SH with high purity (98%) was provided by Chengdu Pfeidel Biotechnology Co., Ltd. Company (Chengdu, Sichuan, China). Information provided by Sigma-Aldrich, Inc., St. Louis, MO. In the state of Missouri in the United States of America, there is a type of town called Louis. Researchers there discovered a chemical called nitrophenyl N-acetyl-β-D-glucamide. In addition, they studied Evans blue and compound 48/80 (C48/80), a well-known initial secretion stimulator. Saline was purchased from Shandong Qilu Pharmaceutical Co., Ltd. (Jinan, Shandong, China). Fluo-3 AM and Pluronic F-127 cells were purchased from Biotium (Fremont, CA, United States). 2-Aminoethyldiphenylboronate (2-APB), somebody 202,190, GO6983, paroxetine hydrochloride, and YM-254 890 were supplied by MCE (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from ExCell Biology, Inc. (Shanghai, China), including human tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Histamine was purchased from Sigma-Aldrich, while histamine · 2HCl (A, A, B, B-D4, 98%) was sourced from Cambridge Isotope Laboratories Ltd. High performance liquid chromatography (HPLC) grade methanol and acetonitrile from Thermo Fisher Scientific (Waltham, MA) were purchased in Cambridge, MA. formic acid, for the liquid chromatography-mass spectrometry (LC-MS) grade, was introduced from Sigma-Aldrich.

In order to study the ligand interactions of receptors, molecular docking experiments were performed with the SURFLEX-DOCKMOde software suite (based on the SYBYL-X2 platform).0; Tripos, St. Louis, Missouri, in the country). The model described earlier provides the basis for the docking model of MRGPRX2. (Cao et al., 2021). The reported MRGPRX2 structure (https://www.rcsb.org/) was used for docking with M-3 and SH.

MRGPRX2 protein (Sino Biological, Inc., Beijing, China) at a concentration of 25 μg/mL was fixed on a CM5 sensor chip (Cytiva Sweden AB, Uppsala, Sweden) by capture-coupling. Then, sequential injections of 0, 0 were performed.0064, 0.032, 0. M-3 of 16, 4, 20 and 100 μm, or 0,0.3125, 0.625, 1.25, 2.5, 5, and 10 μmol of thiol compound were combined with running buffer containing 5% dimethyl sulfoxide (DMSO) in phosphate buffered saline (PBS-P). The interaction of MRGPRX2 with bound small molecules was evaluated at 25°C (General Electric Medical Systems, Fairfield, CT). Binding was performed over 180 s and dissociation was performed over 600 s while maintaining a flow rate of 20 μL per second.

The C57BL/6 strain (6–8 weeks old) was derived from the Xi’an Jiaotong University Laboratory Animal Core (located in Xi’an, China). An experimental model of conditional knockout of MRGPRB2 gene (named MRGPRb2-CKO) was constructed on the basis of C57BL/6 strain, which was verified by PCR. A mast cell deficient mouse strain KitW-sh/W-sh carrying a W-Sash C-kit mutant was provided by the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu). All laboratory rodents were properly housed in the laboratory animal care facilities of Xi’an Jiaotong University and provided with the necessary nutrients and water. All experiments involving consistent procedures on animals were performed by scientific researchers who kept the details of the experiments private.

This study strictly adhered to the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Experimental protocols for the use of mice were approved by the Animal Ethics Committee of Xi’an Jiaotong University (Permit Number: XJTU 2023-157). The study was conducted in accordance with the local legislation and institutional requirements.

The laboratory preparation of MRGPrx2/HEK293 cell line was achieved by PCR and Western blotting. Cells were incubated in DMEM medium containing 10% fetal bovine serum and 100 units of penicillin-streptomycin mixed antibiotics (HyClone, Logan, UT, United States). LAD2 cells provided by researchers Arnold Kirshenbaum and Dean Metcalfe of the National Institutes of Health in Bethesda, Maryland, were propagated in a medium containing STEMPRO-34. The medium incorporates 100 ng/mL of human stem cell growth factor, 100 units of penicillin and streptomycin, 2 mM of L-glutamine, and a nutrient supplement of STEMPRO. The cells were cultured at 37°C in 5% CO2 and maintained a population density of 5 × 10 ^ 5 cells per milliliter.

