Effects Of High Glucose Concentration On Damage Index And Lactic Acid Production Of BRL-3A Rat Hepatocytes And Its Potential Mechanism

Objective: To investigate the effects of high glucose concentration on damage index and lactic acid production of BRL-3A rat hepatocytes and its potential mechanism.Methods:The hepatocytes of BRL-3A rats were cultured in vitro,and intervened with different glucose concentrations(5.5mmol/L,15mmol/L and 25mmol/L),of which5.5mmol/L was used as the control group,and the culture time was 12 h,24h and 48 h respectively.After reaching the intervention time,the fluorescence intensity of the reactive oxygen species(ROS)levels were observed by fluorescence probe,the lactic acid in cell supernatant culture medium,as well as the indexes of liver damage,such as aspartate transaminase(AST)、 alanine transaminase(ALT)and alkaline phosphatase(ALP)were determined.The m RNA expression of Hexokinase 2(HK2),Phosphofructokinase-2/fructose-2,6-bisphosphatase 3(PFKFB3)and signal transducer and activator of transcription 3(STAT3)was detected by RT-PCR.After adding PFKFB3 inhibitor to each group,detecting the lactic acid level and observing the the fluorescence intensity of ROS in each group.Set up a parallel experiment,add STAT3inhibitor(STAT3-IN-1)to BRL-3A cells to detect the levels of ROS fluorescence intensity,lactic acid and hepatocyte damage index.The m RNA expression of STAT3,P-STAT3,HK2 and PFKFB3 were determined by RT-PCR,the protein expression of STAT3,P-STAT3,HK2,PFKFB3 and ALB were further detected by WB.Results:1.Compared with the control group(5.5 mmol/L),the fluorescence intensity of ROS,lactic acid,AST and ALT levels in 15 mmol/L and 25 mmol/L groups were increased(P < 0.05),especially at 48 h,and there was no significant difference in ALP level among the three groups;2.Compared with the control group(5.5 mmol/L),the m RNA expression of HK2,PFKFB3 and STAT3 in 15 mmol/L and 25 mmol/L groups were increased(P < 0.05);3.After adding the PFKFB3 inhibitor,the intracellular lactic acid level decreased,and the ROS level decreased accordingly(P < 0.05);4.After adding STAT3 inhibitor,the levels of ROS fluorescence intensity,lactic acid,AST and ALT increased,and the m RNA expression of HK2 and PFKFB3 decreased in three groups(P < 0.05);5.Compared with the control group(5.5 mmol/L),the protein expression of HK2,PFKFB3,STAT3 and P-STAT3 in 15 mmol/L and 25 mmol/L groups was higher than that in control group,and the protein expression of ALB was lower than that in control group.After adding STAT3 inhibitor,the protein expression of HK2 and PFKFB3 decreased,and the protein expression of ALB increased in three groups(P <0.05).Conclusion:1.Intervention of high glucose in hepatocytes can increase the level of ROS in liver cells and the level of cell damage indicators,and damage the albumin synthesis function of cells;2.The mechanism of high glucose impairing hepatocyte function involves STAT3 regulating the expression of HK2 and PFKFB3,promoting the production of lactic acid and further promoting the production of ROS.