摘要
背景：清营汤（Qingying decoction，QYD）是中国的一种传统处方，已被证明对治疗银屑病有效。然而，其作用机制仍有待阐明：方法：分别从 TCMSP 数据库、HERB 数据库和 SwissTargetPrediction 数据库中获取逍遥散的有效成分和靶点。采用差异表达基因（DEGs）分析和加权基因共表达网络分析（WGCNA）确定与银屑病相关的关键基因。使用 STRING 平台构建了蛋白质-蛋白质相互作用（PPI）网络。基因本体（GO）和京都基因组百科全书（KEGG）分析使用DAVID数据库和R软件的clusterProfiler软件包进行。使用 Cytoscape 3.9.0 软件筛选 QYD 的关键成分和枢纽靶标。分子对接用于检测关键成分与枢纽靶标的结合能力。用五种促炎细胞因子（IL-17 A、IL-22、IL-1α、oncostatin M 和 TNF-α）的混合物（M5）刺激角质形成细胞 HaCaT，建立了银屑病的体外模型。细胞活力和细胞周期分别用细胞计数试剂盒 8（CCK-8）和流式细胞仪测量。实时定量聚合酶链反应（qRT-PCR）用于检测枢纽基因、高增殖标志物角蛋白 6（KRT6）和炎症因子 IL-1β、IL-6 和 TNF-α 的 mRNA 水平。通过 Western 印迹检测了 PI3K/AKT/FoxO 通路相关靶点的蛋白表达水平：结果：本研究共筛选出139种喹乙醇活性成分，1033个靶点，其中59个靶点与银屑病相关基因重叠。槲皮素、木犀草素、山柰酚、β-谷甾醇和甲硫代芒果酮 A 被认为是喹乙醇治疗银屑病的关键成分。CDC25A、TOP2A、NEK2 和 CCNA2 被确定为中心靶点。QYD 可能可以调节细胞周期、T 细胞受体信号通路和代谢通路，从而治疗银屑病。QYD的主要成分与枢纽靶蛋白具有良好的结合亲和力。QYD能明显抑制M5诱导的HaCaT细胞过度增殖和细胞周期进展。M5 刺激可明显上调 CDC25A、TOP2A、NEK2、CCNA2、IL-1β、IL-6 和 TNF-α 的 mRNA 水平，而 QYD 可逆转这一效应。此外，QYD还能抑制M5刺激的HaCaT细胞中PI3K和AKT的磷酸化，并上调p-FOXO1蛋白的表达水平：结论：QYD可通过调节PI3K/AKT/FoxO通路抑制角朊细胞的过度增殖和炎症反应，这表明QYD可能是治疗银屑病的一种有吸引力的处方。Background: Qingying decoction (QYD) is a traditional prescription in China that has been shown to be effective in treating psoriasis. However, its mechanism of action remains to be elucidated.
Methods: The active ingredients and targets of QYD were obtained from TCMSP database, HERB database and SwissTargetPrediction database, respectively. Differential expression gene (DEGs) analysis and weighted gene co-expression network analysis (WGCNA) were used to identify key genes associated with psoriasis. Protein-protein interaction (PPI) network was constructed using STRING platform. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the DAVID database and the clusterProfiler package of R software. Cytoscape 3.9.0 software was used to screen the key components of QYD and the hub targets. Molecular docking was used to detect the binding ability between key components and hub targets. An in vitro model of psoriasis was established by stimulating keratinocyte HaCaT with a mixture of five pro-inflammatory cytokines (IL-17 A, IL-22, IL-1α, oncostatin M, and TNF-α) (M5). Cell viability and cell cycle were measured using cell counting Kit 8 (CCK-8) and flow cytometry, respectively. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect mRNA levels of hub genes, high-proliferation marker keratin 6 (KRT6) and inflammatory factors IL-1β, IL-6 and TNF-α. Protein expression levels of PI3K/AKT/FoxO pathway related targets were detected by Western blot.
Results: A total of 139 active ingredients of QYD were screened in this study, with 1033 targets, 59 of which overlapped with psoriasis-related genes. Quercetin, luteolin, kaempferol, beta-sitosterol and methylophiopogonanone A were considered to be the key ingredients of QYD in the treatment of psoriasis. CDC25A, TOP2A, NEK2 and CCNA2 were identified to be the hub targets. QYD could probably regulate cell cycle, T cell receptor signaling pathway and metabolic pathway to treat psoriasis. The key components of QYD had good binding affinity with hub target proteins. QYD significantly attenuated M5-induced hyperproliferation and cell cycle progression of HaCaT cells. M5 stimulation significantly upregulates the mRNA levels of CDC25A, TOP2A, NEK2, CCNA2, IL-1β, IL-6 and TNF-α, while QYD treatment reversed this effect. In addition, QYD treatment inhibited the phosphorylation of PI3K and AKT in M5-stimulated HaCaT cells and upregulated p-FOXO1 protein expression level.
Conclusion: QYD can inhibit the excessive proliferation and inflammatory response of keratinocytes by regulating the PI3K/AKT/FoxO pathway, suggesting that QYD may be an attractive prescription for psoriasis.
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