[關鍵詞]
[摘要]
目的 利用網絡藥理學、分子對接技術和細胞實驗驗證,探討藏紅花素改善阿爾茨海默?。ˋlzheimer’s disease,AD)的作用機制。方法 運用Super-PRED、DisGenet、Genecards、Uniprot、STRING數據庫構建藏紅花素與AD的相互作用網絡,導入Cytoscape軟件根據連接程度篩選樞紐基因,并進行基因本體(gene ontology,GO)功能及京都基因與基因組百科全書(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析,應用Autodock vina軟件進行分子對接驗證。體外采用脂多糖(lipopolysaccharide,LPS)誘導BV2小膠質細胞炎癥模型,給予藏紅花素干預后,采用CCK-8法檢測細胞活力,qRT-PCR檢測腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、白細胞介素-1β(interleukin-1β,IL-1β)、IL-6 mRNA表達,Western blotting檢測磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)通路相關蛋白表達。結果 網絡藥理學結果顯示,藏紅花素可能通過調控PI3K-Akt信號通路、缺氧誘導因子-1(hypoxia inducible factor-1,HIF-1)信號通路等,作用于熱休克蛋白90AA1(heat shock protein 90 alpha family class A member 1,HSP90AA1)、核因子-κB(nuclear factor-κB,NF-κB)、Toll樣受體4(Toll-like receptor 4,TLR4)、磷脂酰肌醇3-激酶調節(jié)亞基1(phosphatidylinositol 3-kinase regulatory subunit 1,PIK3R1)、磷脂酰肌醇3-激酶催化亞基α(phosphatidylinositol 3-kinase catalytic subunit α,PIK3CA)、E1A結合蛋白p300(E1A-binding protein p300,EP300)等靶點,發(fā)揮治療AD的作用。分子對接結果顯示藏紅花素與PIK3R1靶點對接效果良好。細胞實驗結果顯示,藏紅花素顯著抑制LPS誘導的BV2細胞中炎癥因子表達(P<0.05、0.01),促進PI3K、Akt、GSK-3β的磷酸化(P<0.05)。結論 藏紅花素具有改善小膠質細胞極化、抗神經炎癥的作用,其作用機制與激活PI3K/Akt/GSK-3β通路有關。
[Key word]
[Abstract]
Objective To investigate the mechanism of crocin in improving Alzheimer’s disease (AD) by network pharmacology, molecular docking and cell experiments. Methods The interaction network between crocin and AD was constructed using Super PRED, DisGenet, Genecards, Uniprot and STRING databases. Hub genes were screened based on connectivity using Cytoscape software, and gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed. Molecular docking validation was performed using Autodock vina software. In vitro, a BV2 microglial inflammation model was induced by lipopolysaccharide (LPS). After intervention with crocin, cell viability was detected by CCK-8 assay, mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 was detected by qRT-PCR, Western blotting was used to detect protein expressions related to the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway. Results The results of network pharmacology showed that crocin may act on heat shock protein 90AA1 (HSP90AA1), nuclear factor-κB (NF-κB), Toll-like receptor 4 (TLR4), phosphatidylinositol 3-kinase regulatory subunit 1 (PIK3R1), and phosphatidylinositol 3-kinase catalytic subunit α (PIK3CA) and E1A binding protein p300 (EP300), regulate PI3K-Akt signaling pathway, hypoxia inducible factor-1 (HIF-1) signaling pathway, thereby exert therapeutic effects on AD. The molecular docking results showed that crocin had a good docking effect with PIK3R1. The cell experiment results showed that crocin significantly inhibited the expressions of inflammatory factors in LPS-induced BV2 cells (P < 0.05, 0.01), and promoted the phosphorylation of PI3K, Akt and GSK-3β (P < 0.05). Conclusion Crocin has the effects of improving microglial polarization and anti-neuroinflammation, and its mechanism is related to the activation of PI3K/Akt/GSK-3β pathway.
[中圖分類號]
R285.5
[基金項目]
江蘇省自然科學基金青年項目(BK20240740);南京中醫(yī)藥大學養(yǎng)老服務與管理學院專項研究項目(2024YLFWYGL012)