【摘要】 目的 基于网络药理学与体外细胞实验探讨杜仲治疗肝细胞癌（HCC）的潜在作用机制。方法（1）通过TCMSP数据库检索杜仲的有效成分及作用靶点，通过TCGA数据库下载HCC相关基因表达谱数据和相应患者的临床资料。利用R语言软件进行HCC的差异表达基因分析，将杜仲有效成分的作用靶点和HCC差异表达基因取交集，获得杜仲治疗HCC的潜在作用靶点。针对潜在作用靶点，进行富集分析；筛选有预后预测价值的基因，构建风险评分模型；绘制“杜仲-有效成分-作用靶点-HCC”网络图和蛋白-蛋白相互作用（PPI）网络图，筛选主要有效成分和关键作用靶点，进行分子对接验证。（2）采用CCK-8和划痕实验检测不同浓度槲皮苷对肝癌细胞HCCLM3细胞活力和迁移能力的影响。采用实时荧光定量PCR和Western blot检测不同浓度槲皮苷对Serpin家族E成员1(SERPINE1)mRNA和蛋白表达水平的影响。结果（1）获得60个杜仲治疗HCC的潜在作用靶点，主要涉及对外源刺激的反应、突触后膜、肾上腺素能受体活性、白细胞介素17信号通路和肿瘤坏死因子信号通路等多种生物过程及信号通路。进一步筛选出11个有预后预测价值的基因，风险评分=KCNH2×0.007825+EGF×0.0423204+HMOX1×0.000572+CCNB1×0.020393+SERPINE1×0.000500+NQO1×0.000701+CLDN4×0.000448+SPP1×0.000025+PON1×0.001001+HK2×0.00667+CHEK1×0.018271。“杜仲-有效成分-作用靶点-疾病”网络图显示槲皮苷、山柰酚、表奎宁定和（-）-Tabernemontanine为杜仲治疗HCC的主要有效成分。PPI网络显示，SERPINE1是杜仲治疗HCC的关键作用靶点，同时也是构建风险评分模型的基因。分子对接结果显示，槲皮苷与SERPINE1可相互结合且对接分数最高。（2）与对照组（0μmol/L槲皮苷）相比，高于25μmol/L浓度的槲皮苷可抑制肝癌细胞HCCLM3细胞活性，25μmol/L、100μmol/L、400μmol/L浓度的槲皮苷可抑制肝癌细胞HCCLM3细胞的迁移能力，100μmol/L、400μmol/L浓度的槲皮苷可抑制SERPINE1的mRNA和蛋白表达水平（P<0.05）。结论 杜仲主要有效成分槲皮苷能够抑制肝癌细胞HCCLM3的活性和迁移能力，并下调SERPINE1的表达。更多还原

【Abstract】 Objective To explore the potential mechanism of Eucommiae cortex for the treatment of hepatocellular carcinoma(HCC) based on network pharmacology and in vitro cell experiment. Methods(1) The effective components and effect targets of Eucommiae cortex were retrieved from TCMSP database, and HCC-related gene expression profile data and clinical data of corresponding patients were downloaded from TCGA database. The R language software was adopted to analyze the differentially expressed genes in HCC, and the intersection of the effect targets of effective components of Eucommiae cortex and the differentially expressed genes in HCC was taken to obtain the potential effect targets of Eucommiae cortex for the treatment of HCC. Enrichment analysis was performed for potential effect targets. Genes with prognostic predictive value were screened and a risk score model was constructed. The Eucommiae cortex-effective component-effect target-HCC network and protein-to-protein interaction(PPI) network chart were drawn, and the main effective components and key effect targets were screened for molecular docking validation.(2) CCK-8 assay and scratch assay were used to detect the effects of quercetin at different concentrations on hepatoma cell HCCLM3 cell viability and migration. The effects of quercetin at different concentrations on Serpin family E menber 1(SERPINE1) mRNA and protein expressions were detected by the real-time fluorescent quantitative PCR and Western blot. Results(1) A total of 60 potential effect targets of Eucommiae cortex for the treatment of HCC were obtained. These potential effect targets were primarily involved in various biological processes and signaling pathways such as response to external stimuli, postsynaptic membrane, adrenergic receptor activity, interleukin-17 signaling pathway, and tumor necrosis factor signaling pathway. Through further screening, 11 genes with prognostic predictive value were selected. The risk score was calculated as:risk score=KCNH2×0.007825+EGF×0.0423204+HMOX1×0.000572+CCNB1×0.020393+SERPINE1×0.000500+NQO1×0.000701+CLDN4×0.000448+SPP1×0.000025+PON1×0.001001+HK2×0.00667+CHEK1×0.018271. Eucommiae cortex-effective components-effect targets-disease network diagram revealed that quercetin, kaempferol, epiquinidine, and(-)-Tabernemontanine were the main effective components of Eucommiae cortex for the treatment of HCC. PPI network demonstrated that SERPINE1 served as both the key effect target of Eucommiae cortex for the treatment of HCC and one of the genes included in the risk score model construction. Molecular docking results indicated that quercetin could bind with SERPINE1 and achieved the highest docking score.(2) Compared with the control group(0 μmol/L quercetin), quercetin at concentrations higher than 25 μmol/L inhibited the cell viability of hepatoma cell HCCLM3, and the cell migration ability of hepatoma cell HCCLM3 was inhibited by quercetin at concentrations of 25 μmol/L, 100 μmol/L and 400 μmol/L. Quercetin at 100 μmol/L and 400 μmol/L concentrations inhibited the mRNA and protein expressions of SERPINE1(P<0.05). Conclusion Quercetin, the main effective component of Eucommiae cortex, can inhibit the activity and migration of hepatoma cell HCCLM3 and down-regulate the expression of SERPINE1.更多还原