The Study Of Neonatal Rats Liver Mitochondria Generating Reactive Oxygen Species And Undergoing The Permeability Transition In Response To Hydrophobic Bile Acids

Objectives: Hydrophobic bile acids accumulate in the liver during cholestatic liver diseases are believed to cause hepatocellular toxicity. Mitochondria perturbation play a central role on the toxicity of bile acids. Hepatocellular necrosis and apoptosis are in part induced through mitochondrial permeability transition(MPT) and generation of reactive oxygen species(ROS). In this study we observed the injury of liver mitochondria in response to the hydrophic bile acids and also the protective effects of several inhibitor. The purpose was to approach the mechanism of mitochondria perturbation which induced mitochondrial permeability transition and oxidative stress in neonatal rat's liver by hydrophic bile acids.Methods: We used 6 days old health Wistar neonatal rats(inbreed strain),10 rats as a measuring unit. Rats were anesthetized by diethylether. Then we opened the abdominal wall and cut livers for use. The livers were homogenated to make mitochondria suspension by differential and density gradient centrifugation. The MPT was measured spectrophotometically 5 minutes and morphologically by electron microscopy in neonatal rats'liver mitochondria exposed glycochenodeoxycholic acid(GCDC) in various concentrations with and without cyclosporine A(CsA) and three antioxidants ( Melatonin ,β-Carotine ,α-tocopherol ). Hydroperoxide generation was measured by flow cytometry at 1, 3, 5, 10minutes after the reaction's begining. Comparing the differences among the MPT's and ROS's changes in mitochondria which preincubated with CsA and three antioxidants. All measuring indicatrix were three units'average.Results: GCDC induced the MPT in a time and dose-dependent manner,which was inhibited by cyclosporineA and three antioxidants: melatonin,β-carotine,α-tocopherol. Electron microscope results showed obviously disorganization in mitochondria ultramicrostructure in response to GCDC. GCDC stimulated reactive oxygen species generation in neonatal liver mitochondria in a time and dose-dependent manner, which were significantly inhibited by three antioxidants and cyclosporine A. The disorganization in mitochondria ultramicrostructure were extenuation in anti-oxidant groups comparing the control group and in CsA group comparing anti-oxidant groups by electron microscope observation.Conclusions:1. The isolated mitochondria of neonatal rat liver treated with hydrophobic bile acid GCDC can induced the MPT, ROS generation and morphologic change ,these changes happened rapidly in a time and dose-dependent manner.2. The relationship between the MPT and ROS generation in neonatal rats liver mitochondria in response to GCDC was positive correlation.3. GCDC stimulated ROS generation , MPT and morphologic change which were significantly inhibited by three antioxidants and MPT inhibitor -cyclosporine A.These results can provide a clinical application.