High mobility group box-1 release from H₂O₂-injured hepatocytes due to sirt1 functional inhibition

High mobility group box-1 (HMGB1), recognized as a representative of damage-associated molecular patterns, is released during cell injury/death, triggering the inflammatory response and ultimately resulting in tissue damage. Dozens of studies have shown that HMGB1 is involved in certain diseases, but the details on how injured hepatocytes release HMGB1 need to be elicited.

To reveal HMGB1 release mechanism in hepatocytes undergoing oxidative stress.

C57BL6/J male mice were fed a high-fat diet for 12 wk plus a single binge of ethanol to induce severe steatohepatitis. Hepatocytes treated with H₂O₂ were used to establish an in vitro model. Serum alanine aminotransferase, liver H₂O₂ content and catalase activity, lactate dehydrogenase and 8-hydroxy-2-deoxyguanosine content, nicotinamide adenine dinucleotide (NAD⁺) levels, and Sirtuin 1 (Sirt1) activity were detected by spectrophotometry. HMGB1 release was measured by enzyme linked immunosorbent assay. HMGB1 translocation was observed by immunohistochemistry/immunofluorescence or Western blot. Relative mRNA levels were assayed by qPCR and protein expression was detected by Western blot. Acetylated HMGB1 and poly(ADP-ribose)polymerase 1 (Parp1) were analyzed by Immunoprecipitation.

When hepatocytes were damaged, HMGB1 translocated from the nucleus to the cytoplasm because of its hyperacetylation and was passively released outside both in vivo and in vitro. After treatment with Sirt1-siRNA or Sirt1 inhibitor (EX527), the hyperacetylated HMGB1 in hepatocytes increased, and Sirt1 activity inhibited by H₂O₂ could be reversed by Parp1 inhibitor (DIQ). Parp1 and Sirt1 are two NAD⁺-dependent enzymes which play major roles in the decision of a cell to live or die in the context of stress . We showed that NAD⁺ depletion attributed to Parp1 activation after DNA damage was caused by oxidative stress in hepatocytes and resulted in Sirt1 activity inhibition. On the contrary, Sirt1 suppressed Parp1 by negatively regulating its gene expression and deacetylation.

The functional inhibition between Parp1 and Sirt1 leads to HMGB1 hyperacetylation, which leads to its translocation from the nucleus to the cytoplasm and finally outside the cell.