A. Khader1,2, W. Yang1,2, J. M. Prince1, J. Nicastro1, G. F. Coppa1, P. Wang1,2 1Hofstra North Shore-LIJ School Of Medicine,Surgery,Manhasset, NEW YORK, USA 2Elmezzi Graduate School Of Molecular Medicine,Manhasset, NEW YORK, USA
Introduction: Liver ischemia-reperfusion (I/R) often occurs in trauma, transplantation and prolonged shock state, where it lacks specific treatment. We have recently demonstrated that pharmacologic sirtuin 1 (Sirt1) activation, with SRT1720, is protective in a murine model of liver I/R. However, the detailed mechanism of which is not yet characterized. Sirt1 is an energy-sensing enzyme with multiple roles, including the regulation of energy metabolism, mitochondrial function and autophagy. We therefore hypothesized that SRT1720 protects hepatocytes from hypoxia-induced injury through the activation of mitochondrial biogenesis and the autophagy salvage pathway.
Methods: Rat hepatocyte epithelial H4IIE cells were subjected to 6 h of hypoxia using 5% Oxyrase, followed by reoxygenation in the presence or absence of SRT1720 (500 nM) for 4 or 24 h. Cell survival was determined by cell counts and ATP assay. Cells were stained with MitoTracker green and red FM for measuring mitochondrial mass (mtMass) and membrane potential (MMP), respectively, by flow cytometry. Acridine orange stain was used to assess the formation of autophagic vesicles under fluorescent microscopy. Protein levels were determined by Western blot.
Results: We observed a 27.6% and 21.7% reduction in cell numbers and ATP levels, respectively, at 4 h of reoxygenation (H/R), while SRT1720-treated cells were comparable to the normoxia control. At 4 h of H/R, the mtMass and MMP per cell, as measured by mean fluorescent intensity (MFI), were decreased by 44.6% and 27.6%, respectively, compared to the normoxia control. In contrast, treatment with SRT1720 enhanced both parameters by 3.4- and 5.5-fold, respectively, compared to untreated cells at 4 h of H/R. This enhancement of mtMass and MMP by SRT1720 persisted to 24 h of reoxygenation. Treatment with SRT1720 increased the expression of PGC1α, the master regulator of mitochondrial biogenesis, by 11.0% and 36.7% at 4 h and 24 h of H/R, respectively. In addition, there was a significant enhancement in autophagy in the SRT1720 treated cells as demonstrated by an increase in LC3aII/LC3aI protein ratio to 3.3 compared to 1.7 in the untreated cells at 4 h of H/R. Further, there was an observable increase in autophagic vesicles (bright contrast) at 24 h of reoxygenation which was remarkably increased by SRT1720, as shown by the increase in vesicle numbers and intensity of acridine orange precipitation (Figure).
Conclusion: Pharmacologic activation of Sirt1 improves the survival of hepatocytes under hypoxic stress, which is associated with maintenance of mitochondrial mass and integrity as well as an increase in autophagic activity. Thus, stimulation of mitochondrial function may provide a new protective strategy against liver I/R injury.
