Li Huiting, Pan Yitong, Wu Hongjuan, Yu Shuna, Wang Jianxin, Zheng Jie, Wang Can, Li Jianguo, Jiang Jiying
Department of Anatomy, Weifang Medical University, Weifang, China.
Morphology Lab, Weifang Medical University, Weifang, China.
PeerJ. 2020 Apr 9;8:e8665. doi: 10.7717/peerj.8665. eCollection 2020.
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 (HO)-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 HO-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 HO 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.
为了研究N-乙酰-L-色氨酸（L-NAT）对肝脏缺血再灌注（I/R）损伤的肝保护机制，分别在由BRL细胞和Sprague-Dawley（SD）大鼠构建的体外和体内肝脏缺血再灌注损伤（HIRI）模型中研究了L-NAT的作用。通过细胞计数试剂盒-8（CCK-8）染色评估肝细胞的细胞活力。通过电子显微镜（EM）、定量实时聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法和免疫荧光检测自噬的激活。通过自噬相关蛋白的变化、线粒体结构和功能的变化、自噬蛋白与线粒体追踪染料的共定位来确定线粒体自噬的激活。结果显示，HIRI后肝细胞的形态结构发生显著变化，过氧化氢（HO）诱导的BRL细胞的细胞活力降低。自噬标志物Beclin1、微管相关蛋白1轻链3-II（LC3-II）和自噬相关蛋白-7（ATG-7）在HIRI后高表达，而SQSTM1（P62）的表达降低，这表明I/R后肝细胞自噬被激活。HO诱导后ATP、线粒体DNA（mtDNA）和线粒体跨膜电位（ΔΨm）的降低表明线粒体功能也发生了显著变化。HO诱导的自噬蛋白表达增加、线粒体结构破坏和线粒体功能障碍、Beclin1与线粒体追踪染料共定位增加暗示了过度的线粒体自噬。3-甲基腺嘌呤（3-MA）增加了自噬蛋白的表达，提供了另一证据。重要的是，L-NAT预处理可恢复所有这些变化。总之，目前的研究结果表明，过度的线粒体自噬参与了HIRI过程，L-NAT可能通过抑制线粒体自噬的激活和改善线粒体的结构与功能来保护肝细胞免受HIRI损伤。
