Effects Of Selenium On The Glycometabolism Key Enzymes And Its Mechanisms In Rat Hepatocytes With Oxidative Damage

ObjectiveTo investigate the effect of selenium on the mRNA and protein expression of glucose metabolism key enzymes in rat hepatocytes with oxidative damage,so as to explore the molecular mechanism of selenium on glucose metabolism in diabetes and insulin resistance.Methods1.The oxidative injury model of BRL in vitro:Rat hepatocytes(BRL) were divided into 6 groups.They were normal control groups and six oxidative groups which were injured by H₂O₂ with different concentration(0.05,0.1,0.2,0.5, 1mmol/L,respectively).After 1h,the morphological changes of BRL were observed by the light microscope.Morever,the levels of MTT(OD) and the activity of antioxidant indices of cell in each group were measured.2.Protective effects of selenium on BRL following oxidative injury in vitro: BRL were divided into 5 groups.They were normal control,H₂O₂ damage model, Se-L(0.05μmol/L),Se-M(0.1μmol/L) and Se-H(1μmol/L) groups.BRL were injured by 0.1 mmol/L H₂O₂ for 1h,and then they were incubated with different dosages of selenium for 24 hours.Furthermore,the morphological changes of BRL in each group were observed by light microscope.The activity and antioxidant indices of cell were measured.The mRNA expression level of GSH-Px was detected by real-time quantitative PCR.The effect of selenium on apoptosis rate of BRL was measured by FCM.3.Effects of selenium on glucose metabolism enzymes of BRL following oxidative injury in vitro:The mRNA expression of GK,GS,Akt,ASK1 were detected by real-time PCR.The protein expression of GK and GSK-3 were measured by ELISA,and the protein expression of Akt and ASK1 were measured by western blot. Results1.The oxidative injury model of BRL in vitro:From the morphological changes of BRL,the H₂O₂ groups were significantly different from the normal.The BRL in 0.05～0.2 mmol/L H₂O₂ groups were distorted while the cell boundary was still clear.The cell sharp of 0.5 and 1 mmol/L H₂O₂ groups were distorted apparently and the boundary was not clear.There were some of more of dead cells fall off from the bottom of wells.The levels of MTT(OD) of cells in H₂O₂ groups were significantly lower than those in the normal control group(P＜0.05).Additionally the activities of GSH-Px and SOD in H₂O₂ groups were significantly lower than those in the control group(P＜0.05).And the content of cell MDA in H₂O₂ groups were significantly higher than those in the control group(P＜0.05).2.Protective effects of selenium on BRL following oxidative injury in vitro: From the light of microscope,the recovery of cells was not good in Se-L group.In the Se-M and Se-H groups,the morphological changes of cells were significantly better than those in the H₂O₂ oxidative model group.The distance between cells became shorter and the connection of cells increased.The shape of BRL in Se-H group was similar to the normal control group.The level of MTT(OD),the activities of GSH-Px and SOD of cell in normal control and selenium groups were significantly higher than those in the H₂O₂ oxidative model(P＜0.05).Besides,the contents of MDA,the apoptosis rate of BRL in normal control and selenium groups were significantly lower than those in the H₂O₂ oxidative model(P＜0.05). The mRNA expression of GSH-Px in normal control and selenium groups were significantly higher than those in the H₂O₂ oxidative model(P＜0.05).3.Protective effects of selenium on glucose metabolism enzymes of BRL following oxidative injury in vitro:The mRNA expression of GK,GS,Akt in normal control and selenium groups were significantly higher than those in the H₂O₂ oxidative model(P＜0.05).The protein expression of GK and Akt in normal control and selenium groups were significantly higher than those in the H₂O₂ oxidative model(P＜0.05).The mRNA and protein expression of ASK1 in normal control and selenium groups were significantly lower than those in the H₂O₂ oxidative model(P＜0.05).The protein expression of GSK-3 in H₂O₂ oxidative model and selenium group were significantly lower than those in normal control group(P＜0.05).Conclusions1.Oxidative model of hepatocytes was established by different concentrations of H₂O₂.The oxidative model was identified through morphological observation, MTT(OD) and antioxidant indices detection.Finally the concentration of 0.1mmol/L H₂O₂ was chose as the better dosage to study the protective effects of antioxidant on BRL following oxidative injury in vitro.2.Selenium could increase the mRNA and protein expression of GK,increase the mRNA expression of GS.It suggested that selenium could improve the glycometabolism key enzymes of hepatocytes with oxidative damage to some extent.The potential mechanisms will be listed as follows.(1) Antioxidative pathway:Selenium could increase the activity of SOD,improve the activity and mRNA expression of GSH-Px,decrease the content of MDA in hepatocytes,to protect the hepatocytes from oxidative damage to someextent.(2) PI₃K-Akt-GSK-3 pathway:Selenium could up-regulate the mRNA and protein expression of Akt which was the key molecule in insulin signal pathway,therefore to improve the conduction insulin signals.(3) Cellular apoptosis signal pathway:Selenium could down-regulate the mRNA and protein expression of ASK1,and decrease the apoptosis rate of hepatocytes with oxidative damage,to improve the glycometabolism enzymes of hepatocytes with oxidative injured by H₂O₂ in vitro.