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

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。

刺槐素对鞭毛蛋白诱导的小鼠骨髓巨噬细胞NLRC4炎症小体活化的影响

目的:研究刺槐素(Acacetin)对鞭毛蛋白诱导的小鼠骨髓巨噬细胞Nod样受体家族蛋白4(the nod-like receptor family card domaincontaining protein 4,NLRC4)活化的影响。方法:将小鼠骨髓巨噬细胞分为对照组、脂多糖(LPS)组、LPS+鞭毛蛋白组及LPS+Acacetin+鞭毛蛋白组,对照组加入完全培养基,LPS组用LPS(50 ng/mL)预处理3 h,LPS+鞭毛蛋白组用LPS(50 ng/mL)预处理3 h后加入鞭毛蛋白(10μmol/L)刺激0.5 h;LPS+刺槐素+鞭毛蛋白组用LPS(50 ng/mL)预处理3 h后再加入Acacetin(10μmol/L)处理0.5 h,最后加入鞭毛蛋白(10μmol/L)刺激0.5 h;采用WB检测细胞裂解液Pro-caspase-1和Pro-IL-1β的表达,上清液中caspase-1和IL-1β的蛋白表达;ELISA法检测上清液中IL-1β、IL-18和TNF-α的含量,LDH检测试剂盒检测细胞上清液中LDH的活性。结果:与对照组相比,LPS+鞭毛蛋白组细胞上清液中caspase-1、IL-1β蛋白表达明显增高,差异具有统计学意义(P<0.05),细胞裂解液中Pro-caspase-1、Pro-IL-1β蛋白表达比较,差异无统计学意义(P>0.05);与LPS+鞭毛蛋白组比较,LPS+Acacetin+鞭毛蛋白组细胞上清液caspase-1、IL-1β蛋白表达明显降低,差异具有统计学意义(P<0.05),细胞裂解液中Pro-caspase-1、Pro-IL-1β蛋白表达比较,差异无统计学意义(P>0.05)。ELISA结果显示,与对照组相比,LPS+鞭毛蛋白组细胞上清液中IL-1β、IL-18、TNF-α水平及LDH活性明显升高,差异均具有统计学意义(P<0.05);与LPS+鞭毛蛋白组比较,LPS+Acacetin+鞭毛蛋白组细胞上清液IL-1β释放水平明显降低,差异均具有统计学意义(P<0.05),IL-18、TNF-α的水平及LDH的活性降低,但差异无统计学意义(P>0.05)。结论:刺槐素可有效抑制鞭毛蛋白诱导的小鼠骨髓巨噬细胞NLRC4炎症小体的活化,减少细胞损伤。

Effect of Acacetin on flagellin induced NLRC4 inflammasome activation in mouse bone marrow-derived macrophages

Objective:To investigate the effect of acacetin on flagellin induced NLRC4 inflammasome activation in mouse bone marrow-derived macrophages(BMDMs).Methods:Mouse BMDMs were divided into control group,LPS group,LPS+flagellin group and LPS+acacetin+flagellin group.All groups were added with complete medium,then primed with LPS(50 ng/mL)for 3 hrs except the control group,whereafter,LPS+flagellin group was treated with flagellin(10μmol/L)for 0.5 hr and LPS+acacetin+flagellin group was treated with acacetin(10μmol/L)for 0.5 hr following by flagellin(10μmol/L)for 0.5 hr.Pro-caspase-1,pro-IL-1βin cell lysate and caspase-1,IL-1βin supernatant were detected by Western blot(WB).IL-1β,IL-18 and TNF-αin supernatant were measured by enzyme-linked immunosorbent assay(ELISA).And the activity of LDH in supernatant was assessed by LDH test kit.Results:Compared with the control group,in LPS+flagellin group,the expression of caspase-1,IL-1βprotein in supernatant were significantly increased(all P-values<0.05),but the differences of the expression of pro-caspase-1 and pro-IL-1βprotein in cell lysate were not significant.Compared with LPS+flagellin group,in LPS+acacetin+flagellin group,the expression of caspase-1,IL-1βprotein in supernatant were significantly reduced(all P-values<0.05),while the differences of the expression of pro-caspase-1 and pro-IL-1βprotein in cell lysate were not significant.ELISA showed that compared with the control group,the levels of IL-1β,IL-18,and TNF-αand the activity of LDH in supernatant of LPS+flagellin group were significantly increased(all P-values<0.05).Compared with LPS+flagellin group,in LPS+Acacetin+flagellin group,the level of IL-1βin supernatant was significantly decreased(P<0.05),meanwhile,the decreases of the levels IL-18,TNF-αand the activity of LDH were not significant.Conclusions:We found that Acacetin can effectively inhibit flagellin induced NLRC4 inflammasome activation and reduce cell damage in mouse BMDMs.