Work

Isoxanthohumol inhibits MRGPRX2-mediated mast cell activation reducing inflammation in rosacea

Year: 2025

Type: article

Abstract: To the Editor: Rosacea is a chronic inflammatory skin disorder characterized by facial erythema, papules, and pustules on the cheeks, nose, chin, and forehead. Mast cells (MCs) are implicated in rosacea pathogenesis. Increased Mas-related G protein-coupled receptor X2 (MRGPRX2) expression activates MC degranulation in patients with rosacea. Mice deficient in MRGPRB2, the human counterpart of MRGPRX2, exhibit reduced responses to rosacea-like inflammation. Therefore, inhibiting MRGPRX2 activation, thereby preventing abnormal MC activation, is a potential therapeutic strategy to prevent rosacea-like inflammation.[1] Isoxanthohumol (IXN) is a prenylated flavonoid primarily derived from hops. It possesses anti-inflammatory properties.[2] However, the mechanism by which it inhibits the release of immune response mediators to attenuate inflammation remains unclear. Moreover, its potential to treat rosacea remains unknown. In this study, we aimed to investigate the effects of IXN on rosacea using a rosacea mouse model. The mouse model was treated with IXN 24 h after molding as it showed a mild pathological phenotype [Supplementary Figure 1A, https://links.lww.com/CM9/C543]. Notably, IXN significantly reduced the phenotypic and pathological changes in a dose-dependent manner [Supplementary Figure 1B and C, https://links.lww.com/CM9/C543]. In addition, RNA sequencing was performed to assess the effects of IXN on rosacea. Compared with the control, the IXN-treated mouse model showed 1043 upregulated and 1198 downregulated genes [Supplementary Figure 2A, https://links.lww.com/CM9/C543]. We further analyzed the downregulated genes to determine the specific inhibitory effect of IXN on Mrgprb2. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the downregulated genes revealed that IXN inhibited the calcium, G protein-coupled receptor, and tumor necrosis factor (TNF)-α signaling pathways, which are crucial for inflammation and associated with MRGPRX2 activation in rosacea [Supplementary Figure 2B and C, https://links.lww.com/CM9/C543]. To determine whether IXN inhibits MC activation by downregulating Mrgprb2 expression, Mrgprb2 mRNA levels in vivo before and after IXN treatment were measured through reverse transcription-quantitative polymerase chain reaction. Notably, IXN did not considerably impact the Mrgprb2 mRNA levels [Supplementary Figure 3A, https://links.lww.com/CM9/C543]. RNA-sequencing revealed significantly downregulated tryptase (TPS)-AB1 mRNA levels, indicating that IXN inhibited MC activation. In addition, TNF, matrix metalloproteinase (MMP)-9, interleukin (IL)-1β, interferon (IFN)-γ, and C-X-C motif chemokine ligand (CXCL)-2 mRNA levels were significantly decreased after IXN treatment [Supplementary Figure 3B, https://links.lww.com/CM9/C543]. IXN also significantly decreased the TPS, TNF-α, MMP-9, IL-1β, IFN-γ, and CXCL2 protein levels in a dose-dependent manner [Supplementary Figure 3C, https://links.lww.com/CM9/C543]. Avidin-fluorescein isothiocyanate (FITC) staining revealed that IXN inhibited MC activation in the rosacea lesions of the mouse model. MC responses were also significantly suppressed in the model [Supplementary Figure 4A, https://links.lww.com/CM9/C543]. Moreover, IXN significantly decreased CD4-positive (CD4+) T cell and neutrophil infiltration in a dose-dependent manner [Supplementary Figure 4B and C, https://links.lww.com/CM9/C543]. In vitro, IXN exhibited little cytotoxicity in the Laboratory of Allergic Disease 2 (LAD2) cells at a concentration of 40 μg/mL [Supplementary Figure 5A, https://links.lww.com/CM9/C543] but did not significantly alter the MRGPRX2 levels [Supplementary Figure 5B, https://links.lww.com/CM9/C543]. IXN did not induce β-hexosaminidase release from LAD2 cells [Supplementary Figure 5C, https://links.lww.com/CM9/C543]. Moreover, IXN inhibited LL-37-induced β-hexosaminidase release, and