Abstract:

AIM: To investigate the role of substance P (SP) in the circadian activation of mast cells and its regulatory mechanisms. METHODS: Cell Counting Kit-8 (CCK8) colorimetric assay was used to detect the effects of different concentrations of SP on MCp815 cell activity, and the concentration for subsequent experiments was determined. MCp815 cell degranulation was induced using immunoglobulin E (IgE) to establish a degranulation model. A control group and a BMAL1 knockout group were set up, and both groups of cells were treated with 656.1 nmol/L SP for 30 minutes. Mast cell activation was monitored at different time intervals. Enzyme-linked immunosorbent assay (ELISA) was used to detect the release levels of histamine and tryptase. Quantitative real-time PCR was employed to detect the changes in mRNA levels of SP receptor Mrgprx2, Bmal1, Per2. Chromatin immunoprecipitation and luciferase reporter assays were used to analyze the DNA binding activity of the E-box in the Mrgprx2 gene and the promoter activity in the 2.0 kb upstream region of the transcription start site. C57BL/6 mice were randomly divided into a control group and an SP group, with 10 mice in each group. The mice were kept in either an LD10/10 (10h light/10h dark) cycle or an abnormal LD12/12 (12h light/12h dark) cycle for 8 weeks. HE staining was used to detect the pathological conditions of the nasal mucosa, and Ly6G staining was applied to observe the infiltration of myeloid neutrophils and mast cells in the nasal mucosa. ELISA was used to detect the levels of histamine and tryptase, and qPCR was performed to measure the changes in SP and Mrgprx2 mRNA levels in mouse nasal mucosa. RESULTS: (1) The dose of 656.1 nmol/L SP had no significant effect on mast cell activity and was chosen for use in subsequent experiments. In the control group, at 12 hours post SP treatment, histamine and tryptase secretion levels in mast cells (MC) were significantly higher compared to 24 hours. In the BMAL1 knockout group, there was no difference in the indicators between 12 and 24 hours. At 60 and 72 hours, there were no differences in the levels of histamine and tryptase produced by SP-stimulated MCs in either group. (2) Wild-type mice treated with SP showed more extensive neutrophil infiltration in the nasal cavity at ZT4, accompanied by increased secretion of histamine and tryptase, while at ZT16, this infiltration was less. (3) qPCR results showed that after SP treatment, Mrgprx2 expression in BMMCs at 12 hours was higher than at 24 hours in the control group, but this difference disappeared in BMAL1 knockout BMMCs. At 60 and 72 hours, there was no statistical difference in Mrgprx2 expression between the two groups of cells. At different time points, Mrgprx2 expression levels were consistent with PER2 changes and opposite to BMAL1 changes. The temporal changes in histamine and tryptase release from BMMCs were consistent with these expression patterns. (4) The non-canonical E-box (CATGTGA) in the Mrgprx2 gene showed significant BMAL1 binding activity and a clear time-dependent effect. Luciferase-reporter analysis showed that co-expression of BMAL1 and CLOCK upregulated luciferase activity, but when the E-box was mutated, Mrgprx2 transcription was abolished. (5) Mice housed in an LD10/10 cycle for 8 weeks lost the rhythm of SP-mediated neutrophil infiltration and mast cell Tac1/Mrgprx2 expression, while the rhythmic expression persisted in mice housed in the normal LD12/12 cycle. CONCLUSION: SP may play a key role in regulating the circadian activation of mast cells, with the potential mechanism involving BMAL1/CLOCK directly binding to the non-canonical E-box site in the promoter region of the SP receptor Mrgprx2 to regulate its transcription. Peripheral circadian signals may be an important pathway for MC activation in allergic rhinitis.

Key words: substance P, allergic rhinitis, mast cell activation, circadian rhythm, MRGPRX2