汤阴北艾抗神经炎症药效成分的分离鉴定及其作用机制

Isolation and mechanistic study of anti-neuroinflammatory compounds from Tangyin′s Artemisia vulgaris L.

目的:探究汤阴北艾抗神经炎症的药效成分及作用机制,为北艾抗神经炎症先导化合物的发现及药物研发提供参考。方法:通过开放柱层析和高效液相色谱分离北艾中的抗神经炎症活性化合物,并利用核磁共振(nuclear magnetic resonance,NMR)波谱学方法鉴定其结构和构型;利用CCK8法检测化合物对细胞增殖能力的影响;通过Griess法检测细胞中NO的含量,利用RT-qPCR检测炎症因子TNF-α、IL-6和IL-1β的mRNA表达水平;利用ELISA检测炎症因子TNF-α、IL-6的分泌水平;并采用Western blot检测PI3K/AKT/mTOR和NF-κB信号通路中相关蛋白的表达水平。结果:从北艾抗神经炎症活性部位中分离鉴定出了19个化合物。CCK8法检测结果表明artemvulactone U(AU)对BV-2细胞的生长抑制效果不明显,Griess法检测结果表明AU对NO的抑制能力最为显著。RT-qPCR及ELISA检测结果表明AU能抑制脂多糖(lipopolysaccharide,LPS)诱导的炎症因子TNF-α、IL-6和IL-1β的mRNA表达和分泌。Western blot实验结果发现AU可通过下调INOS和COX-2表达而缓解神经炎症,且其抗炎活性可能是通过影响PI3K/AKT/mTOR和NF-κB信号通路而发挥作用的。结论:北艾石油醚部位中的化合物artemvulactone U具有较好的抗神经炎症活性,具有进一步研究开发的潜力。

Objective:To investigate the active anti-neuroinflammatory components and mechanisms of Artemisia vulgaris L.from Tangyin,providing a theoretical basis for the discovery of lead compounds and the development of anti-neuroinflammatory drugs from this plant.Methods:Terpenoids from Artemisia vulgaris L.were isolated using open column chromatography and high-performance liquid chromatography(HPLC),and their structures and configurations were identified using nuclear magnetic resonance(NMR)spectroscopy.The CCK8 method was utilized to assess the effect of compounds on cellular proliferation capacity.The content of NO in cells was detected by the Griess assay,while the mRNA expression levels of inflammatory cytokines TNF-α,IL-6,and IL-1βwere assessed by RT-qPCR.Protein levels of TNF-αand IL-6 were determined by ELISA.Furthermore,Western Blot analysis was employed to determine the expression levels of relevant proteins within the PI3K/AKT/mTOR and NF-κB signaling pathways.Results:Nineteen compounds were isolated and identified from the active fraction of Artemisia vulgaris L.with anti-neuroinflammatory activity.Among them,artemvulactone U(AU)had no obvious inhibitory effect on the growth of BV-2 cells,as determined by the CCK-8 assay.Notably,AU also demonstrated the most significant inhibition of NO production,as evidenced by the Griess assay.RT-qPCR and ELISA results indicated that AU suppressed the LPS-induced expression of inflammatory cytokines TNF-α,IL-6,and IL-1β.Western blot analysis revealed that AU alleviated neuroinflammation by downregulating the expression of INOS and COX-2.Furthermore,Western blot analysis suggesting that its anti-inflammatory activity may be mediated through modulation of the PI3K/AKT/mTOR and NF-κB signaling pathways.Conclusion:The compound artemvulactone U isolated from the petroleum ether fraction of Artemisia vulgaris L.has good anti-neuroinflammatory activity and holds the potential for further research and development as an anti-inflammatory agent.