基于网络药理学探讨五味子丙素通过抑制血管内皮细胞凋亡参与动脉粥样硬化机制研究

Network-based pharmacological investigation of the involvement of Schisandra C in the mechanism of atherosclerosis through inhibition of vascular endothelial cell apoptosis

关键词:  动脉粥样硬化  五味子丙素  网络药理学  凋亡  ErbB2

Key words:  Atherosclerosis  Schizandrin C  network pharmacology  apoptosis  ErbB2

摘要:

目的 体外探讨五味子丙素(Schizandrin C,SC)防治动脉粥样硬化(Atherosclerosis,AS)的潜在作用机制。方法 网络药理预测SC抗AS作用靶点,构建PPI网络,利用David数据库对靶点进行GO、KEGG分析；HUVECs随机分为六组：对照组(细胞进行常规培养)；模型组(ox-LDL)；SC低剂量组(ox-LDL+1 μM SC)；SC中剂量组(ox-LDL+5 μM SC)；SC高剂量组(ox-LDL+1 μM SC)；阿托伐他汀组(ox-LDL+1 μM AT)；CCK-8法检测细胞毒性；NO试剂盒检测NO释放量；ELISA试剂盒检测细胞IL-6、TNF-α水平；Western blot及IF检测凋亡相关蛋白表达水平；流式细胞术检测细胞凋亡率；最后结合网络药理学结果对相关通路进行验证。结果 共获得SC抗AS的78个靶点,其中ErbB2、AKT1、MTOR为核心靶点,KEGG富集分析主要通路集中于ErbB、PI3K-AKT等信号通路。CCK8结果提示选择低于25 μM SC的药物浓度进行细胞实验；与对照组相比,模型组NO、IL-6、TNF-α水平、细胞凋亡率、Cleaved-caspase3、Cleaved-caspase3、Bax蛋白表达水平显著上升,Bcl-2、p-ErbB2蛋白表达量明显降低(P < 0.01)；与模型组相比,SC和AT组NO、IL-6、TNF-α水平、细胞凋亡率Cleaved-caspase3、Cleaved-caspase3、Bax蛋白表达水平显著降低,Bcl-2、p-ErbB2蛋白表达量明显升高(P <0.05、P < 0.01)。结论 SC可通过抑制细胞凋亡保护ox-LDL诱导的血管内皮细胞损伤进而防治AS。

Abstract:

OBJECTIVE In vitro investigation of the potential mechanism of action of Schizandrin C (SC) against atherosclerosis (AS). METHODS Network pharmacology predicted the target of SC anti-AS action, constructed the PPI network, and used David database for GO and KEGG analysis of the target; HUVECs were randomly divided into six groups: control group (cells were routinely cultured); model group (ox-LDL); SC low-dose group (ox-LDL+1 μM SC); and SC medium-dose group (ox-LDL+5 μM SC); SC high-dose group (ox-LDL+1 μM SC); atorvastatin group (ox-LDL+1 μM AT); cytotoxicity was detected by CCK-8 assay; NO release was detected by NO kit; cellular IL-6 and TNF-α levels were detected by ELISA kit; apoptosis-related protein expression level was detected by Western blot and IF; cellular apoptosis rate; finally, the relevant pathways were validated by combining the results of network pharmacology. RESULTS A total of 78 targets of SC anti-AS were obtained, among which ErbB2, AKT1 and MTOR were the core targets, and the main pathways analysed by KEGG enrichment were focused on the signalling pathways such as ErbB, PI3K-AKT, etc. The CCK8 results suggested that the drug concentration lower than 25 μM SC was selected for the cellular experiments; compared with the control group, the model group showed significantly higher levels of NO, IL-6, TNF-α, cell apoptosis rate, Cleaved-caspase3, Cleaved-caspase3, and Bax protein expression levels were significantly increased, and Bcl-2 and p-ErbB2 protein expression were significantly decreased compared with the model group ( P < 0.01); compared with the model group, the SC and AT groups showed significantly higher levels of NO, IL-6, TNF-α levels, apoptosis rate Cleaved- caspase3, Cleaved-caspase3, and Bax protein expression levels were significantly lower, and Bcl-2 and p-ErbB2 protein expression was significantly higher ( P < 0.05, P < 0.01). CONCLUSION SC may protect against ox-LDL-induced vascular endothelial cell injury and thus prevent AS by inhibiting apoptosis.