Scratching Behavior In Mice Research Articles

Antipruritic effect of ursolic acid through MRGPRX2/MrgprB2-dependent inhibition of mast cell degranulation and reduced TSLP production

Ursolic acid (UA), a pentacyclic triterpene, exhibits diverse pharmacological effects, including potential treatment for allergic diseases. It downregulates thymic stromal lymphopoietin (TSLP) and disrupts mast cell signaling pathways. However, the exact molecular mechanism by which UA interferes with mast cell action remains unclear. Therefore, the current study aimed to uncover molecular entities underlying the effect of UA on mast cells and its potential antipruritic effect, specifically investigating its modulation of key molecules such as TRPV4, PAR2, and MRGPRX2, which are involved in TSLP regulation and sensation.Calcium imaging experiments revealed that UA pretreatment significantly suppressed MRGPRX2 activation (and its mouse orthologue MrgprB2), a G protein-coupled receptor predominantly expressed in mast cells. Molecular docking predictions suggested potential interactions between UA and MRGPRX2/MrgprB2. UA pretreatment also reduced mast cell degranulation through MRGPRX2 and MrgprB2-dependent mechanisms. In a dry skin mouse model, UA administration decreased tryptase and TSLP production in the skin, and diminished TSLP response in the sensory neurons. While PAR2 and TRPV4 activation enhances TSLP production, UA did not inhibit their activity. Notably, UA attenuated compound 48/80-induced scratching behaviors in mice and suppressed spontaneous scratching in a dry skin model.The present study confirms the effective inhibition of UA on MRGPRX2/MrgprB2, leading to reduced mast cell degranulation and suppressed scratching behaviors. These findings highlight the potential of UA as an antipruritic agent for managing various allergy- or itch-related conditions.

LPS exacerbates TRPV4-mediated itch through the intracellular TLR4-PI3K signalling.

Pruritus is often accompanied with bacterial infections, but the underlying mechanism is not fully understood. Although previous studies revealed that lipopolysaccharides (LPS) could directly activate TRPV4 channel and TRPV4 is involved in the generation of both acute itch and chronic itch, whether and how LPS affects TRPV4-mediated itch sensation remains unclear. Here, we showed that LPS-mediated TRPV4 sensitization exacerbated GSK101-induced scratching behaviour in mice. Moreover, this effect was compromised in TLR4-knockout mice, suggesting LPS acted through a TLR4-dependent mechanism. Mechanistically, LPS enhanced GSK101-evoked calcium influx in mouse ear skin cells and HEK293T cells transfected with TRPV4. Further, LPS sensitized TRPV4 channel through the intracellular TLR4-PI3K-AKT signalling. In summary, our study found a modulatory role of LPS in TRPV4 function and highlighted the TLR4-TRPV4 interaction in itch signal amplification.

Region-specific activation in the accumbens nucleus by itch with modified scratch efficacy in mice – a model-free multivariate analysis

Itch is a protective/defensive function with divalent motivational drives. Itch itself elicits an unpleasant experience, which triggers the urge to scratch, relieving the itchiness. Still, it can also result in dissatisfaction when the scratch is too intense and painful or unsatisfactory due to insufficient scratch effect. Therefore, it is likely that the balance between the unpleasantness/pleasure and satisfaction/unsatisfaction associated with itch sensation and scratching behavior is determined by complex brain mechanisms. The physiological/pathological mechanisms underlying this balance remain largely elusive. To address this issue, we targeted the "reward center" of the brain, the nucleus accumbens (NAc), in which itch-responsive neurons have been found in rodents. We examined how neurons in the NAc are activated or suppressed during histamine-induced scratching behaviors in mice. The mice received an intradermal injection of histamine or saline at the neck, and the scratching number was analyzed by recording the movement of the bilateral hind limbs for about 45 min after injection. To experimentally manipulate the scratch efficacy in these histamine models, we compared histamine's behavioral and neuronal effects between mice with intact and clipped nails on the hind paws. As expected, the clipping of the hind limb nail increased the number of scratches after the histamine injection. In the brains of mice exhibiting scratching behaviors, we analyzed the expression of the c-fos gene (Fos) as a readout of an immediate activation of neurons during itch/scratch and dopamine receptors (Drd1 and Drd2) using multiplex single-molecule fluorescence in situ hybridization (RNAscope) in the NAc and surrounding structures. We performed a model-free analysis of gene expression in geometrically divided NAc subregions without assuming the conventional core–shell divisions. The results indicated that even within the NAc, multiple subregions responded differentially to various itch/scratch conditions. We also found different clusters with neurons showing similar or opposite changes in Fos expression and the correlation between scratch number and Fos expression in different itch/scratch conditions. These regional differences and clusters would provide a basis for the complex role of the NAc and surrounding structures in encoding the outcomes of scratching behavior and itchy sensations.

