Methylprednisolone inhibits low-flow hypoxia-induced mitochondrial dysfunction in isolated perfused rat liver.
OBJECTIVE
To investigate the mechanism by which methylprednisolone protects the liver from hypoxia-induced injury.


METHODS
Prospective control study using the isolated rat liver.


METHODS
Animal research facility.


METHODS
Male, fasted, pathogen-free Sprague-Dawley rats.


METHODS
Low-flow hypoxia was produced by reducing afferent perfusate pressure from 10 to 2.5 cm H(2)O; isolated livers were portally perfused for 2 hrs.


RESULTS
We measured mitochondrial membrane potential and hydrogen peroxide production by imaging rhodamine 123 and 2'-7'-dichlorofluorescein fluorescence, respectively. Leakage of mitochondrial enzymes was also monitored by assaying mitochondrial aspartate aminotransferase activity in the outflow perfusate, and the radical-scavenging effect of methylprednisolone was assessed by measuring luminol-dependent hydrogen peroxide chemiluminescence. Apoptosis in liver cells was determined by using terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling. Rhodamine 123 fluorescence was significantly diminished in the hypoxic liver, especially in the region of the terminal hepatic venules, which is indicative of membrane depolarization in the mitochondria in those areas. Hypoxia-induced mitochondrial dysfunction was indicated by leakage of aspartate aminotransferase into the outflow perfusate, and increased 2'-7'-dichlorofluorescein fluorescence indicated increased hydrogen peroxide levels, particularly in the midzone. Pretreatment with 30, 10, or 3 mg/kg of methylprednisolone inhibited the hypoxia-induced mitochondrial membrane depolarization and enzyme leakage, although hydrogen peroxide levels and apoptosis in sinusoidal endothelial cells were unaffected.


CONCLUSIONS
Methylprednisolone does not protect the liver from hypoxia-induced injury by suppressing hydrogen peroxide production. Instead, the beneficial effect of methylprednisolone seems to be related to its ability to protect against mitochondrial membrane depolarization under hypoxic conditions.
Motoyama
Satoru
S
Second Department of Surgery, Akita University School of Medicine, Akita City, Japan.
Saito
Satoshi
S
Minamiya
Yoshihiro
Y
Saito
Reijiro
R
Nakamura
Masakatsu
M
Okuyama
Manabu
M
Imano
Hiroshi
H
Ogawa
Jun-ichi
J
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis; D024442:Aspartate Aminotransferase, Mitochondrial / Q000032:analysis; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D016166:Free Radical Scavengers / Q000494:pharmacology / Q000627:therapeutic use; D006861:Hydrogen Peroxide / Q000032:analysis; D000860:Hypoxia / Q000201:enzymology / Q000209:etiology / Q000517:prevention & control*; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D008099:Liver / Q000098:blood supply*; D008163:Luminescent Measurements; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008775:Methylprednisolone / Q000494:pharmacology / Q000627:therapeutic use*; D008930:Mitochondria, Liver; D028361:Mitochondrial Diseases / Q000201:enzymology / Q000209:etiology / Q000517:prevention & control*; D011446:Prospective Studies; D051381:Rats; D017207:Rats, Sprague-Dawley
D000893:Anti-Inflammatory Agents; D016166:Free Radical Scavengers; D006861:Hydrogen Peroxide; D024442:Aspartate Aminotransferase, Mitochondrial; D008775:Methylprednisolone