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贾子君,周庆兵,张艳,徐凤芹.基于网络药理学和体外实验探讨瓜蒌-薤白药对抗动脉粥样硬化的作用机制[J].湖南中医药大学学报,2023,43(11):2061-2070[点击复制] |
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| 基于网络药理学和体外实验探讨瓜蒌-薤白药对抗动脉粥样硬化的作用机制 |
| 贾子君,周庆兵,张艳,徐凤芹 |
| (中日友好医院, 北京 100029;中国中医科学院西苑医院老年医学研究所, 北京 100091) |
| 摘要: |
| 目的 通过网络药理学和体外实验探讨瓜蒌-薤白药对(Gualou-Xiebai herb pair, GXHP)抗动脉粥样硬化(atherosclerosis, AS)的作用机制。方法 利用TCMSP、DrugBank、GeneCards和Therapeutic Target等数据库筛选并收集GXHP的潜在成分、抗AS效应靶点;通过Cytoscape软件将药物靶点与疾病靶点进行网络化展示,通过网络拓扑算法筛选出关键靶基因;并通过STRING数据平台构建蛋白质-蛋白质相互作用(protein-protein interaction, PPI)网络;京都基因和基因组数据库(Kyoto encyclopedia of genes and genomes, KEGG)分析GXHP抗AS所涉及的核心通路。采用CCK-8法检测不同浓度GXHP对Raw264.7细胞的增殖抑制情况;以氧化型低密度脂蛋白(oxidized-low density lipoprotein, ox-LDL)处理Raw264.7细胞48 h,形成泡沫细胞;将细胞分为5组,分别为空白组(无处理)、模型组(80 μg/mL ox-LDL处理)、GXHP低剂量组(80 μg/mL ox-LDL+0.2 g/L GXHP)、GXHP中剂量组(80 μg/mL ox-LDL+0.6 g/L GXHP)和GXHP高剂量组(80 μg/mL ox-LDL+1.8 g/L GXHP);气相色谱质谱法(gas chromatography-mass spectrometry, GC-MS)和ELISA法分别检测各组总胆固醇(total cholesterol, TC)、游离胆固醇(free cholesterol, FC)以及炎症因子白细胞介素-6(interleukin-6, IL-6)和细胞间黏附分子-1(intercellular adhesion molecule-1, ICAM-1)的表达;Western blot法检测各组MAPK信号通路核心蛋白,包括磷酸化细胞外信号调节激酶(phosphorylated-extracellular signal-regulated kinases, p-ERK)、磷酸化p38丝裂原活化蛋白激酶(phosphorylated-p38 mitogen-activated protein kinase, p-p38MAPK)和磷酸化细胞核因子p65(phosphorylated-p65, p-p65)的表达。结果 确定GXHP含有21种成分,包括槲皮素、亚麻酸乙酯、香叶木素等;21种成分分别对应154个作用靶点和36个核心靶点;KEGG分析发现核心靶点参与多种信号通路,包括脂质和AS、MAPK和PI3K-Akt等信号通路。体外实验结果显示,GXHP浓度在4 g/L时可显著抑制Raw264.7细胞的增殖(P＜0.01),并选用1.8、0.6、0.2 g/L作为后续实验处理浓度。与空白组比较,模型组可见大量橘红色脂质颗粒,TC及FC的表达水平显著升高(P＜0.01),细胞上清液中IL-6、ICAM-1的表达水平显著升高(P＜0.01),p-ERK/ERK、p-p38 MAPK/p38 MAPK和p-p65/p65的相对表达水平明显升高(P＜0.01)。与模型组相比,GXHP高、中剂量组TC及FC的表达明显降低(P＜0.01),GXHP高、中、低剂量组IL-6的表达明显下调(P＜0.01);GXHP高、中剂量组ICAM-1的表达明显下调(P＜0.01),GXHP高、中、低剂量组中p-ERK/ERK、p-p38 MAPK/p38 MAPK和p-p65/p65相对表达水平明显降低(P＜0.05或P＜0.01)。结论 网络药理学分析联合体外实验表明,GXHP可能通过抑制MAPK信号通路实现抗AS效应。 |
| 关键词: 网络药理学 瓜蒌-薤白药对 动脉粥样硬化 泡沫细胞 MAPK信号通路 |
| DOI：10.3969/j.issn.1674-070X.2023.11.020 |
| 投稿时间：2023-02-10 |
| 基金项目:国家自然科学基金项目（81973679）；中国中医科学院科技创新工程（CI2021A01408）；中日友好医院高水平医院临床业务费专项临床研究项目（2023-NHLHCRF-BQ-13）。 |
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| The anti-atherosclerosis mechanism of the classic Gualou-Xiebai herb pair based on network pharmacology and in vitro experiments |
| JIA Zijun,ZHOU Qingbing,ZHANG Yan,XU Fengqin |
| (China-Japan Friendship Hospital, Beijing 100029, China;Institute of Geriatric Medicine, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, China) |
| Abstract: |
| Objective To investigate the mechanism of action of Gualou (Trichosanthis Fructus)-Xiebai (Allii Macrostemonis Bulbus) herb pair (GXHP) against atherosclerosis (AS) by network pharmacological analysis and in vitro experiments. Methods The databases of TCMSP, DrugBank, GeneCards, and Therapeutic Target were taken to screen and collect potential components and anti-AS disease targets of GXHP. Cytoscape was used to network display the drug targets and disease targets, and the key target genes were screened by network topology algorithm. The protein-protein interaction (PPI) network was constructed by STRING data platform. