葛根素对oxLDL诱导的THP-1巨噬细胞TLR4-NF-κB信号转导通路的影响

Effect of puerarin on oxLDL-induced TLR4-NF-κB signal transduction pathway in THP-1 macrophages

目的:研究葛根素对人单核细胞系来源的THP-1巨噬细胞表达的炎症因子及TLR4-NF-κB信号通路的影响,探讨葛根素治疗动脉粥样硬化可能存在的调节机制。方法:100 nmol/L佛波酯(PMA)刺激THP-1细胞24 h,诱导其分化为巨噬细胞,再分别给予oxLDL(50μg/ml)、葛根素低剂量(10μg/ml)和葛根素高剂量(100μg/ml)干预。MTS法检测不同浓度葛根素对THP-1细胞增殖的作用;FCM法检测THP-1细胞凋亡比例;qPCR法检测炎性细胞因子IL-6、TNF-α、IL-1β及TLR4、MyD88、NF-κB mRNA水平;Western blot法及细胞免疫荧光法测定TLR4、MyD88、NF-κB蛋白含量和表达定位。结果:10μg/ml和100μg/ml葛根素对THP-1细胞增殖无抑制作用(P>0.05);低剂量(10μg/ml)和高剂量(100μg/ml)葛根素均能降低oxLDL诱导的THP-1细胞凋亡比例(P<0.01),下调炎性细胞因子IL-6、TNF-α、IL-1βmRNA水平(P<0.05)及TLR4、MyD88、NF-κB mRNA和蛋白质含量(P<0.05);细胞免疫荧光显示TLR4表达于细胞膜,MyD88表达于胞质,NF-κB表达于胞核,且低、高剂量葛根素均能抑制TLR4、MyD88和NF-κB的表达强度。结论:葛根素通过下调oxLDL诱导的THP-1细胞TLR4-NF-κB信号转导通路,进而抑制炎性因子表达,这可能是葛根素抗动脉粥样硬化的机制之一。

Objective:To investigate the effects of puerarin on inflammatory cytokines and TLR4-NF-κB signaling pathway expressed by THP-1 macrophages derived from human monocytic cell line,and to explore the possible regulatory mechanism of puerarin on the treatment of atherosclerosis.Methods:THP-1 cells were stimulated by 100 nmol/L of PMA for 24 h,differentiated into macrophages,and then administered with oxLDL(50μg/ml),low dose of puerarin(10μg/ml)and high dose of puerarin(100μg/ml).MTS assay was used to detect the proliferation of THP-1 cells by different concentrations of puerarin.The apoptosis rate of THP-1 cells was determined by FCM.The mRNA levels of inflammatory cytokines IL-6,TNF-α,IL-1βand TLR4,MyD88 and NF-κB were detected by qPCR.The protein expression of TLR4,MyD88 and NF-κB were detected by Western blot,and located by cellular immunofluorescence in THP-1 macrophages.Results:The 10μg/ml and 100μg/ml of puerarin had no inhibitory effect on the proliferation of THP-1 cells(P>0.05).The low dose(10μg/ml)and high dose(100μg/ml)of puerarin reduced oxLDL-induced apoptosis rate(P<0.01),upregulated IL-6,TNF-αand IL-1βmRNA(P<0.05),and mRNA and protein expressions of TLR4,MyD88 and NF-κB in THP-1 cells(P<0.05).TLR4,MyD88 and NF-κB were located in the cell membrane,cytoplasm and nucleus.And both low and high doses of puerarin inhibited the expression intensity.Conclusion:Puerarin inhibits the expression of inflammatory factors by down-regulating TLR4-NF-κB signal transduction pathway in oxLDL-induced THP-1 cells,which may be one of the mechanisms of puerarin against atherosclerosis.