Trichostatin A Alleviates Liver Injury In Sepsis By Regulating The Foxo3a/autophagy Signaling Pathway

ObjectiveSepsis is a serious life-threatening disease and liver injury is one of the most fatal complications of it.Trichostatin(TSA)has been reported to be involved in inflammation and autophagy in some diseases.We aimed to investigate the role and relationship between TSA,Foxo3a and autophagy in liver injury and inflammation in sepsis.MethodAnimal part:We used cecal ligation and puncture(CLP)method to establish septic mice model to simulate sepsis in vivo.Firstly,the autophagy blocker 3-MA was used to intervene septic mice.Microtubule-associated protein light chain 3(LC3)and P62 proteins were detected by Western blot,serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by biochemical method,tumor necrosis factor α(TNF-α)and interleukin-6(IL-6)were detected by ELISA.Secondly,the mice were randomly divided into control group,TSA group,CLP group and CLP+TSA group.Hematoxylin-eosin(H&E)staining was used to observe the structural damage and inflammatory cell infiltration of liver tissue.ALT and AST were measured by biochemical method,TNF-α and IL-6 were detected by ELISA.Ultrastructure and number of autophagosomes were observed by transmission electron microscope.The immunofluorescence intensity of LC3 was observed under fluorescence microscope,the positive area of LC3 protein was analyzed by immunohistochemistry,the expression level of LC3,P62 and Foxo3a protein was detected by Western blot.Cell part:AML12 cells were pretreated with lipopolysaccharides(LPS)to simulate sepsis in vitro.Firstly,the activity of AML 12 cells under different concentrations of LPS and TSA and different stimulation time were evaluated by CCK8.Secondly,cells were divided into control group,TSA group,LPS group and LPS+TSA group.ALT and AST in cell supernatant were measured by biochemical method.TNF-α and IL-6 were detected by ELISA.LC3,P62 and Foxo3a protein level were evaluated by Western blot.Fluorescence intensity of LC3 and Foxo3a were observed by confocal microscope.Finally,Foxo3a gene was knocked down by siRNA in AML 12 cells,We used Western blot to detect LC3 and P62 protein expressions.ALT and AST were measured by biochemical method and TNF-α and IL-6 were detected by ELISA.ResultsAnimal part:Firstly in CLP group,the expression of LC3 protein was increased and P62 protein was decreased,ALT and AST were increased,TNF-α and IL-6 were also increased.After 3-MA intervention,the expression of LC3 was decreased and P62 was increased,ALT,AST,TNF-α and IL-6 continued to increase.Secondly,compared with CLP group,in CLP+TSA group the expression of LC3 was increased,P62 was decreased,Foxo3a was increased,ALT,AST,TNF-α and IL-6 were decreased.Cell part:When the cells were treated with 10μg/ml LPS and 10μM TSA for 24h,the cell viability began to decline.With the increase of drug concentration and the extension of intervention time,cell viability continued to decline.Secondly,compared with the CLP group,CLP+TSA intervention increased the expression of LC3,decreased the expression of P62,increased the expression of Foxo3a,decreased the expression of ALT,AST,TNF-α and IL-6.Finally,when Foxo3a gene was knocked down in AML12 cells,TSA promoted LC3 less,inhibited P62 less,and improved ALT,AST,TNF-α,and IL-6 less than that in the non-knocked down group under LPS treated.Conclusion1.Autophagy was found in the liver of septic mice and liver cells were damaged.Inhibition of autophagy would aggravate liver injury in septic mice.2.TSA improves liver injury by promoting liver autophagy in septic mice.3.TSA improves cell injury by promoting autophagy of AML 12 hepatocytes under LPS intervention.4.TSA promotes Foxo3a expression in septic mice liver and LPS-treated AML 12 hepatocytes.Foxo3a participates in the regulation of autophagy by TSA to improve liver injury.