A phytosphingosine derivative mYG-II-6 inhibits histamine-mediated TRPV1 activation and MRGPRX2-dependent mast cell degranulation

BackgroundPhytosphingosine and its derivative are known for their skin-protective properties. While mYG-II-6, a phytosphingosine derivative, has shown anti-inflammatory and antipsoriatic effects, its potential antipruritic qualities have yet to be explored. This study aimed to investigate mYG-II-6′s antipruritic properties. MethodsThe calcium imaging technique was employed to investigate the activity of ion channels and receptors. Mast cell degranulation was confirmed through the β-hexosaminidase assay. Additionally, in silico molecular docking and an in vivo mouse scratching behavior test were utilized. ResultsUsing HEK293T cells transfected with H1R and TRPV1, we examined the impact of mYG-II-6 on histamine-induced intracellular calcium rise, a key signal in itch-mediating sensory neurons. Pretreatment with mYG-II-6 significantly reduced histamine-induced calcium levels and inhibited TRPV1 activity, suggesting its role in blocking the calcium influx channel. Additionally, mYG-II-6 suppressed histamine-induced calcium increase in primary cultures of mouse dorsal root ganglia, indicating its potential antipruritic effect mediated by histamine. Interestingly, mYG-II-6 exhibited inhibitory effects on human MRGPRX2, a G protein-coupled receptor involved in IgE-independent mast cell degranulation. However, it did not inhibit mouse MrgprB2, the ortholog of human MRGPRX2. Molecular docking analysis revealed that mYG-II-6 selectively interacts with the binding pocket of MRGPRX2. Importantly, mYG-II-6 suppressed histamine-induced scratching behaviors in mice. ConclusionsOur findings show that mYG-II-6 can alleviate histamine-induced itch sensation through dual mechanisms. This underscores its potential as a versatile treatment for various pruritic conditions.

Cimifugin Relieves Histamine-Independent Itch in Atopic Dermatitis via Targeting the CQ Receptor MrgprA3.

Background: We previously found that cimifugin has a potent antiallergic inflammatory effect in atopic dermatitis (AD). However, whether cimifugin has an antipruritic effect in AD was unknown. Methods: Mouse scratching behavior tests were performed to verify the proposed antipruritic effect of cimifugin on DNFB- or FITC-mediated AD. Chloroquine (CQ)- and compound 48/80-evoked acute itch models were employed to clarify the effect of cimifugin on histamine-dependent or -independent itch. Intracellular calcium changes were assessed in a primary culture of mouse dorsal root ganglia (DRG) in response to pruritogen exposure with or without cimifugin treatment, including CQ, histamine, allyl-isothiocyanate (AITC), and capsaicin. Molecular docking and microscale thermophoresis (MST) assays were performed to predict and verify the binding ability and modes between cimifugin and the CQ receptor MrgprA3, respectively. Results: We found that cimifugin attenuates itch behaviors effectively in FITC-induced AD. Notably, cimifugin significantly alleviated acute itching behaviors induced by CQ but not compound 48/80 in vivo. Moreover, cimifugin remarkably inhibited CQ-evoked calcium influx in DRG cells but had no obvious effect on histamine-induced calcium influx. Nevertheless, cimifugin did not interfere with either AITC-stimulated TRPA1 activation- or capsaicin-stimulated TRPV1 activation-mediated calcium influx in DRG cells. Molecular docking predicted that CQ and cimifugin might share similar binding abilities and binding modes with MrgprA3. MST assay confirmed cimifugin directly targeting MrgprA3. Conclusion: The present study demonstrates that cimifugin has a potent antipruritic effect in AD with a histamine-independent mechanism via targeting the CQ receptor MrgprA3. Thus, cimifugin is a promising candidate antipruritic agent for AD.

