The Protective Effects And Mechanisms Of Isoliquiritigenin On Lipopolysaccharide-induced Acute Respiratory Distress Syndrome In Mice

Objectives:Acute respiratory distress syndrome（ARDS）is a major clinical syndrome characterized by diffuse lung inflammation and intractable hypoxemia.Infection of Gram-negative bacteria is reported to be one of the most important causes for ARDS.As the main pathogenic ingredient of Gram-negative bacteria,lipopolysaccharide can induce both oxidative stress and inflammation,which are important mechanisms in the pathogenesis of ARDS and interact with each other.Therefore,therapies targeting oxidative stress and inflammation may provide a novel perspective for the clinical prevention and treatment against ARDS.In recent decades,monomers of Chinese traditional herbs,especially flavonoid,have been noticed extensively for their excellent antioxidant and anti-inflammatory activities.Isoliquiritigenin,a flavonoid originating from G.uralensis,has diverse biological properties,such as antioxidant,anti-inflammatory and anti-tumor activities.However,there is still no evidence to demonstrate its protective effects on ARDS and the underlying mechanisms.Methods:In the present study,we set up models both in vitro and in vivo to explore the protective effects and mechanisms of isoliquiritigenin in RAW 264.7 cells and ARDS model.In vitro,we selected t-BHP to induce oxidative stress and LPS to induce inflammation respectively in RAW 264.7 cells in order to investigate the antioxidant and anti-inflammatory properties and mechanisms of isoliquiritigenin,as well as the interaction of antioxidant and anti-inflammatory mechanisms.In this process,we applied the methods of MTT assays,ELISA,Western blot and plasmid transfection.In vivo,we selected LPS to induce ARDS in mice to investigate the protective effects of isoliquiritigenin on LPS-induced oxidative and inflammatory lung injuries and the underlying mechanisms.Furthermore,we used gene knockout mice to explore the relationship between antioxidant and anti-inflammatory mechanisms of isoliquiritigenin in ARDS.In this process,we applied the methods of ELISA assays,Western blot and flow cytometry（FCM）.Results:1.In vitro,isoliquiritigenin had potential antioxidant and anti-inflammatory activities,which was reflected in the inhibitory effects on t-BHP-induced cell death and ROS production,as well as LPS-induced iNOS and COX-2 production and cytokines（TNF-α,IL-1βand IL-6）secretion.Furthermore,isoliquiritigenin not only up-regulated AMPK/Nrf2 pathway to mediate antioxidant activity,but also suppressed LPS-activated NF-κB and NLRP3 pathways to mediate anti-inflammatory activity,and the inhibition of NLRP3 was at least partly dependent on Nrf2.2.In vivo,isoliquiritigenin significantly ameliorated LPS-stimulated histopathological injury in lung tissue and severity of lung edema.As well,it decreased the production of ROS,infiltration of inflammatory cells（macrophage and neutrophils）and production of cytokines（TNF-α,IL-1βand IL-6）in BALF.Isoliquiritigenin also decreased the myeloperoxidase（MPO）and MDA production,SOD and GSH consumption,and the protein expressions of iNOS and COX-2 in lung tissue.Furthermore,isoliquiritigenin up-regulated AMPK/Nrf2 pathway and inhibited LPS-activated NF-κB and NLRP3 pathways in LPS-induced ARDS.3.In LPS-induced ARDS,the protection of isoliquiritigenin was fairly weaker in Nrf2^-/-mice compared with WT mice,which was reflected in impaird inhibition of LPS-stimulated pulmonary histopathological injury,ROS production in peritoneal macrophages,and NLRP3 activation in lung tissue,while the inhibition of NF-κB pathway was not affected,indicating its antioxidant,anti-inflammatory and protective effects in LPS-induced ARDS was at least partly dependent on Nrf2.Conclusion:In conclusion,isoliquiritigenin confer protection against LPS-induced ARDS in mice by its antioxidant and anti-inflammatory effects,which might come from the up-regulation of AMPK/Nrf2/ARE pathway,as well as inhibition of LPS-activated NF-κB pathway（Nrf2-independent）and NLRP3 inflammasome（Nrf2-dependent）.