Lipoxin A4 Attenuates MSU Crystals-induced NLRP3 Inflammasome Activation

ObjectiveThe severe inflammation due to monosodium urate(MSU)crystals deposition in the articular and periarticular tissues will induce gout and other related diseases,which has became a common and frequently-occurring disease.The inflammatory response induced by MSU is closely related to the activation of NLRP3inflammasome.Previous studies have shown that MSU can trigger the NLRP3inflammasome activation,thereby promoting IL-1βmaturation and secretion and causing inflammatory effect.There is accumulating evidences demonstrating that taking NLRP3 inflammasome as a target for intervention is a reliable way to interfere with the inflammatory response induced by MSU and a feasible strategy to attenuate inflammation induced by MSU crystal.LXA4 is an endogenous lipoxygenase-derived eicosanoid mediator,and has been associated with fewer drug side effects and strong anti-inflammation properties.LXA4 has a strong ability to suppress inflammatory response,and is well known for being“brake signal”of inflammation.LXA4 is endowed with a strong ability to stifle a variety of inflammatory cells and inflammation-related factors and alleviate inflammatory pathological damage.However,the effet of LXA4 on the ROS/NLRP3inflammasome induced by MSU crystal have not been investigated.For this reason,we employed LXA4 to interfere with MSU-induced inflammatory response in vitro and in vivo,with the purpose of exploring whether LXA4 is provided with an intervention effect on NLRP3 inflammasome activation induced by MSU.In addition,we tried to clarify its specific regulatory mechanism based on oxidative stress response and Nrf2 signal pathway.Our study provides experimental foundation of the future treatment of LXA4 to MSU-related diseases.Method1.Establish the cell model of NLRP3 inflammasome activation induced by MSU crystalsBMDM and PMA differentiated THP-1 macrophages were cultured and primed with LPS,and then stimulated with MSU crystal.The ELISA and Western blot were used to determine the levels of inflammatory cytokines.2.Explore the intervention of LXA4 on NLRP3 inflammasome activationThe cell model of NLRP3 inflammasome activation induced by MSU crystals were pre-intervened by LXA4,and hired ELISA,Western blot,LDH release rate and apoptosis staining to explore the inhibitory effect of LXA4 on NLRP3 inflammasome activation.In addition,the effects of LXA4 on the assembly of NLRP3 inflammasome were analyzed by immunofluorescence and immunoprecipitation.3.Explore the intervention of LXA4 on oxidative stressTotal ROS and mitochondrial ROS,membrane potential and calcium ion levels were detected by specific fluorescent probes,and NADPH oxidase activity was determined using lucigenin-enhanced chemiluminescence.4.Explore whether LXA4 interferes with NLRP3 inflammasome activation by inhibiting Nrf2 pathwayThe Nrf2 specific activator was used to activate Nrf2 and then measured the effect of LXA4 on NLRP3 inflammasome activation.Western blot and immunofluorescence were used to detect the activation of Nrf2,and molecular docking and DARTS experiments were used to analyze the target of LXA4 regulating Nrf2.5.Explore the regulation of LXA4 on Nrf2/Klf9/TXNRD2 signaling pathwayThe expression of Klf9 and TXNRD2 were detected by Western blot and PCR.The chromatin coprecipitation was used to analyze the molecular mechanism of LXA4 regulating Nrf2/Klf9/TXNRD2.6.Explore whether LXA4 works its function dependent on TXNRD2We established TXNRD2-depleted THP-1 cells by small interfering RNA,subsequently analyzed the effect of LXA4 on oxidative stress and NLRP3inflammasome activation by ELISA,Western blot,immunoprecipitation,specific fluorescent probe staining.7.The validation of inhibition of LXA4 on NLRP3 inflammasome in vivoSprague-Dawley rats were used to established model of gouty arthritis with MSU joint injection and LXA4 was pre-injected into joint to prevent gouty arthritis.The joint swelling of rats was detected;HE staining was used to analyze the inflammation of synovial tissue;the level of serum inflammatory factors was measured by ELISA.The NLRP3 inflammasome and Nrf2/Klf9/TXNRD2 protein were measured by Western blot.Result1.MSU crystals triggers NLRP3 inflammasome activationThe concentration of IL-1β,and expression of matured IL-1βand cleaved caspase-1 in culture supernatants of THP-1 and BMDM cells were increased with a stimulation of MSU crystals.2.LXA4 suppresses the activation NLRP3 inflammasome induced by MSU crystalsLXA4 significantly reduced the concentration of IL-1βand expression of matured IL-1β,and cleaved caspase-1 in culture supernatants of THP-1 and BMDM cells with a dose-dependent manner.In addition,LXA4 attenuated the elevated GSDMD-N expression and LDH release,cell death.LXA4 repressed ASC-specks and oligomerization,and ASC-NLRP3 interaction,however,LXA4 barely affected NLRP3,ASC,pro-Caspase-1,pro-IL-1βexpression.3.LXA4 suppresses oxidative stress,the upstream events of NLRP3 inflammasomeLXA4 inhibited ROS production induced by MSU crystals.LXA4 could prevent NADPH oxidase activation and depressed the p22phox expression and hampered the transfer of p47phox from the cytoplasm to membrane.LXA4 decreased MSU crystals-triggered ROS augment in mitochondria,and relieved the depolarization of membrane potential and Ca2~+overload in mitochondria.4.LXA4 interferes with the activation of NLRP3 inflammasome by inhibiting Nrf2LXA4 mitigated the protein level of Nrf2 and relieved the nuclear translocation of Nrf2.In addition,LXA4-induced depression in the release of cleaved caspase-1and matured IL-1βwere reversed by t BHQ,an activator for Nrf2.LXA4 depressed the antioxidants encoded by Nrf2 on the m RNA and protein expression,including HO-1,SOD,and GPx.Keap1,the Binding protein of Nrf2,may be the direct target for LXA4.5.LXA4 regulates Nrf2/Klf9/TXNRD2 signaling pathwayLXA4 reversed this process that MSU crystal increased Klf9 and a decreased TXNRD2.LXA4 depressed the interaction between Nrf2 and promoter of Klf9,and Klf9 and promoter of TXNRD2.6.LXA4 works its function dependent on TXNRD2The knockdown of TXNRD2 reversed the suppressive action of LXA4 on NADPH oxidase activation and mitochondria ROS production.Meanwhile,the knockdown of TXNRD2 also blocked block the inhibitory impact of LXA4 on the activation and assembly of the NLRP3 inflammasome.7.LXA4 can inhibit MSU-induced activation of NLRP3 inflammatory inflammasome in vivoLXA4 markedly relieved acute joint swelling of gouty arthritis rats.LXA4inhibited the massive infiltration of inflammatory cells in joint tissues.In addition,LXA4 could significantly depress serum IL-1βin gouty arthritis rats.LXA4 also significantly inhibited the expression of NLRP3,cleaved caspase-1,and matured IL-1βin the joint of gouty arthritis rats.LXA4 also subverted the increase of Nrf2,Klf9,and depression of TXNRD2 in gouty arthritis rats,which is consistent with in vitro data.ConclusionLXA4 can attenuate the MSU crystals-induced NLRP3 inflammasome activation.The specific mechanism is that LXA4 suppresses Nrf2 activation,and then promotes TXNRD2 expression,which is conducive to LXA4 inhibiting ROS generation and preventing subsequent NLRP3 inflammasome activation.