Arctigenin Attenuated Imiquimod-induced Psoriasis-like Skin Via Down-regulating Keratin17 And Its Mechanism

Psoriasis is a common chronic inflammatory skin disease induced by multi-factors. This disease is featured with high morbidity, complex pathogenesis and difficulty in radical cure. Now it is considered as an autoimmune disease mainly mediated by helper T cells(Th cells). And there are kinds of inflammatory cells and cytokines involved in its pathogenesis.Keratinocyte plays important role in the forming of epidermis, which secretes keratin to promoter this process. What changes occur in keratinocyte in psoriasis? It was reported that the expression profiling of keratinocyte changes in the psoriasis lesion of psoriasis, including significant increase in keratin 17(K17) levels, which is known as proliferation-related psoriasis keratin. Keratin 17 is seldomly expressed in normal skin, but is highly expressed in the skin lesions of psoriasis patients and correlated with the pathogenesis and severity of the disease.There may be a "self-reactive T cells-- cytokines--K17" loop in the pathogenesis of psoriasis. In this loop K17 activates autoreactive T cells to secrete cytokines such as interleukin-22(IL-22), γ- interferon(IFN-γ), etc., causing local inflammation and immune epidermal hyperplasia. It also can increase the expression of K17 KC and further activation of autoreactive T cells, creating a vicious loop,which makes a mutually reinforcement and worsen the development of psoriasis.Most patients of psoriasis are treated with local treatment. Currently psoriasis medications mainly include vitamin D3 analogues, calcipotriol, and corticosteroids. But these drugs did not achieve the expected results. So, how to maximize the local therapeutic effect has become a big challenge for drug development. Arctigenin is a lignans monomer which is isolated from the Compositae Arctium. Modern pharmacology proves that arctigenin has multiple effects like anti-inflammatory, anti-tumor, antioxidant, hypoglycemic and inhibition of T cell proliferation, etc. It’s not known whether it could show any therapeutic effect on skin psoriasis, If the skin reagent of this drug were effective, it may accelerate the clinical application of this monomer, avoiding the toxic effects of this drug in-vivo.Imiquimod is a small molecule immunomodulator of imidazoquinolines class. Its main role is anti-genital warts, anal warts, actinic keratosis and basal cell carcinoma. Imiquimod cream on the skin can cause a variety of immunological changes in the skin, thus it is recognized as the world’s classic model for psoriasis establishment. So, this study used imiquimod to establish the pathogenic model of psoriasis, and obvserved the therapic effect of arctigenin and discussed the potential mechanism.Objectives: To investigate the effect of arctigenin on psoriasis-like skin model induced by imiquimod; and to study the specific molecular mechanisms of arctigenin regulating expression of K17 by keratinocyte.Methods:1)Detecting different penetration enhancers on arctigenin permeability and screening optimal penetration enhancer agents to prepare arctigenin cream;2)Using imiquimod-induced psoriasis-like skin model and investing it in smear arctigenin treatment;3)The organizational structure of each group was observed by HE staining and their disease severity score was analyzed in animal experiments.4)Real-time quantitative polymerase chain reaction(Real-time PCR), western-blot detection were used to detect psoriatic lesions from mice to analysis the impact of k17 expression of arctigenin imiquimod-induced psoriasis-like skin model;5)Using MTT assay to detect changes on KC proliferation level by arctigenin. KC cell cycle and apoptosis was tested by flow cytometry.6)Using IFN-γ and TNF-α stimulating KC cell, building K17 high expression model, after 24 h treatment with arctigenin, by Real-time PCR and western-blot detecting changes on K17 m RNA and protein expression.Results: Arctigenin cream with best permeability was prepared. A mouse model of psoriasis induced by imiquimod was constructed successfully. Arctigenin significantly improved disease severity score rating compared with the control group. The protein expression level of k17 in treatment group was significantly decreased, while AMPK phosphorylation levels gradually increased. The arctigenin inhibited KC cell proliferation in a dose-dependent manner. In flow cytometry experiments, KC cell cycle arrest and apoptosis changed significantly with dose dependence. K17 m RNA and protein expression levels in the cell model group was significantly higher than the control after stimulation by 300 U / m L IFN-γ group and 500 ng / m L TNF-α, and there is significant difference(P <0.05). K17 expression induced by IFN-γ and TNF-α were inhibited in arctigenin group after 24 h of KCs stimulation.Conclusions: Arctigenin could alleviated the psoriasis-like skin development induced by imiquimod. The potential mechanism was by inhibition of the proliferation of KC, which accelarated the apoptosis of KC. Arctigenin can downgrade K17 expression, which may elucidate the regulatory mechanism of activating AMPK, indicating the psoriasis may be associated with K17. This study observed the therapeutic effect of arctigenin on the psoriasis-like skin. We also demonstrated the potential molecular mechanism and provided the theoretic basis of applying the medicine into clinical therapy.