胀果甘草多糖GiP-B1通过TLR4/MyD88/NF-κB信号通路激活巨噬细胞RAW 264.7

Polysaccharide GiP-B1 from Glycyrrhiza inflata activates RAW 264.7 by TLR4/MyD88/NF-κB signaling pathway

为研究胀果甘草多糖GiP-B1通过TLR4/MyD88/NF-κB信号通路对巨噬细胞RAW 264.7免疫功能的影响,体外以TLR4干扰RNA(TLR4-siRNA)转染巨噬细胞RAW 264.724 h,用100μg/mL GiP-B1处理转染和未转染细胞,12 h后分别采用CCK-8和ELISA法检测GiP-B1对巨噬细胞RAW 264.7增殖和分泌炎症因子的影响,并采用RT-PCR和Western blot法检测GiP-B1对巨噬细胞TLR4及其信号通路下游基因MyD88、NF-κB p65、IκBα的mRNA和蛋白表达水平的影响。结果显示,与空白对照组比较,GiP-B1可显著提高巨噬细胞RAW 264.7的增殖,促进白细胞介素1β(IL-1β)、白细胞介素6(IL-6)及肿瘤坏死因子α(TNF-α)的分泌,上调TLR4、MyD88、NF-κB p65、IκBα的mRNA和蛋白表达量(P<0.05);对各组进行TLR4基因沉默后,与正常培养的GiP-B1组比较,转染TLR4-siRNA可抑制GiP-B1诱导的巨噬细胞RAW 264.7的增殖、细胞因子的分泌以及TLR4、MyD88、NF-κB p65、IκBα的mRNA和蛋白表达水平(P<0.05)。以上结果表明,GiP-B1可活化TLR4/MyD88/NF-κB信号通路,促进相关基因表达,使NF-κB进入细胞核调控相关细胞因子的转录水平,进而激活巨噬细胞的免疫功能。

The aim of this study was to investigate the acidic polysaccharide GiP-B1 from Glycyrrhiza inflata on activation of RAW 264.7 through TLR4/MyD88/NF-κB signaling pathway.RAW 264.7 were transfected with TLR4 interfering RNA(TLR4 siRNA)for 24 h in vitro,and then treated with GiP-B1(100μg/mL)for 12 h.The effects of proliferation and secretion of inflammatory factors in RAW 264.7 were detected by CCK-8 and ELISA kits,respectively.RT-PCR and Western blot method was used to detect the effects of GiP-B1 on the mRNA and the protein expression levels of TLR4,MyD88,NF-κB p65 and IκBα.The results showed that compared with the blank control group,GiP-B1 could significantly increase the proliferation of RAW 264.7 and the secretion of interleukin 1 beta(IL-1β),interleukin 6(IL-6),and tumor necrosis factor alpha(TNF-α),and significantly up-regulate the mRNA and protein expressions of TLR4,MyD88,NF-κB p65 and IκBα(P<0.05).After TLR4 gene silencing was performed for each group,compared with the normal cultured GiP-B1 intervention group,the transfection group could inhibited the proliferation and cytokine secretion of RAW 264.7 induced by GiP-B1,which also inhibited the mRNA and protein expression levels of TLR4,MyD88,NF-κB p65 and IκBα(P<0.05).In summary,GiP-B1 activates TLR4/MyD88/NF-κB signaling pathway to promote the expression of related genes,and finally promotes NF-κB into the nuclear to regulate the transcription level of cytokines,and then regulates the macrophage immune function.