摘要
目的评价湖北金粟兰中倍半萜二聚体化合物(+)-chlorahupetenes B[(+)-CHB]对脂多糖(LPS)诱导的小鼠巨噬细胞(RAW264.7)炎症反应的影响及作用机制。方法RAW264.7细胞分为对照组(给予等体积DMSO)、模型组(给予等体积DMSO)、地塞米松磷酸钠注射液(Dex,阳性对照,1μmol·L^(-1))组和(+)-CHB低、中、高浓度(5、10、20μmol·L^(-1))组。各组分别加入相应药物孵育细胞1 h,除对照组外,其余组加入LPS(1μg·mL^(-1))诱导24 h造成炎症应答模型。细胞增殖检测法用于评估细胞活力;Griess反应检测一氧化氮(NO)浓度;酶联免疫分析检测炎症细胞因子肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β、IL-6的生成;实时荧光定量PCR(qRT-PCR)检测IL-1β、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、环氧合酶-2(COX-2)、诱导型一氧化氮合酶(iNOS)、IL-6和TNF-αmRNA表达;Western blotting检测Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核因子κB(NF-κB)p65、p-NF-κBp65、NLRP3、嘌呤能受体(P2X7)蛋白表达水平。结果与模型组比较,(+)-CHB减少了梭形细胞数量,使大部分细胞恢复正常形态;显著抑制NO、TNF-α、IL-6、IL-1β生成(P<0.01),显著降低COX-2、iNOS、IL-6、TNF-α、NLRP3、IL-1βmRNA水平(P<0.05、0.01);显著降低TLR4、MyD88、NF-κB p65、p-NF-κB p65、NLRP3、P2X7蛋白表达水平(P<0.05、0.01)。结论(+)-CHB通过抑制TLR4/MyD88/NF-κB信号通路和P2X7/NLRP3/IL-1β炎症小体轴激活缓解LPS诱导的巨噬细胞炎症反应。
Objective To evaluate the anti-inflammatory activity and mechanism of(+)-chlorahupetenes B,a natural eudesmane-type sesquiterpenoid dimer compound obtained from Chloranthus henryi var.hupehensis,in lipopolysaccharides(LPS)-induced RAW264.7 cell.Methods RAW264.7 cells were divided into control group(given equal volume of DMSO),model group(given equal volume of DMSO),dexamethasone sodium phosphate injection(Dex,positive control,1μmol·L^(-1))group,and(+)-CHB low,medium,and high concentrations(5,10,20μmol·L^(-1))groups.Each group was incubated with corresponding drugs for 1 h.Except for the control group,the other groups were induced with LPS(1μg·mL^(-1))for 24 hours to create an inflammatory response model.Cell proliferation detection method was used to evaluate cell viability.Griess reaction was employed for determining nitric oxide(NO)concentration.Enzyme-linked immunosorbent assay was utilized for detecting inflammatory cytokines including tumor necrosis factorα(TNF-α),interleukin(IL)-1β,and IL-6.Real time fluorescence quantitative PCR(qRT PCR)was performed to analyze IL-1β,NOD like receptor heat protein domain associated protein 3(NLRP3),cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS),IL-6,and TNF-α mRNA expression.Western blotting detection was used to detect the expression levels of Toll like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),nuclear factorκB(NF-κB)p65,p-NF-κB p65,NLRP3,and purinergic receptor(P2X7)proteins.Results Compared with the model group,(+)-CHB reduced the number of spindle cells and restored most of the cells to their normal morphology,significantly inhibited NO and TNF-α,IL-6,IL-1β generation(P<0.01),significantly reduced COX-2,iNOS,IL-6,TNF-α,NLRP3,and IL-1β mRNA levels(P<0.05,0.01),significantly reduced the expression levels of TLR4,MyD88,NF-κB p65,p-NF-κB p65,NLRP3,and P2X7 proteins(P<0.05,0.01).Conclusion These findings suggested that(+)-CHB was an anti-inflammatory compound which could relieve LPS-stimulated macrophage inflammatory response partly by targeting TLR4/MyD88/NF-κB signal pathway and P2X7/NLRP3/IL-1β inflammasome axis.
作者
胡蕴然
庄鹏宇
柳航
钱铭
杨慧
葛卫红
HU Yunran;ZHUANG Pengyu;LIU Hang;QIAN Ming;YANG Hui;GE Weihong(School of Basic Medicine and Clinical Pharmacy,China Pharmaceutical University,Department of Pharmacy,Nanjing Drum Tower Hospital,Nanjing 210008,China;Department of Pharmacy,Affiliated Drum Tower Hospital of Nanjing University Medical School,Nanjing 210008,China;School of Pharmacy,North China University of Science and Technology,Tangshan 063210,China)
出处
《药物评价研究》
CAS
北大核心
2024年第3期505-512,共8页
Drug Evaluation Research
基金
国家自然科学基金青年资助项目(82204720)
中央高校基本科研业务费资助项目(3332022085)。
作者简介
第一作者:胡蕴然(1999-),女,硕士研究生,研究方向为抗炎免疫药理学。E-mail:yunran923@outlook.com;通信作者:杨慧,女,药师,主要研究方向为抗炎免疫药理学、临床药学。E-mail:yanghui@njglyy.com。
