Validation For Attenuation Of Atopic Dermatitis By Specific Inhibition Of Warm-temperature Activated Ca²⁺-permeable TRPV3 Channel

Atopic dermatitis（AD）is a chronic inflammatory skin disease characterized by cutaneous lesions and intense pruritus.All age groups are susceptible to the disease.Children are the most common.The characteristics of AD include dry skin,severe itching and a family history of"heterotopicity".The pathogenesis of the disease has not been fully clarified because of its complex etiology.In recent years,the incidence of AD has increased significantly.Intense itching often causes insomnia.A series of skin characteristic change including xeroderma,ichthyosis,palmprint,peridermal keratosis,skin infection tendency（especially Staphylococcus aureus and herpes simplex virus infection）,eczema,cheilitis,recurrent combination.These symptoms affect the quality of patients’life and their families,and even affect the patient’s self-confidence.The warm temperature-activated Ca²⁺-permeable TRPV3 channel is abundantly in the skin especially keratinocytes.Previous investigations have demonstrated that gain-of-function mutations of warm temperature-activated Ca²⁺-permeable TRPV3 channel（Gly573Cys in WBN/Kob-Ht rats and Gly573Ser in DS-Nh mice）can cause skin inflammation,itching and abnormal hair growth in rodents.These mutations produce AD-like phenotypes such as inflammatory cell infiltration,keratin thickening and high immunoglobulin E.Our and other’s identifications of gain-of-function mutations（G573S,G573C,W692G and G573A）in TRPV3 gene from patients with congenital Olmsted syndrome（OS）characterized by palmoplantar and periorificial keratoderma and severe itching further confirms the causative role of thermoTRPV3 channel in cutaneous sensation and inflammatory skin lesions.These results showed that over-activated TRPV3 is invoved in of skin inflammatory diseases,and specific inhibition of TRPV3 channel may be a new effective target of treating atopic dermatitis or inflammatory dermatosis.Objectives:Topical applications of DNFB were used to establish AD mouse modle.Using osthole,a specific inhibitor of TRPV3 reported by our laboratory,as a tool drug,to treat AD and ear swelling and can alleviate inflammation symptoms by inhibition of TRPV3.We attempted to validate the relationship between inflammation diseases such as AD and TRPV3.Inhibition of TRPV3 genetically and pharmacologically can prevent from inflammation induce by TRPV3 agonist carvacrol.In this study,we attempted to validate cutaneous TRPV3 as an effective therapeutic target for alleviation of AD-like lesions.Methods:Topical application of DNFB resulted in AD and ear swelling mouse model,osthole was applied to alleviate symptoms by inhibiting TRPV3 selectively.Carvacrol was used in TRPV3^-/-mice and WT mice to study whether it caused AD-like and ear swelling symptoms.Furthermore,the effects of DNFB on primary keratinocytes of mice were detected by applying DNFB and Osthole to isolated primary keratinocytes of mice.Western blot（WB）,reverse transcription-polymerase chain reaction（Q-PCR）,immunohistochemistry（IHC）and skin histological sections（H&E staining）were used to evaluate the expression of TRPV3 and inflammation with phenotypic features,recording of ear thickness,swelling scores and dermatitis scores.Results:Topical applications of DNFB resulted in the increased expression of TRPV3.Topical application of TRPV3 inhibitor osthole can reduce skin thickness,down-regulate the expression of TRPV3 and inflammatory factors TNF-αand IL-6,suggesting that inhibiting TRPV3 could alleviate the symptoms of DNFB-induced AD symptoms.In DNFB-induced mouse ear edema model,TRPV3 expression level was up-regulated.TRPV3 inhibitor osthle could reduce ear thickness and relieve the degree of ear edema,which indicated that TRPV3 could alleviate DNFB-induced ear inflammation in mice.In the experiment of TRPV3 agonist carvacrol inducing AD-like symptoms in mice,TRPV3agonist carvacrol could induce AD-like symptoms in WT mice,but it could not induce the same symptoms in TRPV3^-/-mice and mice with local inhibition of TRPV3 by osthle,which indicated that the inhibition of TRPV3 could prevent AD-like skin symptoms in mice.In the experiment of TRPV3 agonist carvacrol inducing ear swelling in mice,the degree of ear swelling induced by carvacrol in WT mice was more serious than that in TRPV3^-/-mice,which further indicated that TRPV3 plays an important role in the development of inflammatory diseases.In mouse keratinocyte experiments,application of DNFB increased the expression of TRPV3 and inflammatory factors,while Osthol reduced the expression of TRPV3 and inflammatory factors.Conclusions:TRPV3 channel is involved in the progressive pathology of AD in mice,including attenuation of symptoms,reducement of ear thickness the degree of ear swelling,down-regulation of the expression of inflammatory factors and prevent inflammation.Topical inhibition of TRPV3 may represent a novel therapeutic strategy for effective treatment:1.Upregulation of TRPV3 in mouse model of AD induced by topical DNFB.2.Reversal of DNFB-induced atopic dermatitis by selective inhibition of TRPV3.3.Upregulation of TRPV3 in ear swelling model of AD induced by topical DNFB.4.Attenuation of DNFB-induced ear swelling by topical TRPV3 inhibitor osthole.5.Suppression of TRPV3 genetically and pharmacologically attenuates skin lesions induced by TRPV3 agonist carvacrol.6.Osthole can inhibit the expression of TRPV3 and inflammatory factors in primary keratinocytes of mice induced by DNFB.7.Topical inhibition of TRPV3 may provide therapeutic potential target for prevention and treatment of atopic dermatitis or related skin diseases.