Effects Of α-lipoic Acid On The Key Enzymes Of Glucose Metabolism And Its Mechanisms In Rat Hepatocytes With Oxidative Damage

ObjectiveTo investigate the effects ofα-LA on the mRNA and protein expression of glucose metabolism key enzymes in rat hepatocytes with oxidative damage, to explore the molecular mechanism ofα-LA on glucose metabolism in diabetes and insulin resistance.MethodsRat hepatocytes (BRL cells) were divided into 6 groups. They were normal control groups and five oxidative groups injured by H2O2 with different concentration (0.05, 0.1, 0.2, 0.4, 0.8mmol/L, respectively). After 1h, the morphological changes of BRL cells were observed by the light microscope. Morever, the levels of MTT (OD) and the activity of antioxidant indices of cells in each group were measured.BRL cells were divided into 5 groups. They were normal control, H2O2 damage model,α-LA-L (50μmol/L),α-LA-M (100μmol/L) andα-LA-H (200μmol/L) groups. BRL cells were incubated with different dosages ofα-LA for 24 hours and then were injured by 0.2 mmol/L H2O2 for 1h. Furthermore, the morphological changes of BRL cells in each group were observed by light microscope.The activity and antioxidant indices of cells were measured and the effect ofα-LA on apoptosis rate of BRL cells was measured by FCM, and the protein expression of Bcl-2 and Bax were measured by western blot.The content of glucose in culture supernatant in each group was detected by oxidasea method, then calculate the consumption of glucose. The mRNA expression of GS, GK, PDH-E2 were detected by real-time PCR. The protein expression of GK, GS and PDH-E2 were measured by ELISA, and the protein expression of Akt was measured by western blot. ResultsFrom the morphological changes of BRL cells, the H2O2 groups were significantly different from the normal. The BRL cells in 0.05-0.2 mmol/L H2O2 groups were distorted while the cell boundary was still clear. The cell shape of 0.4 and 0.8 mmol/L H2O2 groups were distorted apparently and the boundary was not clear. There were 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 MDA in H2O2 groups were significantly higher than those in the control group (P<0.05).From the light of microscope, the recovery of cells was not good inα-LA-L group. In theα-LA-M andα-LA-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 inα-LA-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 andα-LA groups were significantly higher than those in the H2O2 oxidative model (P<0.05). The contents of MDA, the apoptosis rate of BRL in normal control and a-LA groups were significantly lower than those in the H2O2 oxidative model (P<0.05). The protein expression of Bcl-2 in normal control group andα-LA groups were significantly higher than those in H2O2 oxidative model. The protein expression of Bax inα-LA groups were significantly lower than those in H2O2 oxidative model group (P<0.05).The consumption of glucose in the H2O2 oxidative model was significantly lower than that in the normal control (P<0.05). And the consumption of glucose inα-LA groups was higher than that in the H2O2 oxidative model (P<0.05). The protein expression of GK, GS and PDH-E2 in H2O2 oxidative model were significantly lower than those in the normal control andα-LA groups (P<0.05). The mRNA expression of GK, GS, PDH-E2 in normal control and a-LA groups were significantly higher than those in the H2O2 oxidative model (P<0.05). The protein expression of Akt in normal control and a-LA groups were significantly higher than those in the H2O2 oxidative model (P<0.05).Conclusionsa-LA can increase the activity of SOD and GSH-Px, decrease the content of MDA in hepatocytes,a-LA can up-regulate the protein expression of Bcl-2, down-regulate the protein expression of Bax, and decrease the apoptosis rate of hepatocytes with oxidative damage, to protect the hepatocytes from oxidative damage to some extent.a-LA can promote decomposition of glucose,increase the consumption of glucose in cells, and consequently, reduce the content of glucose.a-LA can increase the mRNA and protein expression of GK, GS and PDH-E2. It suggested that a-LA can improve the glucose metabolism key enzymes of hepatocytes following the oxidative injury to some extent. a-LA can up-regulate the protein expression of Akt, the key molecule in insulin signal pathway, to improve the conduction of insulin signals, and consequently, improve the glucose metabolism enzymes of hepatocytes with oxidative injured by H2O2 in vitro.