大豆苷元通过调控NLRP3炎性小体信号通路对高糖诱导的巨噬细胞炎症损伤的影响及机制研究

Daidzein attenuates high glucose-induced inflammatory injury in macrophages by regulating NLRP3 inflammasome signaling pathway

探究大豆苷元(daidxein)调控NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)信号通路对高糖诱导的巨噬细胞炎症反应的抑制作用。cell counting kit(CCK)-8法检测不同浓度大豆苷元对巨噬细胞RAW264.7细胞活力的影响,Western blot法检测不同时间和葡萄糖浓度下巨噬细胞的肿瘤坏死因子(TNF)-α蛋白表达水平,以及高糖诱导下巨噬细胞极化、Toll样受体4(TLR4)-髓分化因子88(MyD88)-NLRP3炎性小体等通路相关蛋白的表达水平,酶联免疫检测试剂盒(ELISA)检测巨噬细胞分泌的TNF-α、白细胞介素(IL)-18、IL-1β炎症因子的表达情况,免疫荧光检测经高糖诱导下的巨噬细胞中细胞核因子(NF)-κB p65的表达水平,DCFH-DA荧光探针检测细胞内活性氧(ROS)的表达情况,qRT-PCR检测巨噬细胞中NLRP3、TNF-α和IL-18 mRNA水平。结果发现,选用30 mmol·L^(-1)葡萄糖浓度诱导48 h可作为巨噬细胞损伤的最佳造模条件;与正常组相比,模型组可以显著改善巨噬细胞极化,增加TNF-α、IL-18、IL-1β炎症因子的分泌,显著增加ROS及NF-κB p65的表达,模型组巨噬细胞NLRP3、TNF-α和IL-18 mRNA与TLR4、MyD88、NLRP3、NF-κB p65、p-NF-κB p65、NF-κB抑制因子(I-κB)、p-I-κB、凋亡相关斑点样蛋白(ASC)、半胱氨酸蛋白酶前体(pro-caspase)-1、pro-IL-1β、cleaved IL-1β和pro-IL-18蛋白表达均显著升高;与模型组相比,大豆苷元10、20、40μmol·L^(-1)剂量组中巨噬细胞各炎症因子,炎症相关基因NLRP3、TNF-α和IL-18 mRNA,炎症相关蛋白TLR4、MyD88、NLRP3、NF-κB p65、p-NF-κB p65、I-κB、p-I-κB、ASC、pro-caspase-1、pro-IL-1β、cleaved IL-1β和pro-IL-18表达水平均显著下调,且ROS的表达明显降低。基于相关文献报道结合该实验结果发现,高糖可诱导巨噬细胞极化,促进炎症因子的分泌,大豆苷元能通过调节NLRP3炎症小体信号通路,抑制巨噬细胞ROS的表达从而减轻高糖诱导的巨噬细胞炎症反应。

This study aims to explore the inhibitory effect of daidzein on macrophage inflammation induced by high glucose via regulating the NOD-like receptor protein 3(NLRP3)inflammasome signaling pathway.The cell counting kit-8(CCK-8)assay was employed to detect the effects of daidzein at different concentrations on the viability of RAW264.7 cells.Western blot was employed to determine the protein level of tumor necrosis factor(TNF)-αin macrophages exposed to different concentrations of glucose for different time periods as well as the expression levels of proteins involved in the polarization and Toll-like receptor 4(TLR4)-myeloid differentiation factor(MyD88)-NLRP3 inflammasome pathway of the macrophages exposed to high glucose.Enzyme-linked immunosorbent assay was employed to measure the levels of TNF-α,interleukin(IL)-18,and IL-1βsecreted by macrophages.The expression level of nuclear factor-kappa B(NF-κB)p65 in macrophages exposed to high glucose was detected by immunofluorescence,and the level of intracellular reactive oxygen species(ROS)was detected by the DCFH-DA fluorescent probe.The mRNA levels of NLRP3,TNF-α,and IL-18 in macrophages were determined by qRT-PCR.The results showed that treatment with 30 mmol·L^(-1) glucose for 48 h was the best condition for the modeling of macrophage injury.Compared with the blank group,the model group showed improved polarization of macrophages,increased secretion of TNF-α,IL-18,and IL-1β,elevated ROS level,and up-regulated expression of NF-κB p65.In addition,the modeling up-regulated the mRNA levels of NLRP3,TNF-α,and IL-18 and the protein levels of TLR4,MyD88,NLRP3,NF-κB p65,p-NF-κB p65,I-κB,p-I-κB,ASC,pro-caspase-1,pro-IL-1β,cleaved IL-1β,and pro-IL-18.Compared with the model group,daidzein(10,20,and 40μmol·L^(-1))lowered the levels of inflammatory cytokines and down-regulated the mRNA levels of NLRP3,TNF-α,and IL-18 as well as the protein levels of TLR4,MyD88,NLRP3,NF-κB p65,p-NF-κB p65,I-κB,p-I-κB,ASC,pro-caspase-1,pro-IL-1β,cleaved IL-1β,and pro-IL-18.In addition,daidzein reduced intracellular ROS.According to the available reports and the experimental results,high glucose can induce the polarization of macrophages and promote the secretion of inflammatory cytokines.Daidzein can inhibit the expression of ROS in macrophages by regulating the NLRP3 inflammasome signaling pathway,thereby reducing the inflammation of macrophages exposed to high glucose.