NF-κB在牙髓卟啉单胞菌脂多糖诱导小鼠成骨细胞白介素6表达中的作用

Effect of NF-κB on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis in MC3T3-E1 cells

目的:探讨核因子κB(NF-κB)在牙髓卟啉单胞菌(P.e)脂多糖(LPS)诱导小鼠成骨细胞株MC3T3-El白介素6(IL-6)表达中的作用。方法:10 mg/L P.e-LPS刺激MC3T3-El细胞不同时间后,应用免疫荧光方法检测NF-κB核易位情况和BAY-117082对NF-κB核易位抑制情况,反转录聚合酶链反应(RT—PCR)和酶联免疫吸附试验(ELISA)检测BAY-117082对MC3T3-El细胞的IL-6基因和蛋白表达的影响。采用SPSS 13.0软件包对结果进行多因素方差分析和Dunnett t检验。结果:在静息状态下,NF-κB定位在胞质中,10 mg/L P.e-LPS刺激MC3T3-El细胞30 min后即引起NF-κB快速的核易位;60 min后,大部分NF-κB重新定位在胞质里,10μmol/L BAY-117082预处理1 h可抑制P.e-LPS引起的NF-κB核易位,降低P.e-LPS诱导MC3T3-El细胞IL-6 mRNA表达水平和IL-6蛋白的分泌水平,其中,蛋白分泌水平从(774.983±6.585)ng/L降低至(377.384±14.620)ng/L(P<0.O1),而BAY-117082单独刺激组与空白对照组无显著差异。结论:P.e-LPS可诱导MC3T3-El细胞中NF-κB的核易位,P.e-LPS可通过激活NF-κB信号途径诱导MC3T3-El细胞产生IL-6。

PURPOSE： To investigate the effect of NF-κB signaling on the expression of interleukin-6（IL-6） induced by lipopolysaccharides（LPS） extracted from Porphyromonas endodontalis（P.e）in MC3T3-El cells.METHODS： MC3T3-E1 cells were pretreated with BAY-117082 for 1 h,and then were treated with 10 mg/L P.e-LPS for different times.The translocation of NF-κB was observed by immunofluorescence.The expression of IL-6 was detected by reverse transcription polymerse chain reaction（RT-PCR） and enzyme-linked immuno sorbent assay（ELISA）.Statistical analysis was performed using multi-way ANOVA and Dunnett’s t test with SPSS 13.0 software package.RESULTS： The staining of NF-κB was mostly in cytoplasm in untreated cells.Rapid translocation of NF-κB into nucleus was observed in the cells stimulated for 30 min and mostly relocalization of NF-κB from nucleus to cytoplasm was observed after 60 min.Pretreatment with 10 μmol/L BAY-117082 for 1h significantly inhibited P.e-LPS-induced translocation of NF-κB.The mRNA and proteins of IL-6 decreased significantly after pretreatment with 10 μmol/L BAY-117082,and the expression of IL-6 proteins was reduced from（774.983±6.585） ng/L to（377.384±14.620） ng/L（P＆lt;0.01）.The group of treatment with BAY-117082 alone had no significant difference from the blank control group.CONCLUSIONS： P.e-LPS can induce translocation of NF-κB in mouse osteoblast MC3T3-El,and P.e-LPS may induce the expression of IL-6 in mouse osteoblast through the signaling of NF-κB.Supported by Science and Technology Program of Liaoning Province（2011225020）.