鱼腥草素钠对脂多糖诱导单核巨噬细胞RAW264.7炎性反应的抑制作用

Inhibitory Effect of Sodium Houttuyfonate on Lipopolysaccharide-Induced Inflammatory Response in Mononuclear Phagocyte RAW264.7

目的探讨鱼腥草素钠(SH)对脂多糖(LPS)诱导小鼠单核巨噬细胞RAW264.7炎性反应的抑制作用及其机制。方法实验分为空白对照组(等体积培养基),模型组(等体积培养基),SH低、中、高剂量组(1.25,5,10μmol/L),以含不同浓度SH的常规培养基培养细胞2 h,后4组细胞加入LPS继续培养24 h。收集细胞上清液,测定一氧化氮(NO)水平,采用酶联免疫吸附(ELISA)法测定肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)及前列腺素E_(2)(PGE_(2))的水平;提取细胞总蛋白,采用Westernblot法检测诱导型一氧化氮合酶(iNOS)、环氧合酶2(COX-2)蛋白及核因子-κB(NF-κB)通路相关蛋白(NF-κB-P-P65蛋白和P-IκB-α蛋白)的表达水平。结果与模型组比较,SH中、高剂量组细胞NO水平显著降低,SH低、中、高剂量组细胞TNF-α,IL-6,PGE_(2)水平和COX-2,NF-κB-P-P65,P-IκB-α蛋白表达水平均显著降低,SH中、高剂量组细胞iNOS蛋白表达水平显著降低(P<0.01)。结论SH能抑制LPS诱导的RAW264.7细胞炎性因子的分泌,其作用机制与抑制炎性蛋白iNOS和COX-2的表达及NF-κB通路相关蛋白的磷酸化有关。

Objective To investigate the inhibitory effect and mechanism of sodium houttuyfonate(SH)on the lipopolysaccharide(LPS)-induced inflammatory response in mononuclear phagocyte RAW264.7 of mice.Methods RAW264.7 cells were divided into the blank control group(equal volume medium),the model group(equal volume medium),the SH low-,medium-and high-dose groups(1.25,5,10μmol/L).Cells were cultured in conventional medium containing different concentrations of SH for 2 h,and then LPS was added into the last four groups for induction(24 h).Cell supernatant was collected to detect the nitric oxide(NO)level,and tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and prostaglandin E_(2)(PGE_(2))levels were detected by the enzyme-linked immunosorbent assay(ELISA).Total cell proteins were extracted,and the expression levels of inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2)and nuclear factor-κB(NF-κB)pathway-related proteins(NF-κB-P-P65 and P-IκB-α)were detected by the Western blot.Results Compared with those in the model group,the NO level in the SH medium-and high-dose groups was significantly lower,the TNF-α,IL-6,PGE_(2)levels,COX-2,NF-κB-P-P65 and P-IκB-αexpression levels in the SH low-,medium-and high-dose groups were significantly lower,and the iNOS expression level in the SH medium-and high-dose groups was significantly lower(P<0.01).Conclusion SH can inhibit the secretion of LPS-induced inflammatory factors in RAW264.7 cells,its mechanism is related to the inhibition of the expression of inflammatory proteins(iNOS and COX-2)and the phosphorylation of NF-κB pathway-related proteins.