Effect And Mechanism Of Allicin On Imiquimod-Induced Psoriasis-Like Skin Lesions In Mice

Objective: Psoriasis is an autoimmune-mediated inflammatory skin disease induced by internal genetics and the external environment,clinically characterized by marked erythema and scaly lesions on the skin.The pathological changes of psoriasis mainly include excessive differentiation and incomplete differentiation of keratinocytes in the skin epidermis,abnormal aggregation in immune cells,and telangiectasis hyperplasia in the dermis.The pathogenesis of psoriasis is not fully clear.The interaction between the proinflammatory cytokine IL-17 released by immune cells and keratinocytes maintains the vicious cycle of inflammation in the skin lesions.Currently,the drugs routinely used in clinical treatment have noticeable side effects,so seeking new drugs with significant curative effects and high safety for psoriasis is necessary.Modern pharmacological studies have shown that allicin has a variety of pharmacological activities,such as antiinflammatory,anti-oxidation,cholesterol-lowering,anti-tumor,and cardio-protection.This study aims to investigate the protective effect of allicin on the pathological model of imiquimod-induced psoriasis-like lesions in mice and to explore the molecular mechanism of allicin against psoriasis.Methods: 1.IMQ was used to induce psoriasis-like skin lesions in mice,randomly selected BALB/c mice were divided into seven groups: control group,model group,vehicle group,allicin-low-dose group,allicin-middle-dose group,allicinhigh-dose group,and positive group.The pharmacodynamic effects of allicin on psoriatic lesions in mice were evaluated by observing the changes of skin on the back of mice,the psoriasis area and severity index(PASI)score,and the morphological changes of skin lesions.2.The mRNA was extracted from the skin lesions of mice in the IMQ model group and the high-dose allicin group for transcriptome sequencing analysis,and the differentially expressed genes were subjected to KEGG and GO enrichment analysis to explore the possible targets and pathways of allicin preliminarily.3.HaCaT and NHEKs were used in this study in vitro,and cell proliferation assay(CCK8)was used to investigate the effects of allicin on the activity of HaCaT and NHEKs cells.Effects of allicin on cell cycle distribution and cell apoptosis of HaCaT and NHEKs were assessed by flow cytometry,while changes in the expression level of the proliferation marker Ki67 protein in the skin tissues were assessed by immunohistochemistry(IHC).Western blot(WB)was used to investigate the effects of allicin on apoptosis-related protein expression of Bcl-2,Bax,and Caspase3/Cleaved caspase3 in vivo and in vitro.The effect of allicin on apoptosis of mice skin tissue was investigated by the notch end labeling technique(TUNEL).4.Investigated the effect of allicin on the interaction between KCs and IL-17 A,a critical inflammatory factor in psoriasis.The mRNA levels of inflammatory factors,chemokines,and antimicrobial peptides in psoriatic skin lesions and KCs were detected by RT-qPCR.After co-culturing HaCaT and NHEKs with IL-17 A and allicin for 48 hours in vitro,the mRNA expression levels of downstream inflammatory chemokines and antimicrobial peptides were detected by RT-qPCR.The effects of allicin on the infiltration of T cells,macrophages,and neutrophils in the skin lesions of mice were investigated by IHC.5.HaCaT and NHEKs cells were co-cultured with inflammatory cytokines such as IL-17 A and allicin in vitro,and the protein expression levels of TRAF6,MAPK,STAT3,and NF-κB(P65)in IL-17 signaling pathway were analyzed by WB after protein extraction,the expression of proteins related to the above pathways was analyzed by extracting proteins from mouse skin lesions and exploring the molecular mechanism in improving psoriasis in vivo and in vitro.Results:1.Topical application of allicin ointment significantly improved the appearance of skin lesions,such as psoriatic erythema and scales,reduced PASI scores,and alleviated the histopathological changes such as epidermal thickening and epidermal under differentiation in mice induced by IMQ.2.Transcriptome sequencing showed that compared with IMQ model group,allicin high-dose group significantly regulated inflammation-related genes.GO enrichment analysis showed that allicin mainly affected biological processes such as immunity and signal transduction,and KEGG enrichment analysis showed that allicin significantly regulated the IL-17 signaling pathway.3.CCK8 results showed that allicin inhibited the viability of HaCaT and NHEKs cells in vitro.Flow cytometry results showed that allicin inhibited the proliferation of KCs by arresting the G2/M phase of the cell cycle.At the same time,allicin induced apoptosis of keratinocytes in a concentration-dependent manner in vitro,and apoptosis was also detected in the skin lesions in situ by TUNEL assay.4.RT-qPCR indicated that allicin significantly reduced the mRNA expression levels of inflammatory factors IL-17 A,IL-17 F,IL-12,IL-20,and IL-22 in the skin lesions of mice.Meanwhile,allicin inhibited the abnormally high expression of chemokines(CXCL8,CCL20)and antimicrobial peptides(S100a8/9)in HaCaT,NHEKs,and skin lesions.IHC showed that allicin might improve inflammation by reducing the local infiltration of CD3+,MPO+,and F4/80+ cells in skin lesions.5.WB showed that allicin inhibited the expression levels of IL-17 signaling pathway-related proteins,including TRAF6,MAPK(ERK,JNK,P38),STAT3,and NF-κB(P65)in IMQ-induced skin lesions,and IL-17A-stimulated HaCaT/NHEKs.Other inflammatory factors(IL-12,IL-20,and IL-22)also partially activated IL-17 signaling pathway-related proteins,indicating that the extensive antiinflammatory effects of allicin jointly inhibited the activation of the inflammatory signaling pathway.Conclusions: 1.Allicin alleviates IMQ-induced psoriatic skin damage in mice by restoring the normalization of epidermal appearance and tissue structure.2.Allicin may interfere with psoriatic lesions in mice by regulating inflammation and the IL-17 signaling pathway.3.Allicin inhibited abnormal proliferation and promoted apoptosis of KCs.4.Allicin reduced the expression of IL-17 A and other inflammatory cytokines,as well as its downstream chemokines and antimicrobial peptides,and inhibited the local inflammatory response of the skin.Allicin plays an anti-psoriasis role by inhibiting the transduction of the IL-17 signaling pathway and breaking the positive feedback between IL-17 A and KCs.