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was taken to analyze the core pathways involved in the GXHP anti-AS. CCK-8 assay was applied to measure the proliferation inhibition of Raw264.7 cells treated with different concentrations of GXHP. Raw 264.7 cells were treated with oxidized-low density lipoprotein (ox-LDL) for 48 hours to form foam cells. The cells were assigned into five groups:blank control (no treatment), model (treat with 80μg/mL ox-LDL), and low- (80 μg/mL ox-LDL and 0.2 g/L GXHP), medium-(80 μg/mL ox-LDL and 0.6 g/L GXHP), and high- (80 μg/mL ox-LDL and 1.8 g/L GXHP) dose GXHP groups. Gas chromatography-mass spectrometry (GC-MS) and ELISA were taken to measure the levels of total cholesterol (TC), free cholesterol (FC), and expressions of inflammatory factor interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in each group. Western blot was used to measure the expressions of core proteins of MAPK signaling pathway in each group, including phosphorylated-extracellular signal-regulated kinases (p-ERK), phosphorylated-p38 mitogen-activated protein kinase (p-p38MARK), and phosphorylated nuclear factor-κB p65 (p-p65). Results It was determined that GXHP contained 21 components, including quercetin, ethyl linolenic acid, diosmetin, etc. The 21 components corresponded to 154 targets of action and 36 core targets, respectively. KEGG analysis showed that the core targets were involved in multiple signaling pathways, including lipid and AS, MAPK, and PI3K-Akt. The in vitro experiments showed that the proliferation of Raw264.7 cells were significantly inhibited (P＜0.01) by GXHP with concentration of 4 g/L, and concentrations of 1.8, 0.6, and 0.2 g/L were selected as the treatment ones for subsequent experiments. Compared with blank group, a large number of orange lipid particles were observed in the model group, the expression levels of TC and FC were significantly higher (P＜0.01), the expression levels of IL-6 and ICAM-1 in the cell supernatant were significantly higher (P＜0.01), and the relative expression levels of p-ERK/ERK, p-p38 MAPK/p38 MAPK, and p-p65/p65 were significantly higher (P＜0.01). Compared with model group, the expressions of TC and FC in high- and medium-dose GXHP groups were significantly reduced (P＜0.01), the expressions of IL-6 in high-, medium- and low-dose GXHP groups were significantly lower (P＜0.01), the expressions of ICAM-1 in high- and medium-dose GXHP groups were significantly down-regulated (P＜0.01), and the relative expression levels of p-ERK/ERK, p-p38 MAPK/p38 MAPK, and p-p65/p65 in high-, medium- and low-dose GXHP groups were significantly lower (P＜0.05 or P＜0.01). Conclusion Network pharmacological analysis combined with in vitro experiments suggested that GXHP may achieve its anti-AS effects by inhibiting the MAPK signaling pathway. |
| Key words: network pharmacology Gualou-Xiebai herb pair atherosclerosis foam cell MAPK signaling pathway |
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