| Part I Blockade of high-mobility group box-1ameliorates acute on chronic liver failure in ratsObjective: Liver failure is a severity case that with high lethality, HMGB1was identified as an extracellular released mediator of inflammation. This study applied monoclonal anti-HMGB1antibody to treat the rats with acute on chronic liver failure (ACLF) to evaluate the therapeutic efficacy of HMGB1blockade.Methods: An ACLF model was induced in rats, and animals were randomly divided into control, model and anti-HMGB1antibody treatment group. The rats received anti-HMGBl antibody (200μg/kg) at12h and24h time point after induction of ACLF. Serum endotoxin, high mobility group box-1(HMGB1), TNF-a and IFN-y levels, changes of liver histology, apoptosis of liver tissue and hepatic levels of HMGB1, caspase3, TLR4and P65were detected.Results:Blockade of HMGB1can significantly decrease levels of endotoxin, HMGB1, TNF-a and IFN-y, ameliorate liver tissue pathological injury and inhibit the apoptosis of liver cells.Conclusion:Blockade of HMGB1can confer protective effect to ACLF rats, even24h after ACLF have been induced. Part II Blockade of TLR4confers protection to rats with acute on chronic liver failureObjective: Acute on chronic liver failure (ACLF) is a severe life-threatening condition with a high fatality. Toll like receptor4(TLR4) signaling pathway and High mobility group box1(HMGB1) play key roles in the pathogenesis of the disease. In this study, anti-TLR4antibody was applied to treat ACLF rats. The aim of this study was to observe the effect of TLR4blockade on rats with acute on chronic liver failure and HMGB1levels.Methods:32male specific pathogen free (SPF) Wistar rats, weighing120to150g were randomly divided into the normal group, the model group, the IgG treatment group and anti-TLR4antibody treatment group. Excepted for the rats in the normal group, an acute on chronic liver failure was induced in all the rats by human serum albumin (HSA), D-galactosamine (D-Gal) and lipopolysaccharide (LPS). The rats in the anti-TLR4antibody treatment group received anti-TLR4antibody100μg/kg by tail vein injection24h after the model had been induced. The rats in the IgG treatment group received rabbit anti-mouse IgG100μg/kg as a control. The rats in the model group received the same dose of physiological saline solution. All the rats were killed24h after the treatment had been given. The liver tissues were dyed by hematoxylin-eosin staining to observe the pathological changes. Serum ALT, TNF-a, IFN-y and HMGB1levels were measured by ELISA assay, and hepatic HMGB1levels was detected by western blotting assay.Results: Compared with the normal group, the liver cell necrosis was observed and serum ALT, TNF-a, IFN-y and HMGB1levels and hepatic HMGB1level were greatly increased in the model group (P<O.05). The pathological changes were improved by blockade of TLR4, serum levels of ALT, TNF-a, IFN-y and HMGB1were also decreased by TLR4antibody as well as HMGB1levels in liver tissue (P<O.05). However, IgG treatment failed to improve the pathological change of the liver tissue, and there was no significant differences between the model group and the IgG treatment group (P>0.05) among serum ALT, TNF-a, IFN-y and HMGB1levels and hepatic HMGB1level.Conclusion:Blockade of TLR4can confer protection to rats with acute on chronic liver failure and decrease HMGB1in sera and liver tissue.Part Ⅲ Inhibitions of NF-κB and TNF-a Result in Differential Effects in Rats with Acute on Chronic Liver Failure Induced by D-Gal and LPSObjective: High mobility group box1(HMGB1) is an inflammatory mediator which is associated with many inflammatory diseases. TLR4-NF-κB signaling pathway is critical in mediating the inflammatory activity of HMGB1. The purpose of this study is to evaluate the effect when NF-κB and TNF-a were inhibited.Methods: In this study, we induced an acute on chronic liver failure model by HSA, D-Gal and LPS in rats. Anti-TNF-a antibody (TNF-a inhibitor) and PDTC (NF-κB inhibitor) were used to treat the liver failure animals. U937cells were differentiated by phorbol myristate acetate (PMA) and activated by LPS. The effect of PDTC was further detected in the differentiated U937cells.Results: TNF-a inhibition is beneficial to the rats in ACLF and NF-κB inhibition failed to protect the rats in ACLF. However, HMGB1levels, cytokine production and activation of TLR4-NF-κB signaling pathway were all suppressed by both TNF-a and NF-κB inhibition. And anti-inflammatory activity of PDTC was proved in U937cell line.Conclusion: Both inhibitions of TNF-a and NF-κB are able to suppress the activation of TLR4and NF-κB signaling pathway. However, NF-κB inhibition with PDTC failed to protect the rats in ACLF induced by HSA, GalN and LPS. |
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