the half maximal inhibitory concentration of IXN was 4.37 ± 2.98 μg/mL [Supplementary Figure 5D, https://links.lww.com/CM9/C543]. IXN inhibited TPS, TNF-α, IL-1β, and CXCL2 release from LAD2 cells. MRGPRX2-mediated MC activation did not induce MMP-9 and IFN-γ release from these cells [Supplementary Figure 5E, https://links.lww.com/CM9/C543]. IXN and MRGPRX2 amino acid residues showed hydrophobic interactions and hydrogen bonding. Specifically, IXN bound to Gln-82, Asn-85, Tyr-89, Thr-106, and Val-265 [Supplementary Figure 6A, https://links.lww.com/CM9/C543]. Subsequently, we conducted a molecular dynamics simulation of the IXN–MRGPRX2 complex. The average root mean square deviation of the complex was less than 0.2 nm, and it maintained stability for 100 ns. These results confirmed that the IXN–MRGPRX2 complex was stable. The amino acid residues showing low root mean square fluctuations in the complex were the same as the key amino acid residues; this was possibly related to the hydrogen bonding and hydrophobic interactions between IXN and MRGPRX2. The key amino acid residues became more stable after IXN was combined with MRGPRX2. Moreover, the radius of gyration of the IXN–MRGPRX2 complex remained stable throughout the 100-ns simulation [Supplementary Figure 6B, https://links.lww.com/CM9/C543]. Surface plasmon resonance (SPR) revealed that IXN inhibited MC activation via MRGPRX2 and that the KD value was (10.01 ± 2.67) × 10−6 mol/L [Supplementary Figure 6C, https://links.lww.com/CM9/C543]. Topical medicines mitigate erythema and papules via unknown mechanisms but also cause irritation and dermatitis, which are primary challenges for rosacea treatment. Immune dysregulation of MC activities, including degranulation and pro-inflammatory cytokine release, are key factors associated with rosacea. To date, various treatments, including intense pulsed light therapy and topical and oral medicines, have been developed to stabilize MCs. These MC stabilizers effectively inhibit mediator release by MCs, thereby alleviating rosacea symptoms, such as erythema and flushing.[3] CD4-positive T-cell and neutrophil infiltration is significantly increased in rosacea, affecting the severity of this condition. This is possibly associated with the release of multiple cytokines and chemokines upon MC activation.[4] Altered MRGPRX2 activation triggers MC activation and degranulation, contributing to inflammation in rosacea. Therefore, drugs targeting MRGPRX2 and regulating its downstream signaling show potential to treat rosacea and other MRGPRX2-related inflammatory diseases.[5] IXN, an extract of the plant material remaining after hop brewing, exhibits the potential to treat osteoporosis and cancer and plays important roles in attenuating inflammation, oxidative stress, and angiogenesis. Except for a few epidemiological studies, the clinical translational potential of IXN in humans remains unknown. Therefore, further investigation of its bioavailability, distribution, degradation, elimination, and safety are necessary to facilitate the clinical use of IXM in humans. In conclusion, as verified by in vivo and in vitro experiments, this study demonstrated that IXN attenuates rosacea-like inflammation by inhibiting MRGPRX2 activation in MCs [Figure 1]. Our findings provide a foundation for future studies on rosacea pathogenesis and highlight IXN as a promising candidate for its treatment.Figure 1: Isoxanthohumol attenuates rosacea-like inflammation by inhibiting MRGPRX2 activation in mast cells. KC: Keratinocyte; MRGPRX2: Mas-related G protein-coupled receptor X2.Acknowledgements The authors are grateful to Lin Han (Biomedical Experimental Center of Xi’an Jiaotong University) for her technical support. Funding This work was supported by grants from the National Natural Science Foundation of China (No. 82304689) and the Natural Science Basic Research Program of Shaanxi (No. 2023-JC-QN-0955). Conflicts of interest None.

Source: Chinese Medical Journal

DOI: 10.1097/cm9.0000000000003730