The Protection Effect And Mechanism Of Gas6 In LPS/D-GalN Induced Acute Liver Injury

BackgroundThe liver plays as a double-edged sword role in sepsis,on one hand it mediated immune response to clear bacteria and toxins,on the other hand it caused inflammation,immunosuppression,and organ damage.Despite advances in the clinical treatment of sepsis,its morbidity and mortality are still high.Liver dysfunction after sepsis is an independent risk factor for death in patients with sepsis.The pathogenesis of sepsis and liver damage is not fully understood.Improving liver function can reduce morbidity and mortality in patients with sepsis.Growth arrest-specific protein 6(Gas6)is a vitamin K-dependent secreted protein composed of 678 amino acids with a relative molecular mass of 75 k Da.It has the similar molecular structure as Protein S(Pro S).It is a common ligand of Tyro3,Axl,Mer,which are member of the TAM receptor tyrosine kinase subfamily(RTKs).Gas6 has a wide range of functions include inhibiting inflammation,reducing TLRs signals,involving in the phagocytosis of apoptotic cells by macrophages during the recovery phase of inflammation.A large number of studies have shown that Gas6 / TAM can exert antiinflammatory effects by directly inhibiting TLRs and inhibiting the expression of inflammation-related genes TNF-?,IL-1?,IFN-?,and NF-?B.To confirm this hypothesis,this project replicated a mouse model of acute liver injury by LPS / D-Gal N.Liver tissue homogenates were tested for expression of Gas6,apoptosis-related proteins,and key proteins of the NF-?B pathway by immunoblotting and other methods.This project will provide new ideas and treatments for the prevention and treatment of liver injury caused by endotoxin,which has significant theoretical and practical value.Research contentPart ?: Construction of a mouse model of LPS/D-GalN induced acute liver injury and expression of Gas6Objective: To construct a mouse model of acute liver injury with sepsis.The molecular biological methods such as RT-PCR were used to detect the expression of Gas6 in patients with acute liver injury in sepsis,and to establish a theoretical basis for further intervention.Methods: SPF C57BL/ 6 mice were randomly divided into 3 groups: control group,3 hours LPS/D-Gal N group,5 hours LPS/D-Gal N group.Blood samples were taken at each time point to detect serum ALT,AST,IL-1?,IL-6,IL-10,TNF-? and MPO levels.RT-PCR was used to determine Gas6 expression.Results:1.Comparison of serum ALT and AST of mice in each groupCompared with the control group,the LPS/D-GalN group had a progressive increase in ALT and AST in modeling serum.Serum ALT and AST increased in the 3 hours after LPS/D-Gal N treatment.Compared with the control group,the difference was statistically significan(P<0.01).The LPS/D-Gal N group had a significant difference compared with the control in the 5 hours after modeling(P<0.01).2.The level of serum inflammatory factors in miceCompared with the control group,the levels of IL-1 ?,IL-6,IL-10 and TNF-? increased with time.The differences of IL-1 ?,IL-6,IL-10 and TNF-? in the 3 hours after operation were statistically significant(P<0.01);the differences of IL-1 ?,IL-6,IL-10 and TNF-? in the 5 hours after operation were statistically significant(P<0.01).3.Liver pathological changes and liver injury pathological scores of mice in each groupThe liver tissue structure of the control group was normal,and the structure was clear.The LPS/D-Gal N group had abnormal structure,hepatocyte edema,and vacuole degeneration.The pathological damage of liver in the LPS/D-Gal N group gradually aggravate after the operation.4.General statusThe control group woke up quickly,moved freely,and ate well;the LPS/D-GalN group awakened slowly,with vertical hairs,chills,and burnout.5.Comparison of Gas6 m RNAThe level of Gas6 m RNA in LPS / D-Gal N group were significantly decreased at 5 hours after operation when compared with the control group(P<0.01).Conclusions:1.The mice in the LPS / D-Gal N group showed sepsis,and liver injury markers serum ALT and AST significantly increased.So does the levels of IL-1?,IL-6,IL-10,TNF-?.The pathological changes of liver tissue were consistent with the symptoms of acute liver injury in sepsis,successfully replicated the acute liver injury model of sepsis.5 hours of mold making can be used as the time point for material collection.2.The expression of Gas6 decreased in acute liver injury of sepsis,and further exogenous rm Gas6 intervention could be given.Part ?: Study on the protection of recombinant mouse Gas6(rm Gas6)on liver function in mice with LPS/D-Gal N-induced acute liver injury and its effect on inflammation indexesObjective: To