Mechanism Of NOD1 Agonist Attenuates Lipopolysaccharide/D-Galactosamine Hydrochloride-induced Acute Liver Failure

Background: Acute liver failure,also called fulminant hepatitis or fulminant liver failure,is a rare but life-threatening disorder.It is a complex process that associated with rapidly progressive multiorgan failure and devastating complications.Complications of fulminant hepatitis can include encephalodema,renal failure,respiratory failure,hemodynamic disorder and coagulopathy.Although liver transplantation is a treatment option for some severe acute liver failure,such treatment is not universally available,and less than 10% of liver transplantation are performed in patients with acute liver failure.Exploration the novel treatment strategies for acute liver failure patients is required.Liver is the largest parenchymal organ with abundant of immune cells.Liver should to maintain an immune homeostasis through the accurate regulation.The core factor of acute liver failure is immune status imbalance,overactive immune cells will lead Inflammatory storm in liver,which result in necrosis and apoptosis of liver cells.In animal models,treatment with lipopolysaccharide(LPS)and the hepatocyte-specific transcriptional inhibitor D-galactosamine hydrochloride(D-GalN)has been widely used to explore the mechanisms of ALF and screen for potential hepatoprotective drugs.LPS activates Kupffer cells through the LPS-LBP-TLR4 complex,which release inflammatory cytokines,especially tumor necrosis factor alpha(TNF-?),in the presence of D-GalN.TNF-? bind the tumor necrosis factor receptor1(TNFR1)on hepatocytes and active the apoptotic signal which lead the abnormal apoptosis of hepatocytes.NOD1 is the member of the NOD-like receptor family,which detects intracellular pathogens(Diaminopimelic acid)to active the immune defense.NOD1 receptor is expressed wildly in body,include immune cells,epithelial cells,parenchyma cells and endothelial cell.Previous studies have shown that NOD1 receptor is high expressed in hepatocytes and may participant the apoptotic gene modulation in some tumor cells.However,the functional significance of NOD1 receptor in acute liver failure has not clarified.It is of great significance to explore the underlying mechanism of NOD1 receptor in prevention and treatment of acute liver failure.Part ? Pretreatment with NOD1 agonist attenuates LPS/D-GalN-induced acute liver failure.Objective:This study aimed to explore the potential effects of NOD1 receptor activity for LPS/D-GalN-induced acute liver failure.Whether the NOD1 agonist injection could inhibit the hepatocellular apoptosis and the potential mechanism.Explore the function of NOD1 receptor in liver which may provide new clinic approach for the treatment of ALF.Methods1.Mice were injected with different doses i.p.(5?g/mouse,10?g/mouse)of C14-Tri-LAN-Gly,the NOD1 receptor agonist,for 6 hours,and then coinjected i.p.with LPS(7.5ug/kg)/D-galN(500mg/kg)to induce ALF.Mice given the same volume of PBS as the vehicle control.Survival curve was followed for 36 h after LPS/D-GalN challenged.2.Mice were injected with C14-Tri-LAN-Gly for 6 hours.Serum ALT level assay and liver tissue H&E staining were used to evaluate the liver damage of NOD1 agonist after LPS/D-GalN challenged.3.Liver tissue were collected 6 hours after LPS/D-GalN challenged.TUNEL staining was used to detect the apoptotic hepatocytes,Western blot assay was used to evaluate the apoptotic relevant proteins in liver.4.Mice were injected with C14-Tri-LAN-Gly for 6 hours,and then injected LPS/D-GalN for 2 hours.Serum TNF-? level was detected by Elisa.Isolated the hepatic mononuclear cells,flow cytometry was used to analysis the distribution liver immune cells and the surface CD69 expression on them.TLR4 expression on F4/80 positive cells also detected by flow cytometry.Resuts:1.Pretreatment with the two different doses of C14-Tri-LAN-Gly could significantly prolong the survival time after LPS/D-GalN challenged relative to control group.2.Serum ALT level was markedly decreased in the C14-Tri-LAN-Gly group and the liver tissue necrosis and inflammation were also markedly decreased in the C14-Tri-LAN-Gly