Effective in vitro evaluation of the risk of histamine release related to valemetostat tosylate using MRGPRX2-expressing cells.

Mas-related G-protein-coupled receptor X2 (MRGPRX2), expressed on mast cells, is associated with drug-induced pseudo-allergic reactions. Although it is well known that there are differences of sensitivity between species in the pseudo-allergic reactions, no platform for evaluating a human risk of the pseudo-allergic reactions observed in nonclinical studies has been established. Valemetostat tosylate, developed as an anti-cancer drug, induced histamine release in a nonclinical study with dogs. The purpose of the current study was to identify the mechanism and assess the human risk of valemetostat-tosylate-induced histamine release using dog and human MRGPRX2-expressing cells. In an experiment with human or dog MRGPRX2-expressing cells, valemetostat tosylate caused activation of human and dog MRGPRX2. Importantly, the EC50 for dog MRGPRX2 was consistent with the Cmax value at which histamine release was observed in dogs. Furthermore, the EC50 for human MRGPRX2 was ca. 27-fold higher than that for dog MRGPRX2, indicating a species difference in histamine-releasing activity. In a clinical trial, histamine release was not observed in patients receiving valemetostat tosylate. In conclusion, an in vitro assay using human and animal MRGPRX2-expressing cells would be an effective platform to investigate the mechanism and predict the human risk of histamine release observed in nonclinical studies.

Identification and functional analysis of first heterozygous frameshift mutation in the GHRH gene in a Chinese boy with isolated growth hormone deficiency.

BACKGROUND: Isolated growth hormone deficiency (IGHD) is a rare genetically heterogeneous disorder caused primarily by mutations in GH1 and GH releasing hormone receptor (GHRHR). The aim of this study was to identify the molecular etiology of a Chinese boy with IGHD. METHODS: Whole-exome sequencing, sanger sequencing and bioinformatic analysis were performed to screen for candidate mutations. The impacts of candidate mutation on gene expression, intracellular localization and protein function were further evaluated by in vitro assays. RESULTS: A novel heterozygous frameshift mutation in the GHRH gene (c.91dupC, p.R31Pfs*98) was identified in a Chinese boy clinically diagnosed as having IGHD. The mutation was absent in multiple public databases, and considered as deleterious using in silico prediction, conservative analysis and three-dimensional homology modeling. Furthermore, mRNA and protein expression levels of mutant GHRH were significantly increased than wild-type GHRH (p < 0.05). Moreover, mutant GHRH showed an aberrant accumulation within the cytoplasm, and obviously reduced ability to stimulate GH secretion and cAMP accumulation in human GHRHR-expressing pituitary GH3 cells compared to wild-type GHRH (p < 0.05). CONCLUSION: Our study discovered the first loss-of function mutation of GHRH in a Chinese boy with IGHD and provided new insights on IGHD pathogenesis caused by GHRH haploinsufficiency.

The blockade of neuropeptide FF receptor 1 and 2 differentially contributed to the modulating effects on fentanyl-induced analgesia and hyperalgesia in mice.

Neuropeptide FF (NPFF) plays a critical role in various physiological processes through the activation of neuropeptide FF receptor 1 and 2 (NPFFR1 and NPFFR2). Numerous evidence has indicated that NPFF exhibits opposite opioid-modulating effects on opioid-induced analgesia after supraspinal and spinal administrations, while the detailed role of NPFFR1 and NPFFR2 remains unclear. In this study, we employed pharmacological and genetic inhibition of NPFFR to investigate the modulating roles of central NPFFR1 and NPFFR2 in opioid-induced analgesia and hyperalgesia, using a male mouse model of acute fentanyl-induced analgesia and secondary hyperalgesia. Our findings revealed that intrathecal (i.t.) injection of the nonselective NPFFR antagonist RF9 significantly enhanced fentanyl-induced analgesia, whereas intracerebroventricular (i.c.v.) injection did not show the same effect. Moreover, NPFFR2 deficient (npffr2-/-) mice exhibited stronger analgesic responses to fentanyl compared to wild type (WT) or NPFFR1 knockout (npffr1-/-) mice. Intrathecal injection of RF9 in npffr1-/- mice also significantly enhanced fentanyl-induced analgesia. These results indicate a crucial role of spinal NPFFR2 in the enhancement of opioid analgesia. Contrastingly, hyperalgesia induced by fentanyl was markedly reversed in npffr1-/- mice but remained unaffected in npffr2-/- mice. Similarly, i.c.v. injection of the selective NPFFR1 antagonist RF3286 effectively prevented fentanyl-induced hyperalgesia in WT or npffr2-/- mice. Notably, co-administration of i.c.v. RF3286 and i.t. RF9 augmented fentanyl-induced analgesia while reducing hyperalgesia. Collectively, these findings highlight the modulating effects of blocking spinal NPFFR2 and supraspinal NPFFR1 on fentanyl-induced analgesia and hyperalgesia, respectively, which shed a light on understanding the pharmacological function of NPFF system in future studies.

