Psoriasis is a common chronic inflammatory skin disease. Keratinocytes hyperproliferation and excessive inflammatory response contribute to psoriasis pathogenesis. The agents able to attenuate keratinocytes hyperproliferation and excessive inflammatory response are considered to be potentially useful for psoriasis treatment. Daphnetin exhibits broad bioactivities including anti-proliferation and anti-inflammatory. This study aims to evaluate the anti-psoriatic potential of daphnetin in vitro and in vivo, and explore underlying mechanisms. HaCaT keratinocytes was stimulated with the mixture of IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α (M5) to establish psoriatic keratinocyte model in vitro. Cell viability was measured using Cell Counting Kit-8 (CCK-8). Quantitative Real-Time PCR (qRT-PCR) was performed to measure the mRNA levels of hyperproliferative marker gene keratin 6 (KRT6), differentiation marker gene keratin 1 (KRT1) and inflammatory factors IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. Western blotting was used to detect the protein levels of p65 and p-p65. Indirect immunofluorescence assay (IFA) was carried out to detect p65 nuclear translocation. Imiquimod (IMQ) was used to construct psoriasis-like mouse model. Psoriasis severity (erythema, scaling) was scored based on Psoriasis Area Severity Index (PASI). Hematoxylin and eosin (H&E) staining was performed to examine histological change in skin lesion. The expression of inflammatory factors including IL-6, TNF-α, IL-23A and IL-17A in skin lesion was measured by qRT-PCR. Daphnetin attenuated M5-induced hyperproliferation in HaCaT keratinocytes. M5 stimulation significantly upregulated mRNA levels of IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. However, daphnetin treatment partially attenuated the upregulation of those inflammatory cytokines. Daphnetin was found to be able to inhibit p65 phosphorylation and nuclear translocation in HaCaT keratinocytes. In addition, daphnetin significantly ameliorate the severity of skin lesion (erythema, scaling and epidermal thickness, inflammatory cell infiltration) in IMQ-induced psoriasis-like mouse model. Daphnetin treatment attenuated IMQ-induced upregulation of inflammatory cytokines including IL-6, IL-23A and IL-17A in skin lesion of mice. Daphnetin was able to attenuate proliferation and inflammatory response induced by M5 in HaCaT keratinocytes through suppression of NF-κB signaling pathway. Daphnetin could ameliorate the severity of skin lesion and improve inflammation status in IMQ-induced psoriasis-like mouse model. Daphnetin could be an attractive candidate for future development as an anti-psoriatic agent.

中文翻译：

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瑞香藤碱抑制人HaCaT角质形成细胞的增殖和炎性反应，并改善咪喹莫特诱发的小鼠牛皮癣样皮肤病变

牛皮癣是一种常见的慢性炎性皮肤病。角质形成细胞过度增殖和过度的炎症反应有助于牛皮癣的发病机理。能够减轻角质形成细胞过度增殖和过度炎症反应的药剂被认为对牛皮癣治疗可能有用。瑞香菌素具有广泛的生物活性，包括抗增殖和抗炎。这项研究旨在评估蜂胶蛋白在体外和体内的抗银屑病潜力，并探讨其潜在机制。用IL-17A，IL-22，抑瘤素M，IL-1α和TNF-α（M5）的混合物刺激HaCaT角质形成细胞，以建立银屑病角质形成细胞体外模型。使用细胞计数试剂盒8（CCK-8）测量细胞活力。进行实时定量PCR（qRT-PCR）以测量过度增生标记基因角蛋白6（KRT6），分化标记基因角蛋白1（KRT1）和炎性因子IL-1β，IL-6，IL-8， TNF-α，IL-23A和MCP-1。用蛋白质印迹法检测p65和p-p65的蛋白水平。进行了间接免疫荧光分析（IFA）以检测p65核易位。咪喹莫特（IMQ）用于构建牛皮癣样小鼠模型。根据牛皮癣区域严重程度指数（PASI）对牛皮癣的严重程度（红斑，鳞屑）进行评分。进行苏木精和曙红（H＆E）染色以检查皮肤病变的组织学变化。通过qRT-PCR测定皮肤病变中IL-6，TNF-α，IL-23A和IL-17A等炎症因子的表达。达啡肽减弱了HaCaT角质形成细胞中M5诱导的过度增殖。M5刺激显着上调IL-1β，IL-6，IL-8，TNF-α，IL-23A和MCP-1的mRNA水平。然而，蜂胶蛋白治疗部分减弱了那些炎性细胞因子的上调。发现达芙那汀能够抑制HaCaT角质形成细胞中的p65磷酸化和核易位。此外，在IMQ诱导的牛皮癣样小鼠模型中，达芙那汀可显着改善皮肤病变的严重程度（红斑，脱屑和表皮厚度，炎性细胞浸润）。达芙那汀治疗减弱了IMQ诱导的小鼠皮肤病变中炎性细胞因子（包括IL-6，IL-23A和IL-17A）的上调。达芙那汀能够通过抑制NF-κB信号通路来减弱HaCaT角质形成细胞中M5诱导的增殖和炎症反应。达啡肽可以改善IMQ诱导的牛皮癣样小鼠模型中皮肤病变的严重程度并改善炎症状态。瑞香菌素可能作为抗银屑病药物在未来的发展中具有吸引力。