Quercetin Attenuates MRGPRX2-Mediated Mast Cell Degranulation via the MyD88/IKK/NF-κB and PI3K/AKT/ Rac1/Cdc42 Pathway

Abstract

Background: CMRF35-like molecule-1 (CLM-1) is a receptor of the CD300 family that inhibits MRGPRX2-mediated mast cell degranulation. Understanding the role and mechanism of CLM-1 agonist has significant implications for the treatment of allergic disease. Quercetin is a natural small molecule compound derived from plants and vegetables that has been shown to prevent histamine release by immune cells.

Objective: This study aims to examine the inhibitory effects of quercetin on MRGPRX2-mediated mast cell degranulation via CLM-1.

Results: We found that C48/80 stimulation resulted in significantly increased release of β-hexosaminidase, histamine and Ca²⁺ in CLM-1-knockdown LAD2 cells than in NC-LAD2 cells. Surface plasmon resonance (SPR) and molecular docking analyses revealed high-affinity binding between quercetin and CLM-1 (K _D = 2.962×10^-5 mol/L) mediated by the formation of hydrogen bonds. In addition, quercetin can selectively bind to CLM-1 on mast cells, leading to SHP-1 phosphorylation and subsequent inhibition of downstream MyD88/IKK/NF-κB signaling. Furthermore, activation of CLM-1 modulated the surface expression of MRGPRX2 by inhibiting F-actin, leading to internalization of the MRGPRX2 receptor via the PI3K/AKT/ Rac1/Cdc42 pathway.

Conclusion: Quercetin is a promising treatment for allergic diseases by acting as a CLM-1 agonist that inhibits MRGPRX2-mediated mast cell degranulation.

Keywords: CLM-1; MRGPRX2; inhibition; mast cell degranulation; quercetin.

Graphical abstract

Figure 1

Quercetin targeting CLM-1 negatively regulates C48/80-mediated MCs activation. Following the knockdown of CLM-1 using siRNA technology or treated with CLM-1 antibody did not affect C48/80 induced release rates of β-hexosamine (A and B), histamine (C) and calcium influx in LAD2 cells (D and E), however the inhibitory effects of quercetin (100 μmol/L) and ceramide (100 μmol/L) were markedly diminished compared with NC-LAD2 cells. The data were expressed as mean ± SEM, and the two-tailed unpaired student T test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.

Figure 2

Quercetin serves as a specific small molecule agonist of CLM-1. Western blot (A and B) and RT-PCR (C) analysis of CLM-1 expression in LAD2 cells after 24-hour pretreatment with quercetin (100 μmol/L) and ceramide (100 μmol/L) and quantification of CLM-1 protein expression was performed using densitometric analysis. The experiments were conducted in triplicate. (D) Molecular docking model of quercetin and CLM-1. (E) SPR binding curve of quercetin and CLM-1 with a K_D value of 2.962×10⁻⁵ mol/L. The data were expressed as mean ± SEM, and the two-tailed unpaired student T test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.

Figure 3

Quercetin activating CLM-1 negatively regulates MRGPRX2 expression. RT-PCR (A) and Western blot (B and C) analysis of MRGPRX2 expression in LAD2 cells after 24-hour pretreatment with quercetin (100 μmol/L) or ceramide (100 μmol/L) and C48/80. The expression of CLM-1 protein in LAD2 cells after 24 h pretreatment with quercetin (100 μmol/L) or ceramide (100 μmol/L) and C48/80 (D–F), and quantification by densitometric analysis (E). The data were expressed as mean ± SEM, and the two-tailed unpaired student T test was used for statistical analysis. *P < 0.05, ***P < 0.001 were considered statistically significant.

Figure 4

Quercetin inhibits the release of mast cell allergic mediators via the MyD88-NF-κB pathway. (A) The expression levels of phosphorylation-SHP-1, SHP-1, MyD88, phosphorylation-IKKα/β, IKKα/β, phosphorylation-IκB, IκB, NF-κB in LAD2 cells treated with quercetin (100 μmol/L) or ceramide (100 μmol/L) and C48/80 for 24 h were analyzed by Western blot. Quantification of phosphorylation-SHP-1 (B), MyD88 (C), phosphorylation-IKKα/β (D), phosphorylation-IκB (E), NF-ΚB (F) protein expression in by densitometric analysis. The data were expressed as mean ± SEM, and the two-tailed unpaired student T test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.

Figure 5

Quercetin attenuate membrane MRGPRX2 expression through F-actin via PI3K/AKT/Rac1/Cdc42 pathway. (A) Western blot analysis of the expression of PI3K in LAD2 cells treated with quercetin (100 μmol/L), ceramide (100 μmol/L), and C48/80 for 24 hours. (B) Quantification of PI3K protein expression was carried out through densitometric analysis. (C) Western blot analysis of the expression AKT phosphorylation in LAD2 cells treated with quercetin (100 μmol/L), ceramide (100 μmol/L), and C48/80 for 24 hours. (D) Quantification of phosphorylation-AKT protein expression was carried out through densitometric analysis. (E) Western blot analysis the expression levels of Rac1/Cdc42 in LAD2 cells treated with quercetin (100 μmol/L), ceramide (100 μmol/L), and C48/80 for 24 hours. (F) Quantified Rac1/Cdc42 protein expression by densitometric analysis. (G) The changes in F-actin in the membrane skeleton structure of LAD2 cells after C48/80 stimulation and the effects of quercetin and ceramide were analyzed using the immunofluorescence method. The DAPI-stained nucleus is shown in blue, and F-actin on the cell membrane is shown in red, with a magnification of 53 times. (H and I) ELISA was used to analyze the effects of quercetin (100 μmol/L) on the MRGPRX2 on the cell membrane and intracellularly in LAD2 cells induced by C48/80. The data were expressed as mean ± SEM, and the two-tailed unpaired student T test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.