Table1_Fangji Dihuang formulation ameliorated DNCB-induced atopic dermatitis-like skin lesions by IL-17 signaling pathway: integrating network analysis and experimental validation.xlsx

Background: The Fangji Dihuang formulation (FJDHF) is a widely recognized Traditional Chinese Medicine (TCM) formula that consists of five plant drugs: Stephaniae Tetrandrae Radix, Cinnamomi Ramulus, Rehmanniae Radix, Saposhnikoviae Radix, and Glycyrrhiza Urensis Fisch. This formulation has been known to exhibit clinical therapeutic effects in the treatment of inflammatory skin diseases. However, there is a lack of pharmacological research on its anti-atopic dermatitis (AD) activity.
Methods: To investigate the potential anti-AD activity of FJDHF, DNCB was used to induce AD-like skin inflammation in the back of mice. Following successful modeling, the mice were administered FJDHF orally. The extent of the inflammatory skin lesions was recorded at day 4, 7, 14 and 28. UHPLC-Q-Exactive Orbitrap MS was used to identify and match the compounds present in FJDHF with ITCM, TCMIP and TCMSID. In silico predictions of potential target proteins of the identified compounds were obtained from SwishTargetPrediction, ITCM and TargetNet databases. AD-related genes were identified from GSE32924 data set, and FJDHF anti-AD hub genes were identified by MCODE algorithm. ClueGo enrichment analysis was employed to identify the core pathway of FJDHF’s anti-AD effect. To further investigate the anti-AD effect of FJDHF, single-cell RNA sequencing data set (GSE148196) from AD patients was analyzed to determine the target cells and signaling pathways of FJDHF in AD. Finally, rt-PCR, flow cytometry, and mouse back skin RNA sequencing were utilized to validate our findings.
Results: FJDHF was found to be effective in improving the degree of the AD-like lesions in the mice. Network pharmacological analysis revealed the core pathway of FJDHF to be the IL-17 signaling pathway, which is interactively associated with cytokines. Single-cell RNA sequencing analysis suggested that FJDHF may play an anti-AD role by influencing dendritic cells. Flow cytometry and rt-PCR results showed that FJDHF can reduce the influence of AD sample of IL-4, IFN-γ and the expression of IL-17. The RNA sequencing of mouse back skin also confirmed our conclusion.
Conclusion: FJDHF may inhibit DNCB-induced AD-like skin inflammation in mice by inhibiting the IL-17 signaling pathway. Thus, FJDHF can be considered as a potential therapeutic agent for AD.

背景：防己地黄方（Fangji Dihuang formulation, FJDHF）是一种广受认可的传统中药（Traditional Chinese Medicine, TCM）方剂，由五味植物药组成：粉防己（Stephaniae Tetrandrae Radix）、桂枝（Cinnamomi Ramulus）、生地黄（Rehmanniae Radix）、防风（Saposhnikoviae Radix）、甘草（Glycyrrhiza Urensis Fisch）。该方剂在炎症性皮肤病的治疗中已被证实具有临床疗效，但目前针对其抗特应性皮炎（atopic dermatitis, AD）的药理学研究仍较为匮乏。
方法：为探究防己地黄方潜在的抗特应性皮炎活性，本研究采用2,4-二硝基氯苯（DNCB）诱导小鼠背部皮肤形成特应性皮炎样炎症模型。造模成功后，对小鼠灌胃给予防己地黄方。分别于造模后第4、7、14及28天记录炎症性皮肤损伤的严重程度。采用超高效液相色谱-静电场轨道阱高分辨质谱（UHPLC-Q-Exactive Orbitrap MS）对防己地黄方中的化学成分进行鉴定，并通过ITCM、TCMIP及TCMSID数据库进行匹配。通过SwishTargetPrediction、ITCM及TargetNet数据库对鉴定得到的化合物进行潜在靶点蛋白的虚拟预测。从GSE32924数据集获取特应性皮炎相关基因，并通过MCODE算法筛选得到防己地黄方抗特应性皮炎的核心靶点基因。采用ClueGo富集分析明确防己地黄方发挥抗特应性皮炎作用的核心通路。为进一步探究防己地黄方的抗特应性皮炎效应，本研究分析了特应性皮炎患者的单细胞RNA测序数据集（GSE148196），以明确防己地黄方在特应性皮炎中作用的靶细胞及信号通路。最终采用实时荧光定量PCR（rt-PCR）、流式细胞术及小鼠背部皮肤RNA测序对研究结果进行验证。
结果：研究发现防己地黄方可有效改善小鼠特应性皮炎样皮肤损伤程度。网络药理学分析显示，防己地黄方发挥抗特应性皮炎作用的核心通路为IL-17信号通路，该通路与细胞因子存在交互调控关联。单细胞RNA测序分析提示，防己地黄方可能通过调控树突状细胞发挥抗特应性皮炎作用。流式细胞术及实时荧光定量PCR结果表明，防己地黄方可降低特应性皮炎样本中IL-4、IFN-γ的表达水平，并抑制IL-17的转录表达。小鼠背部皮肤RNA测序结果进一步验证了上述结论。
结论：防己地黄方可通过抑制IL-17信号通路，缓解2,4-二硝基氯苯诱导的小鼠特应性皮炎样皮肤炎症。因此，防己地黄方可作为治疗特应性皮炎的潜在候选药物。