together these damaging changes drive the loss of neurons. Much of AD development can be attributed to genetic influences, including pathogenic variants of β-amyloid precursor protein, presenilins 1 and 2, and the presence of apolipoprotein 4. However, there are other subtle, yet significant variations within our genome that contribute to protecting us against AD. Variants of the phospholipase Cγ2 and toll-like receptor 4 genes have been associated with reduced risk of developing AD, as well as increased longevity of patients. These neuroprotective variants can guide the development of future therapeutic interventions for AD, as current strategies have proven lackluster.

Transforming growth factor beta orchestrates PD-L1 enrichment in tumor-derived exosomes and mediates CD8 T-cell dysfunction regulating early phosphorylation of TCR signalome in breast cancer.

Tumor cells promote immune evasion through upregulation of programmed death-ligand 1 (PD-L1) that binds with programmed cell death protein 1 (PD1) on cytotoxic T cells and promote dysfunction. Though therapeutic efficacy of anti-PD1 antibody has remarkable effects on different type of cancers it is less effective in breast cancer (BC). Hence, more details understanding of PD-L1-mediated immune evasion is necessary. Here, we report BC cells secrete extracellular vesicles in form of exosomes carry PD-L1 and are highly immunosuppressive. Transforming growth factor beta (TGF-β) present in tumor microenvironment orchestrates BC cell secreted exosomal PD-L1 load. Circulating exosomal PD-L1 content is highly correlated with tumor TGF-β level. The later also found to be significantly associated with CD8+CD39+, CD8+PD1+ T-cell phenotype. Recombinant TGF-β1 dose dependently induces PD-L1 expression in Texos in vitro and blocking of TGF-β dimmed exosomal PD-L1 level. PD-L1 knocked down exosomes failed to suppress effector activity of activated CD8 T cells like tumor exosomes. While understanding its effect on T-cell receptor signaling, we found siPD-L1 exosomes failed to block phosphorylation of src family proteins, linker for activation of T cells and phosphoinositide phospholipase Cγ of CD8 T cells more than PD-L1 exosomes. In vivo inhibition of exosome release and TGF-β synergistically attenuates tumor burden by promoting Granzyme and interferon gamma release in tumor tissue depicting rejuvenation of exhausted T cells. Thus, we establish TGF-β as a promoter of exosomal PD-L1 and unveil a mechanism that tumor cells follow to promote CD8 T-cell dysfunction.

Vav family proteins constitute disparate branching points for distinct BCR signaling pathways.

Antigen recognition by B-cell antigen receptors (BCRs) activates distinct intracellular signaling pathways that control the differentiation fate of activated B lymphocytes. BCR-proximal signaling enzymes comprise protein tyrosine kinases, phosphatases, and plasma membrane lipid-modifying enzymes, whose function is furthermore coordinated by catalytically inert adaptor proteins. Here, we show that an additional class of enzymatic activity provided by guanine-nucleotide exchange factors (GEFs) of the Vav family controls BCR-proximal Ca2+ mobilization, cytoskeletal actin reorganization, and activation of the PI3 kinase/Akt pathway. Whereas Vav1 and Vav3 supported all of those signaling processes to different extents in a human B-cell model system, Vav2 facilitated Actin remodeling, and activation of Akt but did not promote Ca2+ signaling. On BCR activation, Vav1 was directly recruited to the phosphorylated BCR and to the central adaptor protein SLP65 via its Src homology 2 domain. Pharmacological inhibition or genetic inactivation of the substrates of Vav GEFs, small G proteins of the Rho/Rac family, impaired BCR-induced Ca2+ mobilization, probably because phospholipase Cγ2 requires activated Rac proteins for optimal activity. Our findings show that Vav family members are key relays of the BCR signalosome that differentially control distinct signaling pathways both in a catalysis-dependent and -independent manner.

Effects of Apigenin on RBL-2H3, RAW264.7, and HaCaT Cells: Anti-Allergic, Anti-Inflammatory, and Skin-Protective Activities.

