S1P/S1PRs-TRPV4 axis is a novel therapeutic target for persistent pain and itch in chronic dermatitis

inyu Zhang1,2 | Yuan Zhou2 | ChangmingWang2 | JiahuiRen2 | YinWang2 |Pei Liu1 | WeimengFeng1 | XueLi2 | MingxinQi2 | YanYang2 |Chan Zhu2 | FangWang3 | YuxiangMa4 | ZongxiangTang2 | GuangYu1,2

1Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing
University of Chinese Medicine, Nanjing, China
2Key Laboratory for Chinese Medicine of Prevention and Treatment in Neurological Diseases, School of Medicine, Nanjing University of Chinese Medicine, Nanjing, China
3Department of Dermatology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
4School of Life Science, China Pharmaceutical University, Nanjing, China

Abstract
Background and Purpose: While pain and itch are both commonly associated with chronic dermatitis (CD), the molecular mechanisms underlying these debilitating symptoms is not well understood. This study aims to identify novel, endogenous compounds that mediate CD-associated pain and itch.

Experimental Approach: Lesional skin of CD model mice was examined using unbiased metabolomic analysis to identify candidate pain or itch inducing compounds in CD. Sphingosine-1-phosphate (S1P) concentration in CD model skin was analysed using UPLC/MS/MS. Behaviour, calcium imaging and immunofluorescence staining were used to determine the pain and itch effects and mechanisms of the identified CD-related compounds.

Key Results: In the lesional skin of CD model mice, 136 compounds were significantly changed. These compounds are predominately associated with the sphingolipids metabolism pathway. S1P is significantly increased in the lesional skin . The TRPV4 channel was critical for S1P induced itch and pain. Sphingosine kinase 2 (SPHK2), the key enzyme controlling S1P synthesis, was significantly increased in lesional skin. ABC294640, a SPHK2 inhibitor, significantly decreased S1P concentration in lesional CD model skin, as well as in model associated epidermal hyperplasia and chronic pain and itch. In CD patients, SPHK2 expression and S1P concentration were significantly elevated compared to healthy control skin.

Conclusion and Implications: Our results indicate that, in CD, increased S1P induces chronic pain and itch partly through TRPV4. Inhibition of S1P synthesis or the S1P/S1P receptor-TRPV4 pathway are promising treatment strategies for CD associated pain and itch.

KEYWORDS
chronic dermatitis, itch, pain, S1P, TRPV4

Neuronal substance P-driven MRGPRX2-dependent mast cell degranulation products differentially promote vascular permeability

 https://doi.org/10.3389/fimmu.2024.1477072

iPSC-derived human sensory neurons reveal a subset of TRPV1 antagonists as anti-pruritic compounds

Shermaine Huiping Tay, Jeremy Kah Sheng Pang, Winanto Ng, Chong Yi Ng, Zi Jian Khong, Zheng-Shan Chong, Boon Seng Soh & Shi-Yan Ng 

Scientific Reports volume 14, Article number: 31182 (2024) 

Cite this article

Abstract

Signaling interplay between the histamine 1 receptor (H1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in mediating histaminergic itch has been well-established in mammalian models, but whether this is conserved in humans remains to be confirmed due to the difficulties in obtaining human sensory neurons (SNs) for experimentation. Additionally, previously reported species-specific differences in TRPV1 function indicate that use of human SNs is vital for drug candidate screening to have a higher chance of identifying clinically effective TRPV1 antagonists. In this study, we built a histamine-dependent itch model using peripheral SNs derived from human induced pluripotent stem cells (hiPSC-SNs), which provides an accessible source of human SNs for pre-clinical drug screening. We validated channel functionality using immunostaining, calcium imaging, and multielectrode array (MEA) recordings, and confirmed the interdependence of H1R and TRPV1 signalling in human SNs. We further tested the amenability of our model for pre-clinical studies by screening multiple TRPV1 antagonists in parallel, identifying SB366791 as a potent inhibitor of H1R activation and potential candidate for alleviating histaminergic itch. Notably, some of the results using our model corroborated with efficacy and side effect findings from human clinical trials, underscoring the importance of this species-specific platform. Taken together, our results present a robust in vitro human model for histaminergic itch, which can be used to further interrogate the molecular basis of human SN function as well as screen for TRPV1 activity-modifying compounds for a number of clinical indications.

Phytother Res. 2023 Aug;37(8):3572-3582. doi: 10.1002/ptr.7835. Epub 2023 Apr 28.

