P物质及其受体Mas相关G蛋白偶联受体X2对肥大细胞激活昼夜节律的影响

doi:10.12092/j.issn.1009-2501.2026.04.001

摘要/Abstract

摘要：

目的: 探讨P物质（substance P，SP）在肥大细胞昼夜节律性激活中的作用及其调控机制。方法: 采用CCK-8比色法检测不同浓度SP对MCp815细胞活性的影响，确定后续实验的SP浓度。以免疫球蛋白E（immunoglobulin E，IgE）诱导建立MCp815细胞脱颗粒模型。设立空白对照组和BMAL1沉默组；采用656.1 nmol/L SP分别处理两组细胞30 min。在不同时间点检测肥大细胞（mast cell，MC）的激活情况。酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）检测组胺和类胰蛋白酶的释放水平。荧光定量PCR检测SP受体Mrgprx2、Bmal1、Per2的mRNA水平变化。通过染色质免疫沉淀和萤光素酶报告基因分析Mrgprx2基因中E-box中的DNA结合活性以及转录起始位点上游2.0 kb区域的启动子活性。C57BL/6小鼠随机分为对照组和SP组，每组10 只。分别在LD10/10（10 h光/10 h暗）循环和非正常的LD12/12（12 h光/12 h暗）循环下饲养8周。采用HE染色检测小鼠鼻黏膜组织病理情况，Ly6G染色检测鼻黏膜组织髓系中性粒细胞和MC浸润；采用ELISA法检测组胺和类胰蛋白酶水平；qPCR检测小鼠鼻黏膜组织Tac1和Mrgprx2的mRNA水平变化。结果: （1）656.1 nmol/L SP对MC活性无显著影响（P>0.05）。在对照组中，SP处理12 h时，MC中组胺、类胰蛋白酶的分泌水平显著高于24 h时。在BMAL1沉默组中，各指标12 h和24 h时无显著差异。在60 h和72 h时，两组SP刺激MC前后产生的组胺和类胰蛋白酶水平均无显著差异。（2）用SP处理的野生型小鼠在ZT4时鼻腔内中性粒细胞浸润更为广泛，伴随组胺、类胰蛋白酶分泌的增加；而在ZT16时较少。（3）qPCR结果显示，在对照组中，SP处理后，骨髓源性肥大细胞（bone marrow-derived mast cells，BMMCs）在12 h时Mrgprx2表达水平较24 h升高；但在BMAL1沉默的BMMCs中，这种差异消失。而在60 h和72 h时，两组细胞Mrgprx2表达没有统计学差异。在不同时相下，Mrgprx2表达水平同Per2水平变化趋势一致；与BMAL1水平变化趋势相反，并且与BMMCs释放的组胺和类胰蛋白酶的时相水平变化一致。（4）Mrgprx2基因中非经典E-box（CATGTGA）表现出明显的BMAL1结合活性，并展示出明显的时间效应。萤光素酶报告基因分析显示，BMAL1和CLOCK共同表达能使萤光素酶活性上调，而当E-box发生突变时，Mrgprx2无法转录。（5）置于LD10/10循环下饲养8周的小鼠中SP介导的中性粒细胞浸润和MC Tac1/Mrgprx2表达的节律性消失，而在正常LD12/12循环下饲养的小鼠中节律性表达仍存在。结论: SP可能在调节昼夜节律性MC激活中起着关键作用，潜在的作用机制可能是BMAL1/CLOCK通过直接结合SP受体Mrgprx2启动子区域的非经典E-Box位点调控Mrgprx2的转录。外周昼夜节律调控信号可能是变应性鼻炎中MC激活的重要途径。

关键词: P物质, 过敏性鼻炎, 肥大细胞激活, 昼夜节律, MRGPRX2

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

中图分类号:

R965.2

李昕蓉, 黄月, 眭文, 陈宇兰, 吕泽仪, 李雨, 陈曦, 潘卓, 尹漫, 李奇洪, 杨浩. P物质及其受体Mas相关G蛋白偶联受体X2对肥大细胞激活昼夜节律的影响[J]. 中国临床药理学与治疗学, 2026, 31(4): 433-442.

Xinrong LI, Yue HUANG, Wen SUI, Yulan CHEN, Zeyi LYU, Yu LI, Xi CHEN, Zhuo PAN, Man YIN, Qihong LI, Hao YANG. Regulation of peripheral clock to expression of substance P/MRGPRX2 contributes to circadian activity of mast cell[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2026, 31(4): 433-442.

