| Background and purposePsoriasis is a common chronic inflammatory skin disease.It seriously affects thepatients’ health and quality of life.The interaction of keratinocytes,immune cells andproinflammatory cytokine play a significant role in the pathogenesis of psoriasis,but the mechanism has not been entirely defined.As a member of the IL-36 family,thestudy claimed that the expression of IL-36y in the lesions and non-lesion skin of patients with psoriasis was significantly increased,which was related to the severity of the inflammatory response,suggesting that IL-36y may be involved in the pathogenesis of psoriasis as a proinflammatory cytokine.Wnt/β-catenin signaling pathway is involved in the regulation of cell differentiation and proliferation,as well as the pathogenesis of a variety of inflammatory diseases.It was confirmed that the ectopic expression of β-catenin could be seen in the keratinocytes of the basal layer in the lesions of psoriatic patients,suggesting that Wnt/β-catenin signaling pathway may be involved in the pathogenesis of psoriasis.At present,the role of IL-36y and Wnt/β-catenin signaling pathway in the pathogenesis of psoriasis and the interaction between them are not clear Therefore,we study whether IL-36y can regulate the proliferation,differentiation and inflammation of keratinocytes through Wnt/β-catenin signaling pathway,so as to further explore the role of IL-36γ in the pathogenesis of psoriasis and provide new ideas and directions for the pathogenesis and targeted treatment of psoriasis.Methods1.We used CCK-8 to detect the effect of IL-36y on the proliferation of keratinocyteline HaCaT cells.2.The β-catenin protein coding gene CTNNB1 of HaCaT cell line was knocked out by lentivirus transfection,and a stable β-catenin-knockdown HaCaT cell line was constructed3.Western blot was used to detect the expression of Wnt/β-catenin signaling pathway related molecules β-catenin,p-β-catenin,c-myc,cyclinD1 and keratinocyte terminal differentiation indexes K1,K10 and lorincrin after the intervention of IL-36y and endo-IWR-1,an endogenous inhibitor of Wnt/β-catenin signaling pathway.4.The expression and localization of β-catenin and p-β-catenin in HaCaT cells were detected by immunofluorescence.5.The levels of IL-6,IL-8,IL-1β,INF-γ and IL-17A in the supernatant of HaCaT cells were measured by CBA.6.Western blot was used to detect the expression of Wnt/β-catenin signaling pathway related molecules β-catenin,p-β-catenin,cyclinDl and lorincrin after IL-36y intervention on β-catenin-knockdown HaCaT cells7.CBA was used to detect the levels of IL-6,IL-8,IL-1β,INF-γ and IL-17A in the supernatant of β-catenin-knockdown HaCaT cells after the intervention of IL-36y.Results1.In a certain concentration range(1-150ng/ml),IL-36γ promoted the proliferation of HaCaT cells,and in the concentration range of 1-100ng/ml.With the increase of IL-36γ concentration,the cell proliferation was more active.When the concentration of IL-36y was 100ng/ml,the proliferation activity of HaCaT was significantly higher than that of the control group,50ng/ml and 150ng/ml groups,the difference was statistically significant(P=0.0017)2.IL-36y can significantly up-regulate the expression of β-catenin,p-β-catenin and c-myc in HaCaT cells,and significantly down-regulate the expression of K1,K10 and lorincrin of HaCaT cells.The results are statistically significant.3.Endo-IWR-1 can down-regulate the expression of β-catenin in HaCaT and upregulate the expression of K1 and lorincrin,the results are statistically significant;meanwhile,it can inhibit the up-regulation of β-catenin and p-β-catenin protein expression and down-regulation of K1 and lorincrin protein expression in HaCaT cells induced by IL-36γ.4.The levels of IL-6 and IL-8 in the supernatant of HaCaT cells after the intervention of IL-36y increased significantly,and the difference was statistically significant.Compared with the control group and the 50 ng/ml IL-36y group,100 ng/ml IL-36y can significantly increase the level of IL-1β in the supernatant of HaCaT cells.Compared with the control group and 100ng/ml group,the expression of INF-y and IL-17A in the supernatant of HaCaT cells treated with 50ng/ml of IL-36y for 48h was significantlyhigher,the difference was statistically significant.5.Endo-IWR-1 can inhibit the up-regulation of IL-6 induced by IL-36y,and the difference is statistically significant when the concentration of IL-36y was 10ng/ml(P=0.0145)and 1 00ng/ml(P=0.0089);endo-IWR-1 can inhibit the secretion of IL-8 of HaCaT cells compared with the control group and IL-36γ intervention group(P<0.05);endo-IWR-1 could inhibit IL-36y-induced secretion of INF-γ by HaCaT cells,but only when the final concentration of IL-36y was 10ng/ml,the difference between the two groups was statistically significant(P<0.0001).6.After the intervention of IL-36y,the expression of β-catenin protein and p-β-catenin protein in the cell membrane and cytoplasm were increased.7.The knockdown of β-catenin significantly inhibited the up-regulation of β-catenin,p-β-catenin,cyclinD1 expression and down-regulation of lorincrin expression induced by IL-36γ.8.The expression of IL-6 and IL-8 of β-catenin-knockdown HaCaT cells treated with IL-36γ could be increased,but the increase was smaller than that of the negative control group(P<0.05).Conclusion1.IL-36γ can promote the proliferation,inhibit the differentiation and promote the expression of proinflammatory cytokines of HaCaT cells.2.IL-36y can promote the activation of Wnt/β-catenin signaling pathway in HaCaT cells.3.Inhibition of Wnt/β-catenin signaling pathway can promote the differentiation of HaCaT cells,and to some extent inhibit the down-regulation of the differentiation and the up-regulation of the secretion of proinflammatory cytokines of HaCaT cells induced by IL-36γ.4.Knockdown of β-catenin can promote the differentiation of HaCaT cells and inhibit the secretion of proinflammatory cytokines;5.IL-36γ may regulate the proliferation,production of proinflammatory cytokines and differentiation of keratinocytes by Wnt/β-catenin signaling pathway. |
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