Role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation

Abstract

Objective To evaluate the role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation (OLT). Methods Fifty-six pathogen-free healthy adult male Sprague-Dawley rats, aged 8-10 weeks, weighing 220-250 g, were used in the study.Thirty-two rats were selected and assigned into 4 groups (n=8 each)using a random number table: sham operation group (group S), OLT group, hydrogen-rich saline group (group HS) and chloroquine group (group CQ). The other 24 rats severed as the donors.In group S, laparotomy was performed, and the related blood vessels were isolated.The model of OLT was established in OLT, HS and CQ groups.In group OLT, normal saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group HS, hydrogen-rich saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group CQ, autophagy inhibitor chloroquine 60 mg/kg was injected intraperitoneally at 1 h before establishment of the model, and the other treatments were similar to those previously described in group HS.At 6 h of reperfusion, the rats were sacrificed and hippocampi were isolated for determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, for pathological examination (with light microscope), and for detection of cell apoptosis (by TUNEL staining) and expression of autophagy- and apoptosis-related proteins caspase-3, cytochrome c (Cyt c), microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ), Beclin-1 and p53 in hippocampal tissues (by Western blot analysis). Apoptosis index (AI) was calculated. Results Compared with group S, the MDA content and AI were significantly increased, the SOD activity was decreased, and the expression of caspase-3, Cyt c, LC3Ⅱ, Beclin-1 and p53 was up-regulated in OLT, HS and CQ groups (P<0.05). Compared with group OLT, the MDA content and AI were significantly decreased, the SOD activity was increased, the expression of caspase-3 and Cyt c was down-regulated, and the expression of LC3Ⅱ, Beclin-1 and p53 was up-regulated in group HS (P<0.05). Compared with group HS, the MDA content and AI were significantly increased, the SOD activity was decreased, and the expression of caspase-3 and Cyt c was up-regulated, and the expression of LC3Ⅱ, Beclin-1 and p53 was down-regulated in group CQ (P<0.05). Conclusion The mechanism by which hydrogen inhibits apoptosis in hippocampal neurons is related to promotion of autophagy in a rat model of OLT. Key words: Autophagy; Hydrogen; Liver transplantation; Brain injury; Apoptosis