图/表 5

图 1

Fig.1 Mean proliferation multiples of MC after 48 hours of treatment with gradient concentrations of SP ($\bar x $±s, n=3) Overall proliferation of MC under different concentrations of SP. bP<0.05, compared with 0 nmol/L SP.

图 2

Fig.2 SP-mediated rhythmic activation of mast cells ($\bar x $±s, n=3) A: release of Histamine and Tryptase from the 12- or 24-hour cultured (after synchronization) shNC- or shBMAL1-treated BMCMCs. B: release of Histamine and Tryptase from the 60- or 72-hour cultured (after synchronization) shNC- or shBMAL1-treated BMCMCs. C: hematoxylin and eosin (HE) staining of the respiratory epithelia in the mouse nasal cavity (down-panel). Localization of and day-night variation in neutrophils (Ly6G+) in the mouse nasal mucosa (up-panel). D: release of Histamine and Tryptase from the 60- or 72-hour cultured (after synchronization) shNC- or shBMAL1-treated BMCMCs. bP<0.05, compared with 12 h; eP<0.05, compared with mock; hP<0.05, compared with ZT4.

图 3

Fig.3 The expression of SP receptor Mrgprx2 is associated with the circadian genes Bmal1 and Per2 ($\bar x $±s, n=3) A: changes in Mrgprx2 mRNA expression from the 12- or 24-hour cultured (after synchronization) shNC- or shBMAL1-treated BMCMCs. B: changes in Mrgprx2 mRNA expression from the 60- or 72-hour cultured (after synchronization) shNC- or shBMAL1-treated BMCMCs. C: changes in Mrgprx2 protein expression after synchronization shNC- or shBMAL1-treated BMCMCs. The expression of circadian genes Mrgprx2 (D), Bmal1 (E), and Per2 (F) showed circadian oscillations. The release of histamine (G’) and tryptase (H) by shNC or shBMAL1-treated BMCMCs showed rhythmic changes. (I) Changes in Mrgprx2 mRNA expression in mock- or SP-treated BMCMCs after synchronization. bP<0.05, compared with 12 h; eP<0.05, compared with mock; hP<0.05, compared with ZT4.

图 4

Fig.4 CLOCK/BMAL1 regulate the transcriptional rhythmicity of Mrgprx2in mast cells ($\bar x $±s, n=3) A: location of E-boxes in the 2.0 kb promoter region of Mrgprx2 gene. B: chromatin immunoprecipitation analysis shows the binding of BMAL1 to the Mrgprx2 promoter, with the non-classical E-box (CATGTGA) having a higher binding affinity; while the class II E-box (CATTTG) has no binding. C: BMAL1 antibody detection of tissues at different circadian phases shows the effect of circadian rhythm on the binding of BMAL1 to the non-classical E-box (CATGTGA) and (CATTTG) of the Mrgprx2 promoter. D: luciferase reporter assay to identify the role of the non-classical E-box (CATGTGA) in Mrgprx2 transcription. The promoter of Mrgprx2 reporter constructs (pGL3) containing normal or mutant E-boxes were transiently transfected into 293T cells with (+) or without (-) CLOCK and BMAL1 expression constructs. All results were normalized to the luciferase activity in cells transfected with pGL3 reporter containing normal non-classical E-box (CATGTGA) alone (designated value 1). pGL3-Control Vector was used as a positive control.

图 5

Fig.5 Rhythmic changes in neutrophil infiltration and SP/MRGPRX2 expression in the nasal mucosa of mice. Mice were housed under a regular 12-h light/12-h dark (LD12/12) cycle or a 10-h light/10-h dark (LD10/10) cycle for 8 weeks ($\bar x $±s, n=3) A: normalized body weight gain of mice in LD10/10 and LD12/12 condition. B: neutrophils infiltration into the nasal cavity at 10:00 a.m. or 10:00 p.m., and neutrophils were labeled with Ly-6G+. C: Quantitative analysis of Ly-6+ positive cells (n=4-5 per group). Histamine (D) and tryptase (E) levels released by wild-type mice housed under LD12/12 or LD10/10 conditions at 10:00 a.m. or 10:00 p.m., and Tac1 (F) and Mrgprx2 (G) mRNA expression levels in nasal mucosal tissue. bP<0.05, compared with LD12/12; eP<0.05, compared with 10:00 AM.

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