Translocation And Release Of HMGB1 In Hepatocytes During Hepatic Failure

Background: Hepatic failure (HF) is a potentially devastating syndrome with a high mortality rate. In China the most common cause of HF is viral hepatitis, especially HBV infection. In the extracellular milieu, HMGB1 acts as a late mediator and a new therapeutic target of sepsis. Accumulating evidence has established an important role of HMGB1 during HF. In this study we investigated translocation and release of HMGB1 in hepatocytes during HF.Objectives: (1) To identify if HMGB1 and other cytokine release can occur in LPS or TNF-αstimulated hepatocytes as well as RAW264.7 cells without cell death in votro. (2) To observe translocation of HMGB1 in hepatocytes of chronic severe hepatitis B patients. (3) To observe translocation of HMGB1 in hepatocytes of D-GalN+LPS induced-acute hepatic failure. (4) To investigate possible mechanisms of HMGB1 release from hepatocytes.Methods: (1) Different concentrations of LPS or TNF-αwere added to a liver cell line HepG2 cells in vitro. At different time point after treated, cells and supernatant were collected. Cell viability was determined by MTT assay and level of LDH in the supernatant. The apoptotic rats after treated for 24h were determined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay. Levels of HMGB1 in the culture medium and expression of HMGB1 in both the nuclear and cytoplasmic fractions after treated with LPS or TNF-αwere detected by Western blotting. The cytoplasmic translocation of HMGB1 was observed by immunofluorescence. ELISA and human cytokine antibody array assay were used to test cytokines in the supernatant after treated. (2) Liver tissue from chronic severe hepatitis B patients, chronic hepatitis B patients and healthy control were collected and then subjected to immunohistochemical analysis for the location of HMGB1. At the same time HE staining was performed to evaluate the severity of tissue damage. (3) Acute hepatic failure of mouse was induced by intraperitoneally injection of D-GalN (600 mg/kg) and LPS (0.5 mg/kg). Control mice were given the same volume of saline. 3h after treatment, mice were killed. Liver tissue of each mouse was collected for immunohistochemistry staining to observe location of HMGB1 in hepatocytes. (4) Western boltting and immunofluorescence were used to evaluate effects of lemptomycin B or TSA on the cytoplasmic translocation and extracellular release of HMGB1 from HepG2 cells induced by LPS.Results: (1) LPS or TNF-αstimulation in vitro induced release of HMGB1 from HepG2 cells, and the level of HMGB1 in the supernatant increased as a time (reached its peak at 20h in LPS treated group and 24h in TNF-αtreated group) and dose (within a certain range of dosage,≤400ng/mL in LPS treated group and≤25ng/mL in TNF-αtreated group) dependent way. MTT, TUNEL assay and level of LDH in the supernatant demonstrated that there was no significant necrosis or apoptosis after treated. Significant cytoplasmic translocations of HMGB1 in HepG2 and RAW264.7 cells were observed by immunofluorescence after LPS or TNF-αtreated. Levels of HMGB1 in the cytoplasmic fractions increased after treated with LPS or TNF-α, tested by Western blotting using cytoplasmic and nuclear fractions of HepG2 cell cultures. There is no significant release of common pro-inflammation cytokines from LPS, TNF-αor recombinant HMGB1 protein (rHMGB1) treated HepG2 cells identified by ELISA and human cytokine antibody array. (2) There is apparent translocation of HMGB1 in hepatocytes of chronic severe hepatitis B patients, and the translocation rate is 32.84%±7.13%. While in chronic hepatitis B patients and normal liver tissue controls immunohistochemistry staining revealed a nuclear location of HMGB1 in most hepatocytes and no apparent translocation were observed. (3) 3h after D-GalN and LPS treated the traslocation rate in hepatocytes was 27.42%±4.99%. The translocatoion rates were significantly higher compared with saline treated group (P<0.01). (4) Leptomycin B, a CRM1 inhibitor, significantly inhibited LPS-induced release of HMGB1 in HepG2 cells and also the cytoplasmic translocation of HMGB1. TSA, a deacetylace inhibitor, enhanced LPS-induced HMGB1 release in hepatocytes and its cytoplasmic translocation.Conclusions: (1) LPS or TNF-αinduces extracellular release and cytoplasmic translocation of a nuclear protein HMGB1 in HepG2 cells in vitro in a time and dose dependent way. (2) Cytoplasmic translocation of HMGB1 in hepatocytes exists in chronic severe hepatitis B patients, but not in chronic hepatitis B patients and normal liver tissues. (3) Cytoplasmic translocation of HMGB1 in hepatocytes exists in D-Gal+LPS induced-acute hepatic failure in mice. (4) The translocation and release of HMGB1 in LPS stimulated HepG2 cells may have a CRM1 dependent mechanism and can be enhanced by acetylation.