刺槐素通过核因子κB信号通路抑制脂多糖诱导的巨噬细胞损伤

Inhibition of LPS-induced Macrophage Injury by Acacetin via Regulating NF-κB Signaling Pathway

目的:探讨刺槐素(Acacetin)对脂多糖(LPS)诱导的小鼠骨髓巨噬细胞(BMDMs)的抗炎作用机制。方法:CCK-8法检测各浓度刺槐素(5μmol/L,10μmol/L,20μmol/L,40μmol/L)对小鼠骨髓巨噬细胞增殖的影响,筛选最佳剂量;分离培养小鼠骨髓巨噬细胞,LPS(50 ng/mL)预处理BMDMs(0 min,10 min,30 min,60 min),加入刺槐素(10μmol/L)孵育0.5 h。蛋白质印迹法检测p65、磷酸化p65(p-p65)、核因子κB抑制蛋白α(IκBα)、磷酸化IκBα(p-IκBα)的表达,激光共聚焦观察核因子κB核转位情况。结果:CCK-8结果显示,5~40μmol/L的刺槐素对小鼠骨髓巨噬细胞增殖无明显影响,结合课题组前期研究结果,选择10μmol/L刺槐素作为后续研究剂量;与空白对照组比较,LPS不同时间刺激组p-p65、p-IκBα的表达水平均显著增加,给予刺槐素治疗后,能显著降低p-核因子κB p65和p-IκBα的表达;刺槐素能抑制LPS介导的核因子κB p65核转位。结论:刺槐素的抗炎作用与抑制核因子κB信号通路有关,刺槐素可作为一种有潜力的候选抗炎药物。

Objective:To explore the anti-inflammatory mechanisms of acacetin in lipopolysaccharide(LPS)-induced mouse bone marrow-derived macrophages(BMDMs).Methods:The effect of various concentrations of acacetin(5,10,20,and 40 μmol/L) on the proliferation of BMDMs was tested using the CCK-8 method,so as to select the optimal dosage.BMDMs were isolated and cultured,and LPS(50 ng/mL) was used to pretreat BMDMs for 0,10,30,and 60 minutes.Then,BMDMs were added with acacetin(10 μmol/L) and cultured for 0.5 h.Western blotting was used to analyze the expression of phosphorylated p65(p-p65),p65,nuclear factor(NF) κB suppressor protein α(IκBα),and phosphorylated IκBα(p-IκBα).The nuclear translocation of NF κB was detected by laser scanning confocal microscope(LSCM).Results:CCK-8 results showed that acacetin concentrations ranging from 5 to 40 μmol/L had no significant effect on the proliferation of BMDMs.Based on previous studies of the team,10 μmol/L of acacetin was chosen for subsequent experiments.Compared with the blank control group,LPS stimulation significantly increased the expression levels of p-p65 and p-IκBα.Treatment with acacetin significantly reduced the expression levels of p-NF-κB p65 and p-IκBα.Moreover,acacetin inhibited the LPS-induced nuclear translocation of NF-κB p65.Conclusion:The anti-inflammatory effects of acacetin were related to the inhibition of the NF-κB signaling pathway,suggesting acacetin may be a potential anti-inflammatory drug.