目的 結(jié)合網(wǎng)絡(luò)藥理學(xué)與體外實(shí)驗(yàn)驗(yàn)證探討丹皮酚抑制血管內(nèi)皮細(xì)胞間質(zhì)轉(zhuǎn)化（EndMT）的關(guān)鍵靶點(diǎn)及作用機(jī)制。方法 通過PharmMapper、HERB、SymMap、ChEMBL、SwissTargetPredicition數(shù)據(jù)庫獲取丹皮酚的作用靶點(diǎn)；從GeneCards數(shù)據(jù)庫中檢索與EndMT相關(guān)的靶標(biāo)，將兩者進(jìn)行交集分析與可視化；將交集靶點(diǎn)基因上傳至STRING數(shù)據(jù)庫聯(lián)用Cytoscape 3.8.2軟件構(gòu)建蛋白相互作用（PPI）網(wǎng)絡(luò)并篩選核心靶點(diǎn)基因，并進(jìn)行基因本體論（GO）功能富集和京都基因與基因組百科全書（KEGG）信號通路富集分析。采用Autodock Vina軟件對丹皮酚與核心靶點(diǎn)基因進(jìn)行分子對接驗(yàn)證。體外實(shí)驗(yàn)將HUVECs細(xì)胞分為空白組、模型[轉(zhuǎn)化生長因子β1（TGF-β1）]組、丹皮酚低、中、高劑量組（30、60、120 μmol/L），通過蛋白質(zhì)印跡法（Western blotting）篩選TGF-β1誘導(dǎo)EndMT最佳濃度并通過酶聯(lián)免疫吸附測定（ELISA）檢測基質(zhì)金屬蛋白酶2（MMP-2）、基質(zhì)金屬蛋白酶9（MMP-9）的蛋白表達(dá)量。結(jié)果 共篩選出丹皮酚作用靶點(diǎn)414個(gè)，EndMT相關(guān)靶點(diǎn)基因241個(gè)，取交集后得到27個(gè)丹皮酚治療EndMT的潛在作用靶點(diǎn)。核心靶點(diǎn)與京都基因與基因組百科全書（KEGG）通路分析結(jié)果顯示，動脈粥樣硬化信號通路是丹皮酚涉及EndMT的主要通路。分子對接結(jié)果顯示，丹皮酚與MMP-2與MMP-9的結(jié)合能力最強(qiáng)。ELISA實(shí)驗(yàn)證明，丹皮酚可以減少M(fèi)MP-2、MMP-9蛋白的表達(dá)（P＜0.01）。結(jié)論 丹皮酚抑制EndMT的關(guān)鍵靶點(diǎn)是MMP-2與MMP-9，為進(jìn)一步研究丹皮酚抗動脈粥樣硬化的作用機(jī)制提供依據(jù)。

Objective To explore the key targets and mechanism of paeonol inhibiting vascular endothelial interstitial transformation (EndMT) by network pharmacology and in vitro experiments. Methods PharmMapper, HERB, SymMap, ChEMBL, and SwissTargetPredicition were used to predict the target of paeonol. GeneCards was used to search for targets related to EndMT. Paeonol and EndMT targets were used for intersection analysis visualization. The overlapped genes were mapped to the STRING database, and protein-protein interactions (PPI) were built using the STRING database and Cytoscape 3.8.2, and the core target genes were screened. The gene ontology analysis (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed. AutoDock Vina was used to conduct the molecular docking between paeonol and the core target genes. In vitro experiment, HUVECs cells were divided into blank group, model [transforming growth factor β1 (TGF-β1)] group, paeonol low-dose, medium-dose and high-dose groups (30, 60, 120 μmol/L). The optimal concentration of EndMT induced by TGF-β1 was screened by Western blotting, and the protein expressions of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) were detected by enzyme-linked immunosorbent assay (ELISA). Results A total of 414 functional targets of paeonol and 241 EndMT-related targets were screened by network pharmacology analysis. By taking the intersection, a total of 27 potential targets of the regulatory effect of paeonol on EndMT were found. The core target and KEGG pathway analyses showed that the atherosclerotic signaling pathway was the key pathway for paeonol in treatment of EndMT. Molecular docking results showed that paeonol had a better binding ability with MMP-2 and MMP-9. ELISA results indicated that paeonol could reduce the expression of MMP-2 and MMP-9 (P < 0.01). Conclusion The key targets of paeonol inhibiting EndMT are MMP-2 and MMP-9, which provides a basis for further research on the mechanism of paeonol anti-atherosclerosis.

R966

國家自然科學(xué)基金資助項(xiàng)目（82174014）