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 and cell damage in mouse bone marrow-derived macrophages(BMDMs).Methods: Bone marrow macrophages from 3-month-old C57BL/6 mice(25±5g)were used in this study and 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(10 μM)for 0.5 hr and LPS + acacetin + flagellin group was treated with acacetin(10 μM)for0.5 hr following by flagellin(10 μM)for 0.5 hr.The levels of Pro-caspase-1 and Pro-IL-1β in cell lysates and caspase-1 and IL-1β in cell supernatants were measured by Western blot(WB).IL-1β,IL-18 and TNF-α in supernatant were detected by enzyme-linked immunosorbent assay(ELISA)kits.LDH assay kits were used to detect the activity of LDH in cell supernatants.Results:(1)WB showed that the expression levels of caspase-1 and IL-1β protein in the supernatant of cells in the LPS group were increased compared to the control group,but not statistically significant.After stimulation with flagellin,compared with the LPS + group,the expression levels of caspase-1 and IL-1β protein were significantly increased in the LPS + flagellin group(P < 0.05).Compared with the LPS + flagellin group,the expression of caspase-1 and IL-1β protein in cell supernatant was significantly reduced in the LPS +Acacetin + flagellin group(P < 0.05).(2)There was no significant difference in the expression levels of Pro-caspase-1 and Pro-IL-1β protein in the cell lysates between the groups(P>0.05).(3)The Elisa showed that the expression levels of IL-1β and IL-18 in the cell supernatant of the LPS group were increased compared with the control group,but the difference was not statistically significant(P>0.05),while the level of TNF-α was significantly increased and the difference was statistically significant(P<0.05).The levels of IL-1β,IL-18 and TNF-α in the supernatant in the LPS+ flagellin group were significantly higher compared to the LPS group(P<0.05).Compared with the LPS+ flagellin group,the levels of IL-1β in the cell supernatant in the LPS+ Acacetin+ flagellin group were significantly lower(P<0.05),and the levels of IL-18 and TNF-α showed a decreasing trend.(4)The LDH assay showed that the activity of LDH was significantly higher in the LPS group compared to the control group(P<0.05).Compared to the LPS group,in the LPS+ flagellin group,the activity of LDH in the cell supernatant was significantly increased(P<0.05),and in the LPS+Acacetin+ flagellin group,the activity of LDH was significantly increased(P<0.05).Conclusion: Acacetin effectively inhibited flagellin-induced activation of NLRC4 inflammasome in mouse BMDMs,resulting in reduced expression of caspase-1 and IL-1β and secretion of inflammatory factors,decreased cellular damage.