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Fatty liver has lower tolerance against ischemia-reperfusion (I/R). We aimed to ameliorate liver injury following I/R in fatty liver. Astaxanthin (3,3’-dihydroxy-b, b’ carotene-4,4’-dione, ASTX), a carotenoid, exhibits antioxidant and anti-inflammatory activities in several clinical situations. In this study, we aimed to elucidate the protective effect of ASTX on I/R liver injury in fatty liver model.We developed non-alcoholic steatohepatitis model by giving methionine and choline deficient high fat (MCDHF) diet for 3 weeks and divided into 3 groups; sham operation on MCDHF diet mice (MCDHF SO), MCDHF diet and olive oil treatment before I/R (MCDHF I/R), and MCDHF diet and ASTX treatment before IR (MCDHF+ASTX+ I/R). ASTX was orally administrated 3 times at 48h, 24h and 45min before ischemia. All I/R liver injury was induced ischemia for 15min, followed by 3hr reperfusion. Then, the liver was obtained and analyzed. For in vitro study, Reactive oxygen species (ROS), inflammatory cytokines, apoptosis-related proteins and members of the signaling pathway were also examined in isolated Kupffer cells and/or hepatocytes from the steatotic liver.MCDHF I/R group showed higher serum ALT and AST levels, and higher liver tissue TBARS levels, compared to sham operated mice. In ASTX-treated group, serum ALT, AST and TBARS levels were significantly decreased. The number of TUNEL positive apoptotic cells was increased in MCDHF I/R group, but ASTX significantly reduced TUNEL-positive cells number. The number of F4/80 positive cells was increased in MCDHF I/R group, but ASTX significantly reduced F4/80 positive cells. Real-time quantitative RT-PCR showed elevated mRNA levels of inflammatory cytokines in MCDHF I/R group, but decreased levels in MCDHF+ASTX+ I/R group. Moreover, HO-1 and HIF1a expressions were significantly increased by treatment with ASTX.In vitro experiments, Kupffer cells and hepatocyte isolated from fatty liver, when stimulated by 4 hours of hypoxia and 6 hours of reoxygenation (4H6R), inflammatory cytokines were highly induced in Kupffer cells subjected 4H6R, but ASTX pre-treatment for 24 hours markedly reduced the expression of inflammatory cytokines. The expression of HO-1 and HIF-1α, phosphorylation of Akt and mTOR expressions were increased by ASTX. The inflammatory cytokines produced by Kupffer, which were subjected to hypoxia and reoxygenation (HR), were inhibited by ASTX. Expressions of Bcl-2, HO-1 and Nrf2 in hepa- tocytes by HR were increased, whereas Caspases activation, Bax and phosphorylation of ERK, MAPK, and JNK were suppressed by ASTX.These results demonstrated that pretreatment with ASTX has a protective effect and is a safe therapeutic treatment for IRI, including for liver transplantation of the steatotic liver.Grant-in- Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. (17H04277, 15K10043, 17K09411).