After washing the LAD2 cells with calcium imaging buffer twice, they were incubated with M-3 at concentrations of 0, 10, 50, and 100 μM for 30 min at 37. degree.C.CIB consists of a single component. For the chemical formulation provided, it can be rephrased as a solution containing 0.2 mol per liter of sodium bicarbonate, 125 mmol per liter of sodium chloride, 3 mmol per liter of potassium chloride, and 20 mmol per liter of sucrose.5 mM CaCl2,10 mM HEPES,0. Magnesium salt 6 mmol, sugar 40 mmol, neutral 7.4. Fluo-3 AM 4 µM was fused with Pluronic F-127 0 in the culture medium.1%.The cells were washed twice with CIB. Fill each well with C48/80 mixed with CIB at a concentration of 30 μg/mL.Images were captured using an Eclipse Ti-S fluorescence microscope manufactured by Nikon (Tokyo, Japan) at a magnification of 200. Unless otherwise indicated, agents were injected into the wells within 5 s of the initial imaging, and the response was monitored at a frequency of once every 1 s for 120 consecutive seconds. The reaction progress was followed in real time using an Eclipse Ti-S fluorescence microscope manufactured by Nikon (Tokyo, Japan).

Tyrode’s solution comprises 120 mM sodium chloride, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4,10 mM HEPES,5.5 mmol of glucose and 5 mmol of bovine serum albumin. LAD2 cells were seeded into 96-well plates at a density of 2 × 10 ^ 4 cells per well, followed by overnight culture. After removal of the medium, M-3, SH, or C48/80 (used as a positive control) were incorporated into the modified Tyrode solution and incubated for 30 min. A 700 mL plate was pelleted at 400 revolutions per minute for 300 s. Cells were lysed in modified Tyrode’s solution containing 0.5%.In the experiment, 1% Triton X-100 solution was used. The β-hexosaminidase was extracted from the supernatant and cell lysate, and its activity of hydrolyzing nitrophenyl N-acetyl-β-D-glucosamine was quantitatively analyzed.1 m citric acid or sodium citrate buffer solution adjusted to pH 4.5). The samples were incubated at 37°C for 90 min, followed by the addition of a stop buffer (0.1 m sodium carbonate/sodium bicarbonate, pH 11.0). The samples were quantitatively analyzed using a spectrophotometer at a wavelength of 405 nm.

The samples were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) using an LCMS-8040 mass spectrometer manufactured by Shimadzu Corporation (Kyoto, Japan). Using a Venusil HILIC column (2.1 × 150 mm, 3 μm; Agela Technologies, Tianjin, China) to assess histamine in this system. The histamine was eluted at 0.3 mL/min using an isocratic elution buffer containing 0.1% formic acid and 20 mM ammonium formate (77: 23, v/v).

The concentration of chemokines in cell culture supernatants was quantified using the Human Chemokine Assay Kit according to the manufacturer’s guidelines. After incubating the mixture of cells with M-3, SH, C48/80, or their vectors for up to 6–8 h, the modified Tyrode solution was quantitatively analyzed for TNF-α, IL-8, and MCP-1 using a commercially available ELISA kit according to the second procedure described previously. Follow the manufacturer’s guidelines.

