Induction of ischemic tolerance in rat liver via reduced nicotinamide adenine dinucleotide phosphate oxidase in Kupffer cells.
OBJECTIVE
To elucidate the mechanisms of hepatocyte preconditioning by H2O2 to better understand the pathophysiology of ischemic preconditioning.


METHODS
The in vitro effect of H2O2 pretreatment was investigated in rat isolated hepatocytes subjected to anoxia/reoxygenation. Cell viability was assessed with propidium iodide fluorometry. In other experiments, rat livers were excised and subjected to warm ischemia/reperfusion in an isolated perfused liver system to determine leakage of liver enzymes. Preconditioning was performed by H2O2 perfusion, or by stopping the perfusion for 10 min followed by 10 min of reperfusion. To inhibit Kupffer cell function or reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gadolinium chloride was injected prior to liver excision, or diphenyleneiodonium, an inhibitor of NADPH oxidase, was added to the perfusate, respectively. Histological detection of oxygen radical formation in Kupffer cells was performed by perfusion with nitro blue tetrazolium.


RESULTS
Anoxia/reoxygenation decreased hepatocyte viability compared to the controls. Pretreatment with H2O2 did not improve such hepatocyte injury. In liver perfusion experiments, however, H2O2 preconditioning reduced warm ischemia/reperfusion injury, which was reversed by inhibition of Kupffer cell function or NADPH oxidase. Histological examination revealed that H2O2 preconditioning induced oxygen radical formation in Kupffer cells. NADPH oxidase inhibition also reversed hepatoprotection by ischemic preconditioning.


CONCLUSIONS
H2O2 preconditioning protects hepatocytes against warm ischemia/reperfusion injury via NADPH oxidase in Kupffer cells, and not directly. NADPH oxidase also mediates hepatoprotection by ischemic preconditioning.
Tejima
Kazuaki
K
Department of Gastroenterology, University of Tokyo, and Toshiba General Hospital, 6-3-22 Higashiooi, Shinagawa-ku, Tokyo 140-8522, Japan.
Arai
Masahiro
M
Ikeda
Hitoshi
H
Tomiya
Tomoaki
T
Yanase
Mikio
M
Inoue
Yukiko
Y
Nishikawa
Takako
T
Watanabe
Naoko
N
Ohtomo
Natsuko
N
Omata
Masao
M
Fujiwara
Kenji
K
D000818:Animals; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019194:Ischemic Preconditioning / Q000379:methods*; D007728:Kupffer Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D019255:NADPH Oxidases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000503:physiopathology / Q000517:prevention & control*
D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases