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

Huiting Li; Yitong Pan; Hongjuan Wu; Shuna Yu; Jianxin Wang; Jie Zheng; Can Wang; Jianguo Li; Jiying Jiang

doi:10.7717/peerj.8665

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

PeerJ. 2020 Apr 9:8:e8665. doi: 10.7717/peerj.8665. eCollection 2020.

Authors

Huiting Li^#¹, Yitong Pan^#¹, Hongjuan Wu², Shuna Yu¹, Jianxin Wang¹, Jie Zheng³, Can Wang¹, Jianguo Li¹, Jiying Jiang¹

Affiliations

¹ Department of Anatomy, Weifang Medical University, Weifang, China.
² Morphology Lab, Weifang Medical University, Weifang, China.
³ Department of Pathology, Weifang Medical University, Weifang, China.

^# Contributed equally.

Abstract

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.

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

Grants and funding

The study was supported by the National Science Foundation of China (Grant No. 81760567; 81802474), the Key R&D Program of Shandong Province (GG201809200094), the Natural Science Foundation of Shandong Province, China (ZR2018MC012; ZR2014HL020; ZR2014HL021) and the Project Funding for the Development of Medical and Health Science and Technology in Shandong Province (2014WS0464) and the Neurologic Disorders and Regenerative Repair Lab “13th five-year plan” Key Lab of Shandong Higher Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.