白藜芦醇对LPS刺激下RAW264.7细胞向破骨细胞分化的影响

Effect of Resveratrol on Differentiation of RAW264.7 Cells into Osteoclasts under LPS

目的探讨白藜芦醇对LPS刺激下体外破骨细胞形成的作用途径和作用机制。方法小鼠RAW264.7巨噬细胞分3组培养,即空白组(Sham组)、LPS组[空白组+LPS(1μg/ml)]和Res组[LPS组+白藜芦醇(10μmol/L)]。抗酒石酸酸性磷酸酶(TRAP)染色法检测各组成熟破骨细胞数量,酶联免疫吸附试实验(ELISA)法检测各组上清液中TNF-α和IL-1β的含量,荧光定量聚合酶反应法(Q-PCR)检测各组中TNF-α和IL-1βmRNA的表达,Western blot法检测各组核转录因子κBp65(NF-κBp65)与IκBα中磷酸化蛋白的水平。结果TRAP染色:Res组TRAP染色阳性的破骨细胞数目相比LPS组明显减少;ELISA检测:LPS组中各炎性细胞因子表达水平较Sham组显著升高(P<0.05),Res组较LPS组各炎性细胞因子表达显著降低(P<0.05);Q-PCR检测:LPS组TNF-α和IL-1βmRNA的表达水平较Sham组明显升高(P<0.05),Res组中TNF-α、IL-1β的基因表达明显被抑制(P<0.05);Western blot法检测结果显示,Res组IκBα和p65的磷酸化水平相对于LPS组显著降低(P<0.05)。结论白藜芦醇可以通过NF-κB信号通路下调LPS刺激下小鼠RAW 264.7巨噬细胞中TNF-α与IL-1β的表达,进而影响破骨细胞的形成,有望作为治疗无菌性松动引起的炎性骨溶解的关键靶点。

Objective To explore the effect pathway and mechanism of resveratrol on osteoclast formation in vitro stimulated by LPS.Methods Mouse RAW264.7 macrophages were cultured in 3 groups,blank group(Sham group);LPS group:blank group+LPS(1μg/ml);Res group:LPS group+resveratrol(10μmol/L).TRAP staining method was used to detect the number of mature osteoclasts in each group.ELISA method was used to detect the expression of TNF-αand IL-1βin the supernatant of each group.Q-PCR method was used to detect the expression of TNF-αand IL-1βmRNA in each group.Western blot method was used to detect the levels of phosphorylated proteins in nuclear transcription factorκBp65(NF-κBp65)and IκBαin each group.Results TRAP staining:The number of osteoclasts positive for TRAP staining in the Res group was significantly reduced compared with the LPS group.ELISA detection:the expression levels of inflammatory factors in the LPS group were significantly higher than those in the Sham group(P<0.05),the expression of inflammatory factors in the Res group was significantly lower than that in the LPS group(P<0.05).Q-PCR detection:the expression levels of TNF-αand IL-1βmRNA in the LPS group were significantly higher than those in the Sham group(P<0.05),and the gene expression of TNF-αand IL-1βin the Res group was significantly inhibited(P<0.05).Western blot detection:the phosphorylation levels of IκBαand p65 in the Res group were significantly reduced compared to the LPS group(P<0.05).Conclusion Resveratrol can down-regulate the expression of TNF-αand IL-1βin mouse RAW 264.7 macrophages stimulated by LPS through the NF-κB signalling pathway,and then affect the formation of osteoclasts.It is expected to be a key target for the treatment of inflammatory osteolysis caused by aseptic loosening.