Abstract: 【Abstract】 Objective To investigate the possible mechanisms underlying the involvement of the gut microbiota metabolite trimethylamine oxide （TMAO） in the pathogenesis of chronic spontaneous urticaria （CSU）. Methods From June 2023 to June 2024, 67 CSU patients were enrolled from the Dermatology and Cosmetic Center, the Third Affiliated Hospital of Chongqing Medical University, and 69 age-matched healthy controls were also collected at the same time. Serum TMAO levels in both groups were measured using enzyme-linked immunosorbent assay （ELISA）, and D-dimer levels were collected from the CSU patients. A degranulation model was established in rat basophilic leukemia RBL-2H3 cells using anti-DNP IgE/DNP-BSA （IgE/Ag group）; these cells were additionally grouped to be treated with different concentrations of TMAO （IgE/Ag＋10 μmol/L TMAO group, IgE/Ag + 50 μmol/L TMAO group, IgE/Ag＋100 μmol/L TMAO group）; untreated RBL-2H3 cells served as a blank control group. To investigate the effect of the extracellular signal-regulated kinase （ERK） phosphorylation inhibitor U0126 on the action of TMAO, RBL-2H3 cells were divided into another 5 groups: blank group, IgE/Ag group, IgE/Ag + 1 μmol/L U0126 group, IgE/Ag + 100 μmol/L TMAO group, and IgE/Ag + 100 μmol/L TMAO + 1 μmol/L U0126 group. In vivo, a localized allergic reaction model was established in the ears of C57BL/6 mice using anti-DNP IgE/DNP-BSA （IgE/Ag group）, and additional groups included blank group, IgE group, IgE/Ag + solvent （DMSO） group, and IgE/Ag + 10 μg/μl TMAO group. ELISA was performed to detect levels of inflammatory mediators in cell culture supernatants and mouse serum. Toluidine blue staining was employed to observe mast cell degranulation in the cell experiment and mouse ear tissue samples, Evans blue staining to assess vascular permeability in mouse ear tissue samples, and Western blot analysis to detect the ERK phosphorylation levels. The t test was used for comparisons between two groups, and one-way analysis of variance for multiple comparisons. Results Serum TMAO levels were significantly higher in the 67 CSU patients than in the 69 healthy controls （t = 13.27, P < 0.001）. Among the 32 CSU patients with available data about D-dimer, serum TMAO levels were positively correlated with D-dimer levels （r = 0.62, P < 0.001）. In RBL-2H3 cell experiments, degranulation rates were significantly higher in the IgE/Ag + 10, 50, and 100 μmol/L TMAO groups than in the IgE/Ag group; morphologically, RBL-2H3 cells treated with 10, 50, and 100 μmol/L TMAO became increasingly rounded; 50 and 100 μmol/L TMAO significantly promoted the production of β-hexosaminidase （β-Hex）, interleukin-6 （IL-6）, and tumor necrosis factor-α （TNF-α） （all P < 0.01）, and upregulated ERK phosphorylation levels （P < 0.01）; the levels of ERK phosphorylation, IL-6, TNF-α, and β-Hex were significantly lower in the IgE/Ag + U0126 group than in the IgE/Ag group, as well as lower in the IgE/Ag + TMAO + U0126 group than in the IgE/Ag + TMAO group （all P < 0.001）. In the mouse model of localized allergic reaction, the IgE/Ag + TMAO group showed increased vascular permeability, edema degree, and mast cell degranulation, as well as significantly elevated ERK phosphorylation levels and TNF-α expression in mouse ear tissues compared with the IgE/Ag + DMSO group（both P < 0.05）. Conclusion Elevated serum TMAO may participate in the pathogenesis of CSU by upregulating ERK phosphorylation levels in mast cells and skin tissues, thereby promoting IgE/Ag-mediated degranulation of effector cells and production of inflammatory mediators.

Key words: Chronic urticaria, Chronic spontaneous urticaria, Trimethylamine oxide, Extracellular regulated protein kinases, Phosphorylation