Alleviation of allergic conjunctivitis by (±)5(6)-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid in mice.

Background: Allergic conjunctivitis (AC) is a common ophthalmologic disorder that causes symptoms that often reduces a patient's quality of life (QOL). We investigated the effects of the eicosapentaenoic acid metabolite (±)5(6)-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid ((±)5(6)-DiHETE) on AC using a mouse model. Methods: BALB/c mice were sensitized with two injections of short ragweed pollen in alum, challenged fifth with pollen in eyedrops. The clinical signs and tear volume were evaluated at 15min after the final challenge. Histamine-induced ocular inflammation model was prepared by instilling histamine onto the surface of the eye. Fifteen minutes after histamine application, tear volume was measured using the Schirmer tear test. Miles assay was performed to investigate vascular permeability. To cause scratching behavior 10μg of serotonin was injected in the cheek. Results: Repeated topical application of pollen induced conjunctivitis, accompanied by eyelid edema and tearing in mice. Pollen application typically degranulates mast cells and recruits eosinophils to the conjunctiva. Intraperitoneal administration of 300μg/kg of (±)5(6)-DiHETE significantly inhibited pollen-induced symptoms. The administration of (±)5(6)-DiHETE also attenuated mast cell degranulation and eosinophil infiltration into the conjunctiva. To assess the effects of (±)5(6)-DiHETE on the downstream pathway of mast cell activation in AC, we used a histamine-induced ocular inflammation model. Topical application of 4μg/eye histamine caused eyelid edema and tearing and increased vascular permeability, as indicated by Evans blue dye extravasation. Intraperitoneal administration of 300μg/kg or topical administration of 1 μg/eye (±)5(6)-DiHETE inhibited histamine-induced manifestations. Finally, we assessed the effects of (±)5(6)-DiHETE on itching. An intradermal injection of 10μg serotonin in the cheek caused scratching behavior in mice. Intraperitoneal administration of 300μg/kg (±)5(6)-DiHETE significantly inhibited serotonin-induced scratching. Conclusion: Thus, (±)5(6)-DiHETE treatment broadly suppressed AC pathology and could be a novel treatment option for AC.

Oxytocin increases itch sensitivity of mice in late pregnancy and its peripheral receptor mechanism of itch-promoting effect

Oxytocin (OXT) is secreted in a large amount during the middle and late pregnancy. Except for the regulation of functions related to childbirth, OXT is involved in the regulation of cognition, social behavior, addiction, pain and so on. Our aim is to confirm the increase of OXT content in mice in late pregnancy is the main cause of itch during pregnancy and observe whether exogenously administered OXT can induce or increase itch sensitivity. The research shows that itch sensitivity of mice increased significantly in late pregnancy and basically returned to normal one day after delivery. The number of OXT-positive neurons in paraventricular nucleus (PVN) and the content of OXT in serum of the late pregnant mice increased significantly, and decreased sharply after delivery. Intradermal injection of low concentration of OXT (0.2 nmol/L) could not induce scratching behavior in mice, but high concentration of OXT (5 nmol/L, 10 nmol/L) could do this in a dose-dependent manner. Low concentration of OXT significantly increased the itch sensitivity to histamine. Intradermal injection of oxytocin receptor (OXTR) or arginine vasopressin-1a receptor (AVPR1A) antagonist did not affect histamine-induced scratching behavior, but both reversed the increase of itch sensitivity in late pregnant mice or the facilitated itch sensitivity by OXT. Study suggests that both endogenous and exogenous increases in OXT can increase the body's sensitivity to itch, and even induce itch directly. Pruritus during pregnancy is closely related to the increase of OXT content in vivo. In the periphery, the itch-promoting effect of OXT is mediated by OXTR and AVPR1A.

