Objective: To evaluate the effects of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on the expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein as well as enzyme activity in MC3T3-E1 cells and the role of nuclear factor-κB (NF-κB) in the process, so as to investigate the expression of MMP-9 dependent signaling pathways in mouse osteoblasts induced by Pe LPS. Methods: The experiment was conducted in 3 sessions: MC3T3-E1 cells were treated with various concentrations of Pe LPS (0-20 mg/L) and 10 mg/L Pe LPS for different time intervals (0-48 h). The expression of MMP-9 mRNA and protein were detected by real-time reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), while the enzyme activity was detected by gelatin zymography method. The expression of MMP-9 mRNA was also detected in 10 mg/L Pe LPS treated MC3T3-El cells after pretreated with specific NF-κB inhibitor BAY 11-7082 for l h. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. Results: The levels of MMP-9 mRNA and protein increased significantly after the treatment with various concentrations of Pe LPS (0-20 mg/L), which indicated that Pe LPS induced osteoblasts to express MMP-9 in dose dependent manners. The expression of MMP-9 protein increased from (5 395±362) ng/L (blank control group) to (12 684±375) ng/L (20 mg/L group). Maximal induction of MMP-9 mRNA expression was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 24 h. The expression of MMP-9 mRNA in the 20 mg/L group was about 7 times than that in the blank control group. After 24 h, the expression of MMP-9 mRNA decreased. Maximal expression of MMP-9 protein was found in the MC3T3-E1 cells treated with 10 mg/L Pe LPS for 48 h ([35 055±2 346] ng/L) showing the highest enzyme activity. The mRNA of MMP-9 decreased significantly after pretreatment with 10 µmol/L BAY 11-7082 for 1 h. Conclusions: Pe LPS might induce the expression of MMP-9 in MC3T3-E1 cells through the signaling of NF-κB.
目的： 探讨牙髓卟啉单胞菌（Porphyromonas endodontalis，Pe）脂多糖对小鼠成骨细胞基质金属蛋白酶9（matrix metalloproteinase-9，MMP-9）表达的影响及核因子κB（nuclear factor-κB，NF-κB）信号通路是否参与此过程，从分子水平更好地了解根尖周疾病骨破坏的发生机制。 方法： 实验分为3部分，以不同质量浓度的Pe脂多糖[0（空白对照组）、1、5、10、15及20 mg/L]刺激MC3T3-E1细胞24 h；以10 mg/L Pe脂多糖作用于细胞不同时间（0、10、24、48 h）后，采用实时反转录PCR、酶联免疫吸附测定和明胶酶谱法检测MMP-9 mRNA和蛋白的表达以及酶活性变化；采用NF-κB信号通路特异性抑制剂BAY 11-7082预处理1 h，检测其对Pe脂多糖刺激MC3T3-E1细胞后MMP-9 mRNA表达的影响。对结果行单因素方差分析和Dunnett t检验。 结果： 不同质量浓度Pe脂多糖刺激MC3T3-E1细胞后，MMP-9 mRNA和蛋白的表达具有剂量依赖性，蛋白表达量从（5 395±362）ng/L（空白对照组）增加到（12 684±375）ng/L（20 mg/L组）。用10 mg/L Pe脂多糖作用于MC3T3-E1细胞24 h时，MMP-9 mRNA表达量最高，约为空白对照组的7倍，随着作用时间延长，MMP-9 mRNA表达量下降；当10 mg/L Pe脂多糖作用于MC3T3-E1细胞48 h时MMP-9的蛋白表达量最高[（35 055±2 346）ng/L]；酶活性也最高。10 mmol/L的BAY 11-7082预处理细胞1 h后，显著降低了Pe脂多糖诱导的MMP-9 mRNA的表达水平（P<0.01）。 结论： Pe脂多糖可能通过激活NF-κB信号通路诱导小鼠成骨细胞表达MMP-9。.
Keywords: Lipopolysaccharides; Matrix metalloproteinase 9; Osteoblasts; Porphyromonas endodontalis; Signaling pathway.