3’-O-Methylorobol Inhibits the Voltage-Gated Sodium Channel Nav1.7 with Anti-Itch Efficacy in A Histamine-Dependent Itch Mouse Model

Fan Zhang †, Ying Wu †, Shuwen Xue, Shuangyan Wang, Chunlei Zhang * and Zhengyu Cao *

State Key Laboratory of Natural Medicines and Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Development, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China; zhangfan20111112@126.com (F.Z.); wingwycpu@126.com (Y.W.); xsw19850856166@163.com (S.X.); shuangyanwcpu@163.com (S.W.)

* Correspondence: zhangchunlei11@sina.com (C.Z.); zycao1999@hotmail.com (Z.C.); Tel.: +86-25-8618-5955 (C.Z.); +86-25-8618-5955 (Z.C.)

† These authors contributed equally to this work.
Received: 15 November 2019; Accepted: 29 November 2019; Published: 1 December 2019

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Abstract: An itch is a clinical complication that affects millions of patients. However, few treatment options are available. The voltage-gated sodium channel Nav1.7 is predominantly expressed in peripheral sensory neurons and is responsible for the rising phase of action potentials, thereby mediating nociceptive conduction. A gain-of-function mutation of Nav1.7 results in the hyperexcitability of sensory neurons and causes the inherited paroxysmal itch. Conversely, a monoclonal antibody that selectively inhibits Nav1.7 is able to effectively suppress the histamine-dependent itch in mice. Therefore, Nav1.7 inhibitors may possess the potential to relieve the itch. In the present study, using whole-cell voltage-clamp recordings, we demonstrated that 3’-O-methylorobol inhibited Na+ currents in Nav1.7-CHO cells and tetrodotoxin-sensitive Na+ currents in mouse dorsal root ganglion (DRG) neurons with IC50 (half-maximal inhibitory concentration) values of 3.46 and 6.60 μM, respectively. 3’-O-methylorobol also suppressed the tetrodotoxin-resistant Na+ currents in DRG neurons, though with reduced potency (~43% inhibition at 30 μM). 3’-O-methylorobol (10 μM) affected the Nav1.7 by shifting the half-maximal voltage (V1/2) of activation to a depolarizing direction by ~6.76 mV, and it shifted the V1/2 of inactivation to a hyperpolarizing direction by ~16.79 mV. An analysis of 3’-O-methylorobol activity toward an array of itch targets revealed that 3’-O-methylorobol was without effect on histamine H1 receptor, TRPV1, TRPV3, TRPV4, TRPC4 and TRPM8. The intrathecal administration of 3’-O-methylorobol significantly attenuated compound 48/80-induced histamine-dependent spontaneous scratching bouts and the expression level of c-fos in the nuclei of spinal dorsal horn neurons with a comparable efficacy to that of cyproheptadine. Our data illustrated the therapeutic potential for 3’-O-methylorobol for histamine-dependent itching, and the small molecule inhibition of Nav1.7 may represent a useful strategy to develop novel therapeutics for itching.

Keywords: Nav1.7; 3’-O-methylorobol; gating modifier; itch relief; lead compound

Qingpeng Ointment Ameliorates Inflammatory Responses and Dysregulation of Itch-Related Molecules for Its Antipruritic Effects in Experimental Allergic Contact Dermatitis

Xuan Gong1†, Hui Xiong1†, Sisi Liu1, Yutong Liu2, Liang Yin1, Chuyue Tu1, Hua Wang2, Zhongqiu Zhao3,4, Weiwu Chen5 and Zhinan Mei1*

1 School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, China, 2 College of Life Science, South-Central University for Nationalities, Wuhan, China, 3 Center for the Study of Itch, Department of Anesthesiology, Washington University School of Medicine in St. Louis, St. Louis, MO, United States, 4 Barnes-Jewish Hospital, St. Louis, MO, United States, 5 Qizheng Tibetan Medicine Co., Ltd., Lanzhou, China

