目的 基于网络药理学和细胞实验验证探讨地肤子皂苷Ic治疗类风湿性关节炎的作用机制。方法 使用药物靶点数据库以及疾病靶点数据库筛选出地肤子皂苷Ic和类风湿性关节炎的作用靶点，并利用GSE55235数据集筛选出类风湿性关节炎的差异表达靶点。利用韦恩交集获得共同靶点，同时使用Cytoscape构建“药物–靶点–疾病”网络图，对交集靶点进行基因本体（GO）和京都基因与基因组百科全书（KEGG）分析，并将交集靶点导入STRING数据库，以进行蛋白质相互作用（PPI）网络的分析，使用Cytoscape中的CytoNCA插件进行拓扑分析筛选核心靶点，运用AutoDock vina、PyMOL等软件对地肤子皂苷Ic和核心靶点进行对接验证。通过CCK-8法检测地肤子皂苷Ic对RA-FLS细胞的增殖影响；流式染色法检测地肤子皂苷Ic对RA-FLS细胞凋亡的影响；ELISA实验检测检测地肤子皂苷Ic对细胞炎症因子的影响；qRT-PCR实验检测核心靶点的mRNA表达变化；Western blotting实验检测白细胞（IL）-17/核因子（NF-κB）-κB通路上蛋白的表达。结果 通过网络药理学筛出45个地肤子皂苷Ic治疗类风湿性关节炎潜在靶点，KEGG通路富集分析表明IL-17/NF-κB为核心信号通路，PPI网络拓扑分析筛选出蛋白酪氨酸磷酸酶受体C（PTPRC）、脾酪氨酸激酶‌‌（SYK）、过氧化物酶体增殖物激活受体-γ‌‌（PPARG）、表皮生长因子受体（EGFR）、基质金属蛋白酶-9（MMP9）和趋化因子配体5（CCL5）6个核心靶点。分子对接显示地肤子皂苷Ic和6个核心靶点均具有显著的结合性。细胞实验结果表明，地肤子皂苷Ic处理24、48 h后，RA-FLS细胞的活力明显下降（P＜0.01、0.001）；与对照组相比，地肤子皂苷Ic 20、40、60 µmol/L组RA-FLS细胞的凋亡率显著升高（P＜0.001）。与对照组相比，地肤子皂苷Ic各浓度组肿瘤坏死因子-α（TNF-α）含量显著降低（P＜0.05、0.01、0.001）；地肤子皂苷Ic 40、60 µmol/L组IL-1β、IL-17含量显著降低（P＜0.05、0.01、0.001）；地肤子皂苷Ic 60 µmol/L组IL-6含量显著降低（P＜0.01）。与对照组相比，地肤子皂苷Ic各浓度组PPARG mRNA表达显著升高（P＜0.05）；地肤子皂苷Ic 60 µmol/L组PTPRC、SYK、MMP9、CCL5 mRNA表达显著下调（P＜0.05、0.01）；地肤子皂苷Ic 40、60µmol/L组EGFR mRNA表达显著下调（P＜0.05）。与对照组相比，地肤子皂苷Ic各浓度组细胞中Act1、HSP90AA1、TRAF6蛋白相对表达量显著降低；地肤子皂苷Ic 40、60 µmol/L组IL-17A、p-p65/p65蛋白相对表达量显著降低（P＜0.01、0.001）。结论 通过网络药理学以及细胞实验提示地肤子皂苷Ic可能通过调控IL-17/NF-κB信号通路发挥治疗类风湿性关节炎的作用。

Objective To explore the mechanism of momordin Ic in treatment of rheumatoid arthritis based on network pharmacology and cellular experimental validation. Methods To screen the action targets of momordin Ic and rheumatoid arthritis by using drug target databases and disease target databases, to filter the differentially expressed targets of rheumatoid arthritis by utilizing the GSE55235 dataset. T obtained common targets are through Venn intersection, and construct the “drug-target-disease” network diagram by using Cytoscape. Perform GO and KEGG analyses on the intersection targets, and import the intersection targets into the STRING database to conduct the analysis of PPI networks. Core targets are screened through topological analysis using the CytoNCA plugin in Cytoscape. Software such as AutoDock vina and PyMOL is used for docking verification of momordin Ic and core targets. The CCK-8 method is used to detect the effect of momordin Ic on the proliferation of RA-FLS cells; flow cytometry is used to detect the effect of momordin Ic on the apoptosis of RA-FLS cells. ELISA experiments are conducted to detect the effect of momordin Ic on inflammatory factors. qRT-PCR experiments are performed to detect changes in mRNA expression of core targets, and Western blotting experiments are conducted to detect the expression of proteins in the IL-17/NF-κB signaling pathway. Results Through network pharmacology, 45 potential targets of momordin Ic for the treatment of rheumatoid arthritis were screened. KEGG pathway enrichment analysis indicated that IL-17/NF-κB is the core signaling pathway. PPI network topology analysis identified 6 core targets, including PTPRC, SYK, PPARG, EGFR, MMP9, and CCL5. Molecular docking showed that momordin Ic has significant binding affinity with the 6 core targets. The results of the cell experiments indicated that after treatment with momordin Ic for 24 and 48 hours, the viability of RA-FLS cells significantly decreased (P < 0.01, 0.001). Compared with the control group, the apoptosis rate of RA-FLS cells in the 20, 40, and 60 μmol/L momordin Ic groups significantly increased (P < 0.001). Compared with the control group, the content of TNF-α in the momordin Ic groups at various concentrations significantly decreased (P < 0.05, 0.01, 0.001), the content of IL-1β and IL-17 in the momordin Ic 40 and 60 μmol/L groups significantly decreased (P < 0.05, 0.01, 0.001), the content of IL-6 in the momordin Ic 60 μmol/L group significantly decreased (P < 0.01). Compared with the control group, the expression of PPARG mRNA in the momordin Ic groups at various concentrations significantly increased (P < 0.05), the expression of PTPRC, SYK, MMP9, and CCL5 mRNA in the momordin Ic 60 μmol/L group significantly decreased (P < 0.05, 0.01), the expression of EGFR mRNA in the momordin Ic 40 and 60 μmol/L groups significantly decreased (P < 0.05). Compared with the control group, the relative expression levels of Act1, HSP90AA1, and TRAF6 proteins in the cells of the momordin Ic groups significantly decreased; the relative expression levels of IL-17A and p-p65/p65 proteins in the momordin Ic 40 and 60 μmol/L groups significantly decreased (P < 0.01, 0.001). Conclusion Network pharmacology and cell experiments suggest that momordin Ic may exert therapeutic effects on rheumatoid arthritis by regulating the IL-17/NF-κB signaling pathway.

R285;R287.2

忠县科技计划项目(Zxkyxm202231)