核苷酸结合寡聚化结构域1通过核因子κB途径调节牙龈卟啉单胞菌诱导的内皮细胞中血管细胞黏附分子1和细胞间黏附分子1的表达。

Nucleotide-binding oligomerization domain 1 regulates Porphyromonas gingivalis-induced vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression in endothelial cells through NF-κB pathway.

作者信息

Wan M, Liu J, Ouyang X

机构信息

Department of Periodontology, Peking University School and Hospital of Stomatology, Beijing, China.

出版信息

J Periodontal Res. 2015 Apr;50(2):189-96. doi: 10.1111/jre.12192. Epub 2014 May 24.

DOI:10.1111/jre.12192

PMID:24862550

Abstract

BACKGROUND AND OBJECTIVE

Porphyromonas gingivalis has been shown to actively invade endothelial cells and induce vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) overexpression. Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition reporter, and its involvement in this process was unknown. This study focused on endothelial cells infected with P. gingivalis, the detection of NOD1 expression and the role that NOD1 plays in the upregulation of VCAM-1 and ICAM-1.

MATERIAL AND METHODS

The human umbilical vein endothelial cell line (ECV-304) was intruded by P. gingivalis W83, and cells without any treatment were the control group. Expression levels of NOD1, VCAM-1, ICAM-1, phosphorylated P65 between cells with and without treatment on both mRNA and protein levels were compared. Then we examined whether mesodiaminopimelic acid (NOD1 agonist) could increase VCAM-1 and ICAM-1 expression, meanwhile, NOD1 gene silence by RNA interference could reduce VCAM-1, ICAM-1 and phosphorylated P65 release. At last, we examined whether inhibition of NF-κB by Bay117082 could reduce VCAM-1 and ICAM- 1 expression. The mRNA levels were measured by real-time polymerase chain reaction, and protein levels by western blot or electrophoretic mobility shift assays (for phosphorylated P65).

RESULTS

P. gingivalis invasion showed significant upregulation of NOD1, VCAM-1 and ICAM-1. NOD1 activation by meso-diaminopimelic acid increased VCAM-1 and ICAM-1 expression, and NOD1 gene silence reduced VCAM-1 and ICAM-1 release markedly. The NF-κB signaling pathway was activated by P. gingivalis, while NOD1 gene silence decreased the activation of NF-κB. Moreover, inhibition of NF-κB reduced VCAM-1 and ICAM-1 expression induced by P. gingivalis in endothelial cells.

CONCLUSION

The results revealed that P. gingivalis induced NOD1 overexpression in endothelial cells and that NOD1 played an important role in the process of VCAM-1 and ICAM-1 expression in endothelial cells infected with P. gingivalis through the NF-κB signaling pathway.

摘要

背景与目的

牙龈卟啉单胞菌已被证明可主动侵入内皮细胞并诱导血管细胞黏附分子1（VCAM-1）和细胞间黏附分子1（ICAM-1）的过表达。核苷酸结合寡聚化结构域1（NOD1）是一种细胞内模式识别受体，其在这一过程中的作用尚不清楚。本研究聚焦于被牙龈卟啉单胞菌感染的内皮细胞，检测NOD1的表达以及NOD1在VCAM-1和ICAM-1上调中所起的作用。

材料与方法

用人牙龈卟啉单胞菌W83侵入人脐静脉内皮细胞系（ECV-304），未经任何处理的细胞作为对照组。比较处理组和未处理组细胞中NOD1、VCAM-1、ICAM-1、磷酸化P65在mRNA和蛋白质水平上的表达水平。然后检测内消旋二氨基庚二酸（NOD1激动剂）是否能增加VCAM-1和ICAM-1的表达，同时，通过RNA干扰使NOD1基因沉默是否能减少VCAM-1、ICAM-1和磷酸化P65的释放。最后，检测Bay117082抑制核因子κB（NF-κB）是否能降低VCAM-1和ICAM-1的表达。通过实时聚合酶链反应检测mRNA水平，通过蛋白质印迹法或电泳迁移率变动分析（用于检测磷酸化P65）检测蛋白质水平。

结果

牙龈卟啉单胞菌的侵入显示NOD1、VCAM-1和ICAM-1有显著上调。内消旋二氨基庚二酸激活NOD1可增加VCAM-1和ICAM-1的表达，而NOD1基因沉默则明显减少VCAM-1和ICAM-1的释放。牙龈卟啉单胞菌激活了NF-κB信号通路，而NOD1基因沉默则降低了NF-κB的激活。此外，抑制NF-κB可降低牙龈卟啉单胞菌诱导的内皮细胞中VCAM-1和ICAM-1的表达。