Itaconate Attenuates Lipopolysaccharide-induced Murine Acute Lung Injury Via Inhibiting NLRP3 Inflammasome Activation And Oxidative Stress

Background Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are common clinical severe respiratory disorders,and there are no Food and Drug Administration-approved drugs.Itaconate,which is synthesized from the decarboxylation of cis-aconitic acid in the tricarboxylic acid cycle,has been recently emerged as a novel anti-inflammatory and antioxidant endogenous metabolite.However,its role in lipopolysaccharide(LPS)-induced acute lung injury(ALI)remains unknown.ObjectiveLPS instillation intratracheally was used to make a murine ALI model,while a cellular inflammatory model was established by Raw264.7 macrophages stimulated with LPS.The current study aims to investigate the effect of itaconate on LPS-induced ALI and the underlying mechanism.MethodsC57BL/6J mice were randomly divided into four groups: control group,LPS group(2.5mg/kg),LPS+ITA group(30 mg/kg)and LPS+MCC950 treatment group(10 mg/kg).In vitro,Raw264.7 cells were divided into control group,LPS group,ITA group(150 ?M),LPS+ITA(30 ?M)group,LPS+ITA(100 ?M)group and LPS+ITA(150 ?M)group.H&E staining was used to evaluate the pathological changes of lung tissue.Western blot was used to detect the expression of NLRP3,caspase1(p20),IL-1β,oxidative stress-related proteins(nitrotyrosine,SOD2),and mitochondrial fusion proteins(OPA1,Mfn2),mitochondrial fission proteins(DRP1,Fis1)in lung tissues and Raw264.7 cells.Immunohistochemistry was applied to detect the expression of NLRP3,caspase1,IL-1β,8-OHd G and nitrotyrosine in lung tissues.ResultsIn the LPS-induced lung injury model,remarkable alveolar interstitial edema,wall thickening,inflammatory cells infiltration especially neutrophils and pro-inflammatiory cytokines production were identified.However,such LPS-induced effect was strikingly attenuated by itaconate treatment.Meanwhile,LPS exposure strongly increased the activation of NLRP3 inflammasome with higher protein expression of NLRP3,IL-1βand caspase1,elevated oxidative stress related proteins such as nitrotyrosine,SOD2 and8-OHd G,enhanced mitochondrial fusion/fission with increased expression of Optic Atrophy 1(OPA1),Mitofusion 2(Mfn2),Dynamin-related protein 1(DRP1)and Fission 1(Fis1).However,these LPS-induced proteins changes were starkly suppressed by itaconate.Furthermore,MCC950,a NLRP3-inflammasome inhibitor,significantly ameliorated lung tissue injury,and suppressed LPS-induced activation of NLRP3 inflammasome,oxidative stress and mitochondrial fusion/fission.In vitro,we found that LPS stimulation significantly increased the expression of inflammatory cytokines in the cell supernatant,and up-regulated NLRP3 inflammasome activation,oxidative stress,and mitochondrial fission/fusion in Raw264.7 cells,but itaconate treatment(150 ?M)reduced the protein levels of NLRP3,IL-1β,caspase1,nitrotyrosine,OPA1,Mfn2,DRP1,and Fis1.ConclusionCollectively,our results demonstrated that LPS-induced ALI was ameliorated by itaconate,and implied that the possible mechanism may be associated with its inhibitory effect on NLRP3 inflammasome activation and oxidative stress,and therefore suggested that itaconate may be exploited therapeutically as a novel drug candidate for ALI/ARDS.