| Part Ⅰ: Effect of IL-17 A on activation of NF-κB signaling pathway in keratinocytesObjective: To analyze the correlation between the expression of interleukin-17A(IL-17A)and NF-κB signaling pathway-related signature proteins(IKKβ,p-IκBα,p-p65)in psoriasis vulgaris lesions and imiquimod-induced psoriasiform inflammation mouse model lesions;to explore the effect of IL-17 A on the activity of NF-κB signaling pathway in keratinocyte cell line(Ha Ca T)cultured in vitro;to study the intervention effect and potential mechanism of Liangxue Xiaofeng Decoction in imiquimod-induced psoriasis mouse model.Methods: 1.Clinical experiments: Immunohistochemistry was used to detect the expression of IL-17 A,IKKβ,p-IκBα,and p-p65 protein in keratinocytes from skin lesions of 55 patients with psoriasis vulgaris and 30 normal controls.2.Cell experiments: After Ha Ca T cells were stimulated with IL-17 A for 24 h,total protein was extracted,and the expression levels of IKKβ,p-IκBα,and p-p65 protein were detected by Western blot.3.Animal experiments: The expression of IL-17 A,IKKβ,and p-p65 in keratinocytes of mouse model lesions was observed by intervention with Liangxue Xiaofeng Decoction in a mouse model of imiquimod-induced psoriasis-like inflammation.Results: 1.Clinical experiments: Immunohistochemical staining score analysis,the expression levels of IL-17 A,IKKβ,p-IκBα,and p-p65 in normal human keratinocytes were significantly lower than those in keratinocytes in the lesional skin of patients with psoriasis vulgaris(P < 0.01).2.Cell experiments: The expression levels of IKKβ,p-IκBα,and p-p65 proteins were significantly increased in Ha Ca T cells treated with IL-17 A for 24 hours compared with wildtype Ha Ca T cells(P < 0.05).3.Animal experiments: Immunohistochemical staining scores showed that the expressions of IL-17 A,IKKβ,and p-p65 in lesional keratinocytes of mice in the model group were significantly higher than those in the normal control group(P < 0.05 for all);the expressions of IL-17 A,IKKβ,and p-p65 in the low-and medium-dose groups of Liangxue Xiaofeng Decoction were not statistically significant compared with those in the model group(P > 0.05);the expressions of IL-17 A,IKKβ,and p-p65 in the high-dose group of Liangxue Xiaofeng Decoction were significantly lower than those in the model group(P < 0.05).Conclusion: 1.IL-17 A expression and the expression of NF-κB signaling pathway-related signature proteins(IKKβ,P-IκBα,p-p65)were increased in lesional keratinocytes of psoriasis patients,suggesting that IL-17 and NF-κB pathways are involved in the pathogenesis of psoriasis.2.IL-17 A was able to upregulate the expression of IKKβ,p-IKBa,and p-p65 in Ha Ca T cells,suggesting that IL-17 could induce the activation of NF-κB signaling pathway in Ha Ca T cells,indicating that IL-17 could be involved in the pathogenesis of psoriasis by activating NF-κB signaling pathway in keratinocytes.3.Liangxue Xiaofeng Decoction could reduce the expression of IL-17 A,IKKβ,and p-p65 in the lesional skin of a mouse model of imiquimod-induced psoriasiform inflammation,suggesting that Liangxue Xiaofeng Decoction may indirectly inhibit the activation of the NF-κB pathway and improve imiquimod-induced psoriasiform inflammation in mice by reducing the expression of cytokines associated with inflammatory cell infiltration(IL-17A)in keratinocytes.Part Ⅱ: Study on the mechanism of SIRT1 expression in inflammation associated with psoriasis vulgaris.Objective: The expression of silencing information regulator 1(SIRT1)and the acetylation of H3 protein at K9(H3K9Ac)and p65 at K310(ace-k310 p65)in keratinocytes of skin lesions induced by psoriasis vulgaris and imiquimod-induced psoriasis in mice were detected.To study the effect of SIRT1 on the secretion of inflammatory factors by IL-17A-induced Ha Ca T cells,to explore the interaction mechanism between