Silibinin attenuated pseudo-allergic reactions and mast cell degranulation via PLCγ and PI3K/Akt signaling pathway

Yuejin Wang ¹, Shiting Hu ¹, Baowen Dang ¹, Yonghui Zhang ¹, Guodong Zheng ¹, Chenrui Zhao ¹, Yihan Huang ¹, Tao Zhang ¹

Affiliations expand

• PMID: 37115717
• DOI: 10.1002/ptr.7835

Abstract

Anaphylaxis is a type of potentially fatal hypersensitivity reaction resulting from the activation of mast cells. Many endogenous or exogenous factors could cause this reaction. Silibinin is the main chemical component of silymarin and has been reported to have pharmacological activities. However, the anti-allergic reaction effect of silibinin has not yet been investigated. This study aimed to evaluate the effect of silibinin to attenuate pseudo-allergic reactions in vivo and to investigate the underlying mechanism in vitro. In this study, calcium imaging was used to assess Ca²⁺ mobilization. The levels of cytokines and chemokines, released by stimulated mast cells, were measured using enzyme immunoassay kits. The activity of silibinin was evaluated in a mouse model of passive cutaneous anaphylaxis (PCA). Western blotting was used to explore the related molecular signaling pathways. In results, silibinin markedly inhibited mast cell degranulation, calcium mobilization, and preventing the release of cytokines and chemokines in a dose-dependent manner via the PLCγ and PI3K/Akt signaling pathway. Silibinin also attenuated PCA in a dose-dependent manner. In summary, silibinin has an anti-pseudo-allergic pharmacological activity, which makes it a potential candidate for the development of a novel agent to arrest pseudo-allergic reactions.

Keywords: PI3K/Akt; mast cell; pseudo-allergy; silibinin.

https://pubmed.ncbi.nlm.nih.gov/37115717/

The role of PAR2 in regulating MIF release in house dust mite-induced atopic dermatitis

Lingxuan Zhou¹Guohong Zhang¹Kai Zhang¹Ziyan Rao²Zhanli Tang³Yang Wang¹Jiahui Zhao^1,4*

• ¹Department of Dermatology, Peking University First Hospital, Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, National Clinical Research Center for Skin and Immune Disease, National Medical Products Administration (NMPA) Key Laboratory for Quality Control and Evaluation of Cosmetics, Beijing, China
• ²Department of Biomedical Informatics, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing, China
• ³Department of Dermatology, Qilu Hospital of Shandong University, Jinan, China
• ⁴Chinese Institute for Brain Research (CIBR), Beijing, China

Atopic dermatitis (AD) is a chronic disease characterized by relapsed eczema and intractable itch, and is often triggered by house dust mites (HDM). PAR2 is a G-protein coupled receptor on keratinocytes and may be activated by HDM to affect AD processes. We first established a HDM-derived AD mouse model in wild-type (WT) and Par2^-/-mice. Single cell RNA sequencing of the diseased skins found a stronger cellular communication between the ligand macrophage migration inhibitory factor (MIF) from keratinocytes and its receptors on antigen-presenting cells, suggesting the critical role of MIF in AD. HDM-WT mice showed severer skin lesions and pathological changes with stronger immunofluorescence MIF signals in skin sections than HDM-Par2^-/- mice. Primary keratinocytes from WT mice stimulated with HDM or SLIGRL (PAR2 agonist) secreted more MIF in cultured medium and induced stronger immunofluorescence MIF signals than those from Par2^-/- mice. The skin section of HDM-WT mice showed higher immunofluorescence signals of P115 (relating to MIF secretion) and KIF13B (possibly relating to intracellular trafficking of MIF) than that of HDM-Par2^-/- mice. Acetylation of α-tubulin increased after stimulation by SLIGRL in WT keratinocytes but not in Par2^-/- keratinocytes. HDM-WT mice treated with the MIF antagonist ISO-1 displayed improvement of AD-like presentations and lower expressions of IL-4, IL-13, TSLP and Arg1 (a biomarker of M2 macrophage) mRNAs. We conclude that MIF is an important cytokine and is significantly increased in the AD model. PAR2 affects AD changes by regulating the expression, intracellular trafficking, and secretion of MIF in epidermis.

Inhibitory effect of daphnetin on the C48/80-induced pseudo-allergic reaction

Jingyu Zhang ^a, Ling Hong ^b, Ping Zhang ^a, Yanjie Wang ^a, Tie Hong ^a

https://doi.org/10.1016/j.intimp.2023.110874Get rights and content

Highlights

• Daphnetin inhibits inflammatory mediator release by C48/80-activated RBL-2H3 cells.
• Daphnetin inhibits pseudo-allergic reactions induced by C48/80 in mouse models.
• Daphnetin inhibits C48/80 induced mast cell activation by inhibiting phosphorylation of PLCγ, IP3R, PKC, ERK1/2 and P38.

