槲皮素促进脂多糖诱导的原代小胶质细胞M2型极化及机制研究  

Quercetin Promotes M 2 Polarization of Primary Microglia Induced by Lipopolysaccharide and Its Mechanism

作　　者：胡荟琴 李琳[1] 胡小伟 张亚妮 印媛君[1] 方燕[1] 储利胜[1] HU Huiqin;LI Lin;HU Xiaowei(School of Basic Medical Sciences,Zhejiang Chinese Medical University,Hangzhou(310053),China)

机构地区：[1]浙江中医药大学基础医学院,杭州310053

出　　处：《浙江中医药大学学报》2025年第3期259-271,共13页Journal of Zhejiang Chinese Medical University

基　　金：国家自然科学基金项目(82274122、82104426)。

摘　　要：[目的]探讨槲皮素对脂多糖(lipopolysaccharide,LPS)诱导的原代小胶质细胞极化的影响及潜在机制。[方法]分离培养原代大鼠小胶质细胞,设立对照组,模型组,槲皮素低、中、高剂量组。模型组采用LPS诱导小胶质细胞激活,在槲皮素组中,小胶质细胞先用20、40、80μmol·L^(-1)的槲皮素处理1 h,随后在培养基中加入LPS处理24 h。采用CCK-8法检测小胶质细胞活力;采用Griess法测定上清液一氧化氮(nitric oxide,NO)含量;免疫荧光染色钙结合蛋白1(ionized calcium binding adapter molecule 1,Iba1)/CD68检测小胶质细胞激活情况;实时定量聚合酶链式反应(Real time-quantitative polymerase chain reaction,RT-qPCR)检测CD86、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、CD206、精氨酸酶-1(arginase-1,Arg-1)、几丁质酶样蛋白1/2(chitinase like protein 1/2,Ym1/2)、IL-10、转化生长因子-β(transforming growth factor-β,TGF-β)mRNA表达水平;网络药理学和分子对接方法预测槲皮素调控小胶质细胞极化的潜在作用机制;免疫印迹检测CD86、iNOS、CD206、Arg-1、磷酸化磷脂酰肌醇3-激酶(phosphophorylated-phosphatidylinositol 3-kinase,p-PI3K)/磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、磷酸化蛋白激酶B(phosphophorylated-protein kinase B,p-Akt)/蛋白激酶B(protein kinase B,Akt)、磷酸化核因子-κB(phosphophorylated-nuclear factor-κB,p-NF-κB)/核因子-κB(nuclear factor-κB,NF-κB)和磷酸化核因子-κB抑制蛋白α(phosphophorylated-inhibitor of NF-κBα,p-IκB-α)/核因子-κB抑制蛋白α(inhibitor of NF-κBα,IκB-α)的蛋白表达水平。[结果]与LPS组比较,槲皮素组小胶质细胞NO释放量和CD68平均荧光强度显著降低(P<0.01),表明槲皮素抑制LPS诱导的原代小胶质细胞激活;槲皮素抑制CD86和iNOS的mRNA和蛋白表达(P<0.05,P<0.01),降低促炎因子TNF-[ObjectiveObjective]To explore the effect and potential mechanism of quercetin on polarization of lipopolysaccharide(LPS)-induced primary microglia.[MethodsMethods]Primary rat microglia were isolated and cultured,and then randomly divided into control group,model group and quercetin low-dose,medium-dose,high-dose groups.In model group,microglial activation was induced with LPS.In the quercetin groups,microglia were pretreated with quercetin at concentrations of 20,40 and 80μmol·L^(-1) for 1 hour,followed by the addition of LPS to the culture medium for an additional 24 hours.Cell viability was assessed by using the CCK-8 assay.The nitric oxide(NO)content in the supernatant was measured by the Griess assay.Microglia activation was detected by ionized calcium binding adapter molecule 1(Iba1)/CD68 immunofluorescence staining.The expression levels of CD86,inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,CD206,arginase-1(Arg-1),chitinase like protein 1/2(Ym1/2),IL-10 and transforming growth factor-β(TGF-β)mRNA were determined by Real time-quantitative polymerase chain reaction(RT-qPCR).Network pharmacology and molecular docking methods were employed to predict the potential mechanisms of quercetin in regulating microglia polarization.The protein expression levels of CD86,iNOS,CD206,Arg-1,phosphophorylated-phosphatidylinositol 3-kinase(p-PI3K)/phosphatidylinositol 3-kinase(PI3K),phosphophorylated-protein kinase B(p-Akt)/protein kinase B(Akt),phosphophorylated-nuclear factor-κB(p-NF-κB)/nuclear factor-κB(NF-κB)and phosphophorylated-inhibitor of NF-κBα(p-IκB-α)/inhibitor of NF-κBα(IκB-α)were determined by Western blot.[ResultsResults]Compared with LPS group,the NO release and CD68 mean fluorescence intensity of microglia in quercetin groups were significantly reduced(P<0.01),indicating that quercetin inhibited LPS-induced activation of primary microglia.Quercetin inhibited the mRNA and protein expression of CD86 and iNOS(P<0.05,P<0.01),and decreased the m

关 键 词：槲皮素 小胶质细胞 极化 神经炎症 网络药理学 分子对接 PI3K/AKT 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]