The pathogenesis of itchy skin diseases including allergic contact dermatitis (ACD) is complicated and the treatment of chronic itch is a worldwide problem. One traditional Tibetan medicine, Qingpeng ointment (QP), has been used in treatment of ACD in China for years. In this study we used HPLC and LC/MS analysis, combined with a BATMAN-TCM platform, for detailed HPLC fingerprint analysis and network pharmacology of QP, and investigated the anti-inflammatory and antipruritic activities of QP on ACD induced by squaric acid dibutylester (SADBE) in mice. The BATMAN-TCM analysis provided information of effector molecules of the main ingredients of QP, and possible chronic dermatitis-associated molecules and cell signaling pathways by QP. In ACD mice, QP treatment suppressed the scratching behavior induced by SADBE in a dose-dependent manner and inhibited the production of Th1/2 cytokines in serum and spleen. Also, QP treatment reversed the upregulation of mRNAs levels of itch-related genes in the skin (TRPV4, TSLP, GRP, and MrgprA3) and DRGs (TRPV1, TRPA1, GRP, and MrgprA3). Furthermore, QP suppressed the phosphorylation of Erk and p38 in the skin. In all, our work indicated that QP can significantly attenuate the pathological alterations of Th1/2 cytokines and itch-related mediators, and inhibit the phosphorylation of MAPKs to treat the chronic itch.

Role of SNAREs in Atopic Dermatitis–Related Cytokine Secretion and Skin-Nerve Communication

JianghuiMeng¹²JiafuWang¹JoergBuddenkotte³⁴TimoBuhl⁵MartinSteinhoff²³⁴⁶

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptors in atopic dermatitis (AD) is unknown. This study identifies the function of soluble N-ethylmaleimide sensitive factor attachment protein receptor in AD-related cytokine secretion and epidermis-nerve communication. Herein, we report that various cytokines were simultaneously upregulated and coreleased in innate immunity–activated primary human keratinocytes. AD-related cytokines thymic stromal lymphopoietin, endothelin-1, and inflammatory tumor necrosis factor-α activated distinct but overlapping sensory neurons. Tumor necrosis factor-α potentiated thymic stromal lymphopoietin–induced Ca²⁺-influx, whereas endothelin-1 caused itch-selective B-type natriuretic peptide release. In primary human keratinocytes, B-type natriuretic peptide upregulated genes promoting dermatological and neuroinflammatory diseases and conditions. VAMP3, SNAP-29, and syntaxin 4 proved important in driving cytokine release from primary human keratinocytes. Depletion of VAMP3 inhibited nearly all the cytokine release including thymic stromal lymphopoietin and endothelin-1. Accordingly, VAMP3 co-occurred with endothelin-1 in the skins of patients with AD. Our study pinpoints the pivotal role of soluble N-ethylmaleimide sensitive factor attachment protein receptors in mediating cytokine secretion related to AD. VAMP3 is identified as a suitable target for developing broad-spectrum anticytokine therapeutics for controlling itch and atopic skin inflammation.

TLR3 in Chronic Human Itch: A Keratinocyte-Associated Mechanism of Peripheral Itch Sensitization

Short title: Skin TLR3 and chronic human itch.

Keywords: pruritus, toll-like receptor, endothelin, nerve, keratinocyte

The TRPV4 Agonist GSK1016790A Regulates the Membrane Expression of TRPV4 Channels

The TRPV4 Agonist GSK1016790A Regulates the Membrane Expression of TRPV4 Channels

Sara Baratchi1*, Peter Keov1,2,3, William G. Darby1, Austin Lai1, Khashayar Khoshmanesh4, Peter Thurgood4, Parisa Vahidi1, Karin Ejendal5 and Peter McIntyre1
1 School of Health and Biomedical Sciences, RMIT University, Melbourne, VIC, Australia, 2 Molecular Pharmacology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia, 3 St Vincent’s Clinical School, University of New South Wales, Darlinghurst, NSW, Australia, 4 School of Engineering, RMIT University, Melbourne, VIC, Australia, 5 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, United States