New insight into prurigo nodularis: Proadrenomedullin N-terminal 20 peptide mediates mouse mast cell activation via Mrgprb2.

BACKGROUND: Prurigo nodularis (PN) is a chronic inflammatory skin disorder that is characterized by extremely itchy nodules. Proadrenomedullin N-terminal 20 (PAMP) activates mast cell degranulation via Mas-related G protein-coupled receptor X2 (MRGPRX2), which is associated with pruritus in allergic contact dermatitis. However, the mechanisms underlying the action of PAMP and MRGPRX2 in PN remain unclear. OBJECTIVE: To determine the role of PAMP-induced mast cell activation via MRGPRX2 (mouse homologous Mrgprb2) in PN. METHODS: The expression of PAMP and the number of MRGPRX2-expressing mast cells in the skin biopsies of patients with PN, atopic dermatitis (AD), and healthy participants were analyzed using immunohistochemistry and immunofluorescence, respectively. The biphasic response of PAMP9-20 mediated by Mrgprb2 in mouse peritoneal mast cells (PMC) was validated in vitro using qRT-PCR, ELISA, flow cytometry, and siRNA techniques. RESULTS: PAMP expression and the number of MRGPRX2+ mast cells in lesional PN skin, but not in AD, were elevated compared to healthy skin. PAMP9-20 mediates the immediate and delayed phase responses of PMC, such as degranulation, histamine and ß-hexosaminidase release, and secretion of inflammatory factors such as CCL2, TNF-α, and GM-CSF. These effects were inhibited when Mrgprb2 expression was silenced. Silencing Mrgprb2 did not affect the biphasic response of PMC that was induced by IgE-FcεRI activation. CONCLUSIONS: The results show that PAMP mediates mouse mast cell activation via Mrgprb2, which may be involved in the pathogenesis of PN. The PAMP/ Mrgprb2 pathway, independent of classical IgE signaling, could be developed as a candidate drug target for treating PN.

Rfamide-related peptide-3(RFRP-3) receptor gene is expressed in mouse ovarian granulosa cells: Potential role of RFRP-3 in steroidogenesis and apoptosis.

RFRP-3 is a functional ortholog of avian GnIH and regulates reproductive activities in the gonads of animals. However, the role of RFRP-3 in the function of ovarian granulosa cells in mice remains unclear. First, we detected the expression of the RFRP-3 receptor (GPR147) in the ovarian granulosa cells of mice. Second, the effect of RFRP-3 treatment on estradiol and progesterone secretions from granulosa cells was tested by ELISA. Meanwhile, the expression of genes and proteins regulating steroid hormone synthesis was respectively examined by qPCR and western blot. Furthermore, the effect of RFRP-3 treatment on the apoptosis of granulosa cells was analyzed. The results revealed that the GPR147 protein (a RFRP-3 receptor) was expressed in the ovarian granulosa cells of mice. Low and medium doses RFRP-3 treatment significantly reduced progesterone secretion in the granulosa cells (P < 0.05), while RFRP-3 suppressed p450scc, 3ß-HSD, StAR, and FSHR expression in a non-dose-dependent manner. Moreover, RFRP-3 treatment might induce the apoptosis of granulosa cells. Additionally, low doses RFRP-3 significantly reduced p-ERK1/2 protein expression (P < 0.05) in the ovarian granulosa cells. We here, for the first time, confirmed that GPR147 was expressed in the ovarian granulosa cells of mice. Our findings suggested that and RFRP-3 regulates the granulosa cell function through the ERK signaling pathway, which will lay the foundation for uncovering molecular mechanisms by which RFRP-3 regulates follicle development in future.

In the skin lesions of patients with mycosis fungoides, the number of MRGPRX2-expressing cells is increased and correlates with mast cell numbers.

