Network Pharmacology-based Analysis of the Anti-atherosclerosis Mechanism of Scutellarin and Experimental Validation in Vivo
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摘要:
目的 基于网络药理学分析灯盏乙素（Scutellarin） 治疗动脉粥样硬化（atherosclerosis，AS） 潜在的分子生物学机制。 方法 通过PubChem数据库进行灯盏乙素潜在的靶点预测，运用Disgenet，CeneCards筛选与AS关联的靶基因，利用Cytoscape 3.9.0建立疾病-靶点-成份可视化网络，使用String构建蛋白互作网络图，利用David数据库进行GO及KECG的网络构建。另外，高脂喂养复制APOE-/-小鼠AS模型， 用不同浓度的灯盏乙素灌胃治疗，利用RT-PCR方法验证筛选所得核心通路的表达变化。 结果 （1）网络药理学预测结果：灯盏乙素治疗AS的核心靶点共有肿瘤坏死因子-α （TNF-α），AKT丝氨酸/苏氨酸激酶（AKT serine/threonine kinase，MAPK），低密度脂蛋白受体（Low density lipoprotein receptor），载脂蛋白E（apolipoprotein E，APOE）等共106个。灯盏乙素调控PI3K/AKT信号通路（hsa04151）、Ras信号通路（hsa04014）、MAPK信号通路（hsa04010）（P < 0.05）等涉及细胞的增殖，炎症反应，氧化应激，及细胞凋亡等生物学过程来治疗AS；（2） 体内实验验证的结果：和模型组相比，灯盏乙素组呈浓度依耐性地下调PI3K、AKT、mTOR和Bcl-2 的mRNA表达，上调Bax和Caspase-3的mRNA表达，（ P < 0.05）。另外，和模型组相比，灯盏乙素则呈浓度依耐性地下调小鼠血清TNF-α和IL-1β的含量（ P < 0.05）。 结论 经过网络药理学筛选和RT-PCR验证，灯盏乙素通过调控PI3K/AKT/mTOR信号通路抑制APOE-/-小鼠AS模型炎症反应，促进细胞凋亡而治疗AS。 -
关键词:
- 网络药理学 /
- 灯盏乙素 /
- 动脉粥样硬化 /
- APOE-/- 小鼠AS模型 /
- PI3K/AKT/mTOR
Abstract:Objective To analyze the potential molecular biological mechanism of scutellarin for the treatment of atherosclerosis （AS） based on network pharmacology. Methods The PubChem database was used to predict the potential targets of scutellarin, and the Disgenet and CeneCards databases were used to predict and screen the genes related to atherosclerosis, and the Cytoscape 3.7.0 software was used to establish and construct the component-target-disease network visualization network. The GO and KECG pathways were analyzed using the David database. In addition, an APOE-/- mouse AS model was developed and treated with different concentrations of scutellarin, and the expression changes of the core targets were verified by RT-PCR. Results 1. Network pharmacological prediction results: the core targets of Scutellarin for AS treatment were tumor necrosis factor-α （TNF-α）, AKT serine/threonine kinase 1 （MAPK）, low density lipoprotein receptor （LDLR）, apolipoprotein E （APOE）, among other targets, with 106 in total. The key targets of Scutellarin are PI3K/AKT signaling pathway （hsa04151）, Ras signaling pathway （hsa04014） and MAPK signaling pathway （hsa04010） （P < 0.05）, which are involved in cell proliferation, inflammatory response, oxidative stress and apoptosis, to treat AS. 2. Results of in vivo experiments: compared with the model group, the mRNA expression of PI3K, AKT, mTOR and Bcl-2 was down-regulated and the mRNA expression of Bax and Caspase-3 was up-regulated in the Scutellarin group. In addition, compared with model group, Scutellarin down-regulated the contents of TNF-α and IL-1β in serum concentration-dependent （P < 0.05）. Conclusion Through network pharmacology screening and RT-PCR verification, Scutellarin inhibits the process of inflammatory response in a mouse model of AS by regulating the PI3K/AKT/mTOR signaling pathway. -
