牙髓卟啉单胞菌脂多糖对小鼠成骨细胞表达MCP-1的影响

Effects of Porphyromonas endodontalis lipopolysaccharides on the expression of monocyte chemotactic protein-1 in mouse osteoblasts

目的 :探讨牙髓卟啉单胞菌(Porphyromonas endodontalis,P.endodontalis)脂多糖对小鼠成骨细胞表达单核细胞趋化蛋白1(monocyte chemotactic protein-1,MCP-1)m RNA和蛋白的影响,以及是否有p38丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路和核因子κB(Nuclear Factor-κB,NF-κB)信号通路的参与。方法 :以不同质量浓度的P.endodontalis脂多糖(0~50 mg/L)刺激MC3T3-E1细胞24 h;以20 mg/L P.endodontalis脂多糖作用于细胞不同时间(0~48 h)后,采用实时反转录PCR和酶联免疫吸附测定检测MCP-1m RNA和蛋白的表达。采用p38MAPK抑制剂SB203580和NF-κB抑制剂BAY11-7082分别预处理细胞1 h,检测其对P.endodontalis脂多糖刺激MC3T3-E1细胞后MCP-1m RNA表达的影响。采用SPSS 13.0软件包对结果进行单因素方差分析和Dunnett t检验。结果:不同质量浓度的P.endodontalis脂多糖(0~50 mg/L)刺激MC3T3-E1细胞后,MCP-1的m RNA表达和蛋白分泌呈剂量依赖性;观察时间内(0~48 h),20 mg/L P.endodontalis脂多糖作用于MC3T3-E1细胞后,MCP-1 m RNA的表达和蛋白分泌呈时间依赖性;10 mol/L的SB203580和BAY11-7082分别预处理细胞1 h,可以降低P.endodontalis脂多糖诱导MCP-1 m RNA的表达,且SB203580的抑制作用强于BAY11-7082。结论:P.endodontalis脂多糖可能通过激活p38MAPK和NF-κB信号通路诱导成骨细胞表达MCP-1m RNA和蛋白。

PURPOSE： To investigate the effects of lipopolysaccharide（LPS） extracted from Porphyromonas endodontalis（P.endodontalis） on expression of monocyte chemotactic protein-1（MCP-1） m RNA and protein in MC3 T3-E1 cells and the role of p38 mitogen-activated protein kinase（MAPK） and nuclear factor-κB（NF-κB）in the process. METHODS：MC3 T3-E1 cells were treated with different concentrations of P.endodontalis LPS（0-50 mg/L） and 20 mg/L P.endodontalis LPS for different hours（0-48 h）. The expression of MCP-1 m RNA was detected by real-time reverse transcription PCR（RT-PCR） and protein was detected by enzyme-1 inked immunosorbent assay（ELISA）. MC3 T3-E1 cells were pretreated with SB203580（inhibitor of p38 MAPK） and BAY11-7082（inhibitor of NF-κB） for 1 h, and then were treated with 20 mg/L P.endodontalis LPS for 24 h, the expression of MCP-1 m RNA was also detected by RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS： The level of MCP-1 m RNA and protein increased significantly after treatment with different concentrations of P.endodontalis LPS（0-50 mg/L）,which indicated that P.endodontalis LPS induced osteoblasts to express MCP-1 in a dose dependent manners. During the observation time（0-48 h）, the impact of 20 mg/L P.endodontalis LPS on induction of MCP-1 in MC3 T3-E1 cells exhibited a time-dependent manner. The expression of MCP-1 m RNA decreased significantly after pretreated with 10 mol/L SB203580 and BAY11-7082 for 1 h,and the inhibitory effect of SB203580 was stronger than BAY11-7082.CONCLUSIONS： P.endodontalis LPS may induce the expression of MCP-1 m RNA and protein in MC3 T3-E1 cells through the signaling pathway of p38 MAPK and NF-κB.