【目的】观察支气管上皮细胞(BEAS-2B细胞)和嗜中性粒细胞(Neutrophils细胞)接触共培养体系中基质金属蛋白酶组织抑制剂-2(tissue inhibitors of metalloproteinases-2,TIMP-2)合成,探讨不同剂量葛根素对共培养体系TIMP-2的抑制作用。【方法】应用蛋白芯片筛查不同培养体系炎性细胞因子表达;应用ELISA方法定量测量TIMP-2浓度;应用Real-time PCR方法检测TIMP-2基因表达。【结果】BEAS-2B细胞和Neutrophils细胞联合培养上清液中TIMP-2浓度升高,基因表达水平明显上调(P<0.05)。经葛根素干预可显著抑制两种细胞联合培养TIMP-2浓度及基因表达(P<0.05)。【结论】Neutrophils细胞与BEAS-2B细胞联合培养可显著增加细胞培养上清液中炎性细胞因子TIMP-2浓度,上调BEAS-2B细胞中TIMP-2基因表达;葛根素可下调TIMP-2浓度及表达。
Objective： To investigate the effect of puerarin on intedeukin （IL）-8 mRNA expression and the protein release in the co-culture of human bronchial epithelial （BEAS-2B） cells and human neutrophils. Methods： BEAS- 2B cells and neutrophills were cultured separately and co-cultured with puerarin （50, 100, and 200 ug/mL） for a predetermined time. Cytokines in culture supematant were evaluated by protein array and IL-8 quantified by enzymelinked immunosorbent assay （ELISA）. IL-8 mRNA expression was evaluated by real-time quantitative polymerase chain reaction （real-time qPCR）. Results： The co-culture of BEAS-2B cells and neutrophils exhibited synergistic effects on IL-8 mRNA expression in BEAS-2B cells, but not in neutrophils after 12 h incubation （P〈0.01）, as compared with that in BEAS-2B cells or neutrophils alone. IL-8 protein release in the culture supernatant was obviously elevated when BEAS-2B cells were co-cultured with human neutrophils as compared with that in the supematant of BEAS-2B cells or neutrophils alone after incubated for 2, 6, 12, and 18 h （P〈0.01）. Treatment with puerarin could significantly down-regulate the expression of IL-8 mRNA in BEAS-2B cells and IL-8 release in the supematant of the co-culture of BEAS-2B cells and neutrophils （P〈0.01）. Conclusion： Puerarin could exhibit anti-inflammatory activity by suppressing IL-8 production from the co-culture of human bronchial epithelial cells and neutrophils.
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.
