发酵金针菇多糖通过核转录因子-κB-NOD样受体家族含pyrin结构域蛋白3信号通路抑制巨噬细胞炎症反应

Fermented Flammulina velutipes Polysaccharide Inhibits Macrophage Inflammatory Response through Nuclear Factor-κB-NOD-Like Receptor Family Pyrin Domain-Containing Protein 3 Signaling Pathway

为探究金针菇多糖(FVP)和发酵金针菇多糖(FFVP)对小鼠单核巨噬细胞(RAW264.7)炎症反应的影响与机制,以脂多糖(LPS)构建RAW264.7炎症模型,设置CON组(正常培养基)、LPS组(正常培养基+1μg/mL LPS)、FVP组(正常培养基+1μg/mL LPS+25、50或100μg/mL FVP)和FFVP组(正常培养基+1μg/mL LPS+25、50或100μg/mL FFVP),通过测定RAW264.7的细胞活力、吞噬能力、活性氧(ROS)和一氧化氮(NO)含量以及炎症因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-18(IL-18)和肿瘤坏死因子-α(TNF-α)的含量与mRNA相对表达量,比较FVP和FFVP抑制巨噬细胞炎症反应的作用;以核转录因子-κB(NF-κB)抑制剂BAY11-7082处理RAW264.7,通过Western blot检测磷酸化核转录因子-κB抑制蛋白α(p-IκBα)、NOD样受体家族含pyrin结构域蛋白3(NLRP3)、半胱天冬蛋白酶-1(Caspase-1)和IL-1β的蛋白相对表达量,探究FVP和FFVP对LPS诱导的巨噬细胞炎症反应可能的作用机制。结果表明:FVP和FFVP均能抑制LPS引起的RAW264.7 ROS和NO含量升高,浓度为100μg/mL时,两者差异显著(P<0.05);相比LPS组,FVP和FFVP分别使RAW264.7的吞噬能力提升37.65%和47.06%;FVP和FFVP降低炎症因子IL-1β、IL-6、IL-18和TNF-α的含量及mRNA相对表达量,并呈剂量依赖性。Western blot结果表明以BAY11-7082与FVP或FFVP同时处理,RAW264.7的p-IκBα、NLRP3、Caspase-1和IL-1β的蛋白相对表达量显著降低(P<0.05),说明FVP/FFVP可通过降低IκBα的磷酸化抑制NLRP3信号通路的激活。综上可知,FVP和FFVP均能增强RAW264.7的细胞活力和吞噬能力,降低ROS和NO含量,通过抑制NF-κB-NLRP3信号通路的激活抑制巨噬细胞炎症反应;相同浓度下,FFVP的抗炎效果优于FVP。

To investigate the effects and mechanism of Flammulina velutipes polysaccharide(FVP)and fermented Flammulina velutipes polysaccharide(FFVP)on the inflammatory response of mouse mononuclear macrophages(RAW264.7),we constructed an inflammatory model of RAW264. 7 with lipopolysaccharide(LPS). CON group(normal medium),LPS group(normal medium+1μg/mL LPS),FVP group(normal medium+1μg/mL LPS+25,50 or 100μg/mL FVP)and FFVP group(normal medium+1μg/mL LPS+25,50 or 100μg/mL FFVP)were set,cell viability,phagocytosis ability,the contents of reactive oxygen species(ROS)and nitric oxide(NO)as well as the contents and mRNA relative expression levels of inflammatory cytokines interleukin-1β (IL-1β),interleukin-6(IL-6),interleukin-18(IL-18)and tumor necrosis factor-α(TNF-α)in RAW264.7 were determined to compare the inhibitory effects of FVP and FFVP on the inflammatory response of macrophages. RAW264.7 were treated with nuclear factor-κB(NF-κB)inhibitor BAY11-7082,and the protein relative expression levels of phosphorylated nuclear factor-κB inhibitorα (p-IκBα),NOD-like receptor family pyrin domain-containing protein 3(NLRP3),cysteinyl aspartate specific proteinase-1(Caspase-1)and IL-1βwere detected by Western blot to explore the mechanism of FVP and FFVP on LPS-induced inflammatory response of macrophages. The results showed that both FVP and FFVP could inhibit the increase of ROS and NO contents in RAW264.7 induced by LPS,and the difference was significant at 100μg/mL(P<0.05). Compared with LPS group,FVP and FFVP increased phagocytosis ability of RAW264.7 by 37.65% and 47.06%,respectively. FVP and FFVP decreased the contents and mRNA relative expression levels of inflammatory cytokines IL-1β,IL-6,IL-18 and TNF-αin a dose-dependent manner.Western blot results showed that the protein relative expression levels of p-IκBα,NLRP3,Caspase-1 and IL-1βin RAW264.7 were significantly decreased by BAY11-7082 combined with FVP or FFVP(P<0.05),indicating that FVP/FFVP could inhibit the activation of NLRP3 signaling pathway by reducing IκBαphosphorylation. In conclusion,both FVP and FFVP can enhance cell viability and phagocytosis ability of RAW264.7,reduce ROS and NO contents,and inhibit macrophage inflammatory response by inhibiting NF-κB-NLRP3 signaling pathway activation. The anti-inflammatory effect of FFVP is better than that of FVP at the same concentration.[Chinese Journal of Animal Nutrition,2022,34(2):1205-1216]