Specific siRNAs were used to knock down MRGPRX2, while the siRNA as a control was selected to serve as a Non-treatment group. Derived from DNA drug manufacturers., Ltd. In Shanghai, researchers have successfully constructed an intelligent double-stranded siRNA library, which targets MRGPRX2 and contains non-specific siRNA.Final delivery of siRNA was by Lipofectamine^® 2,000-mediated transfection (from Invitrogen, Carlsbad, Calif., United States) at a concentration of 1 μM according to the manufacturer’s guidelines. After 48 h of culture, the expression level of MRGPRX2 in the cells was significantly reduced, followed by quantitative analysis of β-hexosaminidase activity and cytokine secretion. The sequence of the siRNA was as follows: negative control siRNA, forward, 5′-UUC​UCC​GAA​CGU​GUC​ACG​UTT-3′, and reverse 5′-ACG​UGA​CAC​GUU​CGG​AGA​ATT-3’; MRGPRX2 knockdown siRNA, forward, 5′-GUA​CAA​CAG​UGA​AUG​GAA​ATT-3′, and reverse, 5′-UUU​CCA​UUC​ACU​GUU​GUA​CTT-3’.

We seeded LAD2 cells in 96-well plates at 2 × 10⁴ cells/well and they were treated as described next. M-3 and SH lone or in combination were analyzed at the indicated amounts using the aforementioned from β-hexosaminidase. The concentration gradient of M-3 or SH was predetermined according to the efficacy of each drug on mast cell activation. The rate of release rate of β-hexosaminidase from mast cells was analyzed at constant dilution ratios of the two drugs (2 μM, 4 μM, and 8 μM for both M-3 and SH). The rate of release of β-hexosaminidase was assayed as described above after treatment for 30 min. Using the online SynergyFinder software (https://synergyfinder.fimm.fi), the rate of release of β-hexosaminidase was considered as the percentage inhibition ranging from 0 to 1. The drug synergy score was calculated along with the Inhibition Index using response surface modeling and the zero interaction potency (ZIP) calculation method (Ianevski et al., 2022; Zheng et al., 2022). ZIP synergy scores >0 were considered synergistic and are denoted in red, and scores >10 were considered strongly synergistic (Zhang et al., 2022).

The rectal temperature of experimental mice was monitored by FT3400 animal thermometer (produced by Nanjing Kevin Biotechnology Company., Ltd. Nanjing, China). Mice were injected intravenously with 0.200 μL contains 5 mg per milliliter of M-3 substance. By using a bioactivity assessment device, the body temperature of the individual is measured, and a temperature sensor is implanted in the anus to obtain a rectal temperature reading. Temperature checks are taken every 5 min, and the process lasts for an hour.

Adult mice (6–8 weeks) were anesthetized with 50 mg/kg pentobarbital sodium intraperitoneally. 15 min after the induction of anesthesia, the rats were given an intravenous injection of 200 μL of 4% Evans Blue. A Vernier caliper was used to measure the thickness of the paws before any injection. After 5 min, one paw was injected with 0.5 mg/ml M-3 or SH or 30 μg/mL C48/80. A reasonable amount of saline was injected into the second paw as a negative control. The paw thickness was measured and recorded after 15 min. The mice were then euthanized with CO₂. Paw tissues were collected, dried at 65°C, and weighed separately. 1 mL of acetone (7: 3, v/v) solution of acetone (7: 3, v/v) was added and incubated for 6 h at 56°C. The tissues are then ground up, disturbed in an ultrasound machine for 10 min, and centrifuged for 20 min at 3,000 rpm. The supernatants were aliased equally into 96 well plates (200 μL/well), and the optical density (OD) was measured at 620 nm with a microplate reader.

LAD2 cells with a density of 5 × 10⁶ cells/mL were inoculated into 6-well plates. M-3 (50 μmol/L), SH (50 μmol/L), and PAMP-12 (2 μmol/L) was added to the cells for 3 h. After incubation, remove the cell suspension into the corresponding 1.5 mL EP tubes, centrifuge to discard the medium, wash twice with RNase-Free PBS, and centrifuge to obtain the cell precipitate; lysed the cells by adding Trizol solution, and then placed at room temperature for 5–10 min to be used until the cells were completely lysed, and then transferred to a freezing tube to be preserved at −80°C.