TRPV4 is a non-selective cation channel that tunes the function of different tissues including the vascular endothelium, lung, chondrocytes, and neurons. GSK1016790A is the selective and potent agonist of TRPV4 and a pharmacological tool that is used to study the TRPV4 physiological function in vitro and in vivo. It remains unknown how the sensitivity of TRPV4 to this agonist is regulated. The spatial and temporal dynamics of receptors are the major determinants of cellular responses to stimuli. Membrane translocation has been shown to control the response of several members of the transient receptor potential (TRP) family of ion channels to different stimuli. Here, we show that TRPV4 stimulation with GSK1016790A caused an increase in [Ca2+]i that is stable for a few minutes. Single molecule analysis of TRPV4 channels showed that the density of TRPV4 at the plasma membrane is controlled through two modes of membrane trafficking, complete, and partial vesicular fusion. Further, we show that the density of TRPV4 at the plasma membrane decreased within 20min, as they translocate to the recycling endosomes and that the surface density is dependent on the release of calcium from the intracellular stores and is controlled via a PI3K, PKC, and RhoA signaling pathway.
Keywords: TRPV4, membrane trafficking, endothelial cells, GSK1016790A, calcium

Facilitation of MrgprD by TRP-A1 promotes neuropathic pain

Facilitation of MrgprD by TRP-A1 promotes neuropathic pain

Changming Wang,*,†,‡,§ Leying Gu,*,†,‡,§ Yonglan Ruan,*,†,‡,§ Xiao Geng,*,†,‡,§ Miao Xu,{ Niuniu Yang,*,k Lei Yu,*,† Yucui Jiang,*,† Chan Zhu,*,†,‡,§ Yan Yang,*,†,‡,§
Yuan Zhou,*,†,‡,§ Xiaowei Guan,*,†,‡,§ Wenqin Luo,# Qin Liu,**,††,‡‡ Xinzhong Dong,§§,{{ Guang Yu,*,†,‡,§,1 Lei Lan,{,2 and Zongxiang Tang*,†,‡,§
*School of Medicine and Life Sciences, †Key Laboratory of Chinese Medicine for Prevention and Treatment of Neurological Diseases, ‡State Key Laboratory Cultivation Base for Traditional Chinese Medicine Quality and Efficacy, and §Key Laboratory of Drug Target and Drug for Degenerative Disease of Jiangsu Province, Nanjing University of Chinese Medicine, Nanjing, China; {Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China; kDepartment of Traditional Chinese and Western Medicine, Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou University, Yangzhou, China; #Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA; **Department of Anesthesiology, ††Center for the Study of Itch, and ‡‡Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, USA; and §§The Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, and {{Howard Hughes Medical Institute, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA
ABSTRACT: Neuropathic pain remains a therapeutic challenge because of its complicated mechanisms. Mas-related GPCR D (MrgprD) is specifically expressed in small-diameter, nociceptive neurons of dorsal root ganglia (DRGs) and is implicated in pain modulation. However, the underlying mechanism of MrgprD involved in neuropathic pain remains elusive. In this study, we used behavioral experiments and physiologic examination methods to investigate the role of MrgprD in chronic constriction injury (CCI)–induced neuropathic pain. We found that MrgprD is necessary for the initiation of mechanical hypersensitivity and cold allodynia, but not for heat allodynia. Moreover, we demonstrated that transient receptor potential cation channel (TRP)-A1 was the ion channel down- stream of MrgprD, and the b-alanine–induced calcium signal was attributed mostly to TRP-A1 function. We further showed that PKA serves as a downstream mediator of b-alanine–activated MrgprD signaling to activate TRP-A1 in DRG neurons and in human embryonic kidney 293 cells, to coexpress MrgprD and TRP-A1 plasmids. Finally, we found that the b-alanine–induced pain behavior was increased, whereas the itching behavior was unchanged in CCI models compared with sham-injured animals. Knockout of TRPA1 also attenuated the b-alanine–induced pain behavior in CCI models. In conclusion, MrgprD is essential in cold allodynia in CCI-induced neuropathic pain through the PKA–TRP-A1 pathway. TRP-A1 facilitates MrgprD to development of neuropathic pain. Our findings reveal a novel mechanism of neuropathic pain formation and highlight MrgprD as a promising drug target for the treatment of neuropathic pain.—Wang, C., Gu, L., Ruan, Y., Geng, X., Xu, M., Yang, N., Yu, L., Jiang, Y., Zhu, C., Yang, Y., Zhou, Y., Guan, X., Luo, W., Liu, Q., Dong, X., Yu, G., Lan, L., Tang, Z. Facilitation of MrgprD by TRP-A1 promotes neuropathic pain. FASEB J. 33, 1360–1373 (2019). www.fasebj.org
KEY WORDS: MrgprA1 • dorsal root ganglia (DRG) • protein kinase A (PKA)