Apigenin (4′,5,7-trihydroxyflavone, flavonoid) is a phenolic compound that is known to reduce the risk of chronic disease owing to its low toxicity. The first study on apigenin analyzed its effect on histamine release in the 1950s. Since then, anti-mutation and antitumor properties of apigenin have been widely reported. In the present study, we evaluated the apigenin-mediated amelioration of skin disease and investigated its applicability as a functional ingredient, especially in cosmetics. The effect of apigenin on RAW264.7 (murine macrophage), RBL-2H3 (rat basophilic leukemia), and HaCaT (human immortalized keratinocyte) cells were analyzed. Apigenin (100 μM) significantly inhibited nitric oxide (NO) production, cytokine expression (interleukin (IL)-1β, IL6, cyclooxygenase (COX)-2, and inducible nitric oxide synthase [iNOS]), and phosphorylation of mitogen-activated protein kinase (MAPK) signal molecules, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK) in RAW264.7 cells. Apigenin (30 μM) also inhibited the phosphorylation of signaling molecules (Lyn, Syk, phospholipase Cγ1, ERK, and JNK) and the expression of high-affinity IgE receptor FcεRIα and cytokines (tumor necrosis factor (TNF)-α, IL-4, IL-5, IL-6, IL-13, and COX-2) that are known to induce inflammation and allergic responses in RBL-2H3 cells. Further, apigenin (20 μM) significantly induced the expression of filaggrin, loricrin, aquaporin-3, hyaluronic acid, hyaluronic acid synthase (HAS)-1, HAS-2, and HAS-3 in HaCaT cells that are the main components of the physical barrier of the skin. Moreover, it promoted the expression of human β-defensin (HBD)-1, HBD-2, HBD-3, and cathelicidin (LL-37) in HaCaT cells. These antimicrobial peptides are known to play an important role in the skin as chemical barriers. Apigenin significantly suppressed the inflammatory and allergic responses of RAW264.7 and RBL cells, respectively, and would, therefore, serve as a potential prophylactic and therapeutic agent for immune-related diseases. Apigenin could also be used to improve the functions of the physical and chemical skin barriers and to alleviate psoriasis, acne, and atopic dermatitis.

Kaempferol ameliorates secretagogue-induced pseudo-allergic reactions via inhibiting intracellular calcium fluctuation.

To investigate the inhibitory effects of Kaempferol, a natural flavonol active compound, on pseudo-allergic reactions (in vivo and in vitro), particularly on the mechanism underlying its effect in human mast cells. Compound 48/80 (C48/80)-induced immunoglobulin E (IgE)-independent passive cutaneous anaphylaxis (PCA) model and systemic anaphylaxis were applied to investigate the anti-allergic activity of Kaempferol. The degranulation assay, calcium imaging and the secretion of cytokines and chemokines were used to evaluate the inhibitory effect on mast cell activation. Western blot analysis was performed to investigate intracellular calcium fluctuation-related signalling pathways. Kaempferol dose-dependently attenuated C48/80-induced mice hind paw swelling, dye extravasation and skin mast cell degranulation, and rehabilitated the hypothermia, as well as reduced the serum concentrations of histamine, tryptase, tumour necrosis factor-alpha (TNF-α), interleukin-8 (IL-8) and monocyte chemo-attractant protein-1 (MCP-1). Furthermore, Kaempferol suppressed C48/80-triggered human MC degranulation and calcium fluctuations by inhibiting phospholipase Cγ (PLCγ) phosphorylation and subsequent cytokines synthesis pathways. The inhibition of the process of PLCγ phosphorylation to Ca2+ mobilization represents a major strategy in Kaempferol-suppressed pseudo-allergic reactions. Thus, Kaempferol could be considered as a therapeutic drug candidate for non-IgE-mediated allergic reactions or inflammations.

The histone deacetylase inhibitor valproic acid attenuates phospholipase Cγ2 and IgE-mediated mast cell activation.

Mast cell activation through the high-affinity IgE receptor (FcεRI) plays a central role in allergic reactions. FcεRI-mediated activation triggers multiple signaling pathways leading to degranulation and synthesis of different inflammatory mediators. IgE-mediated mast cell activation can be modulated by different molecules, including several drugs. Herein, we investigated the immunomodulatory activity of the histone deacetylase inhibitor valproic acid (VPA) on IgE-mediated mast cell activation. To this end, bone marrow-derived mast cells (BMMC) were sensitized with IgE and treated with VPA followed by FcεRI cross-linking. The results indicated that VPA reduced mast cell IgE-dependent degranulation and cytokine release. VPA also induced a significant reduction in the cell surface expression of FcεRI and CD117, but not other mast cell surface molecules. Interestingly, VPA treatment inhibited the phosphorylation of PLCγ2, a key signaling molecule involved in IgE-mediated degranulation and cytokine secretion. However, VPA did not affect the phosphorylation of other key components of the FcεRI signaling pathway, such as Syk, Akt, ERK1/2, or p38. Altogether, our data demonstrate that VPA affects PLCγ2 phosphorylation, which in turn decreases IgE-mediated mast cell activation. These results suggest that VPA might be a key modulator of allergic reactions and might be a promising therapeutic candidate.