Shikonin Ameliorate Alcohol-aggravated Imiquimod-induced Mice Psoriasiform Dermatitis Via PKM2-TH17 Pathway

Introduction:Psoriasis is one of the most common chronic inflammatory skin disease that affects approximately 125 million people worldwide.Moreover,the severity of psoriasis greatly affects the life quality and psychological health of patients.The pathogenesis of psoriasis is complex and not fully elucidated.Excessive activation of the adaptive immune system plays an important role in pathogenesis of psoriasis,including the skewing of Th1/Th17 immune response as the central role and the secretion of effector cytokines,mainly represented by TNF-α,IL-23 and IL-17.IL-17 plays the critical role in the pathogenesis of psoriasis,and biologics targeting IL-17A have shown remarkable effects in the treatment of psoriasis.The important cellular sources of IL-17A are TH17 cells,mast cells,innate lymphoid cells,neutrophils,et al.For several years,Th17 was recognized as the primary source of IL-17A.CD4+T cells differentiate into TH17 cells require the first signals of TCR receptor recognition from antigen-presenting cells and the co-stimulatory molecule（the second signal）,combined with certain cytokines environment,such as IL-23,IL-6,TGF-βand IL-1β.IL-23/IL-17 axis plays a pivotal role in psoriasis by activating the Th17 cells.After activation by autoantigens（such as LL37）and other microorganisms（such as fungi）,DCs produce IL-23.Binding to its receptors,IL-23 can trigger the phosphorylation of receptor-associated JAKs,and followed by activating the transcription factor retinoid-related orphan receptor-γt（ROR-γt）and signal transducer and activator of transcription 3（STAT3）.Finally,CD4+T differentiate into TH17 and produce IL-17A,TNF-α,IL-22,making a positive feedback loop.Moreover,the differantiation of TH17 undergoes a series of metabolic reprograms,such as disorder of glycolysis,Glutaminase（GLS）,fatty acids metabolism.Therefore,regulation of energy metabolism has also become a potential target for autoimmune diseases.Even when oxygen concentration is ample,the differantiation of TH17 rely on glycolysis,known as the Warburg effect,which is first found by Dr.Warburg in most cancer cells.Pyruvate kinase 2（PKM2）,as a rate-limiting enzyme of glycolysis,is involved in the pathogenesis of many tumors.In addition,PKM2 plays a biological role by regulating the phosphorylation of many transcription factors,such as HIF-α,STAT3.PKM2 is upregulated in most cancer cells,as well as in psoriatic lesions.Moreover,PKM2 has been found to regulate TH1/TH17 differentiation in recent years.Therefore,PKM2 is a potencial new target for the treatment of tumors and autoimmune diseases.Environmental factors can contribute to the severity and triggering of psoriasis,such as infection,smoking,alcohol intake.One investigation showed that nearly 50%of patients with psoriasis have a history of excessive alcohol drinking.Excessive alcohol consumption can increase the risk of anxiety and depression for psoriasis patients.Alcohol and its metabolite acetone may promote keratinocyte proliferation in vitro and disrupt skin barrier function;alcohol can increase the expression of IL-6,TNF-α,IFN-γ,tiggering TH1 responses in coculture system of keratinocyte and T-lymphoma cells;chronic alcohol drinking can worsen the lesion of mice psoriasiform dermatitis induced by IMQ,and increase the m RNA expression of IL-17.Besides,one study of lung fibroblasts suggested that alcohol can promote Th17 development in vivo and in vitro,but the mechanism is largely unclear.In light of the important role of IL-17/TH17 in the pathogenesis of psoriasis,this study used imiquimod（IMQ）-induced murine model,and used naive CD4+T cells differentiate into TH17 as cell model,to study the effect of alcohol on psoriasis and the expression of TH17/IL-17.What’s more,we further explored the underlying mechanisms of elevated IL-17 by alcohol from the perspective of energy metabolism,and find the possible therapeutic targets.We found that alcohol can promote TH17 differentiation and increase PKM2 expression.As an inhibitor of PKM2,shikonin has been found to alleviate imiquimod-induced psoriasis model in previous studies.Our study further study the effect of shikonin on the worsening of psoriasis-like skin lesions by alcohol.Methods:This study were divided into two parts:the vivo and in vitro experiments.The vivo study were divided into two parts:one part is on the alcohol aggravated the