Anatomical and functional dichotomy of ocular itch and pain

Anatomical and functional dichotomy of ocular itch and pain

Cheng-Chiu Huang1,7,9, Weishan Yang1,9, Changxiong Guo1, Haowu Jiang1, Fengxian Li 1,2, Maolei Xiao1, Steve Davidson3, Guang Yu 4, Bo Duan5,8, Tianwen Huang5, Andrew J. W. Huang 6 and Qin Liu1,6*

Itch and pain are refractory symptoms of many ocular conditions. Ocular itch is generated mainly in the conjunctiva and is absent from the cornea. In contrast, most ocular pain arises from the cornea. However, the underlying mechanisms remain unknown. Using genetic axonal tracing approaches, we discover distinct sensory innervation patterns between the conjunctiva and cornea. Further genetic and functional analyses in rodent models show that a subset of conjunctival-selective sensory fibers marked by MrgprA3 expression, rather than corneal sensory fibers, mediates ocular itch. Importantly, the actions of both histamine and nonhistamine pruritogens converge onto this unique subset of conjunctiva sensory fibers and enable them to play a key role in mediating itch associated with allergic conjunctivitis. This is distinct from skin itch, in which discrete popu- lations of sensory neurons cooperate to carry itch. Finally, we provide proof of concept that selective silencing of conjunctiva itch-sensing fibers by pruritogen-mediated entry of sodium channel blocker QX-314 is a feasible therapeutic strategy to treat ocular itch in mice. Itch-sensing fibers also innervate the human conjunctiva and allow pharmacological silencing using QX-314. Our results cast new light on the neural mechanisms of ocular itch and open a new avenue for developing therapeutic strategies.

In this work, we establish a role for the bioactive lipid sphingosine 1-phosphate (S1P) and its
receptor S1PR3 in inflammatory thermal pain and itch. S1P injection elicited robust heat hypersensitivity, acute pain, and itch behaviors in mice, which were wholly dependent on S1PR3. S1P-evoked pain was entirely dependent on TRPV1 ion channels, whereas S1P evoked itch was dependent on TRPA1. Consistent with our in vivo findings, we found that S1P activated subsets of both heat- and itch-sensitive dorsal root ganglion (DRG) neurons, and that activation was solely dependent on S1PR3, and not S1PR1. Our data support a model in which S1P elicits neuronal activation via two pathways in distinct subsets of somatosensory neurons. Gβγ-dependent activation of TRPA1 mediates S1PR3-evoked calcium responses in one subpopulation, whereas PLC-dependent activation of TRPV1 mediates S1PR3-evoked calcium responses in the other subpopulation. In the Complete Freund’s Adjuvant (CFA) -induced inflammatory pain model, S1PR3-deficient mice failed to develop heat hypersensitivity, whereas mechanical hypersensitivity developed normally. Pharmacological blockade of S1PR3 activity or S1P production rapidly and selectively ameliorated heat hypersensitivity in wild-type mice treated with CFA. These data definitively identify S1PR3 as a mediator of thermal pain and itch, and, furthermore, demonstrate for the first time that elevated peripheral S1P is an essential driver of chronic inflammatory pain.

