妇科千金胶囊的抗炎作用与其调节NF-κB信号通路的关系

Anti-inflammatory Effect of Fuke Qianjin Capsule and Its Relationship with Regulating NF-κB Signal Pathway

目的研究妇科千金胶囊总浸膏(Fuke Qianjin capsule extract, FKE)体外抗炎活性及其抗炎作用机制。方法采用MTT法检测不同浓度FKE单独使用和FKE叠加100 ng/mL脂多糖(lipopolysaccharide, LPS)使用时对小鼠巨噬细胞(RAW 264.7)活性的影响;采用ELISA法检测不同浓度FKE对LPS刺激后RAW 264.7细胞炎症因子一氧化氮(NO)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)释放的影响;通过RT-PCR法检测不同浓度FKE对炎症相关蛋白环氧合酶2 (COX-2)、IL-6、诱导型一氧化氮合酶(iNOS)、TNF-α转录水平的影响;采用Western blot检测不同浓度FKE对炎症相关蛋白IκBα、iNOS表达的影响;利用激光共聚焦技术检测FKE对NF-κB p65核转位的影响;采用双荧光素酶报告基因检测不同浓度FKE对核转录因子-κB (nuclear factor-κB, NF-κB)的影响。结果 MTT测定结果表明,与对照组相比,FKE在50~800滋g/mL浓度范围内对RAW 264.7细胞活性的影响无显著差异(P>0.05);ELISA检测结果显示,FKE能明显降低LPS刺激诱导的RAW 264.7细胞NO含量,在400滋g/mL浓度时,呈现出对IL-6、TNF-α的抑制作用(P<0.05);Western blot检测结果表明,FKE对IκBα的表达无影响,但对iNOS活性具有一定的抑制作用;激光共聚焦检测发现,与对照组比较,FKE部分抑制p65核转位;FKE在400滋g/mL时,可明显抑制NF-κB的激活(P<0.05)。结论 FKE具有明显减轻细胞炎症反应的作用,其机制可能与FKE抑制炎症通路关键信号分子NF-κB激活从而导致TNF-α、IL-6、iNOS等释放减少有关。

Objective To evaluate the in vitro anti-inflammatory and action mechanism of anti-inflammatory effect of Fuke Qianjin Capsule extract(FKE).Methods MTT assay was used to detect the effects of different concentrations of FKE alone and FKE plus 100 ng/mL lipopolysaccharide(LPS) on the activity of mouse macrophages RAW 264.7 cells.ELISA method was applied to examine the effects of FKE with different concentrations on the release of inflammatory cytokines, like nitric oxide(NO),interleukin-6(IL-6), and tumor necrosis factor α(TNF-α) in RAW 264.7 cells after LPS stimulation.The effects of FKE with different concertrations on the transcription levels of inflammatory-related proteins, such as COX-2, IL-6, iNOS, and TNF-αwere observed by RT-PCR.Western blot was used to analyze the effects of FKE with different concentrations on the expression of inflammatory-related proteins of IκBα and iNOS.The effect of FKE with different concentration on nuclear translocation of NF-κB p65 was detected by laser confocal technology.The effect of FKE with different concentrations on nuclear factor-kB(NF-κB)was detected by the dual-luciferase reporter system.Results MTT measurement showed that FKE had no significant effect on the activity of RAW 264.7 cells in the concentration of 50~800 μg/mL when compared with the control group(P>0.05).ELISA detecation showed that FKE can significantly reduce the NO content of RAW 264.7 cells induced by LPS.FKE with 400 μg/mL showed an inhibitory effect on IL-6 and TNF-α levels(P<0.05);Western blot detection showed that FKE significently inhibited the activity of iNOS, but without effects on IκBα.Laser confocal analysis showed that FKE partially inhibited the nuclear transpotation of p65 when compared with the control group.FKE at 400 μg/mL could obviously inhibit the activation of NF-κB(P<0.05).Conclusion FKE showed an obvious effect on alleviating inflammation, whose mechanism might be related to FKE’s inhibition of activation of NF-κB, a key signaling molecule in the inflammatory pathway, resulting in reduced release of TNF-α, IL-6, iNOS, etc.