The Mechanism For Scutellarin-mediated Suppression Of NLRP3 Inflammasome Activation In Macrophages And Its Protective Action Against Bacterial Sepsis

Aim:Scutellarin is a flavonoid isolated from Erigeron breviscapus(Vant.)Hand.-Mazz.and has a wide range of pharmacological effects including improving cerebral circulation and increasing cerebral blood Flow,reducing cerebral vascular resistance,improving the permeability of the blood-brain barrier,increasing the flow of nutrient myocardium,enhancing the phagocytic immune function of macrophages,and counteracting the platelet aggregation induced by adenosine diphosphate.Clinically,scutellarin is mainly used in the treatment of cerebral vascular disease after paralysis as well as ischemic and hypoxic cardiovascular diseases.In addition,in recent years,some studies have shown that scutellarin can inhibit inflammatory-related signaling and the expression of inflammatory cytokines to exert its anti-inflammatory effects.However,the effect of scutellarin on the activation of NLRP3 inflammasome has not been reported yet.Therefore,in this research,we used LPS-primed mouse e macrophages as an inflammatory cell model in vitro and mouse bacterial sepsis model by intraperitoneal inoculation of viable Escherichia coli(E.coli)to investigate the effects of scutellarin on the activation of NLRP3 inflammasome and pyroptosis and the its action against bacterial sepsis.Methods:The effects of scutellarin on LPS+ATP or LPS+nigericin induced pyroptosis in murine macrophages were analyzed by propidium iodide staining assay.The levels of IL-1β,caspase-1 and HMGB1 in cell lysates and culture supernatants were analysis by Western blotting.Soluble cytokine IL-1β in culture supernatants of murine macrophages was detected by cytometric beads array(CBA).ATP or nigericin-induced ASC speck formation and ASC oligomerization in murine macrophages were analysed by immunofluorescence and cross-linking.Mouse model of bacterial sepsis was established by intraperitoneal injection of a lethal dose of viable Escherichia coli,and oral administration of scutellarin was used to observe its effect on survival rate of mice and the changes of serum IL-1β and inflammatory cells in the liver.Results:Scutellarin treatment inhibited ATP or nigericin induced caspase-1 activation and mature IL-1β release,and also suppressed NLRP3 inflammasome activation induced pyroptotic cell death and ASC speck formation in murine macrophages.Scutellarin’s inhibitory effects on ATP-induced pyroptosis and ASC speck formation were markedly reversed by the adenylate cyclase inhibitor MDL12330 A and selective protein kinase A(PKA)inhibitor H89.In vivo activity of scutellarin was assayed in a mouse model of bacterial sepsis.Oral administration of scutellarin significantly improved the survival of mice with bacterial sepsis.In line with this,scutellarin treatment significantly reduced serum IL-1β levels and attenuated the infiltration of inflammatory cells in the liver of E.coli-infected mice.Conclusion:Scutellarin inhibited NLRP3 inflammasome activation and pyroptosis by modulating the PKA activity in macrophages,thereby suppressing over inflammatory responses and exhibiting anti-septic activity.