FSLLRY-NH2, a protease-activated receptor 2 (PAR2) antagonist, activates mas-related G protein-coupled receptor C11 (MrgprC11) to induce scratching behaviors in mice

AimsProtease-activated receptor 2 (PAR2), a type of G protein-coupled receptor (GPCR), plays a significant role in pathophysiological conditions such as inflammation. A synthetic peptide SLIGRL-NH2 (SLIGRL) can activate PAR2, while FSLLRY-NH2 (FSLLRY) is an antagonist. A previous study showed that SLIGRL activates both PAR2 and mas-related G protein-coupled receptor C11 (MrgprC11), a different type of GPCR expressed in sensory neurons. However, the impact of FSLLRY on MrgprC11 and its human ortholog MRGPRX1 was not verified. Hence, the present study aims to verify the effect of FSLLRY on MrgprC11 and MRGPRX1. MethodsThe calcium imaging technique was applied to determine the effect of FSLLRY in HEK293T cells expressing MrgprC11/MRGPRX1 or dorsal root ganglia (DRG) neurons. Scratching behavior was also investigated in wild-type and PAR2 knockout mice after injecting FSLLRY. Key findingsIt was surprisingly discovered that FSLLRY specifically activates MrgprC11 in a dose-dependent manner, but not other MRGPR subtypes. Furthermore, FSLLRY also moderately activated MRGPRX1. FSLLRY stimulates downstream pathways including Gαq/11, phospholipase C, IP3 receptor, and TRPC ion channels to evoke an increase in the intracellular calcium levels. The molecular docking analysis predicted that FSLLRY interacts with the orthosteric binding pocket of MrgprC11 and MRGPRX1. Finally, FSLLRY activated primary cultures of mouse sensory neurons, and induced scratching behaviors in mice. SignificanceThe present study has revealed that FSLLRY is capable of triggering itch sensation through activation of MrgprC11. This finding highlights the importance of considering the unexpected activation of MRGPRs in future therapeutic approaches aimed at the inhibition of PAR2.

Allantoin induces pruritus by activating MrgprD in chronic kidney disease.

Chronic kidney disease (CKD) is a disease with decreased, irreversible renal function. Pruritus is the most common skin symptom in patients with CKD, especially in end-stage renal disease. The underlying molecular and neural mechanism of CKD-associated pruritus (CKD-aP) remains obscure. Our data show that the level of allantoin increases in the serum of CKD-aP and CKD model mice. Allantoin could induce scratching behavior in mice and active DRG neurons. The calcium influx and action potential reduced significantly in DRG neurons of MrgprD KO or TRPV1 KO mice. U73122, an antagonist of phospholipase C, could also block calcium influx in DRG neurons induced by allantoin. Thus, our results concluded that allantoin plays an important role in CKD-aP, mediated by MrgprD and TrpV1, in CKD patients.

A new system to study mouse scratching behavior

A new system to study mouse scratching behavior

Rg3-enriched Korean red ginseng alleviates chloroquine-induced itch and dry skin pruritus in an MrgprA3-dependent manner in mice