The experiment was entrusted to Lianchuan Biotechnology Co., Ltd. for subsequent on-line analysis and basic data analysis. The samples were tested and analyzed for correlation, differentially expressed genes, differentially expressed gene function and pathway annotation and KEGG enrichment analysis. After data acquisition, bioinformatics analysis was performed on the data.

LAD2 cells were seeded at a density of 2 × 10⁴ cells/well in a 96-well plate. Then they were treated with 50 μL 2-APB (45 μM), SB202190 (10 μM), and CCT129957 (3 μM), respectively. 30 min later, the cells were added 50 μM M-3additionally. Then the cells were incubated for 30 min. And the β-hexosaminidase release ratio were detected as described in 2.8.

The data set is expressed in the form of mean ± SD. Independent-sample t-tests were performed using IBM’s SPSS statistical software in Armonk, New York, to assess the statistical differences between different samples. Differences were considered significant at p < .05 (^* p < .05, ^** p < .01, and ^*** p < .001, depending on the experiment).

As more protein and nucleic acid structures have become available, molecular docking is considered for compound screening on the basis of its increased accuracy (Shoichet et al., 2002). To investigate the differences in the interactions between M-3, SH, and MRGPRX2, molecular docking simulations were performed using the Surflex-Dock Mode of the SYBYL-X 2.0 program package. The simulations investigated ligand–protein interactions between M-3, SH, and MRGPRX2. According to the docking results, M-3 and MRGPRX2 formed hydrogen bonds with ARG220 (2.41 Å and 2.61 Å, respectively), THR224 (1.90 Å), and ARG127 (2.41 Å) (Figure 1A). M-3 bound to MRGPRX2 (4.8309) to form hydrogen bonds. SH and MRGPRX2 formed hydrogen bonds with ARG220 (1.98 Å and 1.98 Å, respectively) and THR224 (1.89 Å and 2.52 Å, respectively) (Figure 1B). Binding of SH to MRGPRX2 (5.1807) to form hydrogen bonds was evident. These results indicate that M-3, similar to SH, has a good binding capacity with MRGPRX2.

In order to carry out follow-up experimental research, M-3 compound were synthesized by chemical methods (Figure 1C). To a solution of 1A (Figure 1C) (2.00 g, 6.07 mmol, 1.00 eq) in DCM (20.0 mL) was added to MCPBA (2.62 g, 12.14 mmol, 80% purity, 2.00 eq) portion-wise at 0°C. The mixture was stirred at 0°C–25°C for 1 h. LC-MS showed 1A was consumed completely. These two reactions were then combined together. The reaction mixture was added NH₃.H₂O to pH∼9, followed by extraction with three 50-mL volumes of CHCl₃. The combined organic layers were washed with three 100-mL volumes of 10% Na₂SO₃ solution, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue, designated M-2. M-2 (4.19 g, crude) was a brown oil. It was used in the next step without further purification.

M-2 (2.09 g, 6.05 mmol, 1.00 eq) was added portion-wise at 0°C to a solution of FeSO₄.7H₂O (10.09 g, 36.31 mmol, 6.00 eq) in 60.0 mL methanol. The mixture was stirred at 0°C for 4 h. LC-MS showed that M-2 was completely consumed, and the desired compound was detected. These two reactions were then combined. The reaction mixture was added EDTA (0.60 M, 59.60 mL, 5.91 eq) at 0°C for 4 h. The mixture then received NH₃·H₂O until the pH was approximately 9, and was then extracted with three 100-mL volumes of DCM. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue designated M-3. M-3 was purified using preparative HPLC using a Phenomenex Luna C18 column (100 × 30 mm × 5 um) with a phase of 10 mM NH₄HCO₃ acetonitrile in water). M-3 (76.4 mg, 199.87 µmol, 1.65% yield, 82.5% purity) was obtained as an off-white solid and checked by H-nuclear magnetic resonance and LC-MS.