investigate the role of exogenous rm Gas6 on the liver function of mice with acute liver injury in sepsis,and to detect the effect of rm Gas6 on inflammatory factors.Methods: SPF C57BL/6 mice were randomly divided into 3 groups: control group,LPS/D-Gal N group,LPS/D-Gal N+rm Gas6 group.According to the results of the first part,the model was selected for 5 hours as the end point.10 mice were taken from each group,and blood and liver samples were collected.Blood samples were taken to detect serum ALT and AST.The levels of IL-1 ?,IL-6,IL-10 and TNF-? were detected by ELISA and RT-PCR respectively.Liver tissue was stained with HE and the pathological damage of liver was observed.Results:1.The liver pathological damage of mice in sepsis acute liver injury group was significantly worse when compared with the control group.(P<0.01);compared with the sepsis group,the pathological damage of liver of mice in LPS/D-Gal N+Gas6 group significantly decreased(P<0.01).2.Compared with the control group,the expression of ALT and AST in LPS / D-Gal N group increased significantly(P<0.01).Compared with LPS/D-Gal N group,ALT and AST levels in LPS/D-Gal N+rm Gas6 group were significantly decreased(P<0.01).3.Serum inflammatory factors were detected.Compared with the control group,the levels of IL-1 ?,IL-6,IL-10,TNF-? in the liver of LPS / D-Gal N group were significantly increased(P<0.01).Compared with LPS / D-Gal N group,the levels of IL-1 ?,IL-6,IL-10,TNF-? in liver tissue of LPS / D-Gal N + rmgas6 group were significantly decreased.4.The expression levels of IL-1?,IL-6,IL-10,TNF-? m RNA in the liver tissue of LPS / D-Gal N group were significantly increased when compared with the control group(P<0.01).The expressions of IL-1?,IL-6,IL-10,TNF-? m RNA in liver tissues of LPS/D-Gal N+rm Gas6 were significantly down-regulated when compared with LPS / DGal N group.Conclusions:1.rm Gas6 can improve acute liver injury in sepsis.2.rmGas6 can down-regulate IL-1?,IL-6,IL-10,TNF-? in blood and IL-1?,IL-6,IL-10,TNF-? m RNA levels in mice liver tissue.3.rm Gas6 has a better protective effect on the liver.Part ?: The protective mechanism of recombinant mouse Gas6 on liver function in mice with LPS/D-Gal N acute liver injuryObjective: To investigate the effect of rm Gas6 on liver cell apoptosis in mice with acute liver injury in sepsis,and to explore the effect of rm Gas6 on liver NF-?B signal transduction pathway in mice with acute liver injury in sepsis.The aim was to discover a potential mechanism for the protection of rm Gas6 in acute liver injury in sepsis.Method: We chose C57 BL / 6 mice which belonged to SPF and were divided into 3 groups randomly: control group,LPS/D-Gal N group,LPS/D-Gal N + rm Gas6 group.LPS/D-Gal N for 5 hours was selected as the end point of modeling.Taken 10 mice from each group and take blood and liver samples.TUNEL staining was used to observe the apoptosis of liver cells.Western blot was used to detect the expression of cleaved caspase-3 and p65,I?B? in NF-?B pathway.Bax and Bcl-2 were detected by western blot and RT-PCR respectively.Results:1.Effect of rm Gas6 on liver tissue apoptosis in sepsisCompared with the control group,the expression of cleaved caspase-3 protein in the liver tissue of the LPS/D-Gal N group increased significantly(P<0.05),and cleaved caspase-3 level in the liver tissue was decreased after intervention with rm Gas6(P<0.05).Compared with the control group,Bax protein expression in liver tissue of LPS/D-Gal N group increased significantly(P<0.05),and Bax level of liver tissue decreased after rm Gas6 intervention(P<0.05).Compared with the control group,the expression of Bcl-2 protein in the liver tissue of the LPS/D-Gal N group was significantly decreased(P<0.05),and the rm Gas6 could increase the level of Bax protein in the liver tissue(P<0.05).The same trend was detected for m RNAs of Bax and Bcl-2 in liver tissues.2.Effect of rm Gas6 on NF-?B pathway in acute liver injuryCompared with the control group,in LPS/D-GalN group,the expression of phosphorylated I?B?,phosphorylated p65 and p65 in the nucleus increased significantly,suggesting NF-?B activated.Compared with the LPS/D-Gal N group,the rm Gas6 group showed the expression of phosphorylated I?B?,phosphorylated p65 and p65 in the nucleus decreased.The above results indicate that rm Gas6 can inhibit the activation of NF-?B pathway and improve the acute liver injury in sepsis.Conclusions:1.rm Gas6 can improve the apoptosis of hepatocytes in the model of sepsis acute liver injury.2.In the model of sepsis acute liver injury,NF-?B signaling pathway was activated.rm Gas6 can inhibit the activation of NF-?B pathway,so as to reduce the acute liver injury.