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廖兴华, 庞河, 吴航, 魏波.基于网络药理学探究黄芩苷抗骨肉瘤的作用机制及其与SP600125协同效应的研究[J].湖南中医药大学学报,2024,44(10):1845-1856[点击复制] |
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| 基于网络药理学探究黄芩苷抗骨肉瘤的作用机制及其与SP600125协同效应的研究 |
| 廖兴华,庞河,吴航,魏波 |
| (广东医科大学附属医院骨科中心, 广东 湛江 524001) |
| 摘要: |
| 目的 本研究通过网络药理学和体外实验探讨黄芩苷治疗骨肉瘤的潜在靶点和作用机制以及其与SP600125协同效应的研究。方法 运用PharmMapper、Swiss Target Prediction、 Genecards、OMIM和TTD数据库检索黄芩苷和骨肉瘤的潜在靶点。通过STRING平台构建蛋白质-蛋白质相互作用网络图和筛选核心靶点。使用R语言进行GO和KEGG富集分析。在GEO数据库筛选出骨肉瘤中的差异表达基因。采用分子对接方法评估其与核心靶点的结合潜力。利用CCK-8细胞增殖实验、细胞迁移实验、细胞凋亡检测、透射电子显微镜和Western blot验证其作用机制。结果 通过数据库确定46个黄芩苷治疗骨肉瘤的潜在靶点。通过蛋白质-蛋白质相互作用网络筛选出23个核心靶点。GO和KEGG富集分析表明，磷脂酰肌醇三激酶（phosphatidylinositol 3-kinase, PI3K）/蛋白激酶B（protein kinase B, PKB/AKT）通路和丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）通路和凋亡通路在黄芩苷治疗骨肉瘤中起着关键作用。核心靶点与骨肉瘤差异性表达基因比对的结果发现，AKT1、热休克蛋白90α （heat shock protein 90 alpha, HSP90AA1）、膜联蛋白A5（annexin A5, ANXA5）、细胞周期检验点激酶1（checkpoint kinase 1, CHEK1）和尿激酶型纤溶酶原激活剂（urokinase-type plasminogen activator, PLAU）在骨肉瘤组织中的表达高于正常骨组织。分子对接揭示黄芩苷与AKT1、HSP90AA1、ANXA5、CHEK1和PLAU靶点有较好的结合活性。体外细胞实验表明，黄芩苷抑制HOS和143B骨肉瘤细胞的增殖和迁移，并促进细胞凋亡。此外，联合使用c-Jun氨基末端激酶（c-Jun N-terminal kinase, JNK）抑制剂SP600125进一步增强了黄芩苷的抗骨肉瘤作用。透射电子显微镜显示，黄芩苷增加骨肉瘤细胞内自噬小体的数量，但与SP600125联合时，黄芩苷诱导的自噬小体的增多会受到抑制。Western blot分析结果表明，黄芩苷抑制了AKT1蛋白的磷酸化和p-AKT/AKT表达水平，并与SP600125共处理可增强该抑制作用。另外，黄芩苷可诱导骨肉细胞内LC3-II和p62以及p-JNK/JNK的表达，但与SP600125联用会显著抑制此效应。结论 黄芩苷通过多靶点和多通路的相互作用发挥抗骨肉瘤效应。此外，SP600125协同增强了黄芩苷治疗骨肉瘤的效应，为骨肉瘤的治疗提供了研究依据和理论支持。 |
| 关键词: 骨肉瘤 黄芩苷 网络药理学 蛋白激酶B1 自噬 SP600125 |
| DOI：10.3969/j.issn.1674-070X.2024.10.018 |
| 投稿时间：2024-01-17 |
| 基金项目:广东省自然科学基金项目(2020A1515010003);2021年度广东省科技专κ金("大专项+任务清单")竞争性分配项目(2021A05234)。 |
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| Mechanism of action of baicalin against osteosarcoma and its synergistic effects with SP600125 based on network pharmacology |
| LIAO Xinghua, PANG He, WU Hang, WEI Bo |
| (Orthopedic Center, Hospital of Guangdong Medical University, Zhanjiang, Guangdong 524001, China) |
| Abstract: |
| Objective To investigate the potential targets and mechanism of action of baicalin in treating osteosarcoma and its synergistic effects with SP600125 by network pharmacology and in vitro experimentation. Methods Using PharmMapper, Swiss Target Prediction, Genecards, OMIM, and TTD databases, potential targets for baicalin and osteosarcoma were retrieved. A protein-protein interaction network was constructed and core targets were screened through the STRING platform. GO and KEGG enrichment analyses were performed using R language. Differentially expressed genes in osteosarcoma were identified from the GEO database. Molecular docking was employed to assess the binding potential with the core targets. The mechanism of action was validated through CCK8 cell proliferation assays, cell migration assays, apoptosis detection, transmission electron microscopy, and Western blot. Results Through database analysis, 46 potential targets for baicalin in the treatment of osteosarcoma were identified. Among these, 23 core targets were screened via the protein-protein interaction network. GO and KEGG enrichment analyses indicated that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) pathway, the mitogen-activated protein kinase (MAPK) pathway, and the apoptosis pathway play pivotal roles in the treatment of osteosarcoma with baicalin. By comparing the core targets with differentially expressed genes in osteosarcoma, it was found that AKT1, heat shock protein 90 alpha (HSP90AA1), annexin A5 (ANXA5), checkpoint kinase 1 (CHK1), and urokinase-type plasminogen activator (PLAU) were expressed at higher levels in osteosarcoma tissues than in normal bone tissues. Molecular docking revealed that baicalin exhibited good binding activity with AKT1, HSP90AA1, ANXA5, CHK1, and PLAU targets. In vitro cell experiments demonstrated that baicalin inhibited the proliferation and migration of HOS and 143B osteosarcoma cells while promoting cell apoptosis. Furthermore, the combination with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 further enhanced the anti-osteosarcoma effects of baicalin. Transmission electron microscopy showed that baicalin increased the number of autophagosomes within osteosarcoma cells, but this increase was inhibited when combined with SP600125. Western blot analysis indicated that baicalin inhibited the phosphorylation of AKT1 protein and reduced the p-AKT/AKT expression level, and cotreatment with SP600125 enhanced this inhibitory effect. Additionally, baicalin induced the expression of LC3-II, p62, and p-JNK/JNK in osteosarcoma cells, but this induction was significantly suppressed when combined with SP600125. Conclusion Baicalin exerts anti-osteosarcoma effects through interaction with multiple targets and pathways. Furthermore, SP600125 synergistically enhances the therapeutic efficacy of baicalin in treating osteosarcoma, providing research evidence and theoretical support for the treatment of this disease. |
| Key words: osteosarcoma baicalin network pharmacology protein kinase B1 autophagy SP600125 |
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