Hongli An

Chronic urticaria (CU) is a skin allergy caused by the excessive activation of mast cells. The main etiology of CU is a type I allergic reaction mediated by immunoglobulin (Ig)E. This study mainly explored the therapeutic effect of quercetin in ovalbumin (OVA)‐induced CU mice and investigated its target and mechanism in vitro. The CU symptom‐alleviating effect of quercetin was assessed by the CU model. The possible molecular mechanisms of quercetin were initially inferred through bioinformatic and multi‐database analyses. Quercetin targets were examined using mast cell activation experiments with CD300f knockdown. RT‐PCR and western blot experiments were performed to verify the molecular mechanisms of quercetin. Quercetin relieved wheal and scratching times on the back skin of mice as well as reduced eosinophilic infiltration and mast cell degranulation in the skin lesions and inhibited the release of IgE, histamine, TNF‐α, MCP‐1, and IL‐13 in the serum of mice. In addition, it exhibited potential therapeutic effects on CU through the PI3K‐Akt signaling pathway. Meanwhile, quercetin upregulated CD300f in the skin of CU, activated CD300f, and induced downstream SHP‐1 phosphorylation. Of note, quercetin bound to CD300f to prevent IgE‐mediated LAD2 cell β‐hexosaminidase release, histamine release, Ca²⁺ influx, mast cell degranulation, and F‐actin cytoskeleton remodeling by inhibiting the AKT/IKK/NF‐κB inflammatory pathway. The study results suggest that quercetin alleviates CU by activating the CD300f/SHP‐1 signaling pathway. In addition, it activates CD300f to inhibit IgE‐mediated mast cell degranulation and F‐actin cytoskeleton remodeling by inhibiting the AKT/IKK/NF‐κB inflammatory pathway.

Background: Mast cells (MCs) are effectors of anaphylactoid reactions. Mas‐related G‐protein‐coupled receptor X2 (MRGPRX2) receptor mediates the direct activation of MCs in anaphylactoid disease. Siglec‐6 negatively regulates MC activation and is a promising target in the development of antianaphylactoid reaction drugs. While caffeine exhibits an inhibitory effect against anaphylactic shock, the molecular mechanisms underlying these activities remain unknown. Objectives: Our objective was to investigate the inhibitory effect of caffeine and its underlying molecular mechanism in MRPGRX2‐induced MC activation and anaphylactoid reactions. Methods: Local and systemic anaphylactoid reactions in mice and in vitro MC activation experiments were conducted to investigate the effects of caffeine on anaphylactoid reactions. Molecular docking and surface plasmon resonance (SPR) experiments were used to predict and verify the molecular target of caffeine activity. siRNA silencing and western blot analyses were utilized to investigate the molecular mechanisms underlying caffeine activity. Results: Caffeine inhibited local and systemic anaphylactoid reactions in mice and attenuated MRGPRX2‐induced MC activation. Release of β‐hexosaminidase, histamine, and Ca ²⁺ in siRNA‐Siglec‐6‐laboratory allergic disease 2 (LAD2) cells was significantly higher than in NC‐LAD2 cells. The binding affinity between caffeine and Siglec‐6 protein is with a calculated K _D of 1.76 × 10 ⁻⁷ mol/L. Caffeine increased Siglec‐6 expression, phosphorylation of SHP‐1, and dephosphorylation of PLC‐γ1, IP3R, and ERK1/2 in the MRGPRX2 signaling pathway. Western blot demonstrated that phosphorylated SHP‐1 (p‐SHP‐1) protein levels showed no increase, and MRGPRX2, phosphorylated PLCγ1 (p‐PLCγ1), and phosphorylated ERK1/2 (p‐ERK1/2) were abolished with caffeine treatment in Siglec‐6‐knockdown cells than in NC‐knockdown cells. Caffeine suppressed the m‐3M3FBS‐induced upregulation of p‐PLCγ1 and p‐ERK1/2 levels. Conclusions: We have demonstrated that caffeine is an agonist of Siglec‐6 and that subsequent activation of the ITIM motif of Siglec‐6 phosphorylates SHP‐1. This arrests MRGPRX2/PLC‐γ1/IP3R signal transduction, thereby attenuating anaphylactoid reactions, including anaphylactic shock.

Immunologic contact urticaria (ICU) is characterized by the wheal and flare reaction from direct contact with a chemical or protein agent, which involves a type I hypersensitivity mediated by allergen‐specific immunoglobulin E (sIgE). Myricetin (Myr), a bioactive flavonoid, exhibits antiinflammatory activities. Our results showed that treatment with Myr could alleviate ICU symptoms, including a decrease in the number of wheals and scratching, and inhibit ear swelling in the IgE/DNFB‐induced mice. The serum level of IgE, histamine, interleukin (IL)‐4, TNF‐α, and MCP‐1 were reduced in Myr‐treated mice. Myr also attenuated mast cells (MCs) degranulation and H‐PGDS, TSLP, IL‐33, PI3K, Akt, and NF‐κB mRNA levels in ICU model. The IgE‐mediated anaphylaxis mouse models demonstrated anti‐allergic effects of Myr. In vitro analysis showed that Myr reduced IgE‐induced calcium (Ca²⁺) influx, suppressed degranulation, and chemokine release in LAD2 cells (human primary mast cells). Myr can significantly inhibited PLCγ1, Akt, NF‐κB, and p38 phosphorylation. In conclusion, the study demonstrated that Myr alleviate ICU symptoms and inhibit mast cell activation via PI3K/Akt/NF‐κB signal pathway.

Melatonin is an important bioactive molecule in plants. Two synthetases, N-acetylserotonin methyltransferase (ASMT) and serotonin N-acetyltransferase (SNAT) are involved in the final two steps of melatonin synthesis. Melatonin participates in responses to a variety of biotic and abiotic stresses in plants, but few studies have addressed the roles of endogenous melatonin in pathogen resistance. We investigated the role of endogenous melatonin in resistance to Botrytis cinerea infection in an Arabidopsis thaliana model system. Plant lines that overexpressed ASMT or SNAT through genetic manipulation showed upregulated expression of resistance genes PR1 and PR5, transcription factor gene WRKY33, and jasmonic acid (JA) defense pathway marker gene PDF1.2, and downregulated transcription factor gene MYC2 in JA signaling pathway. Higher melatonin content also enhanced the activity of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD), increased JA content, reduced plant disease symptoms, and reduced lesion size in leaves. These findings indicate that endogenous melatonin enhances plant resistance to B. cinerea infection. In contrast, ASMT and SNAT gene silencing lines showed opposite results and were more susceptible to B. cinerea. Thus, it can be demonstrated that melatonin functions as an effective regulator of plant stress resistance at the genetic level. A schematic model is presented for its role in resistance to B. cinerea infection. Our findings also helped to elucidate the associated signal transduction pathways and interactions between melatonin and other plant hormones.