目的通过网络药理学预测落新妇苷治疗骨关节炎的潜在机制，并通过实验进行验证。方法从PubChem获得落新妇苷分子化学式C₂₁H₂₂O₁₁，通过PharmMapper和Swiss Target数据库筛选出与落新妇苷相关的靶点；在GeneCards、TTD、Disease Gene Search Engine 3个数据库筛选出与骨关节炎疾病密切相关的靶点基因；将落新妇苷-靶点基因和骨关节炎-靶点基因的网络合并，筛选出共同作用靶点，导入String数据库，导入Cytoscape进行可视化分析；将靶点基因上传到DAVID（https://david.ncifcrf.gov/）进行基因功能和KEGG通路富集分析。建立白细胞介素（IL）-1β诱导的人软骨细胞模型，造模同时给予落新妇苷低、高质量浓度（12.5、25.0 μg/mL）干预，CCK8法检测药物干预1、2、3 d软骨细胞活性；Western blotting法和实时荧光定量RCR法检测药物干预24 h后肿瘤坏死因子（TNF）-α和G1/S-特异性周期蛋白-D1（CCND1）蛋白和mRNA表达。结果通过挖掘数据获得377个与落新妇苷相关的潜在靶点和2 758个骨关节炎的潜在基因；对网络进行对接，获得43个潜在靶基因，随后构建PPI网络；关键基因为TNF、癌基因同源物（HRAS）、肿瘤标志物热休克蛋白90α（HSP90AAS）、淀粉样蛋白前体蛋白（APP）和CCND1；关键通路为AGE/RAGE信号通路、肿瘤聚糖信号通路、MAPK信号通路、P13K-AKT信号通路、细胞增殖、凋亡等。与模型组比较，落新妇苷低、高质量浓度组人软骨细胞吸光度（A_{450 nm}）值均显著升高，差异有统计学意义（P<0.05），TNF-α蛋白和mRNA表达均显著降低（P<0.05），CCND1蛋白和mRNA表达均显著升高（P<0.05）。结论落新妇苷可能通过直接作用于STAT1、STAT3、RAS等靶点，干预AGE/RAGE通路，从而调节TNF-α和CCND1的表达，进而促进软骨细胞的增殖和抑制炎症因子的产生。

Objective To predict the potential mechanism of astilbin in the treatment of osteoarthritis through network pharmacology and verify it by experiments. Methods The molecular formula C₂₁H₂₂O₁₁ of astilbin was obtained from PubChem, and the targets related to astilbin were screened by PharmMapper and Swiss Target database. Three databases were used to screen out the target genes closely related to osteoarthritis. The networks of astilbin target gene and osteoarthritis target gene were combined to screen out the common targets, which were imported into String database and Cytoscape for visual analysis. The target genes were uploaded to David(https://david.ncifcrf.gov/) Gene function and KEGG pathway enrichment were analyzed. Human chondrocyte model induced by IL-1β was established, and astilbin was given at low and high doses (12.5 and 25.0 μg/mL) at the same time. CCK8 method was used to detect the activity of chondrocytes on day 1, 2 and 3. Western blotting method and real-time fluorescence quantitative RCR method were used to detect the expression of TNF-α and CCND1 protein and mRNA. Results By mining data, 377 potential targets related to astilbin and 2 758 potential genes for osteoarthritis were obtained. The network was then docked to obtain 43 potential target genes, and then a PPI network was constructed. The key genes are TNF (tumor necrosis factor), HRAS (oncogene homolog), HSP90AAS (tumor marker heat shock protein 90α), APP (amyloid precursor protein), and CCND1 (G1/S-specific cyclin-D1). The key pathways are the AGE/RAGE signaling pathway and tumor clustering. Sugar signaling pathway, MAPK signaling pathway, P13K-AKT signaling pathway, cell proliferation, apoptosis and other related pathways. Compared with model group, the absorbance (A_{450 nm}) of human chondrocytes in astilbin low-dose and high-dose groups were significantly increased (P < 0.05), the expression of TNF-α protein and mRNA were significantly decreased (P < 0.05), and the expression of CCND1 protein and mRNA were significantly increased (P < 0.05). Conclusion Astilbin may directly target STAT1, STAT3, RAS and other targets, and this target interferes with the AGE/RAGE pathway, thereby regulating the expression of TNF-α and CCND1, and proving the efficacy of astilbin in the treatment of osteoarthritis.

R965

陕西省自然科学基础研究计划项目（2019JM-493）