SIRT1 and NF-κB signaling pathway,and to investigate the role and mechanism of SIRT1 in the intervention of imiquimod-induced psoriatic-like inflammation in mice model by cooling blood and cooling wind decoction.Methods: 1.Clinical trial: immunohistochemistry was used to detect the expression of SIRT1 protein in keratinocytes of skin lesions in 55 patients with psoriasis vulgaris and 30 normal skin tissues,the acetylation level of H3 protein at K9 site and the acetylation level of p65 at K310 site.2.Cell experiment: the experiment was divided into the normal control group,the IL-17A(5ng/ml)group and the SIRT1 sh RNA group.The protein expression of SIRT1 and IL-23,the acetylation level of H3 at K9 and the acetylation level of p65 at K310 were detected by Western blot.3.Animal experiments: establish a mouse model of imiquimod induced psoriatic-like inflammation;To observe the expression of SIRT1 protein and the acetylation levels of H3 proteins at K9 and p65 at K310 in the mouse model of imiquimod-induced psoriatic inflammation with the intervention of Liangxuexiaofeng decoction.Results: 1.Clinical trials: the immunohistochemical study score showed that formed in 55 cases of patients with psoriasis vulgaris horniness cells,the SIRT1 expression and H3 protein at a K9 site acetylation level horniness cells was significantly lower than normal(P < 0.05),but as a result of progress of different symptoms in patients have different degree of skin thickening of stratum spinosum,found that as the lesion area in patients with spine layer thickening,SIRT1,H3K9 Ac is to reduce the increased;Ace-k310 p65 in all patients with psoriasis vulgare was significantly higher than that in the control group(P<0.05).2.Cell experiment: IL-17 A stimulation can reduce the expression of SIRT1 and the acetylation level of H3 protein at K9 site in Ha Ca T cells(P <0.05),and increase the expression of IL-23 protein and the acetylation level of p65 at K310 site(P <0.05).Compared with wild-type Ha Ca T cells,SIRT1 Knockdown by sh RNA significantly increased the expression of IL-23 protein and the acetylation level of p65 at K310(P <0.05).3.Animal experiments: immunohistochemical staining score analysis showed that the expression of SIRT1 and the acetylation level of H3 protein at K9 site in the skin lesions of mice in the model group were significantly lower than that of the normal control group(P<0.05),while the acetylation level of p65 at K310 site was significantly higher than that of the normal control group(P<0.05).The expression of SIRT1,the acetylation of H3 protein at K9 site and the acetylation of p65 at K310 site in the low and medium dose groups of Liangxuexiaofeng decoction were not statistically significant compared with the model group(P > 0.05).The expression of SIRT1 and acetylation of H3 protein at K9 site in the high-dose group of Liangxuexiaofeng decoction were significantly higher than those in the model group(P<0.05),and ace-k310 p65 was significantly lower than those in the model group(P<0.05).Conclusion: 1.The expression of keratinocytes SIRT1 and the acetylation level of H3 protein at K9 site in patients with psoriasis vulgaris were significantly lower than those in the normal group,and the acetylation level of p65 protein at K310 site was significantly higher than that in the normal group,suggesting that the abnormal ace-k310 p65,SIRT1 and H3K9 Ac were involved in the pathogenesis of psoriasis vulgaris.2.The increase of SIRT1 in the expression of ace-k310 p65 may be one of the reasons for the sustained activation of NF-κB signaling pathway.3.The inhibition of p65 acetylation by the increase of SIRT1 expression and the inhibition of NF-κB signaling pathway continued activation may be one of the mechanisms of the treatment of imiquimod-induced psoriatic inflammation. |
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