Abstract

Pseudo-allergic reaction is an allergic reaction mediated by nonimmunoglobulin E (IgE), which does not require prior contact with antigen sensitization and directly leads to mast cell degranulation. Daphnetin (DAP) is known for its anti-inflammatory effects, but there are few studies on the effect of DAP on pseudo-allergy and its mechanism. To investigate the effect of DAP on pseudo-allergy and its mechanism, we inflicted pseudo-allergy on RBL-2H3 cells using C48/80 in vitro. Moreover, to assess the antipseudo-allergy effect of C48/80 in vivo, mouse models of local anaphylaxis, systemic anaphylaxis, and itch were used. The in vitro results show that DAP inhibits degranulation and chemokine release; furthermore, DAP reduced the activation of the PLC-IP3R and MAPK signaling pathways induced by C48/80. Additionally, our in vivo results showed that DAP inhibited C48/80-induced local anaphylaxis and inhibited eosinophil aggregation, vasodilation and mast cell degranulation. In systemic anaphylaxis, DAP inhibits the decrease in body temperature and reduces the release of His, TNF-a and IL-8. In C48/80-induced itch, the number of scratches in mice was reduced. Our results demonstrate that DAP can play a suppressive role in the pseudo-allergy induced by C48/80, providing information for the cure of disorders linked to pseudo-allergic reactions.

Keywords: Pseudo-allergic reaction, Daphnetin,C48/80,RBL-2H3 cells,PLC/IP3R

Cell. 2024 Oct 24:S0092-8674(24)01149-8.

 doi: 10.1016/j.cell.2024.10.001. Online ahead of print.

Structure-guided discovery of bile acid derivatives for treating liver diseases without causing itch

Jun Yang ¹, Tianjun Zhao ², Junping Fan ³, Huaibin Zou ⁴, Guangyi Lan ⁵, Fusheng Guo ¹, Yaocheng Shi ³, Han Ke ³, Huasheng Yu ⁶, Zongwei Yue ¹, Xin Wang ⁷, Yingjie Bai ⁷, Shuai Li ⁸, Yingjun Liu ⁸, Xiaoming Wang ⁸, Yu Chen ⁹, Yulong Li ¹⁰, Xiaoguang Lei ¹¹

• PMID: 39476841
• DOI: 10.1016/j.cell.2024.10.001

Abstract

Chronic itch is a debilitating symptom profoundly impacting the quality of life in patients with liver diseases like cholestasis. Activation of the human G-protein coupled receptor, MRGPRX4 (hX4), by bile acids (BAs) is implicated in promoting cholestasis itch. However, the detailed underlying mechanisms remain elusive. Here, we identified 3-sulfated BAs that are elevated in cholestatic patients with itch symptoms. We solved the cryo-EM structure of hX4-Gq in a complex with 3-phosphated deoxycholic acid (DCA-3P), a mimic of the endogenous 3-sulfated deoxycholic acid (DCA-3S). This structure revealed an unprecedented ligand-binding pocket in MRGPR family proteins, highlighting the crucial role of the 3-hydroxyl (3-OH) group on BAs in activating hX4. Guided by this structural information, we designed and developed compound 7 (C7), a BA derivative lacking the 3-OH. Notably, C7 effectively alleviates hepatic injury and fibrosis in liver disease models while significantly mitigating the itch side effects.

Keywords: 3-sulfonated bile acids; MRGPRX4; OCA; bile acids; cholestatic pruritus; cryo-EM structure; deoxycholic acid; farnesoid X receptor; liver diseases; non-alcoholic steatohepatitis.

Acta Pharmacol Sin 2018 Mar;39(3):331-335.

 doi: 10.1038/aps.2017.152. Epub 2017 Nov 2.

A pivotal role for the activation of TRPV3 channel in itch sensations induced by the natural skin sensitizer carvacrol

Ting-Ting Cui ¹, Gong-Xin Wang ¹, Ning-Ning Wei ¹, KeWei Wang ¹

• PMID: 29094727
• PMCID: PMC5843833
• DOI: 10.1038/aps.2017.152

Abstract

Itching is an intricate, common symptom of dermatologic and systemic diseases, and both TRPV3 and TRPA1 channels have been suggested to function as downstream effector targets. But the relative contributions of TRPV3 and TRPA1 to itch sensation in vivo remain unclear. To dissect the role of TRPA1 or TRPV3 in the cutaneous sensation of itching, we took the advantage of a natural compound carvacrol from oregano, and examined its effect on the induction of scratching behavior in mice. We showed that the intradermal injection of carvacrol (0.01%, 0.1% and 1%, 50 μL) induced scratching in a concentration-dependent manner. But in TRPV3-knockout mice, the scratching induced by carvacrol (1%, 50 μL) was markedly decreased by approximately 64% (from 275 scratching bouts down to 90) within 60 min. Further analysis revealed that TRPV3-knockout caused a reduction of scratching bouts for approximately 40% in the first 20 min (the initial phase), whereas the scratching bouts were reduced by approximately 90% in the last 40 min (the sustained phase). These results were in consistence with those in our whole-cell recordings in HEK-293T cells expressing either TRPA1 or TRPV3: carvacrol exhibited similar potencies in activating either TRPA1 or TRPV3, but carvacrol-activated TRPA1 current showed a rapid desensitization, which was reduced by approximately 90% within 5 min before a complete washout, whereas carvacrol-induced TRPV3 current showed a slow desensitization that caused less than 30% of current reduction in 10 min and left a significant residual TRPV3 current after washout. Our results demonstrate that carvacrol from plant oregano is a skin sensitizer or allergen; TRPV3 is involved in the initial phase and the sustained phase of pruritus, whereas TRPA1 likely contributes to the initial phase.