Persistent Extracellular Signal-Regulated Kinase Activation by the Histamine H4 Receptor in Spinal Neurons Underlies Chronic Itch

Kun Huang1,2,5, Dan-Dan Hu1,3,5, Dong Bai1,2,5, Ze-Yang Wu1,3, Yi-Yang Chen4, Yi-Jun Zhang1,3, Xin Lv4, Qing-Xiu Wang2 and Ling Zhang1,3

Persistent Extracellular Signal-Regulated Kinase Activation by the Histamine H4 Receptor in Spinal Neurons Underlies Chronic Itch

Transient extracellular signal-regulated kinase (ERK) activation in the spinal cord triggers histamine-induced acute itch. However, whether persistent ERK activation plays an important role in chronic itch development remains unclear. This study investigated the role of spinal ERK activation in chronic itch. The results showed that repetitive DNFB painting on the nape of mice evoked not only initial scratching but also sustained, spontaneous scratching. In addition, DNFB induced itching rather than nociception, as demonstrated using a cheek model. Furthermore, ERK was persistently activated in the spinal cord of DNFB-treated mice, and the intrathecal inhibition of phosphorylation of ERK suppressed both spontaneous itching and ERK activation. ERK activation was observed in neurons but not in glia cells during chronic itch development. Finally, DNFB- induced spontaneous itching behavior and ERK activation were largely inhibited by the histamine H4 receptor antagonist JNJ7777120 but not by the H1 receptor antagonist chlorpheniramine. Our results indi- cate that persistent ERK activation via the histamine H4 receptor in spinal neurons underlies DNFB-induced chronic itch.
Journal of Investigative Dermatology (2018) -, -e-; doi:10.1016/j.jid.2018.02.019

Cooling Relief of Acute and Chronic Itch Requires TRPM8 Channels and Neurons
Radhika Palkar1,3, Serra Ongun1,2, Edward Catich1,3, Natalie Li1, Neil Borad1, Angela Sarkisian1 and David D. McKemy1,2,3

1Neurobiology Section, Department of Biological Sciences, University of Southern California, Los Angeles, California; 2Molecular and Computational Biology Graduate Program, Department of Biological Sciences, University of Southern California, Los Angeles, California; and 3Neuroscience Graduate Program, Department of Biological Sciences, University of Southern California, Los Angeles, California
Correspondence: David D. McKemy, Neurobiology Section, Department of Biological Sciences, University of Southern California, 3641 Watt Way, HNB 201, Los Angeles, CA 90089. E-mail: mckemy@dornsife.usc.edu
Abbreviations: Cqx, chloroquine; a-Me5-HT, a-methyl 5-HT
Received 16 August 2017; revised 8 December 2017; accepted 20 December 2017; accepted manuscript published online 27 December 2017; corrected proof published online XXX

Cooling Relief of Acute and Chronic Itch Requires TRPM8 Channels and Neurons

Cooling or the application of mentholated liniments to the skin has been used to treat itch for centuries, yet remarkably little is known about how counter-stimuli such as these induce itch relief. Indeed, there is no clear consensus in the scientific literature as to whether or not cooling does in fact block the transduction of itch signals or if it is simply a placebo effect. This gap in our understanding led us to hypothesize that cooling is antipruritic and, like cooling analgesia, requires function of the cold-gated ion channel TRPM8, a receptor for menthol expressed on peripheral afferent nerve endings. Using a combination of pharmacologic, genetic, and mouse behavioral assays, we find that cooling inhibits both histaminergic and non-histaminergic itch pathways, and that inhibition of itch by cooling requires TRPM8 channels or intact and functional TRPM8-expressing afferent neurons. The cold mimetic menthol is also effective in ameliorating itch in a TRPM8-dependent manner. Moreover, we find that chronic itch can be ameliorated by cooling, demonstrating that this counter-stimulus activates a specific neural circuit that leads to broad itch relief and a potential cellular mechanism for treatment of chronic itch.
Journal of Investigative Dermatology (2018) -, -e-; doi:10.1016/j.jid.2017.12.025