| Objective Peroxisome proliferator-activated receptor-α(PPAR-α) is one of the three subtypes of the nuclear receptor PPAR family. Wy14643 is a selective PPAR-αagonist, which protects against important organs ischemia-reperfusion injury associated with inhibiting of oxidative stress and inflammatory response. Our previous studies indicated that PPAR-αprobably protect against liver ischemia/reperfusion (I/R) injury. But effects of different doses of Wy14643 on on primary hepatocytes subjected to hypoxia/reoxygenation (H/R) injury in rats have not been reported. In the present study, we determined whether PPAR-αactivation by the selective agonist Wy14643 had a protective role in H/R injury of hepatocytes in vitro. The purpose of this study was to investigate the effects of different doses of Wy14643 on hepatocytes H/R injury in rats and its mechanism.Methods Male Sprague-Dawley rats(weighing 250-300g)were used in these experiments. Primary rat hepatocytes were isolated and cultured. Hepatocytes were isolated and maintained overnight at 37°C in a humidified incubator containing 95% air and 5% CO2 (referred to as normoxic conditions). The next day, hypoxic conditions were attained by exposure to 95% N2 and 5% CO2 gas mixture in a humidified incubator for 4 h. Hypoxic exposure was confirmed in each experiment by measuring the ambient PO2 of the gas above the monolayers. Reoxygenation of hypoxic cultures was achieved with normoxia conditions for another 10 h, which ambient values should return to prehypoxic levels within 5 min. Six groups of culture hepatocytes (6 wells each) were separated as follows: (1), The control group was exposed to normoxic medium for 14 h. (2), The H/R injury group was exposed to hypoxic (4 h) and then reoxygenation (10h) conditions as decribed above. (3), model H/R hepatocytes treated with different doses of Wy14643 (10×10-6, 30×10-6 and 100×10-6 mol/L respectively). The content of aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) in the culture medium and supeoxide dismutase (SOD), glutathione (GSH) in hepatocytes were measured at the end of the experiments. The appearance and ultramicrostructure change of hepatocytes were observed by electronic microscope. The PPARα-mRNA expression was measured by reverse transcription-polymerase chain reaction (RT-PCR). Cell viability was assayed by MTT method. Finally, all data were expressed as mean±SD. The statistical significance of differences between groups was analyzed using the one-way analysis of variance (ANOVA) and methods of LSD with the SPSS11.5 for windows XP statistical software package. The P values less than 0.05 were considered statistically significant.Results Compared with control group, the H/R group showed more hypoxia/reoxygenation induced injury. H/R induced a significant incresase ALT, AST in the culture medium and MDA in the hepatocytes (P<0.01). However, SOD and GSH activity in hepatocytes were lower in H/R group than control group (P<0.01). ALT and AST in culture medium activities in Wy14643 groups(10×10-6, 30×10-6 and 100×10-6 mol/L respectively) were all lower than those in H/R group (P<0.01 or P<0.05). In Wy14643 group (10×10-6 mol/L), SOD and GSH activities in the hepatocytes was not decreased significantly (P>0.05), MDA in the hepatocytes was not increased signicantly (P>0.05). In Wy14643 group (30×10-6 mol/L), SOD and GSH activities in the hepatocytes decreased significantly (P<0.05), as well as MDA in the hepatocytes increased signicantly (P<0.05). In Wy14643 group (100×10-6 mol/L), SOD and GSH activities in the hepatocytes decreased significantly (P<0.01), as well as MDA in the hepatocytes increased signicantly (P<0.01).Quantitative analysis showed that the mRNA expressions of PPARαin the H/R group was significantly decreased when compared with the control group (P<0.01). However, when comparing the Wy14643 group (10×10-6, 30×10-6, 100×10-6 mol/L) with H/R group, mRNA expressions of PPARαwas increased after the addition of Wy14643 (P<0.01 or P<0.05).Under electron microscope it was showed that the occluded hepatocytes from the H/R group was markedly damaged with mitochondrion swollen, vacuolar degenerationand, and mitochondrial crista destruction, marked decrease of rough endoplasmic reticulum and nucleus structure destruction compared with control group. It was demonstrated that hepatic injury was a significant amelioration in Pretreatment groups with Wy14643 (10×10-6, 30×10-6, 100×10-6 mol/L).Conclusion A PPAR agonist, Wy14643, exerts significant protective effect against H/R injury in primary hepatocytes via PPARαactivation and attenuating oxidative stress. In addition, the protective effects may be associated with doses of Wy14643...
|
PDF Full Text Request