J Ethnopharmacol 2025 Jan 30;337(Pt 2):118882.

 doi: 10.1016/j.jep.2024.118882. Epub 2024 Oct 2.

Investigation of the anti-inflammatory, anti-pruritic, and analgesic effects of sophocarpine inhibiting TRP channels in a mouse model of inflammatory itch and pain

Hekun Zeng ¹, Zhe Zhang ², Dan Zhou ³, Ranjing Wang ⁴, Alexei Verkhratsky ⁵, Hong Nie ⁶

• PMID: 39366497
• DOI: 10.1016/j.jep.2024.118882

Abstract

Ethnopharmacological relevance: Sophocarpine is a bioactive compound extracted from the dried root of Sophorae Flavesentis Aiton, a plant that has been used for thousands of years for various conditions including skin itch and pain. Its antipruritic and analgesic effects are suggested in publications, while the molecular mechanisms underneath interacting with TRP channels are not understood.

Aim of the study: We investigated the anti-inflammatory, antipruritic, and analgesic effects of sophocarpine in a murine inflammatory itch and pain model to elucidate the underlying mechanisms.

Materials and methods: We evaluated sophocarpine’s anti-pruritic and analgesic effects by monitoring mice’s scratching and wiping behaviors, and the anti-inflammatory effect by measuring psoriasis area and severity index (PASI) score. The mRNA and protein expression of TRPA1/TRPV1 was analyzed by real-time quantitative polymerase chain reaction and western blotting. We further investigated the relationship between sophocarpine and TRPA1/TRPV1 in mice administered allyl-isothiocyanate (AITC) or capsaicin and by molecular docking.

Results: We found that sophocarpine decreased scratching bouts, wipes, and the PASI score, reduced the TNF-α and IL-1β in the skin and TRPA1 and TRPV1 in the trigeminal ganglion. Pretreatment of sophocarpine decreased AITC-induced scratching bouts and wipes and capsaicin-induced wipes. We also found potential competitive bindings between sophocarpine and AITC/capsaicin to TRPA1/TRPV1.

Conclusions: Sophocarpine is a potential competitive inhibitor of TRPA1 and TRPV1 channels eliciting strong anti-inflammatory, anti-pruritic, and analgesic effects, suggesting its significant therapeutic potential in treating diseases with inflammatory itch and pain.

Keywords: Analgesic; Anti-inflammatory; Anti-pruritic; Sophocarpine; TRPA1; TRPV1.

Sci Adv 2024 Sep 27;10(39):eadp6038. doi: 10.1126/sciadv.adp6038. Epub 2024 Sep 25.

Kappa opioids inhibit spinal output neurons to suppress itch

Tayler D Sheahan ¹, Charles A Warwick ¹, Abby Y Cui ¹, David A A Baranger ², Vijay J Perry ¹, Kelly M Smith ¹, Allison P Manalo ¹, Eileen K Nguyen ¹, H Richard Koerber ¹, Sarah E Ross ^1 3

• PMID: 39321286
• PMCID: PMC11423883
• DOI: 10.1126/sciadv.adp6038

Abstract

Itch is a protective sensation that drives scratching. Although specific cell types have been proposed to underlie itch, the neural basis for itch remains unclear. Here, we used two-photon Ca²⁺ imaging of the dorsal horn to visualize neuronal populations that are activated by itch-inducing agents. We identify a convergent population of spinal interneurons recruited by diverse itch-causing stimuli that represents a subset of neurons that express the gastrin-releasing peptide receptor (GRPR). Moreover, we find that itch is conveyed to the brain via GRPR-expressing spinal output neurons that target the lateral parabrachial nuclei. We then show that the kappa opioid receptor agonist nalfurafine relieves itch by selectively inhibiting GRPR spinoparabrachial neurons. These experiments provide a population-level view of the spinal neurons that respond to pruritic stimuli, pinpoint the output neurons that convey itch to the brain, and identify the cellular target of kappa opioid receptor agonists for the inhibition of itch.