| Acute on chronic Liver failure (ACLF) is a severe life-threatening entity with a high mortality rate. It was confirmed that intestinal endotoxemia play a significant role in the progresses of the liver failure. High mobility group box-1(HMGB1) protein is involved in the process of endotoxemia. Sodium butyrate has been shown to ameliorate hepatic, renal and intestinal mucosal injury, and down-regulate expression of several proinflammatory mediators in a wide variety of animal models of critical illnesses. On the basis of above-mentioned research background, this study firstly aims to explore the roles of HMGB1in the pathogenesis of ACLF in rats, and also to investigate the protective effects of sodium butyrate treatment on ACLF in rat model. These reports were made up of four parts as follows.Part I Establishment of Acute on Chronic Liver Failure in RatsObjective:Acute-on-chronic liver failure(ACLF) is a serious clinical syndrome with a high mortality rate, and there is no recognized animal model of ACLF available to demonstrate the possible mechanism of it. This study aims to investigate the approaches for making ACLF model in rats. Methods:Firstly immunological liver fibrosis in Wistar rats was induced by injection with human serum albumin(HSA), then the rats were attacked by intraperitoneal injection with D-galactosamine and lipopolysaccharide to induce acute liver failure on the basis of chronic liver injury. Serum ALT, AST, TBIL TNF-a, IFN-y and HMGB1were measured and histology from liver, lung, kidney and intestine were detected respectively at different time point after induction of ACLF. The survival duration of ACLF rats was also observed. Results:Most of the model rats developed into liver fibrosis or cirrhosis after injection of human serum albumin for6weeks, and90percent of them died of liver failure after attacked by D-galactosamine and lipopolysaccharide. Liver histopathology showed massive or submassive necrosis and regenerated nodules in pseudolobule in model rats. The kinetic changes of ALT, AST and TBIL were compatible with massive necrosis of liver while serum TNF-a, IFN-y and HMGB1increased significantly(P<0.05). Conclusion:ACLF rat model could be induced by injection with human serum albumin and D-galactosamine and lipopolysaccharide, which is practical and available for further studies such as pathophysiological mechanism, evolution of ACLF and evaluation of drugs and so on. Part ? Protective Effects of Sodium Butyrate on Acute-on-chronic Liver Failure in RatsObjective:Acute-on-chronic liver failure (ACLF) is a serious clinical syndrome with a high mortalityrate. Sodium butyrate has been shown to alleviate organ injury in a wide variety of preclinical models of critical illnesses. The aim of this study was to investigate the protective effect of sodium butyrate on ACLF in rats. Methods:An ACLF model was induced in Wistar rats. All rats were randomly divided into normal, model, sodium butyrate treatment group. After induction of ACLF, the rats received sodium butyrate (500mg/kg) at12h or24h time point in treatment groups respectively. Serum endotoxin, high mobility group box-1(HMGB1), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), tumor necrosis factor alpha (TNF-?), interferon gamma (IFN-y) were determined respectively. The expression of HMGB1and nuclear factor kappa B (NF-?B) protein(P65) in liver tissue were detected by western blotting after induction of ACLF. The histology from liver was examined and the survival duration of rats was also observed. Results:Liver histology showed more severe pathological injury in model group compared with normal while serum endotoxin, HMGB1, ALT, TNF-a, IFN-y were increased significantly (P<0.05). However, sodium butyrate administration improved histopathology from liver, reduced the levels of endotoxin and inflammatory cytokines in serum, suppressed HMGB1and NF-?B (P65) proteins in liver tissue, and prolonged the survival duration regardless of treating time point at12h or24h after induction of ACLF(P<0.05). Conclusion:Sodium butyrate administration can protect acute-on-chronic liver failure in rats, and is a potential therapeutic agent for severe liver injury.Part III Sodium Butyrate Decreases Intestinal Permeability on Acute-on-chronic Liver Failure in Rats.Objective:The pathogenesis and progression of acute liver failure are closely associated with intestinal endotoxemia because of the high permeability of the intestinal wall. Sodium butyrate has been shown to protect liver failure effectively. The current study aimed to explore the relationship between proinflammatory cytokines and intestinal permeability, and to investigate whether sodium butyrate administration might prevent the release of multiple proinflammatory cytokines and decrease intestinal permeability. Methods:The ACLF rat model was induced by injection with human serum albumin(HSA), lipopolysaccharide(LPS) and D-galactosamine. The rats were randomly divided into control, model and sodium butyrate treatment group. Samples were obtained respectively at12h or24h time point after ACLF induction. The histology of intestinal tissue was accessed. Serum endotoxin, D-lactate, diamine oxidase(DAO), tumor necrosis factor-alpha(TNF-a), interferon-y(IFN-y) and high mobility group box-1(HMGB1) were evaluated respectively. Results:The rats in model group showed severe damage to intestinal mucosa after ACLF induction. Sodium butyrate alleviate significantly intestinal injury. In addition, serum endotoxin, D-lactate, DAO, TNF-a, IFN-y and HMGB1increased significantly in the model group compared with the control group(P<0.05). There was a positive correlation between intestinal permeability and proinflammatory cytokines. Sodium butyrate reduced significantly serum endotoxin, D-lactate, DAO, TNF-a, IFN-y and HMGB1(P<0.05). Conclusion:Sodium butyrate has protective and therapeutic effects on intestinal mucosa, decreases intestinal permeability, and inhibits the release of multiple proinflammatory cytokines in rats with ACLF.Part IV Sodium Butyrate Prevents High Mobility Group Box-1Protein Release from Caco-2Cells.Objective:To investigate the effect of sodium butyrate on HMGB1releasing from Caco-2cells Methods:Caco-2cells was induced with50ug/L LPS for0h-48h. The effects of sodium butyrate on HMGB1releasing from Caco-2cells was observed while U937cells served as control. The levels of HMGB1in cell culture supernatant were determined. The expression of HMGB1mRNA and protein and nuclear factor kappa B (NF-?B) protein (P65) in Caco-2cells were detected by RT-PCR and western blotting respectively. Results:MTT and Hochest tests showed no significant differences on cell necrosis and apoptosis in the both cells (P<0.05). RT-PCR showed that the levels of HMGB1mRNA in LPS induced groups was not significant increasing at24h and48h time point in Caco-2cells (P>0.05). Significant cytoplasmic translocation of HMGB1in both Caco-2cells and U937cells were observed after intervention by LPS. The levels of HMGB1after intervention in the treated groups significantly increased at24h in Caco-2cells (P<0.05), which was later than that in U937cells (12h). Time-dose dependences on HMGB1level were observed in both U937and Caco-2cells. However, HMGB1level in U937cells were significantly higher than that in Caco-2cells at a certain time point. The cytoplasmic translocation of HMGB1in both Caco-2cells were suppressed by sodium butyrate treatmen (P<0.05)t. Conclusion:Cytoplasmic translocation and extracellular release of HMGB1from Caco-2cells were independent to cell death, which could be induced by LPS. While compared to the secretion process of immune cells, the secretion of HMGB1from Caco-2cells underwent later and less amounts that could be suppressed by sodium butyrate. |
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