Supplementary MaterialsSupplemental Information 1: qRT-PCR gene sequences peerj-08-8665-s001. activation of autophagy was detected Oxaliplatin (Eloxatin) by electron microscopy (EM), quantitative real-time PCR (qRT-PCR), Western blotting and immunofluorescence. The activation of mitophagy was determined by the switch of autophagy related protein, switch of mitochondrial structure and function, co-location of autophagy protein and MitoTracker. Results showed that this 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 (m) after H2O2-induced revealed that function of mitochondrial experienced 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 Oxaliplatin (Eloxatin) L-NAT may protect hepatocytes against HIRI by inhibiting activation of mitophagy and improving the structure and function of mitochondria. through inhibiting the disruption of hepatocytes, improving the cell viability, attenuating the inflammation and the expression of RIP2, Caspase-1 and IL-1(Wang et al., 2019). However, the relationship between mitophagy and the hepatoprotective of L-NAT are not fully understood. In this study, we investigated the effects of L-NAT on hepatocytes morphology, the structure and function of mitochondria, and activation of autophagy during the period of HIRI, which may provide experimental evidence for application of L-NAT on HIRI. Material and Methods Chemicals L-NAT, Beclin1, microtubule- associated protein 1 light chain 3-II (LC3-II), autophagy related protein 7 (ATG-7), SQSTM1 (P62) antibodies and 3-Methyladenine (3-MA) were purchased from Sigma-Aldrich (St.Louis, MO, USA) and GAPDH antibody was obtained from Proteintech Group (Chicago, USA). Secondary anti-rabbit antibody was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). The enhanced chemiluminescence (ECL) system was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). RIPA lysis was purchased from Solarbio (Beijing, China). The cell counting kit-8 (CCK-8) was purchased from 7sea-Biotech (Shanghai, China). ATP assay kit was purchased from Beyotime Biotechnology (Shanghai, China) and MitoTracker Red kit came from Yeasen Biotech (Shanghai, China) and DAPI came from Life Technologies. Animals Healthy male Sprague-Dawley (SD) rats weighing Rabbit Polyclonal to NEIL3 200-220 g were purchased from Pengyue experimental animal center in Jinan, China (Weifang Medical University or college Medical Ethics Committee provided full approval for this research (No. 2017253)). They were randomly divided into sham group, I/R group, and I/R + L-NAT group, with 6 rats in each group. Rats were fasted for 12 h before surgery, and were given free access to water. In I/R + L-NAT group, L-NAT (10 mg/kg) was intraperitoneal injected 30 min before modeling (Wang et al., 2019). The rats were anesthetized by intraperitoneal injection of ketamine, the left and middle branches of the hepatic pedicle were occluded with a non-traumatic vascular clamp in I/R + L-NAT group and I/R group. The success of the model was apparent once the liver color switched from reddish to dark purple. After 45 min of ischemia, the clip was removed to allow hepatic reperfusion. In sham group, the rats underwent the same surgery but no vessel clamps were placed. According to the previous literature of our laboratory, the most obvious liver function damage was after 6 h of reperfusion, so we required the liver tissue after 6 h of reperfusion. The Animal Ethics Committee of the University or college approved all working protocols. Cell culture and treatment The rat hepatocyte BRL cell collection was purchased from the Chinese Academy of Sciences Cell Lender (Shanghai, China). BRL cells were cultured in a 37?C incubator which is a humidified environment of 95% air flow/5% CO2. The cells were divided into three groups: control group, H2O2 group and H2O2+ L-NAT group. Referring to Oxaliplatin (Eloxatin) the previous literature in our laboratory, oxidative damage model of BRL cells were prepared by pre-treatment with 200 M H2O2 for 6 h. And H2O2+ L-NAT group was pre-treated with 10 M.