Study On The Ameliorative Effect Of Cannabinoid Receptor Type 2 Agonist On Psoriasis-like Skin Damage And Its Underlying Mechanism

Background and Objective:Psoriasis is a chronic disease mediated by immune abnormalities.Inflammation and oxidative stress play important roles in the pathogenesis of psoriasis,directly or indirectly leading to dermatitis symptoms and abnormal proliferation and keratinization of keratinocytes in patients with psoriasis.Recent studies indicate that cannabinoid receptor type 2(CB2R)is an attractive target for the treatment of a variety of inflammatory and oxidative stress-mediated diseases.CB2R is widely expressed in skin tissues and has great potential for treating inflammatory skin diseases such as psoriasis.However,the exact role of CB2R activation in psoriasis and the associated mechanisms remain poorly understood.In this study,the imiquimod(IMQ)-induced BALB/c mouse model of psoriasis and the tumor necrosis factor(TNF)-α-induced immortalized human keratinocyte(Ha Ca T cell)model were used as study subjects,and the CB2R-specific agonist GW842166X(GW)as the experimental drug.We investigated the interventional effects of CB2R activation on inflammation and oxidative stress in psoriasis-like lesions in vivo and in vitro and its possible underlying mechanisms.Methods:1.Cell experiment:Ha Ca T cells were divided into 4 groups:(1)Control group:no drug added;(2)TNF-αgroup:50 ng/m L TNF-αadded;(3)GW group:50 n M GW added;(4)TNF-α+GW group:50 ng/m L TNF-αand 50 n M GW added.(1)After 12 hours of incubation,the CCK-8 method was used to detect the cells in each group at the absorbance of 450 nm,and the cell viability was calculated.(2)After 48 hours of incubation,the expression level of i NOS in each group was detected by flow cytometry.2.Animal experiment:6～8-week-old BALB/c female mice were randomly grouped into(1)Control group,(2)IMQ group,and(3)IMQ+GW group.According to the groups,10mg/kg of GW agent or its solvent in a volume of 100μL was injected intraperitoneally at regular intervals every day,and 50 mg of vaseline or 5%IMQ cream was evenly applied to the exposed area of the mice’s back skin 6 hours later for 7 days.The psoriasis lesion severity index(PASI)of the mice’s back skin lesions was recorded daily.The mice were executed at the end of the experiment,and the skin tissues of the experimental areas were taken for HE staining to observe the histopathological changes.The expression levels of markers related to epidermal proliferation(PCNA,K17),inflammatory cells(CD11c,MPO,IL-17),NF-κB(IκB,Rel B),and Keap1/Nrf2 signaling pathway(Nrf2,HO-1)were observed by immunohistochemistry.Immunofluorescence staining was performed to detect M1macrophages(F4/80~+i NOS~+cells)and M2 macrophages(F4/80~+CD206~+cells)in the skin.The expression levels of CB2R and i NOS were determined using indirect immunofluorescence,and gene expression levels of cytokines(IL-17A,IL-22,IL-6,TNF-α,IL-1β)were detected by RT-q PCR.The ratios of dendritic cells,neutrophils,macrophages,and IL-17A~+γδT cells of the spleens were detected by flow cytometry.Results:1.GW attenuated the increased TNF-α-induced cell viability in Ha Ca T cells.2.GW suppressed the enhanced i NOS expression induced by TNF-αin Ha Ca T cells.3.GW improved the symptoms,elevated PASI scores,pathological changes,and excessive proliferation of the epidermal layer in the skin of IMQ-induced mice.4.GW reduced the infiltration of monocytes/neutrophils,dendritic cells,and IL-17~+cells,high expression levels of IL-17A,IL-22,and IL-6,and increased the proportion of M2macrophages.5.GW reduced the infiltration of monocytes/neutrophils,dendritic cells,IL-17~+cells and high expression of IL-17A,IL-22,IL-6,and increased the proportion of M2 macrophages.6.GW decreased the high expression level of CB2R and i NOS in skin lesions of psoriasis-like mice induced by IMQ.7.GW reversed the reduced expression of Nrf2 and HO-1 and the increased expression of IκB and Rel B in the skin lesions of IMQ-induced mice.Conclusions:1.Activation of CB2R prevents TNF-α-induced hyperproliferation of Ha Ca T cells,probably by suppressing their inflammation and oxidative stress.2.Activation of CB2R systematically treats the psoriasis-like skin inflammation manifested by IMQ-induced mice and the massive infiltration of pro-inflammatory cells in the spleen,and promotes the polarization of macrophages toward an anti-inflammatory phenotype.The specific activation of CB2R reduced the level of oxidative stress in the skin,thereby inhibiting the NF-κB pathway and activating the Keap1/Nrf2 pathway,which may be involved in regulating the treatment process.