BackgroundPrevious studies have found that Korean red ginseng extract (KRG) has antipruritic effects, which can be attributed to the presence of Rg3, one of the most potent ginsenosides. Therefore, Rg3-enriched KRG extract (Rg3EKRG) is anticipated to have enhanced antipruritic effects. The present study was conducted to examine the effects of Rg3EKRG in acute chloroquine (CQ)-induced and chronic dry skin pruritus. MethodsCalcium imaging technique was used in HE293T cells expressing MrgprA3 and TRPA1 (“MrgprA3/TRPA1”) and in primary cultures of mouse dorsal root ganglia (DRG) neurons. Mouse scratching behavior tests were performed on dry skin models. To verify the altered expression of itch-related genes, real-time RNA sequencing analysis and PCR were performed on DRG sections obtained from dry skin models. ResultsRg3EKRG suppressed CQ-induced intracellular calcium changes to a greater degree than KRG. Rg3EKRG dose-dependently inhibited CQ-induced responses in MrgprA3/TRPA1 cells. Rg3EKRG likely targeted MrgprA3 rather than TRPA1 to exert its inhibitory effect. Further, Rg3EKRG strongly inhibited the scratching behavior in mice induced by acute CQ injection. Importantly, DRG neurons obtained from dry skin mice models showed increased mRNA levels of MrgprA3, and treatment with Rg3EKRG alleviated chronic dry skin conditions and suppressed spontaneous scratching behaviors. ConclusionThe results of the present study imply that Rg3EKRG has a stronger antipruritic effect than KRG, inhibiting both acute CQ-induced and chronic dry skin pruritus in an MrgprA3-dependent manner. Therefore, Rg3EKRG is a potential antipruritic agent that can suppress acute and chronic itching at the peripheral sensory neuronal level.

An Anterior Cingulate Cortex-to-Midbrain Projection Controls Chronic Itch in Mice

Itch is an unpleasant sensation that provokes the desire to scratch. While acute itch serves as a protective system to warn the body of external irritating agents, chronic itch is a debilitating but poorly-treated clinical disease leading to repetitive scratching and skin lesions. However, the neural mechanisms underlying the pathophysiology of chronic itch remain mysterious. Here, we identified a cell type-dependent role of the anterior cingulate cortex (ACC) in controlling chronic itch-related excessive scratching behaviors in mice. Moreover, we delineated a neural circuit originating from excitatory neurons of the ACC to the ventral tegmental area (VTA) that was critically involved in chronic itch. Furthermore, we demonstrate that the ACC→VTA circuit also selectively modulated histaminergic acute itch. Finally, the ACC neurons were shown to predominantly innervate the non-dopaminergic neurons of the VTA. Taken together, our findings uncover a cortex–midbrain circuit for chronic itch-evoked scratching behaviors and shed novel insights on therapeutic intervention.

Scratch-AID: a deep-learning based system for automatic detection of mouse scratching behavior with high accuracy.

Mice are the most commonly used model animals for itch research and for development of anti-itch drugs. Most labs manually quantify mouse scratching behavior to assess itch intensity. This process is labor-intensive and limits large-scale genetic or drug screenings. In this study, we developed a new system, Scratch-AID Automatic Itch Detection), which could automatically identify and quantify mouse scratching behavior with high accuracy. Our system included a custom-designed videotaping box to ensure high-quality and replicable mouse behavior recording and a convolutional recurrent neural network (CRNN) trained with frame-labeled mouse scratching behavior videos, induced by nape injection of chloroquine (CQ). The best trained network achieved 97.6% recall and 96.9% precision on previously unseen test videos. Remarkably, Scratch-AID could reliably identify scratching behavior in other major mouse itch models, including the acute cheek model, the histaminergic model, and a chronic itch model. Moreover, our system detected significant differences in scratching behavior between control and mice treated with an anti-itch drug. Taken together, we have established a novel deep learning-based system that is ready to replace manual quantification for mouse scratching behavior in different itch models and for drug screening.

Anti-pruritic effect of isothiocyanates: Potential involvement of toll-like receptor 3 signaling.

The innate immune system has an emerging role as a mediator of neuro-immune communication and a therapeutic target for itch. Toll-like receptor 3 (TLR3) plays an important role in itch, as shown in TLR3 knock-out mice. In this study, to evaluate effects of TLR3 inhibitors on histamine-independent itch, we used two kinds of isothiocyanate (ITC). Both phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) inhibited Poly I:C (PIC)-induced signaling in the RAW264.7 cell line. We then investigated the anti-pruritic effect of these compounds on PIC- and chloroquine (CQ)-induced scratching behavior. PEITC and SFN both suppressed PIC-evoked scratching behavior in mice, and PEITC also inhibited CQ-induced acute itch. Finally, we examined the oxazolone-induced chronic itch model in mice. Surprisingly, oral dosing of both compounds suppressed scratching behaviors that were observed in mice. Our findings demonstrate that TLR3 is a critical mediator in acute and chronic itch transduction in mice and may be a promising therapeutic target for pruritus in human skin disorders. It is noteworthy that SFN has potential for use as an antipruritic as it is a phytochemical that is used as a supplement.

