Study On The Anti-inflammatory Mechanism Of Wedelolactone By Inhibiting NLRP3 Inflammasome Activation And Pyroptosis

PurposeHerba ecliptae is one of the commonly used traditional Chinese medicines in clinic.Wedelolactone is one of the main active components of herba ecliptae.Wedelolactone has been shown to play good anti-inflammatory and anti-tumor activities,and it has been reported that wedelolactone can inhibit the activation of NLRP3(NOD-like receptor family pyrin domain containing 3)inflammasome,but the molecular mechanism of anti-inflammatory effect has not been elucidated.Therefore,this paper aims to explore the molecular mechanism of wedelolactone inhibiting the activation of NLRP3 inflammasome and pyroptosis in macrophages,and its effect on monosodiumurate(MSU)-induced peritonitis and gouty arthritis.Methods1.The macrophages were presensitized with bacterial lipopolysaccharide(LPS)for 4 h,then treated with different concentrations of wedelolactone(10,20,40μmol/L)for 0.5 h,and then treated with ATP,Nigericin,or MSU(NLRP3 stimuli)for a certain time to induce inflammasome activation.The expression of NLRP3 inflammasome activating marker protein(Caspase 1 p20,IL-1β)in cell lysate was analyzed by Western blotting assay,and the content of IL-1β in medium supernatant was detected by enzyme linked immunosorbent assay(ELISA)to study whether wedelolactone could inhibit the activation of NLRP3 inflammasome.2.Wedelolactone treated LPS-primed macrophages with ATP,Nigericin,or MSU to induce NLRP3 inflammasome activation.The percentage of PI-positive cells was analyzed by staining with propidium iodide(PI)and Hoechst 33342 reagent.The supernatant of culture medium was collected to detecte lactate dehydrogenase(LDH)release.The expression of GSDMD was analyzed by Western blotting.To investigate the effect of wedelolactone on pyroptosis.3.The formation of ASC speck was observed by immunofluorescence staining of ASC antibody,and the formation of ASC oligomer was detected by chemical cross-linking method to explore the effect of wedelolactone on ASC oligomer.4.LPS-primed macrophages were treated with wedelolactone and H89,an inhibitor of protein kinase A(PKA),and then NLRP3 inflammasomes were activated with nigericin.The molecular mechanism of wedelolactone inhibiting the activation of inflammasome was studied by PI and Hoechst 33342 staining,LDH release detection,ELISA analysis of IL-1β,Western blot and ASC speck analysis by cell immunofluorescence staining.5.To explore the anti-inflammatory activity of wedelolactone in vivo,we used MSU crystal to induce inflammation in mice,including injecting MSU crystal into abdominal cavity to induce peritonitis,and injecting MSU crystal into articular cavity to induce gouty arthritis.Results1.Wedelolactone(10,20,40μmol/L)reduced IL-1β and caspase-1 p20 expression in a dose-dependent manner compared with LPS plus ATP,Nigericin,or MSU induced NLRP3 inflammasome activation.Wedelolactone was also shown to inhibit the release of mature IL-1β into the culture supernatant by ELISA.2.The results of PI and Hoechst 33342 staining showed that wedelolactone could reduce the proportion of PI positive cells.Wedelolactone could also inhibit the release of LDH into the cell supernatant.Western blotting result showed that the expression of N-terminal of pyroptosis executive protein GSDMD(GSDMD-NT)could be inhibited by wedelolactone in a dose-dependent manner.3.Wedelolactone inhibits the formation of ASC speck induced by LPS and ATP,Nigericin or MSU.The results of Western blot showed that wedelolactone decreased the expression of ASC oligomer when NLRP3 inflammasome was activated.4.In PI staining and LDH release tests,PKA inhibitor H89 reverses the effect of wedelolactone on reduced cell death rates in NLRP3 inflammasome activation models.Western blot result showed that H89 could antagonize the inhibitory effect of wedelolactone on the expression of IL-1β and caspase-1 p20.ELISA result of IL-1βconfirmed the antagonism of H89 to wedelolactone.Immunofluorescence result showed that H89 blocked the inhibitory effect of wedelolactone on ASC speck formation.The results of immunoprecipitation(IP)suggested that wedelolactone inhibited the activation of NLRP3 inflammasome mainly by upregulation of phosphorylation of NLRP3 at Ser/Thr residues,while H89 reduced the phosphorylation level of wedelolactone stimulated NLRP3 at Ser/Thr.5.Wedelolactone reduces the ratio of neutrophils(CD11b+Ly6G+)and the expression of inflammatory cytokine IL-1β in peritoneal lavage fluid of MSU crystal induced peritonitis mice.In addition,wedelolactone reduced the secretion of IL-1β and TNF-α in the tissue culture supernatant of MSU crystal-induced gout arthritis mice.Hematoxylin eosin(HE)staining showed that wedelolactone reduced leukocyte infiltration in joint tissue.Immunohistochemical results showed that wedelolactone could reduce the expression of endogenous full-length and activated Caspase-1 P20 and inflammatory factor IL-1β.ConclusionsWedelolactone plays an anti-inflammatory role in vivo by enhancing PKA signaling,promoting phosphorylation of NLRP3 on Ser/Thr residues,and inhibiting activation of NLRP3 inflammasome and pyroptosis.