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

上海口腔医学 ›› 2013, Vol. 22 ›› Issue (4): 378-383.

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

于雅琼^1,2，郭佳杰^1,2，仇丽鸿^1,2，吕游^1,2，贾舸³，郭艳⁴

(1.中国医科大学口腔医学院牙体牙髓病科，辽宁沈阳 110002；2.辽宁省口腔医学研究所牙体牙髓病研究室，辽宁沈阳 110002；3.合肥市口腔医院牙体牙髓病科，安徽合肥 230001；4.中国医科大学口腔医学院中心实验室，辽宁沈阳 110002)

收稿日期:2013-02-20 修回日期:2013-03-19 出版日期:2013-08-10 发布日期:2013-08-10
通讯作者: 仇丽鸿,Tel:024-22895932,E-mail:drqlh@yahoo,com
作者简介:于雅琼(1987-)，女，医师，博士研究生，E-mail:yuyaqiong1987@126.com
基金资助:
辽宁省科学技术计划项目(2011225020)

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

YU Ya-qiong^1,2， GUO Jia-jie^1,2，QIU Li-hong^1,2，LV You^1,2，JIA Ge³，GUO Yan⁴

1.Department of Endodontics, School of Stomatology, China Medical University. Shenyang 110002, Liaoning Province; 2.Lab of Endodontics, Liaoning Provincial Institute of Stomatology. Shenyang 110002, Liaoning Province; 3.Department of Endodontics,Stomatological Hospital of Hefei City.Hefei 230001,Anhui Province; 4.Central Laboratory,School of Stomatology,China Medical University.Shenyang 110002,Liaoning Province,China

Received:2013-02-20 Revised:2013-03-19 Online:2013-08-10 Published:2013-08-10
Supported by:
Supported by Science and Technology Program of Liaoning Province(2011225020).

摘要/Abstract

摘要： 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，10 μmol/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。

关键词: 牙髓卟啉单胞菌, 脂多糖, 成骨细胞, 白细胞介素6, 核因子κB

Abstract: 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<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.

Key words: Porphyromonas endodontalis；Lipopolysaeeharides；Osteoblast, Intedeukin-6；Nuclear factor kappa B

中图分类号:

R780.2

于雅琼, 郭佳杰, 仇丽鸿, 吕游, 贾舸, 郭艳. 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[J]. 上海口腔医学, 2013, 22(4): 378-383.

YU Ya-qiong, GUO Jia-jie, QIU Li-hong, LV You, JIA Ge, GUO Yan. Effect of NF-κB on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis in MC3T3-E1 cells[J]. Shanghai Journal of Stomatology, 2013, 22(4): 378-383.

参考文献

[1] Sassone LM, Fidel RA, Faveri M, et al. A microbiological profile of unexposed and exposed pulp space of primary endodontic infections by checkerboard DNA-DNA hybridization[J]. J Endod, 2012, 38(7): 889-893.
[2] Cao H, Qi Z, Jiang H, et al. Detection of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia in primary endodontic infections in a Chinese population[J]. Int Endod J, 2012, 45(8): 773-781.
[3] R??as IN, Alves FR, Santos AL, et al. Apical root canal microbiota as determined by reverse-capture checkerboard analysis of cryogenically ground root samples from teeth with apical periodontitis[J]. J Endod, 2010, 36(10): 1617-1621.
[4] Montagner F, Jacinto RC, Signoretti FG, et al. Clustering behavior in microbial communities from acute endodontic infections[J]. J Endod, 2012, 38(2): 158-162.
[5] 杨谛, 李任, 仇丽鸿, 等. 牙髓卟啉单胞菌内毒素对成骨细胞表达IL-1βmRNA和IL-6mRNA的影响[J]. 上海口腔医学, 2009, 18(2): 194-197.
[6] 杨谛，仇丽鸿, 李任, 等. 牙髓卟啉单胞菌内毒素诱导成骨细胞表达炎症因子的信号通路研究[J]. 华西口腔医学杂志, 2010, 28(2): 135-138.
[7] 杨文东, 李志新, 高学军. IL-1β mRNA和TNFα mRNA在慢性根尖周病损组织中的表达[J]. 牙体牙髓牙周病学杂志, 2007, 17(4): 187-191.
[8] Gazivoda D, Dzopalic T, Bozic B, et al. Production of proinflammatory and immunoregulatory cytokines by inflammatory cells from periapical lesions in culture[J]. J Oral Pathol Med, 2009, 38(7): 605-611.
[9] 贾舸, 仇丽鸿, 李任, 等. 牙髓卟啉单胞菌脂多糖对成骨细胞CD14表达的影响[J]. 上海口腔医学, 2010, 19(5): 521-524.
[10] 贾舸，仇丽鸿, 李任, 等.CD-14和Tool样受体在牙髓卟啉单胞菌脂多糖诱导成骨细胞白细胞介素6表达中的作用[J]. 中华口腔医学杂志, 2011, 46(9): 531-536.
[11] 贾舸, 薛明, 李任, 等. 不同卟啉单胞菌脂多糖对小鼠成骨细胞CD14、TLRs表达的影响[J].上海口腔医学,2011,20(6):598-602.
[12] 吕游, 贾舸, 仇丽鸿, 等. 牙髓卟啉单胞菌脂多糖对成骨细胞p38丝裂原活化蛋白激酶和细胞外信号调节激酶1、2表达的影响[J]. 上海口腔医学, 2012, 21(4): 389-392.
[13] 吕游, 仇丽鸿. 口腔致病茵脂多糖诱导的MyD88依赖性信号转导途径相关机制研究进展[J].中国实用口腔科杂志,2011,4(12): 755-758.
[14] Diya Zhang, Lili Chen, Shenglai Li, et al. Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1β,TNF-α and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS[J]. Innate Immun, 2008, 14(2): 99-107.
[15] Chen D, Nie M, Fan MW, et al. Anti-inflammatory activity of curcumin in macrophages stimulated by lipopolysaccharides from Porphyromonas gingivalis[J]. Pharmacology,2008,82(4):264-269.
[16] Choi EY, Jin JY, Lee JY, et al. Melatonin inhibits Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-6 in murine macrophages by suppressing NF-κB and STAT1 activity[J]. J Pineal Res, 2011, 50(2): 197-206.
[17] Chang J, Zhang C, Tani-Ishii N, et al. NF-κB activation in human dental pulp stem cells by TNF and LPS[J]. J Dent Res, 2005, 84(11): 994-998.
[18] 赵秀敏, 艾红军, 常新. 成骨细胞和破骨细胞的传导通路和相关因子[J]. 中国实用口腔科杂志, 2009, 2(3): 176-179.
[19] Dunmyer J, Herbert B, Li Q, et al. Sustained mitogen-activated protein kinase activation with Aggregatibacter actinomycetem comitans causes inflammatory bone loss[J]. Mol Oral Microbiol, 2012, 27(5): 397-407.
[20] Zhou Q, Amar S. Identification of signaling pathways in macrophage exposed to Porphyromonas gingivalis or to its purified cell wall components[J]. J Immunol, 2007, 179(11): 7777-7790