After obtaining the synthesized M-3, SPR was used to study its affinity with MRGPRX2. The results indicated the strong and dose-dependent binding of M-3 to MRGPRX2 (K _D = 7.583 × 10⁻⁶ mol/L; Figure 1D-a). The calculated interaction strength between SH and MRGPRX2 was K _D = 6.812 × 10⁻⁷ mol/L (Figure 1D-b). The collective findings indicate that both metabolite M-3 and its prototype, SH, can activate MRGPRX2 and may cause hypersensitivity.

MRGPRX2 is the human ortholog of mouse Mrgprb2. MRGPRX2/HEK293 cells which were treated with 10, 50, or 100 μM M-3 produced ramarkable dose-dependent increases in intracellular Ca²⁺ mobilization (Figure 2A), similar to the C48/80 positive control. When LAD2 cells were treated in the same manner, M-3 produced a ramarkable dose-dependent increase in intracellular Ca²⁺ migration (Figure 2B). We used LAD2 mast cells as a model to detect changes of the secretion of β-hexosaminidase (a degranulation marker), histamine (mediators of increased vascular permeability) and TNF-α, IL-8, and MCP-1. The treatment of LAD2 cells with 10, 50, or 100 μM M-3 for 30 min and the levels of secreted β-hexosaminidase, histamine, TNF-α, IL-8, and MCP-1 were examined. C48/80 (30 μg/mL) was used as the positive control to verify whether the M-3-induced activation of mast cells promoted degranulation. The three concentrations of M-3 and 30 μg/mL C48/80 promoted the secretion of both β-hexosaminidase (Figure 2C-a) and the anaphylactic mediator histamine (Figure 2C-b). Release of TNF-α, IL-8, and MCP-1 was substantially increased (Figure 2C-c, d, e) in dose-dependent manners.

LAD-2 cells in which MRGPRX2 was knocked down by siRNA treatment were used to validate the interaction between the MRGPRX2 receptors and M-3. Treatment with 0, 10, 50, and 100 μM M-3, or 30 μg/mL C48/80 reduced the release rate of β-hexosaminidase release rate, treated by the same concentration of M-3, respectively (Figure 3A). To compare the differences in mast cell activation by the same concentration of M-3 and SH, we treated LAD2 cells with 10, 50, and 100 μM M-3 and SH, respectively (Figure 3B). M-3 induced release of β-hexosaminidase was greater than SH at 10 and 50 μM. As an active metabolite of SH, M-3 coexists with SH in vivo. Accordingly, we investigated in a series of orthogonal experiments whether the two have a synergistic effect in promoting β-hexosaminidase release from mast cells and detected the effect on histamine release at the same time. M-3 and SH were used at different concentration ratios to stimulate the release of β-hexosaminidase and histamine from mast cells. Using SynergyFinder software to calculate The ZIP synergy scores at the detected numerical value, to judge the synergistic effect of M-3 and SH. The ZIP synergistic score revealed a synergistic effect of M-3 and SH on β-hexosaminidase (Figures 3C, D) and histamine (Figures 3E, F) release from LAD2 cells. The findings support the view that the M-3 metabolite can act synergistically with the prototype SH to activate mast cells in vivo and aggravate the prototype drug to trigger anaphylactic reactions.

The body temperature of mice decreased rapidly and recovered slowly when anaphylaxis occurred. To investigate whether M-3 causes systemic allergic symptoms in mice, body temperature was monitored for 60 min after intravenous injection of M-3. Within 60 min of injection, the body temperature of the mice dropped sharply, slowly recovered, and eventually returned to normal (Figure 4). The results indicate that M-3 can induce anaphylaxis.