Background: Mycosis fungoides (MF) is an indolent T-cell lymphoma that mainly affects the skin and presents with itch in more than half of the patients. Recently, the expression of Mas-related G protein-coupled receptor X2 (MRGPRX2), a receptor of mast cell (MC) responsible for the IgE-independent non-histaminergic itch, has been shown in lesional skin of patients with pruritic skin diseases, including chronic urticaria, prurigo, and mastocytosis. As of yet, limited knowledge exists regarding the MRGPRX2 expression in the skin of patients with MF. Objectives: To investigate the number of MRGPRX2-expressing (MRGPRX2+) cells in the skin of patients with MF and its correlation with clinical and laboratory characteristics of the disease. Methods: MRGPRX2 was analyzed in lesional and non-lesional skin of MF patients and healthy skin tissues by immunohistochemistry. Co-localization of MRGPRX2 with the MC marker tryptase was assessed by immunofluorescence. Public single-cell RNAseq data was reanalyzed to identify the MRGPRX2 expression on the distinct cell types. Results: In lesional skin of MF patients, MRGPRX2+ cell number was higher than in non-lesional skin and healthy control skin (mean:15.12 vs. 6.84 vs. 5.51 cells/mm2, p=0.04), and correlated with MC numbers (r=0.73, p=0.02). MC was the primary cell type expressing MRGPRX2 in MF patients. The ratio of MRGPRX2+ MCs to MRGPRX2+ cells in lesional and non-lesional skin correlated with the severity of disease (r=0.71, p=0.02 and r=0.67, p=0.03, respectively). Conclusions: Our findings point to the role of MRGPRX2 and MC in the pathogenesis of MF that should be investigated in further studies.

Neuropeptide B (NPB) and NPB receptor 2b (NPBWR2b) in the ricefield eel Monopterus albus: expression and potential involvement in the regulation of gonadotropins.

The neuropeptide B/W signaling system is composed of neuropeptide B (NPB), neuropeptide W (NPW), and two cognate receptors, NPBWR1 and NPBWR2, which are involved in diverse physiological processes, including the central regulation of neuroendocrine axes in vertebrates. The components of this signaling system are not well conserved during vertebrate evolution, implicating its functional diversity. The present study characterized the ricefield eel neuropeptide B/W system, generated a specific antiserum against the neuropeptide B/W receptor, and examined the potential roles of the system in the regulation of adenohypophysial functions. The ricefield eel genome contains npba, npbb, and npbwr2b but lacks the npw, npbwr1, and npbwr2a genes. The loss of npw and npbwr1 probably occurred at the base of ray-finned fish radiation and that of npbwr2a species specifically in ray-finned fish. Npba and npbb genes are produced through whole-genome duplication (WGD) in ray-finned fish. The ricefield eel npba was expressed in the brain and some peripheral tissues, while npbb was predominantly expressed in the brain. The ricefield eel npbwr2b was also expressed in the brain and in some peripheral tissues, such as the pituitary, gonad, heart, and eye. Immunoreactive Npbwr2b was shown to be localized to Lh and Fsh cells but not to Gh or Prl cells in the pituitary of ricefield eels. Npba upregulated the expression of fshb and cga but not lhb mRNA in pituitary fragments of ricefield eels cultured in vitro. The results of the present study suggest that the NPB system of ricefield eels may be involved in the neuroendocrine regulation of reproduction.

Signaling mechanism of growth hormone-releasing hormone receptor.

The hypothalamic peptide growth hormone-releasing hormone (GHRH) stimulates the secretion of growth hormone (GH) from the pituitary through binding and activation of the pituitary type of GHRH receptor (GHRH-R), which belongs to the family of G protein-coupled receptors with seven potential membrane-spanning domains. Splice variants of GHRH-Rs (SV) in human tumors and other extra pituitary tissues were identified and their cDNA was sequenced. Among the SVs, splice variant 1 (SV1) possesses the greatest similarity to the full-length GHRH-R and remains functional by eliciting cAMP signaling and mitogenic activity upon GHRH stimulation. A large body of work have evaluated potential clinical applications of agonists and antagonists of GHRH in diverse fields, including endocrinology, oncology, cardiology, diabetes, obesity, metabolic dysfunctions, Alzheimer's disease, ophthalmology, wound healing and other applications. In this chapter, we briefly review the expression and potential function of GHRH-Rs and their SVs in various tissues and also elucidate and summarize the activation, molecular mechanism and signalization pathways of these receptors. Therapeutic applications of GHRH analogs are also discussed.

Neuromedin U programs eosinophils to promote mucosal immunity of the small intestine.