Desensitization of TRPV1 Involved in the Antipruritic Effect of Osthole on Histamine-Induced Scratching Behavior in Mice.

Osthole has been isolated from the fruits of Cnidium monnieri (L.) Cusson, which has been used in Chinese traditional medicine to treat pruritic disorders for a long time. However, the antipruritic mechanism of osthole is not fully understood. In the present study, using calcium imaging, molecular docking, and animal scratching behavior, we analyzed the pharmacological effects of osthole on transient receptor potential vanilloid 1 (TRPV1). The results showed that osthole significantly induced calcium influx in a dose-dependent manner in dorsal root ganglion (DRG) neurons. Osthole-induced calcium influx was inhibited by AMG9810, an antagonist of TRPV1. Osthole and the TRPV1 agonist capsaicin-induced calcium influx were desensitized by pretreatment with osthole. Furthermore, molecular docking results showed that osthole could bind to TRPV1 with a hydrogen bond by anchoring to the amino acid residue ARG557 in the binding pocket of TRPV1. In addition, TRPV1 is a downstream ion channel for the histamine H1 and H4 receptors to transmit itch signals. Osthole attenuated scratching behavior induced by histamine, HTMT (histamine H1 receptor agonist), and VUF8430 (histamine H4 receptor agonist) in mice. These results suggest that osthole inhibition of histamine-dependent itch may be due to the activation and subsequent desensitization of TRPV1 in DRG neurons.

STAT3 maintains skin barrier integrity by modulating SPINK5 and KLK5 expression in keratinocytes.

Skin barrier dysfunction induces skin inflammation. Signal transducer and activator of transcription 3 (STAT3) is known to be involved in Th17-mediated immune responses and barrier integrity in the cornea and intestine; however, its role in the skin barrier remains largely unknown. In this study, we elucidated the potential role of STAT3 in the skin barrier and its effect on kallikrein-related peptidase 5 (KLK5) and serine protease inhibitor Kazal-type 5 (SPINK5) expression using a mouse model with keratinocyte-specific ablation of STAT3. Keratinocyte-specific loss of STAT3 induced a cutaneous inflammatory phenotype with pruritus and intense scratching behaviour in mice. Transcriptomic analysis revealed that the genes associated with impaired skin barrier function, including KLK5, were upregulated. The effect of STAT3 on KLK5 expression in keratinocytes was not only substantiated by the increase in KLK5 expression following treatment with STAT3siRNA but also by its decreased expression following STAT3 overexpression. Overexpression and IL-17A-mediated stimulation of STAT3 increased the expression of SPINK5, which was blocked by STAT3siRNA. These results suggest that the expression of SPINK5 and KLK5 in keratinocytes could be dependent on STAT3 and that STAT3might play an essential role in the maintenance of skin barrier homeostasis.

Riboflavin Inhibits Histamine-Dependent Itch by Modulating Transient Receptor Potential Vanilloid 1 (TRPV1).