psoriasis mouse model;the other part is on the shikonin alleviation of the worsen of psoriasis-like skin lesions by alcohol.6 to 8 weeks of BALB/c mice（18-20 g）were used for this study.In the first part,mice were housed one week（20-22g）,then randomly divided into 6 groups:blank group,model group,different concentration of alcohol（20%,30%,40%,50%）.Blank were treated with water and vaseline,other groups were all treated with imiquimod.Model mice were treated with water and imiquimod;Alcohol were used with different concentrations of ethanol and imiquimod.The imiquimod were given for 7 days.After 7days,six mice of each group were killed,skin lesion,spleen and blood were collected for further study.Skin tissues were collected for HE staining and IHC.Blood were used to measure the expression of related inflammatory cytokines by ELISA.FACS was used to measure the ration of CD4/CD8/IL-17 in the spleen.Western Blot was used to measure the expression of IL-17 and the related protein in skin.After 7 days,the group of 20%alcohol were divided into two groups:one was treated with water,the other was treated with alcohol.The rest of mice were treated with water and alcohol for another 7 days.On the 10th and 14th day,skin tissues were collected for HE staining.Photos,Psoriasis Area and Severity Index（PASI）score,H&E staining,IHC were used to assess the effect of alcohol in psoriasis inflammatory responses.In the second part,mice were randomly divided into 4 groups:Model,Alcohol,Alcohol+shikonin and Shikonin.Six mice for each group,and shikonin was dissolved by DMSO to 20mg/ml,and then diluted by edible oil to the required concentration of10mg/ml.Model group were treated with 10mg/kg edible oil containing 5%DMSO and200ul dd H₂O by intragastric injection.Alcohol group were treated with 200ul 20%alcohol and 10mg/kg edible oil containing 5%DMSO.Alcohol+shikonin were treated with 20%alcohol and 10mg/kg shikonin by intragastric injection.Shikonin group were treated with 10mg/kg shikonin and 200ul dd H₂O.Mice were killed at the 6th day,skin lesion and blood were collected for further study.Photos,Psoriasis Area and Severity Index（PASI）score,H&E staining were used to assess the severity of the lession.Western Blot was used to measure the expression of IL-17 and related protein in skin.ELISA was used to measure the expression of IL-17 in the blood.Naive CD4+T cells were isolated from wild-type C57BL/6 mice and cultured with alcohol and shikonin or not under TH17-cell differentiation conditions.After incubation for 48hs,cells were stimulated with BD cell stimulation cocktail for 4-6h,and FACS was used to measure the ration and proliferation of TH17.ELISA was used to measure the expression of IL-17 in the culture supernatants.Western Blot was used to measure the expression of PKM2,STAT3,p-STAT3.Results:1)Alcohol can aggravate the progression of psoriasis and slow the regression of skin lesions,increase the infiltration of inflammatory cells,promote inflammatory cytokines expression.After six days of treatment with imiquimod,model group showed typical psoriasis-like dermatitis,erythema,scaly and thickening,and subside on the seventh day.It is noted that mice taking high concentration alcohol group（40%,50%）died during the experiment.Compared with the model group,the alcohol group（20%,30%）showed erythema,scaly earlier and regression slowly,especially the scale.Histopathology of skin lesions at day 7 showed higher epidermal hyperplasia in alcohol group.Immunohistochemical results showed that epidermal CD3-positive cell infiltration and IL-6,HIF-αand VEGF expression were higher in alcohol group.2)Alcohol can increase the protein expression of PKM2-TH17 pathway-related molecules in the skin lesion,and increase the differentiation of TH17 in spleen.RT-PCR,WB and ELISA showed that IL-17A in skin lesions and serum were increased in alcohol group.Moreover,alcohol increase the ratio of TH17 and PKM2 expression in spleen.The expression of PKM2 was increased in skin lesions of alcohol group 3)In vitro,alcohol can promote the differentiation of TH17,and shikonin can reverse this trend.4)Shikonin can alleviate the worsening of psoriasis-like skin lesions by alcohol,and reduce the expression of IL-17.Conclusion:Alcohol can aggravate the IMQ-induced psoriasis-like skin lesions,and promote the differentiation of TH17,increase the expression of IL-17.Shikonin can alleviate the worsening of psoriasis-like skin lesions by alcohol through PKM₂-TH17pathway.