Eosinophils are granulocytes that play an essential role in type 2 immunity and regulate multiple homeostatic processes in the small intestine (SI). However, the signals that regulate eosinophil activity in the SI at steady state remain poorly understood. Through transcriptome profiling of eosinophils from various mouse tissues, we found that a subset of SI eosinophils expressed neuromedin U (NMU) receptor 1 (NMUR1). Fate-mapping analyses showed that NMUR1 expression in SI eosinophils was programmed by the local microenvironment and further enhanced by inflammation. Genetic perturbation and eosinophil-organoid coculture experiments revealed that NMU-mediated eosinophil activation promotes goblet cell differentiation. Thus, NMU regulates epithelial cell differentiation and barrier immunity by stimulating NMUR1-expressing eosinophils in the SI, which highlights the importance of neuroimmune-epithelial cross-talk in maintaining tissue homeostasis.

PACAP activates MRGPRX2 on meningeal mast cells to drive migraine-like pain.

Migraine ranks among the most prevalent disorders worldwide, leading to disability and decreased quality of life in patients. Recently, neurogenic inflammation has been recognized as a potential underlying pathology contributing to the migraine pain pathway. Mast cells reside in the meninges and have been implicated in contributing to the pathophysiology of migraine. Here we report for the first time that the mouse Mas-Related G-protein-coupled Receptor B2 (MrgprB2), is expressed on meningeal connective tissue mast cells and contributes to Pituitary Adenylate Cyclase Activating Peptide (PACAP)-induced migraine-like pain behavior. We also found that PACAP was able to dose-dependently lead to enzyme release from human mast cells via activation of MRGPRX2; the human homolog of MrgprB2. Using a transgenic MRGPRX2 mouse, we observed significant increases in PACAP-induced migraine-like pain behavior in MRGPRX2+ mice vs mice lacking the receptor. These results reveal both MrgprB2 and MRGPRX2 as important contributors to neuropeptide-induced migraine pain.

Functional Analysis of Pheromone Biosynthesis Activating Neuropeptide Receptor Isoforms in Maruca vitrata.

Insect sex pheromones are volatile chemicals that induce mating behavior between conspecific individuals. In moths, sex pheromone biosynthesis is initiated when pheromone biosynthesis-activating neuropeptide (PBAN) synthesized in the suboesophageal ganglion binds to its receptor on the epithelial cell membrane of the pheromone gland. To investigate the function of PBAN receptor (PBANR), we identified two PBANR isoforms, MviPBANR-B and MviPBANR-C, in the pheromone glands of Maruca vitrata. These two genes belong to G protein-coupled receptors (GPCRs) and have differences in the C-terminus but share a 7-transmembrane region and GPCR family 1 signature. These isoforms were expressed in all developmental stages and adult tissues. MviPBANR-C had the highest expression level in pheromone glands among the examined tissues. Through in vitro heterologous expression in HeLa cell lines, only MviPBANR-C-transfected cells responded to MviPBAN (≥5 µM MviPBAN), inducing Ca2+ influx. Sex pheromone production and mating behavior were investigated using gas chromatography and a bioassay after MviPBANR-C suppression by RNA interference, which resulted in the major sex pheromone component, E10E12-16:Ald, being quantitatively reduced compared to the control, thereby decreasing the mating rate. Our findings indicate that MviPBANR-C is involved in the signal transduction of sex pheromone biosynthesis in M. vitrata and that the C-terminal tail plays an important role in its function.

The pharmacologic and toxicologic characterization of the potent and selective KRAS G12D inhibitors ERAS-4693 and ERAS-5024.

Two potent and selective KRASG12D inhibitors, ERAS-4693 and ERAS-5024, were generated as possible clinical candidates to treat patients harboring G12D mutations in solid tumors. Both molecules exhibited strong anti-tumor activity in the KRASG12D mutant PDAC xenograft mouse models while ERAS-5024 also showed tumor growth inhibition when administered on an intermittent dosing regimen. Acute dose-limiting toxicity consistent with an allergic reaction was observed for both molecules shortly after administration at doses just above those which demonstrated anti-tumor activity, indicative of a narrow therapeutic index. A series of studies were subsequently conducted to identify a common underlying mechanism for the observed toxicity, including CETSA® (Cellular Thermal Shift Assay) as well as several functional off-target screens. Both ERAS-4693 and ERAS-5024 were identified to agonize MRGPRX2 which has been linked to pseudo-allergic reactions. In vivo toxicologic characterization of both molecules included repeat-dose studies in the rat and dog. Dose-limiting toxicities were observed in both species with ERAS-4693 and ERAS-5024 and plasma exposure levels at the maximum tolerated doses were generally below that which caused strong anti-tumor activity, supporting the initial observation of a narrow therapeutic index. Additional overlapping toxicities included a reduction in reticulocytes and clinical pathological changes suggestive of an inflammatory response. Furthermore, increases in plasma histamine were observed in dogs administered ERAS-5024, supporting the hypothesis that MRGPRX2 agonism may be the cause of the pseudo-allergic reaction. This work highlights the importance of balancing both the safety and efficacy of KRASG12D inhibitors as this class of molecules begins to enter clinical development.

