目的通過網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)落新婦苷治療骨關(guān)節(jié)炎的潛在機(jī)制，并通過實(shí)驗(yàn)進(jìn)行驗(yàn)證。方法從PubChem獲得落新婦苷分子化學(xué)式C₂₁H₂₂O₁₁，通過PharmMapper和Swiss Target數(shù)據(jù)庫篩選出與落新婦苷相關(guān)的靶點(diǎn)；在GeneCards、TTD、Disease Gene Search Engine 3個(gè)數(shù)據(jù)庫篩選出與骨關(guān)節(jié)炎疾病密切相關(guān)的靶點(diǎn)基因；將落新婦苷-靶點(diǎn)基因和骨關(guān)節(jié)炎-靶點(diǎn)基因的網(wǎng)絡(luò)合并，篩選出共同作用靶點(diǎn)，導(dǎo)入String數(shù)據(jù)庫，導(dǎo)入Cytoscape進(jìn)行可視化分析；將靶點(diǎn)基因上傳到DAVID（https://david.ncifcrf.gov/）進(jìn)行基因功能和KEGG通路富集分析。建立白細(xì)胞介素（IL）-1β誘導(dǎo)的人軟骨細(xì)胞模型，造模同時(shí)給予落新婦苷低、高質(zhì)量濃度（12.5、25.0 μg/mL）干預(yù)，CCK8法檢測(cè)藥物干預(yù)1、2、3 d軟骨細(xì)胞活性；Western blotting法和實(shí)時(shí)熒光定量RCR法檢測(cè)藥物干預(yù)24 h后腫瘤壞死因子（TNF）-α和G1/S-特異性周期蛋白-D1（CCND1）蛋白和mRNA表達(dá)。結(jié)果通過挖掘數(shù)據(jù)獲得377個(gè)與落新婦苷相關(guān)的潛在靶點(diǎn)和2 758個(gè)骨關(guān)節(jié)炎的潛在基因；對(duì)網(wǎng)絡(luò)進(jìn)行對(duì)接，獲得43個(gè)潛在靶基因，隨后構(gòu)建PPI網(wǎng)絡(luò)；關(guān)鍵基因?yàn)門NF、癌基因同源物（HRAS）、腫瘤標(biāo)志物熱休克蛋白90α（HSP90AAS）、淀粉樣蛋白前體蛋白（APP）和CCND1；關(guān)鍵通路為AGE/RAGE信號(hào)通路、腫瘤聚糖信號(hào)通路、MAPK信號(hào)通路、P13K-AKT信號(hào)通路、細(xì)胞增殖、凋亡等。與模型組比較，落新婦苷低、高質(zhì)量濃度組人軟骨細(xì)胞吸光度（A_{450 nm}）值均顯著升高，差異有統(tǒng)計(jì)學(xué)意義（P<0.05），TNF-α蛋白和mRNA表達(dá)均顯著降低（P<0.05），CCND1蛋白和mRNA表達(dá)均顯著升高（P<0.05）。結(jié)論落新婦苷可能通過直接作用于STAT1、STAT3、RAS等靶點(diǎn)，干預(yù)AGE/RAGE通路，從而調(diào)節(jié)TNF-α和CCND1的表達(dá)，進(jìn)而促進(jìn)軟骨細(xì)胞的增殖和抑制炎癥因子的產(chǎn)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

陜西省自然科學(xué)基礎(chǔ)研究計(jì)劃項(xiàng)目（2019JM-493）