多种真菌多糖近年来因其免疫调节活性成为保健食品领域研究的热点。本实验以食药用真菌蛹虫草提取物蛹虫草多糖(Cordyceps militaris polysaccharides,CMP)为研究材料,初步探究CMP对小鼠巨噬细胞RAW264.7免疫活性的调节机制。噻唑蓝实...多种真菌多糖近年来因其免疫调节活性成为保健食品领域研究的热点。本实验以食药用真菌蛹虫草提取物蛹虫草多糖(Cordyceps militaris polysaccharides,CMP)为研究材料,初步探究CMP对小鼠巨噬细胞RAW264.7免疫活性的调节机制。噻唑蓝实验结果显示CMP无细胞毒性,且100、200μg/mL CMP可以明显增强RAW264.7细胞活性;中性红法、Griess法和酶联免疫吸附检测结果表明25～200μg/mL的CMP以剂量依赖方式增强RAW264.7细胞吞噬活性并增加一氧化氮(nitric oxide,NO)和白介素-1β(interleukin-1β,IL-1β)分泌,肿瘤坏死因子-α(tumor necrosisfactor-α,TNF-α)的分泌呈先升高后降低的趋势,在100μg/mL时达到最高值;抑制剂中和实验结果显示当Toll样受体4(Toll-like receptors 4,TLR4)和甘露糖受体(mannose receptor,MR)受到抑制时,CMP诱导的RAW264.7免疫反应显著降低,这表明TLR4和MR均为CMP激活巨噬细胞的受体;此外,Western blot结果显示丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号通路也参与CMP诱导的RAW264.7细胞分泌NO、TNF-α和IL-1β。表明TLR4和MR/MAPK信号传导途径在CMP诱导巨噬细胞RAW264.7产生免疫应答时发挥了重要作用。展开更多
Objective： In this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these ad...Objective： In this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase （p38 MAPK） and nuclear factor kappa B （NF- K B） in modulating adhesion molecules expressions. Methods： Neutrophils and BEAS-2B cells （one human bronchial epithelial cell line） were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction （real-time qPCR）. Phosphorylated p38 MAPK and inhibitor K B were analyzed by Western blot. Results： In co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrephils were enhanced significantly （P〈0.05）. Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor K B in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin （P〈0.05）. Conclusions： Co- culture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF- K B pathways, and exhibiting its anti-inflammation activity.展开更多
目的初步分析BC02(BCG Cp G DNA combination adjuvants system 02)复合佐剂构成成分在巨噬细胞激活过程中的协同加强作用。方法采用夹心ELISA和RT-PCR法分别检测BC02复合佐剂及其构成成分[Al(OH)3佐剂及BC01(BCG Cp G DNA compound adj...目的初步分析BC02(BCG Cp G DNA combination adjuvants system 02)复合佐剂构成成分在巨噬细胞激活过程中的协同加强作用。方法采用夹心ELISA和RT-PCR法分别检测BC02复合佐剂及其构成成分[Al(OH)3佐剂及BC01(BCG Cp G DNA compound adjuvants system 01)佐剂]对小鼠巨噬细胞RAW264.7分泌的TNF-α及单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)细胞因子水平和m RNA转录水平的影响。信号通路蛋白磷酸化芯片检测经BC01和BC02刺激45 min后,RAW264.7细胞中核转录因子-κB(nuclear factor-κB,NF-κB)和促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的变化情况。结果 BC02对巨噬细胞的刺激存在剂量和时间依赖性,最佳浓度和培养时间分别为1/10人用剂量[(7.5μg/m L BC01+20μg/m L Al(OH)3]和24 h。Al(OH)3无机盐佐剂能协同增强BC01生物佐剂对RAW264.7细胞的刺激活性,同时,TLR-9抑制剂ODN 2088与NLRP3抑制剂MCC 950单独或联合处理RAW264.7细胞,均能显著降低BC02刺激RAW264.7细胞分泌TNF-α和MCP-1细胞因子的能力。信号通路蛋白磷酸化芯片结果表明,BC02复合佐剂显著上调NF-κB信号通路中NF-κB-p105/p50、NF-κB-p65及NF-κB-p100/p52等关键蛋白分子的磷酸化水平;同时,也能显著上调MAPK信号通路中p38 MAPK、c-Jun、SPAK/JNK及EIK-1等关键蛋白分子的磷酸化水平。结论 BC02复合佐剂中BC01生物佐剂与Al(OH)3无机盐佐剂成分在激活小鼠巨噬细胞参与固有免疫应答中具有协同加强作用。展开更多
基金Supported by the National Natural Sciences Foundation of China(No.30873419)
文摘Objective： In this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase （p38 MAPK） and nuclear factor kappa B （NF- K B） in modulating adhesion molecules expressions. Methods： Neutrophils and BEAS-2B cells （one human bronchial epithelial cell line） were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction （real-time qPCR）. Phosphorylated p38 MAPK and inhibitor K B were analyzed by Western blot. Results： In co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrephils were enhanced significantly （P〈0.05）. Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor K B in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin （P〈0.05）. Conclusions： Co- culture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF- K B pathways, and exhibiting its anti-inflammation activity.
文摘目的初步分析BC02(BCG Cp G DNA combination adjuvants system 02)复合佐剂构成成分在巨噬细胞激活过程中的协同加强作用。方法采用夹心ELISA和RT-PCR法分别检测BC02复合佐剂及其构成成分[Al(OH)3佐剂及BC01(BCG Cp G DNA compound adjuvants system 01)佐剂]对小鼠巨噬细胞RAW264.7分泌的TNF-α及单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)细胞因子水平和m RNA转录水平的影响。信号通路蛋白磷酸化芯片检测经BC01和BC02刺激45 min后,RAW264.7细胞中核转录因子-κB(nuclear factor-κB,NF-κB)和促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的变化情况。结果 BC02对巨噬细胞的刺激存在剂量和时间依赖性,最佳浓度和培养时间分别为1/10人用剂量[(7.5μg/m L BC01+20μg/m L Al(OH)3]和24 h。Al(OH)3无机盐佐剂能协同增强BC01生物佐剂对RAW264.7细胞的刺激活性,同时,TLR-9抑制剂ODN 2088与NLRP3抑制剂MCC 950单独或联合处理RAW264.7细胞,均能显著降低BC02刺激RAW264.7细胞分泌TNF-α和MCP-1细胞因子的能力。信号通路蛋白磷酸化芯片结果表明,BC02复合佐剂显著上调NF-κB信号通路中NF-κB-p105/p50、NF-κB-p65及NF-κB-p100/p52等关键蛋白分子的磷酸化水平;同时,也能显著上调MAPK信号通路中p38 MAPK、c-Jun、SPAK/JNK及EIK-1等关键蛋白分子的磷酸化水平。结论 BC02复合佐剂中BC01生物佐剂与Al(OH)3无机盐佐剂成分在激活小鼠巨噬细胞参与固有免疫应答中具有协同加强作用。
