Influences Of Berberine On ATP-induced Inflammasome Activation In Macrophages And The Underlying Mechanism

Aim:Berberine is an isoquinoline alkaloid purified from Rhizoma coptidis and has been reported to possess many pharmacological properties including antioxidant,antidiabetic,anti-inflammatory and antibacterial infection activities.Although the direct bactericidal effect of berberine has been documented,its influence on the antibacterial functions of macrophages is largely unknown.As inflammasome activation in macrophages is important for the host defense against bacterial infection.Pattern recognition receptors(PRRs),which reside in the plasma membrane or intracellular of macrophages can detect unique microbial structures termed pathogen associated molecular patterns(PAMPs)expressed on pathogenic microorganisms.They induce inflammatory responses leading to the expression of critical inflammasome components(such as NLRP3 and pro-IL-1β)and the activation of macrophages.When the activated macrophages are further triggered with danger signals(such as extracellular ATP and monosodium urate crystal),these above proteins assemble into cytosolic protein complexes named inflammasomes to induce the inflammatory form of cell death termed pyroptosis,which is required for the release of mature IL-1β as well as other cytokines.These cytokines not only expand inflammatory responses,but also promote the functions of macrophages by increasing their phagocytic and bacterial killing abilities.Thus we aimed to investigate the role of berberine to augment ATP-induced inflammasome activation and pyroptosis and the underlying mechanism,which will facilitate the understanding of the immunopharmacological action and indirect antimicrobial effects of berberine against bacterial infection by influencing the functions of macrophages as well as other innate immune cells.Methods:(1)Macrophages: J774 A.1 murine macrophage cell line and two additional types of primary murine macrophages were used.One was thioglycollate(TG)-elicited peritoneal macrophages(TGPMs),and the other was bone marrow-derived macrophages(BMDMs).(2)Inflammasome activation: Murine macrophages were primed with LPS for 4 h and then pre-treated with graded concentrations of berberine for 1 h,followed by incubation with ATP in the absence of LPS.Propidium iodide staining was used to detect the cell death.Western blotting assay was used to detect the effect of berberine on the expression levels of the inflammasome activation associated markers.Soluble mature interleukin(IL)-1β levels were also detected by using a bead-based immunoassay(CBA).(3)Analysis of action mechanism of berberine: AMP-activated protein kinase(AMPK)specific inhibitor compound C and siRNA knockdown of AMPKα expression were used to block AMPK signaling.(4)Bacterial infection model: Mice were injected intraperitoneally with live E.coli,and intragastrically administered with berberine.CBA or western blotting was used to assess the level of IL-1β.Flow cytometric analysis was adopted to analyze the percentages of neutrophils in the peritoneal cavity of mice.In addition,histological section was used to explore inflammatory cell infiltration.The bacterial burden was assayed by LB agar plates.The survival of mice was also recorded.Results:(1)Berberine could markedly augment ATP-induced inflammasome activation,pyroptosis and mature IL-1β release in a dose-dependent manner in macrophages.(2)Berberine could enhance AMPK signaling in macrophages upon ATP treatment.(3)AMPK signaling blockade by specifc inhibitor compound C or AMPKα knockdown markedly reversed berberine-mediated augmentation of ATP-induced inflammasome activation and pyroptosis.(4)Berberine enhanced bacterial killing and clearance by macrophages in vitro and in vivo,while decreasing the bacterial burden(CFUs)in the peritoneal cavity.Furthermore,berberine treatment enhanced IL-1β release and neutrophil recruitment in the peritoneal cavity and colon,leading to increased the survival of mice upon bacterial infection.Conclusion:Berberine could enhance bacterial killing and clearance by augmenting inflammasome activation and intensifying the function of macrophages,thus indirectly exhibiting antibacterial effects.These effects of berberine on macrophages might be attributed to its role in enhancing AMPK activity in these cells.