黄酮化合物对巨噬细胞中白细胞介素介导的TLR4/MyD88/NF-κB信号转导通路的调节作用OA北大核心CSTPCD
Regulatory Effect of Flavonoid Compounds on Interleukins in Macrophage via TLR4/MyD88/NF-κB Signaling Pathway
目的 利用巨噬细胞,评价黄酮类化合物(华良姜素和木犀草素)对促炎、抗炎白细胞介素(IL)因子表达以及Toll样受体 4/髓样分化因子/核因子-κB(TLR4/MyD88/NF-κB)信号转导的调节作用.方法 采用脂多糖(LPS)诱导小鼠单核巨噬细胞RAW264.7,黄酮设低、中、高浓度组分别与LPS共处理,建立细胞炎症和抗炎模型.利用酶联免疫吸附法测定培养液中一氧化氮(NO)和IL表达.沉默IL-6,用反转录聚合酶链反应法检测IL-10/IL-17 mRNA沉默IL-10,检测IL-6/IL-12 mRNA 变化.蛋白免疫印迹法检测 Toll 样受体 4(TLR4)、磷酸化 p65(p-p65)、髓样分化因子(MyD88)、磷酸化 IκBα(p-IκBα)蛋白.结果 华良姜素、木犀草素抑制 NO 半数抑制浓度(IC50)分别为 0.60 和8.93 μmol·L-1.LPS诱导可降低抗炎因子IL-4、IL-10 表达,并且提高促炎因子 IL-6、IL-12、IL-16、IL-17 和 IL-23 表达(P＜0.01).与LPS组比较,黄酮分别处理可剂量依赖地中和促炎因子上调和抗炎因子的下调作用(P＜0.05 或P＜0.01).沉默IL-10 上调IL-6/IL-12 mRNA在空白对照组、LPS诱导和LPS+黄酮组细胞中表达;沉默IL-6下调IL-10/IL-17 mRNA表达(P＜0.05 或 0.01).与空白对照组比较,LPS诱导激活细胞表达TLR4、p-p65、MyD88、p-IκBα蛋白,而经黄酮处理后蛋白表达均被抑制(P＜0.05 或 0.01).结论 2 种黄酮类化合物均具有抗炎活性,可中和巨噬细胞的促炎、抗炎IL因子.IL-10 反向调节IL-6/IL-12,IL-6正向调节IL-10/IL-17.巨噬细胞的双重IL中和作用通过抑制TLR4/MyD88/NF-κB信号通路来实现.
Objective To validate regulatory effects of two flavonoid(kumatakenin and luteolin)in macrophage con-cerning inflammatory/anti-inflammatory interleukins(IL)expression and TLR4/MyD88/NF-κB signaling.Methods Murine RAW264.7 macrophage cellular inflammation was induced by lipopolysaccharide(LPS),coupling synergistically anti-inflammato-ry treatment with different flavonoid concentrations.Expression levels of nitric oxide(NO)and IL were determined in cell culture supernatant using enzyme-linked immunosorbent assay.An effect of small-interfering RNA(siRNA)-mediated IL-6 knockdown on IL-10/IL-17 mRNA expressions was assayed by reverse transcription polymerase chain reaction(RT-PCR),and IL-6/IL-12 mR-NA regulation is performed by IL-10 knockdown.Western blotting(WB)compares a relative ratio of TLR4,p-p65,MyD88,and p-IκBα productions in macrophages blank,LPS-induced,and LPS+flavonoid-co-treated.Results IC50 values of kumatakenin and luteolin are 0.60 and 8.93 μmol·L-1,respectively,concerning the NO inhibition in LPS-induced cells.Compared to blank,LPS inducement leads to a decrease of anti-inflammatory IL-4 and IL-10,whereas the increase of inflammatory IL-6,IL-12,IL-16,IL-17,and IL-23(P＜0.01).Cellular co-treatment with LPS+kumatakenin or LPS+luteolin significantly blocks a regulatory effect of ILs in a flavonoid dose-dependent manner(P＜0.05 or P＜0.01).IL-6 and IL-12 mRNA are increased in the IL-10 siRNA,LPS+IL-10 siRNA,and LPS+IL-10 siRNA+flavonoid treatment groups,compared to the no-target siRNA negative control.IL-6 knockdown can down-regulate IL-10 and IL-17 mRNA expressions(P＜0.05 or P＜0.01).WB result shows that expression of TLR4,p-p65,MyD88,and p-IκBα is significantly increased in the LPS-induced group,while the expression level is inhibited when LPS cotreatment with flavonoid(P＜0.05 or P＜0.01).Conclusions Kumatakenin and luteolin are active against LPS-induced inflammation.They neutralize both pro-inflammatory and anti-inflammatory ILs in macrophages.IL-10 negatively regu-lates IL-6 and IL-12 expressions,while IL-6 positively regulates IL-10 and IL-17.Dual-neutralization of ILs occurs via inhibition of the TLR4/MyD88/NF-κB signaling in inflammatory macrophage.
冯逸佳;李庆芳;董壮壮;张圆琳;乔元彪;李青山
山西中医药大学中药与食品工程学院,基于炎性反应的重大疾病创新药物山西省重点实验室,晋中 030619
药学
黄酮化合物华良姜素木犀草素白细胞介素核因子-κB
Flavonoid compoundsKumatakeninLuteolinInterleukinNuclear factor-κB
《医药导报》 2024 (005)
689-695 / 7
山西省医学科技创新团队领军项目(2020TD02);山西中医药大学博士科研启动基金资助项目(2022BK01).
评论