Baicalin-mediated Inhibition Of NLRP3 Inflammasome Activation In Macrophages And The Underlying Action Mechanism

Aim:Baicalin is a flavonoid isolated from the root of Scutellaria baicalensis Georgi,a well-known Chinese medicinal plant.It has been demonstrated that baicalin possesses many bioactivities and pharmacological effects,including anti-inflammatory,anti-tumor,antibacterial,antiviral and hepatoprotective activities.Although baicalin has been show to exhibit anti-inflammatory activity by down-regulating the expression of inflammatory cytokines via suppressing the NF-κB signaling pathway,its action on NLRP3 inflammasome activation and the underlying action mechanism is currently unclear.The NLRP3 inflammasome has critical roles in combating against pathogenic infections,excessive or constitutive activation of NLRP3 inflammasome has been implicated in many inflammatory diseases in the contexts of infections.Excessive or continuous activation of NLRP3 inflammasome is associated with a variety of inflammatory diseases.Activation of NLRP3 inflammasome leads to the excessive secretion of inflammatory cytokines like IL-1β,and also culminating in cell death(or pyroptosis)leading to multiple organ damage and septic death during bacterial infections.Thus we aimed to investigate the role of baicalin on NLRP3 inflammasome activation and the underlying action mechanism to provide experimental evidence for its application in the prevention and treatment of NLRP3-related inflammatory diseases.Methods:(1)A cellular model of NLRP3 inflammasome activation was established by using ATP or nigericin as a stimulator in LPS-primed macrophages.(2)The expression levels of IL-1β,caspase-1 and HMGB1 in cell lysates and culture supernatants were evaluated by Western blotting.(3)Soluble IL-1β in the culture supernatants was determined by bead-based immunoassay(CBA).(4)Effects of baicalin on cell death induced by LPS plus ATP were analyzed by propidium iodide(PI)staining.(5)Formation of ASC specks or ASC oligomers was analyzed by immunofluorescence microscopy or chemical cross-linking,respectively.(6)Phosphorylation of NLRP3 on PKA-specifc sites was measured by immunoprecipitation.(7)Mice were injected intraperitoneally with viable Escherichia coli as a model of bacterial sepsis,and CBA was used to assess the level of IL-1β in sera and the survival of mice was also analyzed.Result:Baicalin treatment dose-dependently inhibited adenosine triphosphate(ATP)-or nigericin-induced release of mature IL-1β,active caspase-1p10 and HMGB1 protein from LPS-primed macrophages.The formation of ASC specks and ASC oligomers was robustly inhibited by baicalin in the macrophages upon ATP or nigericin stimulation,indicating that baicalin was able to inhibit NLRP3 inflammasome activation.Mechanistically,all these inhibitory effects of baicalin were partly reversed by MDL12330 Aor H89,which are both inhibitors for the PKA signaling pathway.Moreover,baicalin strongly enhanced PKA-mediated phosphorylation of NLRP3,whereas the PKA inhibitor H89 blocked baicalin-induced NLRP3 phosphorylation on PKA-specifc sites,demonstrating that baicalin at least partly inhibited NLRP3 inflammasome activation by augmenting the PKA activity.In addition,animal experiments showed that when administered pre and post exposure to E.coli infection baicalin treatment signifcantly improved mouse survival in bacterial sepsis.Baicalin administration also signifcantly reduced IL-1β levels in the sera of bacterial infected mice.Conclusion:Baicalin inhibited NLRP3 inflammasome activation in macrophages at least partly through augmenting PKA signaling.Meanwhile,baicalin increased the survival of mice with bacterial sepsis probably by reducing the secretion of inflammatory cytokines including IL-1β,highlighting its therapeutic potential for the treatment of NLRP3-related inflammatory diseases.