Data Sheet 1_Aqueous extracts of Portulaca oleracea L. alleviate atopic dermatitis by restoring skin barrier function.pdf

BackgroundPortulaca oleracea L. (PO) is an edible plant with a long medicinal history in traditional Chinese medicine for various inflammatory diseases, including skin disorders such as atopic dermatitis (AD). However, the anti-inflammatory effects and AD-alleviating mechanisms of PO remain unclear.
MethodsPO aqueous extract was prepared from a water-soluble portion and then mixed with carbomer to obtain a hydrogel, which provided stable drug permeation and absorption in mouse skin. Amantadine acetate was identified as an abundant ingredient and further predicted to be a Janus kinase 1 (JAK1) inhibitor via molecular binding simulation. Mice with AD, established by repeated sensitization with 2,4-dinitrochlorobenzene (DNCB), were topically treated with PO hydrogel. Aurantiamide acetate was applied to HaCaT keratinocytes prior to inflammatory challenge in the presence or absence of JAK1-siRNA. Transcriptional and translational gene expressions associated with cutaneous inflammation or skin barriers were assessed by qPCR and Western blotting, respectively. Enzyme-linked immunosorbent assays were performed to detect immunoglobulin E and proinflammatory factors in skin tissue or serum. The phosphorylation of JAK1, signal transducer and activator of transcription (STAT)3, and STAT6 in keratinocytes and skin were analyzed by Western blotting.
ResultsIn DNCB-sensitized mice, the PO hydrogel ameliorated skin lesions, lowered symptom scores, and reduced epidermal thickness by suppressing proinflammatory factor generation, oxidative stress, and the expression of CD4+. The PO hydrogel promoted the expression of caspase-14 and filaggrin, thereby helping restore skin barrier function in AD. The PO hydrogel and/or aurantiamide acetate inhibited the enzymatic activity of JAK1 and downstream (STAT)3/STAT6 signaling pathways in vitro and in vivo.
ConclusionPO significantly ameliorated skin lesions and restored epidermal barrier function in AD mice. This was achieved by suppressing JAK1 enzymatic activity and JAK1-mediated STAT signaling pathways.

背景：马齿苋（Portulaca oleracea L.，PO）是一种药食两用植物，在传统中医药中拥有悠久的药用历史，可用于治疗多种炎症性疾病，包括特应性皮炎（atopic dermatitis，AD）这类皮肤病症。然而，马齿苋的抗炎功效及其缓解特应性皮炎的作用机制仍未明确。
方法：从水溶性组分中制备得到马齿苋水提取物，随后与卡波姆（carbomer）混合制得水凝胶，该水凝胶可在小鼠皮肤中实现稳定的药物渗透与吸收。通过分子对接模拟发现，乙酸金刚烷胺（Amantadine acetate）是马齿苋中含量丰富的成分之一，并被预测为Janus激酶1（Janus kinase 1，JAK1）抑制剂。采用2,4-二硝基氯苯（2,4-dinitrochlorobenzene，DNCB）反复致敏构建特应性皮炎小鼠模型，随后将马齿苋水凝胶局部涂抹于模型小鼠。在预先使用或不使用JAK1小干扰RNA（JAK1-siRNA）处理的情况下，将乙酸橙酰胺（Aurantiamide acetate）施加于HaCaT角质形成细胞，随后进行炎症刺激。分别通过实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction，qPCR）与蛋白质免疫印迹（Western blotting）检测与皮肤炎症或皮肤屏障相关的基因转录与翻译水平表达情况。采用酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测皮肤组织与血清中的免疫球蛋白E（immunoglobulin E，IgE）及促炎因子水平。通过蛋白质免疫印迹分析角质形成细胞与皮肤组织中JAK1、信号转导与转录激活因子（signal transducer and activator of transcription，STAT）3及STAT6的磷酸化水平。
结果：在DNCB致敏的特应性皮炎小鼠模型中，马齿苋水凝胶通过抑制促炎因子生成、氧化应激及CD4+细胞的表达，改善了皮肤皮损、降低了症状评分并减轻了表皮增厚。马齿苋水凝胶可上调半胱天冬酶-14（caspase-14）与丝聚蛋白（filaggrin）的表达，从而帮助恢复特应性皮炎小鼠的皮肤屏障功能。马齿苋水凝胶与/或乙酸橙酰胺可在体内外抑制JAK1的酶活性及其下游STAT3/STAT6信号通路。
结论：马齿苋可显著改善特应性皮炎小鼠的皮肤皮损并恢复表皮屏障功能，其作用机制为抑制JAK1的酶活性及其介导的STAT信号通路。