group.3.TUNEL staining showed the apoptotic hepatocytes were markedly decreased in C14-Tri-LAN-Gly group.Western blot assay showed cleaved caspase-3,an important apoptotic protein,was significantly downmodulated in the C14-Tri-LAN-Gly group.4.Elisa showed the serum or liver tissue TNF–? level in C14-Tri-LANGly treated mice have no significant difference relative to control group.Flow cytometry analysis showed that no significant difference of hepatic immune cells distribution and CD69 expression on them.Moreover,TLR4 expression on Kupffer cells has no markedly changed in C14-Tri-LAN-Gly treated mice.Conclusions: NOD1 agonist pretreatment could protect mice from LPS/D-GalN-induced ALF.Moreover,the protective effect is independent to TNF–? secretion and the TLR4 expression on the surface of Kupffer cells in liver.Part ? NOD1 agonist protect mice from LPS/D-GalN-induced ALF through the A20 up-modulated expression in liverObjective:1.Use the TNF–?/D-GalN-induced ALF model to explore the mechanism of NOD1 agonist protective function in ALF.2.Detect the anti-apoptotic genes expression in liver to explore the molecular mechanism of NOD1 agonist protective function in ALF.3.Further investigated the mechanism of NOD1 agonist in ALF.Methods:1.Mice were pretreated with C14-Tri-LAN-Gly for 6 hours,and then co-injected i.p.with mouse rTNF–?(0.4ug/mouse)/D-galN(500mg/kg)to induce ALF.Mice given the same volume of PBS as the vehicle control.Survival curve was followed for 36 hours after rTNF–?/D-GalN challenged.Serum ALT level,liver tissue H&E staining,liver tissue TUNEL staining and liver cleaved-caspase3 expression were used to evaluate the hepatocellular damage as well as apoptosis.2.Mice were treated with C14-Tri-LAN-Gly or same volume of PBS for 3 hours,and then isolated the primary hepatocytes and marked with antiTNFR1.Flow cytometry were used to analysis the TNFR1 expression on the surface of primary hepatocytes.3.RT-qPCR and Western blot were used to detect the anti-apoptotic genes mRNAs and proteins expression in liver tissue.4.Mice were injected with C14-Tri-LAN-Gly or same volume of PBS alone.RT-qPCR and Western blot were used to detect A20,an anti-apoptotic genes,mRNA and protein expressions in liver tissue.5.Mice were hydrodynamically injected with shRNA to specifically against murine A20 three days before C14-Tri-LAN-Gly challenge.And then to investigate the protective function of C14-Tri-LAN-Gly after A20 downmodulation by ALT assay and TUNEL staining.6.C14-Tri-LAN-Gly was treat to the primary hepatocytes,and RTqPCR and Western blot were used to detect A20 mRNA and protein expressions.7.Mice were injected with clodronate-liposomes i.v.to deplete the Kupffer cells.RT-qPCR and Western blot were used to detect A20 mRNA and protein expressions.Results:1.Similar in the LPS/D-GalN model,C14-Tri-LAN-Gly could also protect mice from rTNF-?/D-GalN induced ALF regard to survival time,serum ALT level,H&E staining,TUNEL staining and cleaved-caspase3 expressing.2.Flow cytometry analysis indicated that the TNFR1 expression on the surface of primary hepatocytes was not influenced by C14-Tri-LAN-Gly after LPS/D-GalN challenged.3.RT-qPCR and Western blot assay indicated that the anti-apoptotic gene A20 in liver was markedly increased in C14-Tri-LAN-Gly treatment mice.4.Mice treat with C14-Tri-LAN-Gly along could also significantly increase the expression of A20 in liver.5.In comparison to control shRNA,shA20 down-modulated A20 expression in vivo by ~70%,Down-modulated A20 abolished the protective effect of C14-Tri-LAN-Gly pretreatment in LPS/D-GalN induced ALF,which serum ALT was significantly increased in shA20 injected mice and the hepatocellular apoptosis also increased in shA20 injected mice.6.Real-time PCR assay showed A20 expression was not markedly different between PBS and C14-Tri-LAN-Gly group,Western blot also showed the expression of A20 was not changed after in vitro activation.7.The A20 mRNA and protein expression was markedly reduced in Kupffer cell-depleted mice.Conclusions: A20 upregulation play the core role in NOD1 agonist protective effects in LPS/D-GalN-induced ALF,which was dependent on Kupffer cells in liver.