Inhibition of excessive mitophagy by N-acetyl-L-tryptophan confers hepatoprotection against Ischemia-Reperfusion injury in rats

In order to investigate the mechnism of hepatoprotective of N-acetyl-L-tryptophan (L-NAT) against ischemia-reperfusion (I/R) injury, the effects of L-NAT were investigated in hepatic ischemia-reperfusion injury (HIRI) models both in vitro and in vivo, which were made by BRL cells and Sprague-Dawley (SD) rats, respectively. The cell viability of hepatocyte was assessed by cell counting kit-8 (CCK-8) staining. The activation of Autophagy was detected by electron microscopy (EM), quantitative Real-Time PCR (qRT-PCR), Western blotting and immunofluorescence. The activation of Mitophagy was determined by the change of Autophagy related protein, change of mitochondrial structure and function, co-location of Autophagy protein and MitoTracker. Results showed that the morphological structures of hepatocytes were changed significantly after HIRI, and the cell viability of hydrogen peroxide (H₂O₂)-induced BRL cells was decreased. Autophagy markers Beclin1, microtubule associated protein 1 light chain 3-II (LC3-II) and Autophagy related protein-7 (ATG-7) were highly expressed and the expression of SQSTM1 (p62) was decreased after HIRI, which suggested that Autophagy of hepatocytes was activated after I/R. The reduction of ATP, mitochondrial DNA (mtDNA) and the mitochondrial transmembrane potential (ΔΨm) after H₂O₂-induced revealed that function of mitochondrial had also undergone significant changes. The increased expression of Autophagy protein, destructure of mitochondria and mitochondrial dysfunction, the increased co-location of Beclin1 and MitoTracker induced by H₂O₂ implied the excessive Mitophagy. The expression of the Autophagy protein was increased by 3-Methyladenine (3-MA), providing another piece of evidence. Importantly, all changes were restored by L-NAT pretreament. In conclusion, the present findings demonstrate that excessive Mitophagy involved in the process of HIRI and L-NAT may protect hepatocytes against HIRI by inhibiting activation of Mitophagy and improving the structure and function of mitochondria.

H2O2; Hepatic ischemia-reperfusion injury; Mitophagy; N-acetyl-L-tryptophan.