Transcriptome sequencing of the central nervous system to identify the neuropeptides and neuropeptide receptors of Antheraea pernyi.

Neuropeptides and neuropeptide receptors are crucial regulators for the behavior, lifecycle, and physiology of insects and are mainly produced and released from the neurosecretory cells of the central nervous system (CNS). In this study, RNA-seq was employed to investigate the transcriptome profile of the CNS which is composed of the brain and ventral nerve cord (VNC) of Antheraea pernyi. From the data sets, a total of 18 and 42 genes were identified, which respectively encode the neuropeptides and neuropeptide receptors involved in regulating multiple behaviors including feeding, reproductive behavior, circadian locomotor, sleep, and stress response and physiological processes such as nutrient absorption, immunity, ecdysis, diapause, and excretion. Comparison of the patterns of expression of those genes between the brain and VNC showed that most had higher levels of expression in the brain than VNC. Besides, 2760 differently expressed genes (DEGs) (1362 up-regulated and 1398 down-regulated ones between the B and VNC group) were also screened and further analyzed via gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses. The results of this study could provide comprehensive profiles of the neuropeptides and neuropeptide receptors of A. pernyi CNS and lay the foundation for further research into their functions.

Identification and expression of short neuropeptide F and its receptors in the tick Ixodes ricinus.

In Europe, the tick Ixodes ricinus is the most important vector of numerous pathogens that are transmitted during blood feeding on their vertebrate hosts. To elucidate mechanisms controlling blood intake and associated transmission of pathogens we identified and described expression of short neuropeptide F (sNPF) and its receptors which are known to regulate feeding in insects. Using in situ hybridization (ISH) and immunohistochemistry (IHC) we stained numerous neurons producing sNPF in the central nervous system (CNS; synganglion), while a few peripheral neurons were detected anteriorly to the synganglion, and on the surface of the hindgut and leg muscles. Apparent sNPF expression was also found in enteroendocrine cells individually scattered in anterior lobes of the midgut. In silico analyses and BLAST search for sNPF receptors revealed two putative G protein-coupled receptors (sNPFR1 and sNPFR2) in the I. ricinus genome. Aequorin-based functional assay in CHO cells showed that both receptors were specific and sensitive to sNPF in nanomolar concentrations. Increased expression levels of these receptors in the gut during blood intake suggest that sNPF signaling may be involved in regulation of feeding and digestion processes of I. ricinus.

The G protein-coupled receptor neuropeptide receptor-15 modulates larval development via the transforming growth factor-ß DAF-7 protein in Caenorhabditis elegans.

G protein-coupled receptors (GPCRs) are a major class of membrane receptors that modulate a wide range of physiological functions. These receptors transmit extracellular signals, including secreted bioactive peptides, to intracellular signaling pathways. The nematode Caenorhabditis elegans has FMRFamide-like peptides, which are one of the most diverse neuropeptide families, some of which modulate larval development through GPCRs. In this study, we identified the GPCR neuropeptide receptor (NPR)-15, which modulates C. elegans larval development. Our molecular genetic analyses indicated the following: 1) NPR-15 mainly functions in ASI neurons, which predominantly regulate larval development, 2) NPR-15 interacts with GPA-4, a C. elegans Gα subunit, and 3) NPR-15, along with GPA-4, modulates larval development by regulating the production and secretion of the transforming growth factor-ß (TGF-ß)-like protein DAF-7. The present study is the first report to demonstrate the importance of a GPCR to the direct regulation of a TGF-ß-like protein.

DOCK2 regulates MRGPRX2/B2-mediated mast cell degranulation and drug-induced anaphylaxis.