Riboflavin, also known as vitamin B2, isfound in foods and is used as a dietary supplement. Its deficiency (also called ariboflavinosis) results in some skin lesions and inflammations, such as stomatitis, cheilosis, oily scaly skin rashes, and itchy, watery eyes. Various therapeutic effects of riboflavin, such as anticancer, antioxidant, anti-inflammatory, and anti-nociceptive effects, are well known. Although some studies have identified the clinical effect of riboflavin on skin problems, including itch and inflammation, its underlying mechanism of action remains unknown. In this study, we investigated the molecular mechanism of the effects of riboflavin on histamine-dependent itch based on behavioral tests and electrophysiological experiments. Riboflavin significantly reduced histamine-induced scratching behaviors in mice and histamine-induced discharges in single-nerve fiber recordings, while it did not alter motor function in the rotarod test. In cultured dorsal root ganglion (DRG) neurons, riboflavin showed a dose-dependent inhibitory effect on the histamine- and capsaicin-induced inward current. Further tests wereconducted to determine whether two endogenous metabolites of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), have similar effects to those of riboflavin. Here, FMN, but not FAD, significantly inhibited capsaicin-induced currents and itching responses caused by histamine. In addition, in transient receptor potential vanilloid 1 (TRPV1)-transfected HEK293 cells, both riboflavin and FMN blocked capsaicin-induced currents, whereas FAD did not. These results revealed that riboflavin inhibits histamine-dependent itch by modulating TRPV1 activity. This study will be helpful in understanding how riboflavin exerts antipruritic effects and suggests that it might be a useful drug for the treatment of histamine-dependent itch.

Comparative Study on Different Skin Pruritus Mouse Models

The animal model is an important tool to study the mechanism of disease formation. Different animal models of pruritus have been adopted based on the purpose of researchers in the study of the itching mechanism. Although the symptoms of various models are quite different, scratching behavior is a key indicator. Therefore, it is necessary to find an animal model that can quickly induce animal scratching and maintain the stability of scratching behavior. In this study, we compared animal models of pruritus induced by four substances and found that the scratching behavior of mice induced by urushiol not only reached the plateau stage quickly but also showed more stability in the plateau phase than that induced by 2,4-dinitrofluorobenzene, oxazolone, and imiquimod. Meanwhile, in the animal model induced by urushiol, the changes of epidermal thickening and inflammatory cell aggregation were also more obvious. In addition, pruritus induced by urushiol is prevalent all over the world, especially in the United States and Europe, involving outdoor groups such as firefighters, forest loggers, and farmers. Therefore, we believe that the urushiol-induced animal model is an ideal choice for the study of the itch formation mechanism and the development of antipruritic drugs.

A group of cationic amphiphilic drugs activates MRGPRX2 and induces scratching behavior in mice

Mas gene-related G protein-coupled receptors (MRGPRs) are a G protein-coupled receptor family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells mediating IgE-independent signaling and pseudoallergic drug reactions. Our aim was to increase knowledge about the function and regulation of MRGPRX2/MRGPRB2, which is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus. To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds by utilizing a high-throughput calcium mobilization assay. The identified hit compounds were analyzed for their pseudoallergic and pruritogenic effects in mice and human. We found a class of commonly used drugs activating MRGPRX2 that, to a large extent, consists of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated by using the 3 representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we were able to show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2 (Laboratory of Allergic Diseases-2). Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior following intradermal injection into C57BL/6 mice but less so in MRGPRB2-mutant mice, as well as wheal-and-flare reactions following intradermal injections in humans. Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions such as drug-induced pruritus to prevent severe drug hypersensitivity reactions.

Serotonin and noradrenaline modulate chronic itch processing in mice

The roles of serotonin and noradrenaline in the modulation of chronic pruriceptive processing currently remain unclear. To clarify the contribution of serotonin and noradrenaline to chronic itch, the effects of the administration of antidepressants or noradrenaline reuptake inhibitors were evaluated in the present study. A pretreatment with milnacipran, a serotonin and noradrenaline reuptake inhibitor, and mirtazapine, a noradrenergic and specific serotonergic antidepressant, attenuated the induction of spontaneous scratching behavior in mice with chronic itch. The administration of a serotonin reuptake inhibitor, such as fluvoxamine and paroxetine, but not escitalopram, or a noradrenaline reuptake inhibitor, such as atomoxetine and nisoxetine, ameliorated the induction of spontaneous scratching behavior in mice with chronic itch. Furthermore, this attenuation was reversed by the administration of yohimbine, a selective α2-adrenoceptor antagonist, or methysergide, a non-selective serotonin receptor antagonist. These results suggest that elevated serotonin and noradrenaline levels are involved in the attenuation of scratching behavior induced by chronic itch, and serotonin receptors and an α2-adrenoceptor play a crucial role in chronic pruriceptive processing.