The Protective Role Of Ethyl Pyruvate On Hepatic Ischemia-reperfusion Injury And Its Mechanism

PartⅠThe protective effect of ethyl pyruvate on hypoxia/reoxygenation of L-02 hepatocyte and its mechanismObjective To elucidate the protective effect of ethyl pyruvate on hypoxia/reoxygenation of L-02 hepatocyte and its underlying mechanism.Methods We set the model of hypoxia/reoxygenation of L-02 hepatocyte in Billups-Rothenberg device, which experienced hypoxic phase for 1 hr and reoxygenational phase for 1 hr. Depending on different interventions, the model was divided into 3 groups:control group (Con group), Lactate Ringer's (LR group), and ethyl pyruvate (EP group). First, we confirmed the optional concentration of EP for the vitro study thru the study of concentration gradient using HO/PI method. Then, we examined concentration of AST and ALT in supernatant. Thru method of HO/PI, we observed necrotic cells and apoptotic cells among different groups, and counted exact cells with fluorescence microscope. We also analyzed the extent of necrosis and apoptosis using Flow Cytometry method. Depending on method of Western blot, we tested the expression of HMGB1 in supernatant and determined its optical density value. Using the same method, we also tested the expression of Caspase9, Caspase3 and PARP of hepatocyte and determined their optical density values. We observed structure of mitochondria via transmission electron microscope. Finally, we measured the concentration of ATP in hypatocyte using luciferase luminescence method.Results The population of necrotic cells was lowest at concentration of 2 mM of EP among concentration gradient, P value<0.05. The concentration of AST and ALT in EP group were 34.6±0.5 IU/L and 28.5±0.6 IU/L respectively, which were lower than those in Con group and LR group, P value<0.05. Via method of HO/PI, we observed viable cells, necrotic cells and apoptotic cells. The population of viable cells in EP group was higher than that of other groups, P value>0.05. The population of necrotic cells in EP group was lower than that of other groups, P value<0.05. The population of apoptotic cells in EP group was higher than that of other groups, P value<0.05. We obtained the same results using Flow Cytometry method. Thru Western blot method, the expression of HMGB1 in EP group, which value of optical density was 0.400.05, was weaker than those in other groups, P value<0.05. Using the same method, the expression of Caspase9, Caspase3 and PARP of hepatocyte in EP group, which values of optical density respectively were 0.92±0.01,0.25±0.02, and 0.87±0.01, were stronger than those in other groups, P value<0.05. We observed that the injured heptacytes among 3 groups experienced structure changes of mitochondria using transmission electron microscope, which became swelling and bleb. Finally, the concentration of ATP in Con group, LR group and EP group were 1.78±0.3umol/g,1.73±0.4μmol/g, and 3.476±0.5μmol/g respectively, P value<0.05.Conclusions EP played the role of protecting L-02 hepatocyte underlying hypoxia/reoxygenation. In this vitro model, EP decreased the population of necrotic cells and increased the population of apoptotic cells. The fact that EP improved intracellular ATP level might activate the pathway of apoptosis, depending on the necroapoptosis theory. Simultaneously, reduction of necrotic cells could lower expression of HMGB1, which is an important pro-inflammatory factor.Part IIThe role of ethyl pyruvate during hepatic ischemia-reperfusion injury and its mechanism in rat modelObjective To explore the protective effect of EP during liver ischemia-reperfusion injury and its mechanism in rat model.Methods We established the model of liver ischemia-reperfusion injury in SD rats, which were divided into three groups depending on intervention:sham group, LR group and EP group. We monitored blood level of AST and ALT serially at 0,1,3, and 6 hrs postreperfusion, for determining the peak injury of ischemia/reperfusion and EP's protective effect. From the morphological view, we used HE/TUNEL method to observe the extent of necrosis and apoptosis and to determine cell population of injured hepatic tissue. Detected by Western blot method, apoptosis-related factors (Caspase9, Caspase3 and PARP protein) were measured, which optical density values were calculated. Using luciferase luminescence, we detected the liver tissue ATP levels in the various groups.Results At 3 hours poetreperfusion, concentrations of AST and ALT in LR group were 2773.4±16.4 IU/L and 1436.4±15.2 IU/L respectively and those in EP group were 2356.3±15.4 IU/L and 964±9.7 IU/L respectively, which were higher than those at other time site, P values<0.05. At the same time, aminotransferase's concentrations in EP group were lower than that in LR group, P value<0.05. Depending on HE/TUNEL judgment, proportion of necrotic cells in LR group and EP group were 45±0.4% and 22±0.3% respectively, P value<0.05; proportion of apoptotic cells in LR group and EP group were 0.610±0.01% and 0.449±0.02% respectively, P value> 0.05; apoptotosis/necrosis ratio in LR group and EP group were 0.014 and 0.020 respectively, P value<0.05. Using Western blot method, the expression of Caspase9, Caspase3 and PARP of hepatic tissue in EP group, which values of optical density respectively were 1.32±0.04,2.5±0.06, and3.27±0.05 respectively, were stronger than those of LR group, P value<0.05. By luciferase luminescence method, ATP levels of injured liver in LR group and EP group were 1.79±0.3μmol/g and 2.76±0.4μmol/g respectively, P value<0.05.Conclusions EP had protective effect in liver IRI in rat model. EP reduced the necrotic cells in this model. The proportion of apoptotic cells increased relatively. The fact that EP improved intracellular ATP level might activate the pathway of apoptosis, according to the necroapoptosis theory.