BACKGROUND: Drug-induced anaphylaxis is triggered by the direct stimulation of mast cells (MCs) via Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MRGPRB2). However, the precise mechanism that links MRGPRX2/B2 to MC degranulation is poorly understood. Dedicator of cytokinesis 2 (DOCK2) is a Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 regulates migration and activation of leukocytes, its role in MCs remains unknown. OBJECTIVE: We aimed to elucidate whether-and if so, how-DOCK2 is involved in MRGPRX2/B2-mediated MC degranulation and anaphylaxis. METHODS: Induction of drug-induced systemic and cutaneous anaphylaxis was compared between wild-type and DOCK2-deficient mice. In addition, genetic or pharmacologic inactivation of DOCK2 in human and murine MCs was used to reveal its role in MRGPRX2/B2-mediated signal transduction and degranulation. RESULTS: Induction of MC degranulation and anaphylaxis by compound 48/80 and ciprofloxacin was severely attenuated in the absence of DOCK2. Although calcium influx and phosphorylation of several signaling molecules were unaffected, MRGPRB2-mediated Rac activation and phosphorylation of p21-activated kinase 1 (PAK1) were impaired in DOCK2-deficient MCs. Similar results were obtained when mice or MCs were treated with small-molecule inhibitors that bind to the catalytic domain of DOCK2 and inhibit Rac activation. CONCLUSION: DOCK2 regulates MRGPRX2/B2-mediated MC degranulation through Rac activation and PAK1 phosphorylation, thereby indicating that the DOCK2-Rac-PAK1 axis could be a target for preventing drug-induced anaphylaxis.

Inhibition of Mast Cell Degranulation in Atopic Dermatitis by Celastrol through Suppressing MRGPRX2.

Background: Atopic dermatitis is a common dermatological disease, and mast cell degranulation is believed to be related with the progression of atopic dermatitis. Mas-related G protein-coupled receptor-X2 (MRGPRX2), and calcium release-activated calcium channel protein 1-2 (ORAI-1, ORAI-2) are involved in mast cell degranulation. Celastrol is an active monomer of Tripterygium wilfordii, and it presents an antiatopic role. Methods: 2,4-Dinitrofluorobenzene (DNFB) and compound 48/80 (C 48/80) were used to establish a slow and acute scratching animal model, respectively. Hematoxylin-eosin and toluidine blue staining was used to investigate tissue injury. Inflammatory factor concentration was measured with ELISA. The expression of MRGPRX2, ORAI-1, and ORAI-2 was detected with immunohistochemistry (IHC) staining. Gene expression profiling and microRNA array were performed to investigate gene differential expression. Results: Celastrol greatly inhibited atopic dermatitis-related tissues injury, mast cell production, histamine release, scratching level, inflammatory factor expression, and activation of MRGPRX2/ORAI axis in the DNFB-induced atopic dermatitis model. The influence of Celastrol on atopic dermatitis was remarkably reversed by overexpression of MRGPRX2. Conclusion: We found that the improvements of atopic dermatitis caused by Celastrol were reversed by treatment with MRGPRX2OE, indicating that Celastrol might affect atopic dermatitis through MRGPRX2. This study might provide a novel thought for the prevention and treatment of atopic dermatitis by regulating MRGPRX2.

Hydroquinone-induced skin irritant reaction could be achieved by activating mast cells via mas-related G protein-coupled receptor X2.

Hydroquinone (HQ) is one of the most effective drugs to treat hyperpigmentary disorders, but often causes skin irritation in clinic. Mast cell plays an important role in contact dermatitis and triggering pseudo-allergic reactions via MRGPRX2. Whether HQ-induced skin irritant reaction through activating mast cells via MRGPRX2 remains unknown. To investigate the role of mast cells in HQ-induced skin irritant reaction and verify whether MRGPRX2 participated in the HQ effect on mast cells which contributed to the pathogenesis of skin irritant reaction, a mouse model of HQ-induced skin irritation was established to observe the local and systemic inflammation associated with mast cell receptor MrgprB2. Human mast cell LAD2 was used to verify the effect of HQ on mast cells via MRGPRX2 by knocking down with siRNA. As a result, mast cells were involved in the development of HQ-induced irritant reaction, and local inflammation is closely related to mast cell receptor MrgprB2. HQ could activate mast cells via MRGPRX2, causing changes in calcium concentration, degranulation and release of inflammatory cytokines which lead to skin irritant reaction. In conclusion, HQ-induced skin irritant reaction could be skin pseudo-allergic reactions achieved by activating mast cells via MRGPRX2.