Study of the extracellular release of HMGB1 in human liver cell line-HepG2 cells induced by TNF-α

Objective:To investigate whether HMGB1 release occur in TNF-α stimulated HepG2 cells without cell death.Methods:HepG2,RAW264.7 and HEK293 cells were treated with TNF-α(25 ng/ml)for 4 h,8 h,12 h,16 h,20 h and 24 h respectively,at the same time points cells without TNF-α treatment was designed as controls.At each time point cell viability was determined by MTT assay and levels of HMGB1 in the culture medium were detected by Western blot.The cytoplasmic translocation of HMGB1 at 24 h after TNF-α treatment was observed by immunofluorescence.The apoptotic rates of HepG2,RAW264.7 and HEK293 cells after treated with TNF-α were determined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling(TUNEL)assay.Results:The survival rates of HepG2,RAW264.7 and HEK293 cells were 91.99%±0.30%,91.28%±0.56% and 90.85%±0.72% respectively after exposure to TNF-α for 24 hours.Compared with control groups and TNF-α treated HEK293 cells,the levels of HMGB1 in TNF-α treated HepG2 and RAW264.7 cells were significantly higher at 12 h,16 h,20 h and 24 h(P0.05).The levels of HMGB1 in the culture medium increased in a time-dependent pattern and reached peak at 24 hours after TNF-α treatment.Significant cytoplasmic translocations of HMGB1 in HepG2 and RAW264.7 cells were observed at 24 h after TNF-α treatment.TUNEL test demonstrated that the percentage of apoptotic cells in TNF-α treated HepG2,RAW264.7 and HEK293 cells was very low and did not increased compared with that of control groups(P0.05).Conclusion:Cytoplasmic translocation and extracellular release of HMGB1 in HepG2 cells can be induced by TNF-α,which is not accompanied with cell death and apoptosis.