Key words:
- Network pharmacology /
- Scutellarin /
- Atherosclerosis /
- APOE-/- mice AS model /
- PI3K\AKT\mTOR
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图 3 灯盏乙素-动脉粥样硬化-靶点网络
Figure 3. Intersection of Scutellarin predicted targets and atherosclerotic targets
图 4 灯盏乙素治疗动脉粥样硬化潜在靶点的PPI网络
Figure 4. PPI network of potential targets of Scutellarinl on anti-AS
图 6 灯盏乙素治疗动脉粥样硬化潜在靶点的GO富集分析
Figure 6. GO enrichment analysis of potential targets of Scutellarin for anti-atherosclerosis
图 7 灯盏乙素治疗AS潜在靶点的KEGG通路分析
Figure 7. KEGG pathway analysis of potential targets of Scutellarin on anti- atherosclerosis
图 8 HE染色结果 （200×）
A：正常对照组；B：模型组；C：Scutellarin 7 mg/kg组；D：Scutellarin 14 mg/kg组；E：Scutellarin 28 mg/kg组；F：ATV 10 mg/kg组。
Figure 8. HE staining results （200×）
图 9 灯盏乙素对PI3K/AKT/mTOR信号通路及炎症因子的影响[（
$\bar x \pm s $ ），n = 6）]A-a：PI3K的mRNA 表达；A-b：AKT的mRNA 表达；A-c：mTOR的mRNA 表达。B-a：Bax的mRNA 表达；B-b：Bcl-2的mRNA 表达；B-c：Caspase-3的mRNA 表达。C： 小鼠血清TNF-α的含量。D：小鼠血清IL-1β的含量。与空白对照组相比较，*P < 0.05，** P < 0.01;与模型组比较，#P < 0.05，#P < 0.01。
Figure 9. Effects of Scutellarin on PI3K/AKT/mTOR signaling pathway-related genes and inflammatory factors[（
$\bar x \pm s $ ），n = 6）]表 1 实验涉及的部分数据库
Table 1. Main databases involved in the experiment
名称 网址 用途 Pubchem https://pubchem.ncbi.nlm.nih.gov/ 化合物相关信息下载 Swiss http://www.swisstargetprediction.ch/ 药物相关靶点检索 Pharm mapper http://www.lilab-ecust.cn/pharmmapper/ 药物相关靶点检索 Genecards https://www.genecards.org/ 疾病相关靶点检索 Disgenet https://www.disgenet.org/ 疾病相关靶点检索 Venny https://bioinfogp.cnb.csic.es/tools/venny/ 疾病-药物靶点交集图 Uniprot https://www.uniprot.org/ 靶点名称翻译 David https://david.ncifcrf.gov/ KEGG、GO分析 String https://string-db.org/ 蛋白质相互作用网络 微生信平台 http://www.bioinformatics.com.cn/ 数据处理 Cytoscape v3.9.0软件 https://cytoscape.org/ 数据处理及绘图 
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表 2 引物序列
Table 2. The primer sequence
引物名称 长度（bp） 引物序列5′→3′ PI3K 20 Forward primer：CTTATGTCCTTGGCATTGGT Reverse primer：CAAAGTCTATGTGGAAGAGCTG AKT1 20 Forward primer：TCACCCAGTGACAACTCAG Reverse primer：AAACTCGTTCATGGTCACAC Bcl-2 19 Forward primer：TGACTGAGTACCTGAACCG Reverse primer：TAGTTCCACAAAGGCATCC Bax 19 Forward primer：CTGCAGAGGATGATTGCTG Reverse primer：GTCTGCAAACATGTCAGCT Caspase-3 21 Forward primer：AACTCTTCATCATTCAGGCCT Reverse primer：CCATATCATCGTCAGTTCCAC β-actin 20 Forward primer：CTTCCTGGGTATGGAATCCT Reverse primer：TCTTTACGGATGTCAACGTC mTOR 20 Forward primer：GCAGATTTGCCAACTATCTTCGG Reverse primer：CAGCGGTAAAAGTGTCCCCTG
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