摘要
目的:探讨山楂叶金丝桃苷(hyperoside,Hyp)对脂多糖(lipopolysaccharides,LPS)诱导巨噬细胞RAW264.7炎症反应的抑制作用及其可能的作用机制。方法:采用MTT法观察Hyp对脂多糖(LPS)诱导的RAW264.7巨噬细胞的影响,通过Griess法和ELISA法分别测定上清中一氧化氮(nitric oxide,NO)含量和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)、诱导型一氧化氮合酶(induciblenitric oxide synthase,iNOS)水平,通过RT-PCR法检测细胞中TNF-α、IL-1β、IL-6和iNOS mRNA水平,通过Western blot法检测p38、Sirt6的蛋白表达和核转录因子κB(nuclear factor-κB,NF-κB)p65的磷酸化水平,通过流式细胞术分析Hyp对细胞周期和凋亡的影响。结果:Hyp浓度在100μmol/L以内对RAW264.7细胞增殖无影响,确定0~100μmol/L为Hyp的安全浓度范围。Hyp较模型组显著降低促炎因子NO、TNF-α、IL-1β、IL-6和iNOS含量,Hyp能够明显减少TNF-α、IL-1β、IL-6和iNOS mRNA水平。此外,Hyp较模型组显著上调Sirt6表达,下调p38、NF-κB p65的磷酸化水平。分子对接结果表明,山楂叶提取物Hyp能够与p38MAPK结合并通过氨基酸残基Asp168、Lys53,His107等发生相互作用。此外,Hyp能够显著抑制LPS诱导的细胞凋亡并使其阻滞在S期。结论:Hyp能够抑制LPS诱导巨噬细胞促炎因子的释放,其作用可能与调控p38MAPK/Sirt6/NF-κB信号通路有关。
Objective:To investigate the inhibitory effects of hyperoside(Hyp)on RAW264.7 induced by lipopolysaccharide(LPS)and its possible mechanism.Methods:MTT assay was used to observe the effect of Hyp on RAW264.7 induced by LPS.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)and induced nitric oxide synthase(iNOS)in the supernatant were determined by Griess assay and enzyme-linked immunosorbent assay(ELISA).The mRNA levels of TNF-α,IL-1β,IL-6 and iNOS in the cells were detected by RT-PCR.Protein expression of P38 and Sirt6 and phosphorylation of nuclear transcription factorκB(NF-κB)p65 were determined by Western blot.The effects of Hyp on cell cycle and apoptosis were analyzed by flow cytometry.Results:The proliferation of RAW264.7 cells was not affected when Hyp concentration was within 100μmol/L.The safe concentration range of Hyp was 0~100μmol/L.The levels of NO,TNF-α,IL-1β,IL-6 and iNOS in Hyp group were significantly lower than those in the model group.Hyp significantly reduces TNF-α,IL-1β,IL-6 and iNOS mRNA levels.In addition,Hyp significantly up-regulated Sirt6 expression and down-regulated phosphorylation levels of P38 and NF-κB p65 compared with the model group.Molecular rendezvous results showed that Hyp from hawthorn leaves could bind to p38 MAPK and interact with it through amino acid residues Asp168,Lys53,His107,etc.In addition,Hyp can significantly inhibit LPS-induced apoptosis and block it in the S phase.Conclusion:Hyp inhibits LPS-induced release of proinflammatory cytokines in macrophages,which may be related to the regulation of the p38 MAPK/Sirt6/NF-κB signaling pathway.
作者
杨莺
姚新月
李海波
YANG Ying;YAO Xin-yue;LI Hai-bo(Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110032,China;Graduate School,Liaoning University of Traditional Chinese Medicine,Shenyang 1108473,China;Basic Medical College,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China)
出处
《中国中医基础医学杂志》
CAS
CSCD
北大核心
2021年第6期944-949,1040,共7页
JOURNAL OF BASIC CHINESE MEDICINE
基金
国家自然科学基金面上项目(81603605)-山楂依赖肠道菌群通过卵磷脂代谢调脂的作用研究
国家中医药管理局第四批全国中医(临床、基础)优秀人才研修项目(国中医药人教函[2017]248号)。
作者简介
杨莺(1976-),女,辽宁沈阳人,主任医师,博士研究生,从事动脉粥样硬化及相关心血管疾病的中医药临床与研究;通讯作者:李海波(1968-),男,辽宁朝阳人,教授,博士研究生,从事中药抗氧化研究,Tel:024-31207308,E-mail:735658485@qq.com。