In order to assess the role of M-3 and SH in the formation of pseudo-allergic reactions, we adopted a passive skin allergic model in mice. After intravenous injection, M-3 or SH were injected subcutaneously for 15 min. C48/80 was used as positive control. The pseudo allergic reaction was characterized by hind paw edema and Evans blue stain leakage into the paw. Subcutaneous saline injection (vehicle) did not cause inflammation of the hind paws (swelling and extravasation). On the contrary, M-3 or SH could induce extensive extravasation and swelling in mice. In a previous study, SH-induced pseudo-anaphylactic reactions associated with Mrgprb2 expression in mast cells (Liu et al., 2017). Therefore, we examined whether the development of hind paw inflammation following M-3 injection was related to mast cells, using Kit ^W-sh/W−sh mice. In C57BL/6-Kit ^W-sh/W−sh mice, M-3 induced hindpaw inflammation was nearly absent, suggesting that M-3 was related to mast cells. Next, MrgprB2-CKO mice were used to examine the relationship between the inflammation of the hind paw and the Mrgprb2 expression in the mast cells. Compared with the wild-type Mrgprb2 mice, the Mrgprb2-CKO mice showed virtually no hind paw inflammation (Figure 5A). In addition, M-3 induced telangiectasis in C57BL/6 mice (Figure 5B) and mast cell degranulation (Figure 5C). These effects, however, were practically absent in the Kit ^W-sh/W−sh and Mrgprb2-CKO mice. These results indicate that the activation of mast cell mediators and subsequent vasodilation induced by M 3 may be associated with Mrgprb2, similar to that of SH.

PAMP (9-20) (PAMP-12) is a major product of the precursors of adrenomedullin. PAMP-12 can induce MRGPRX2-mediated mast cell degranulation reactions (Che et al., 2021). To investigate whether exogenous small-molecule compounds (M-3 and SH) and endogenous short peptides (PAMP-12) stimulate MRGPRX2 at different sites of action, leading to different effects, we analyzed the differentially expressed genes using RNA-sequencing (RNA-seq) in vitro after treatment of LAD2 cells with M-3, SH, and PAMP-12. Kyoto Encyclopedia and Genes and Genomes pathway enrichment analysis showed that M-3 and SH enriched the highest number of genes in the cytokine-cytokine receptor interaction pathway, whereas PAMP-12 enriched the highest number of genes in metabolic pathways. In addition, M-3 had a higher number of enriched genes in metabolic pathways and PAMP-12 had a higher number of enriched genes in the cytokine-cytokine receptor interaction pathway (Figure 6A). Next, we analyzed the major RNA-seq data for these pathways to identify genes that were differentially expressed (Figure 6B). Genes co-regulated by M-3 and SH in the cytokine-cytokine receptor interaction pathway were concentrated in the chemokine CC subfamily (CCL4, CCL18, CCL4L2, CCL3L3, CCL7, and CCL1) as well as the interleukin family, which invovles IL1B, IL1RN, IL17RE, IL7R, IL4R, and IL27RA (Figure 6B-a). Genes co-regulated by M-3 and PAMP-12 in the cytokine-cytokine receptor interaction pathway were similarly concentrated in the chemokine CC subfamily (CCL1, CCL4, CCL18, and CCL4L2) and the interleukin family (IL27RA, IL1B, IL1RN, IL11RA, and IL17RE) (Figure 6B-b). Unlike the M-3 and SH small-molecule compounds, the genes regulated by PAMP-12 were mainly involved in metabolic pathways (Figure 6B-c).

Phospholipase C gamma breaks down phosphatidylinositol-4,5-bisphosphate to form dissolved IP3. The release of calcium ions is triggered by the interaction between IP3 and IP3R, forming the “first wave”. This release is transient from endoplasmic reticulum storage. After pre-incubation of LAD2 cells with the IP3R inhibitor, the release of β-hexosaminidase from LAD2 cells triggered by M-3 was blocked (Figure 7A). In addition, we determined the level of β-hexosaminidase release after preincubation of LAD2 cells with PLC-γ and P38 inhibitors, and it was found that PLC-γ and P38 inhibitors similarly inhibited M-3-induced β-hexosaminidase release (Figures 7B, C).