Study On The Role And Mechanism Of CB2R Mediated Neuro-Immune Pathway In Psoriatic Inflammation And Itch

Objective:Psoriasis is a frequent,chronic,disfiguring,inflammatory skin disease.In addition to the typical manifestations of erythema,scaling,and skin thickening,itch has been reported in approximately 41%～90%of patients.The pathogenesis of psoriasis is still not fully understood,so there is still no specific and targeted treatment.Cannabinoid receptor 2(CB2R)is a G protein-coupled receptor that is mainly located on immune organs and immune cells,and can be involved in the pathophysiological processes of immune regulation and sensory conduction,and the activation of it can inhibit the scratching behavior of rats induced by acute prurigogens.Therefore,the selective agonists of CB2R may become a new potential candidate for anti-inflammatory and antipruritic drugs by interfering with the occurrence and progression of inflammatory skin diseases.However,to date,the role of CB2R in psoriasis and chronic itch is unclear.The cellular biological mechanism of its antipruritic effect still needs to be elucidated.This study intends to explore the role of CB2R in inflammation and itch in psoriatic dermatitis(Ps D)mouse model and its possible mechanism.Methods:(1)Following daily treatment with topical imiquimod(IMQ)cream for 5～7consecutive days to construct Ps D and chronic itch mouse model in C57 BL/6J wild-type(WT)and CB2R gene knockout(KO)mice.(2)The Psoriasis Area and Severity Index(PASI)score of each mouse was assessed every day,and photographed at the same time.(3)Hematoxylin and eosin staining was used to observe the histological changes.(4)Quantitative real-time polymerase chain reaction was performed to verify the m RNA expression level of various cytokines,chemokines and transcription factors,and western blot was employed to determine the changes in the protein expression of CB2R and nerve growth factor(NGF).(5)Immunohistochemistry was used for semi-quantitative counting of CD4~+T cells.(6)Immunofluorescence staining was used to observe the localization of CB2R in skin and the changes in epidermal nerve fiber density.(7)The expression of Th1/Th2/Th17 cells related cytokines in serum of mice was detected by cytometric bead array.(8)The proportion of Th17/Treg cells in mouse spleen was analyzed using flow cytometry.(9)The times of daily spontaneous scratching were recorded by video playback method for 1 h.(10)Using toluidine blue staining to observe the number and degranulation rate of mast cells,immunofluorescence staining labeled c-kit~+was used to further verify the number of mast cells.(11)The selective agonist JWH-133 and antagonist AM-630 of CB2R were injected intradermally to observe their effects on inflammation and itch in Ps D mice,all the methods were used as same as before.(12)Video recording was used to observe the spontaneous scratching behavior of mice after intradermal injection of recombinant cytokines IL-17A and IL-23.Results:Compared with the skin tissues of control group mice,the expression of CB2R was upregulated in psoriatic mouse model,mainly located in the spinous layer and granular layer of the epidermis,and localized in the cytoplasm.But in psoriasis patients,CB2R was expressed in both epidermis and dermis.Compared with WT mice,CB2R deficiency exacerbated IMQ-induced Ps D by upregulating the expression of proinflammatory cytokines(IL-17A,IL-17C,IL-23A,TNF-α,IL-1β),chemokine(CXCL-1),downregulating the expression of anti-inflammatory cytokine IL-10,increasing the infiltration of CD4~+T cells and the differentiation ratio of Th17/Treg cells,regulating the release of key cytokines of Th1/Th2/Th17 cells.Intradermal injection of CB2R selective agonist JWH-133 significantly reversed inflammation by inhibiting the production of pro-inflammatory cytokines and down-regulating the Th17/Treg cell ratio.Compared with WT mice,CB2R deficiency aggravated IMQ-induced scratching behavior in Ps D mice,and pretreatment with CB2R agonist significantly reversed scratching behavior.Obvious proliferation and prolongation of nerve fibers and high expression of NGF were observed in Ps D and CB2R KO mouse model group.CB2R deficiency did not increase the number and degranulation rate of mast cells in the skin of psoriatic mouse model,nor did it upregulate the m RNA expression levels of IL-31 and TSLP.Compared with control mice,intradermal injection of 1μg of IL-17A and IL-23 did not increase the scratching behavior in mice.Conclusion:The results of this study indicate that CB2R plays an essential role in the pathophysiology of psoriatic inflammation and itch,providing a new potential therapeutic target for the selection of anti-inflammatory and antipruritic drugs.