Glycyrrhizic Acid Alleviates LPS-induced Acute Lung Injury Through Activation Of Autophagy

Objective: To investigate the role and mechanism of autophagy in reducing lipopolysaccharide(LPS)induced Acute lung injury(ALI)by glycyrrhizic acid(GA)in vivo and in vitro.Methods:(1)In vitro: RAW264.7 cells were treated with LPS(0,0.1,0.5,1,5 ?g/m L),GA(0,200,400 ?g/m L),and autophagy inhibitor,3-Methyladenine(3-MA,0,1,3 m M),for 24 h.CCK8 assays were used to determine the optimal concentrations of LPS,GA and 3-MA for further experiment.After pretreatment with or without 3-MA(1 m M)for 0.5 h,cells were treated with GA(200 ?g/m L)for 1 h,and incubated with LPS(1 ?g/m L)for 24 h.Cell viability was measured by CCK8.We analyzed the protein levels of LC3,Beclin-1,P62,TNF-?,IL-1?,HMGB1,p-PI3 K,p-AKT,and p-m TOR by Western blotting.The expression of LC3 and HMGB1 were detected by immunofluorescence assays.The numbers of autophagosomes were observed by transmission electron microscopy.(2)In vivo: Forty-eight male Balb/c mice,7-8 weeks of age with body weight ranging from 20-25 g,were randomly divided into four groups and treated as following: Control group,lipopolysaccharide induction group(LPS group),glycyrrhizic acid treatment group(GA + LPS group),and autophagy inhibition group(3-MA + GA + LPS group).The model of ALI was induced by LPS(10 mg/kg,i.p.),GA(200 mg/kg,i.p.),and/or 3-MA(15 mg/kg,i.p.)administration were used to test the possible effect of GA on ALI and the role of Autophagy in it.Lung tissues of mice were harvested after 24 h.Lung wet weights were measured to calculate the lung coefficients of mice.The histopathological changes of lung tissue were showed and evaluated by Hematoxylin eosin(HE)stain.The levels of inflammatory cytokines TNF-?,IL-1? and autophagy genes LC3 and Beclin-1 were detected by q RT-PCR.We analyzed the level of HMGB1 protein by immunohistochemistry.The protein levels of TNF-?,IL-1?,HMGB1,LC3,Beclin-1,P62,p-PI3 K,p-AKT,and p-m TOR were detected by Western blotting.Results: 1.GA attenuates LPS-induced RAW264.7 cells injury.2.GA upregulates autophagy-related proteins LC3 and Beclin-1 and downregulates the expression of P62 in vivo and in vitro.3.GA decreases the TNF-?,IL-1?,and HMGB1 proteins expression in vivo and in vitro.4.GA alleviates the pathological changes of ALI induced by LPS.5.GA reduces the phosphorylation levels of PI3 K,AKT,and m TOR in vitro and in vivo.Conclusion: 1.GA improves LPS-induced ALI.2.GA inhibits the expression of inflammatory factors,TNF-?,IL-1?,and HMGB1,by inducing autophagy in ALI.3.The PI3K/AKT/m TOR pathway could be involved in GA upregulated autophagy in the inhibition of ALI.