Antipruritic Effect of Natural Coumarin Osthole through Selective Inhibition of Thermosensitive TRPV3 Channel in the Skin

Abstract

Coumarin osthole is a dominant bioactive ingredient of the natural plant commonly used for traditional Chinese herbal medicines for therapies and treatments including antipruritus and antidermatitis. However, the molecular mechanism underlying the action of osthole remains unclear. In this study, we report that osthole exerts an antipruritic effect through selective inhibition of Ca-permeable and thermosensitive transient receptor potential vanilloid 3 (TRPV3) cation channels that are primarily expressed in the keratinocytes of the skin. Coumarin osthole was identified as an inhibitor of TRPV3 channels transiently expressed in HEK293 cells in a calcium fluorescent assay. Inhibition of the TRPV3 current by osthole and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV3-expressing HEK293 cells and mouse primary cultured keratinocytes. Behavioral evaluation demonstrated that inhibition of TRPV3 by osthole or silencing by knockout of the 3 gene significantly reduced the scratching induced by either acetone-ether-water or histamine in localized rostral neck skin in mice. Taken together, our findings provide a molecular basis for use of natural coumarin osthole from the plant in antipruritic or skin care therapy, thus establishing a significant role of the TRPV3 channel in chronic itch signaling or acute histamine-dependent itch sensation.

Pharmacological Inhibition of the Temperature-Sensitive and Ca2+-Permeable Transient Receptor Potential Vanilloid TRPV3 Channel by Natural Forsythoside B Attenuates Pruritus and Cytotoxicity of Keratinocytes

“…HEK293 cells were transfected with cDNAs of TRP channels before they were seeded at a density of approximately 40,000 cells per well in a 96-well black-walled plate (Thermo Fisher Scientific, Waltham, MA) for overnight culture at 37°C with 5% CO 2 . To measure intracellular calcium, cells were incubated with the calcium fluorescent dye provided in the Cal-520 PBX Calcium Assay Kit at 37°C for 1.5 hours; relative fluorescence units were measured in the FlexStation 3 assay under an excitation wavelength at 485 nm and an emission wavelength at 515 nm at an interval of 1.6 seconds (Sun et al, 2018).…”

“…One scratching bout was counted when a mouse raised its right hindlimb to the injection site and continuously scratched for any time length before it returned the limb to its mouth or the floor (Wilson et al, 2013). All behavioral experiments were conducted in a double-blind manner (Sun et al, 2018).…”

“…To access the effect of forsythoside B on itch scratching, vehicle (0.3% DMSO) or forsythoside B (3, 30, and 300 mM) was injected intradermally 30 minutes before intradermal injection of the agonist carvacrol. For video recordings of acute itch induced by histamine, vehicle (0.03% DMSO) or forsythoside B (0.3, 3, or 30 mM) was injected intradermally 30 minutes before intradermal injection of 100 mM pruritogen histamine (Cui et al, 2018;Sun et al, 2018).…”

“…The histamine level in the skin of DS-Nh mice carrying a gain-of-function TRPV3 mutation is substantially higher compared with agematched DS mice (Asakawa et al, 2006). Similarly, TRPV3 knockout mice also exhibit attenuation of pruritus evoked by histamine (Sun et al, 2018). Thus, we further tested the effect of forsythoside B on histamine-dependent acute itch evoked by histamine.…”

“…TRPV3 channel dysfunction caused by genetic gain-of-function mutations or pharmacological activation is implicated in the pathogenesis of skin inflammation, dermatitis, and chronic itch (Asakawa et al, 2006;Imura et al, 2009;Lin et al, 2012;Cui et al, 2018). In rodents, a spontaneous gain-of-function mutation of the TRPV3 gene in WBN/Kob-Ht rats (Gly573 substituted by Cys) and DS-Nh mice (Gly573 substituted by Ser) causes the development of skin lesions with pruritus and dermatitis (Asakawa et al, 2006;Imura et al, 2009;Yoshioka et al, 2009;Yamamoto-Kasai et al, 2013), and silencing of TRPV3 attenuates itch sensation or inflammatory pain (Bang et al, 2012;Yamamoto-Kasai et al, 2012;Sun et al, 2018). We previously reported three gain-offunction mutations of TRPV3 (W692G, G573C, and G573S) in Chinese patients with genetic Olmsted syndrome (OS) featured with mutilating palmoplantar keratoderma with dermatitis, periorificial keratotic plagues, and severe itching (Lin et al, 2012).…”