硒代蛋氨酸通过NF-κB和MAPK通路抑制P.g-LPS诱导的RAW264.7细胞iNOS和COX-2表达

Selenomethionine Inhibited Expression of iNOS and COX-2 in P.g -LPS Induced RAW264.7 Cells through NF-κB and MAPK Pathways

目的:探讨硒代蛋氨酸(Selenomethionine,SeMet)对牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharide,P.g-LPS)诱导的RAW264.7细胞的作用和机制。方法:使用P.g-LPS诱导小鼠巨噬细胞RAW264.7体外构建炎症细胞模型,CCK-8法检测不同浓度SeMet对RAW264.7细胞活性的影响。以不同浓度的SeMet(10、25、50μmol/L)干预细胞1 h后,再使用P.g-LPS诱导细胞24 h。RT-PCR法检测诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和环氧合酶2(cyclooxygenase-2,COX-2)相关基因表达,ELISA法检测iNOS和COX-2相关蛋白分泌情况。Western blot检测NF-κB和MAPK信号通路相关蛋白表达情况。结果:浓度低于50μmol/L的SeMet对RAW264.7细胞活性没有显著影响。对RAW264.7使用SeMet预处理后,SeMet明显抑制P.g-LPS诱导的iNOS和COX-2相关基因表达和蛋白分泌增加,其作用与SeMet浓度有关,浓度越高,抑制作用越明显。其作用机制是通过抑制NF-κB通路的p65向核内转移,以及抑制MAPK通路的p38、ERK和JNK磷酸化发挥作用,并且其抑制作用呈浓度依赖性。结论:SeMet抑制P.g-LPS诱导的RAW264.7细胞iNOS和COX-2表达,其作用机制可能与SeMet抑制NF-κB p65核内转移和抑制MAPK通路p38、ERK和JNK的磷酸化有关。

Objective:To demonstrate the effects and mechanism of selenomethionine(SeMet)on porphyromonas gingivalis lipopolysaccharide(P.g-LPS)induced RAW264.7.Methods:P.g-LPS was used to stimulate the RAW264.7 cell line.CCK-8 was used to demonstrate the cell viability of RAW264.7 cells induced by SeMet on different concentration.After RAW264.7 was pretreated with SeMet(10,25,50μmol/L)for 1 h and then cultured with or without 1μg/mL P.g-LPS for 24h,RT-PCR was used to clarify the gene expression of iNOS and COX-2,ELISA assay was used detect the protein secretion,and Western blot was used to determine the relative protein expression of NF-κB and MAPK signaling pathway.Results:SeMet lower than 50μmol/L had no significant influence on RAW264.7 cell viability.After the RAW264.7 cells were pretreated with SeMet,the relative gene expression and protein secretion of iNOS and COX-2 were significantly inhibited by SeMet.The higher the concentration,the greater the inhibition.The mechanism was via inhibiting nuclear transcription of NF-κB p65 and phosphorylation of p38,ERK,and JNK of MAPK signaling pathway.The inhibiting effects were concentration-dependent.Conclusion:SeMet inhibited the expression of iNOS and COX-2 on RAW264.7 induced by P.g-LPS.The mechanism was related to inhibition of nuclear transcription of NF-κB p65 and phosphorylation of p38,ERK,and JNK of MAPK signaling pathway.