The Imbalance Of Mitochondria Fission/Fusion Participate In The Experimental Study Of Astrocytes Injury Induced By High Glucose And Hypoxia

Objective To investigate whether mitochondrial division/fusion imbalance was involved in the damage of astrocytes induced by high glucose and hypoxia by simulating the model of diabetes and cerebral ischemia injury in vivo using the high glucose combined with oxygen glucose deprivation?OGD?model.Methods In this study,astrocytes were cultured in vitro.In order to investigate the effects of high glucose and/or hypoxic on astrocytes and their mechanisms,they were grouped as follows:?1?Normal glucose concentration without hypoxia group?NG-Con?:Astrocytes were cultured in DMEM high glucose medium and normal oxygen incubator?37 C,5%CO²?.?2?High glucose culture without hypoxia group?HG-Con?:Astrocytes were cultured in DMEM high glucose complete medium with 50 mmol/L glucose and normal oxygen incubator?37°C,5%CO₂?;?3?Oxygen-glucose deprivation with normal glucose concentration group?NG-OGD?:Astrocytes were cultured in DMEM high glucose medium for 24 hours,place it in a hypoxic incubator?37°C,1%O₂,5%CO₂?for 6 hours,and then put it back into normal oxygen incubator 0,6,12,24h;?4?Oxygen-glucose deprivation with high glucose concentration group?HG-OGD?:Astrocytes were cultured in DMEM high glucose medium containing 50mmol/L glucose for 24 hours,and placed in hypoxic incubator?37°C,1%O₂,5%CO₂?6h,and then back to normal oxygen incubator 0,6,12,24h.The effects of different concentrations of glucose on the proliferation activity of astrocytes were examined by CCK-8 assay.Morphological changes of astrocytes after high glucose and?or?hypoxia intervention were observed by inverted microscope.Flow cytometry was used to detect the apoptosis of astrocytes after high glucose and hypoxia.the content of ROS was detected by DCFH-DA in different treatment groups.Immunofluorescence,immunocytochemistry and Western blotting were used to observe the expression of mitochondrial division/fusion factors?Fis1,Drp1,Opa1,Mfn2?in different treatment groups.Results?1?CCK-8 assay was used to detect the effect of different concentrations of glucose on the proliferation of astrocytes.The results showed that the cell proliferation activity firstly increased and then decreased as the glucose concentration increased.?2?The density and morphological changes of astrocytes were observed.Both in normal glucose and high glucose concentration,the longer the reoxygenation time after hypoxia,the lower the cell density.Compared with the normal glucose concentration culture group,the density of astrocytes in the corresponding high-glucose culture group was further reduced,the cell gap was sparse,and a large number of cells became round;especially in the high-glucose culture HG-OGD/R 24h group,a large number of round dead cells and cell debris were observed to be suspended in the medium.?3?Flow cytometry showed that the apoptotic rate of astrocytes increased after hypoxia and reoxygenation in the normal glucose concentration culture group and the high glucose culture group,the increase of apoptosis rate is more pronounced in the high-glucose culture HG-OGD/R 12h group.?4?The results of DCFH-DA assay for ROS production showed that compared with normal glucose concentration without hypoxia group?NG-Con?,a large amount of ROS production was detected in the normal glucose concentration NG-OGD/R 24h group;however,after high glucose culture,ROS production increased more significantly in the high glucose HG-OGD/R 24h group.?5?The result of immunocytochemistry or immunocytofluorescence,western blotting were shown that compared with the normal glucose concentration without hypoxia group?NG-Con?,the expression of Fis1 and Drp1 protein increased gradually with the prolonged reoxygenation time after hypoxia,while the expression of the fusion factors Opa1 and Mfn2 protein decreased gradually.After high glucose cultured,the expression of Fis1 and Drp1 protein increased further with the prolonged reoxygenation time after hypoxia,while the expression levels of Opa1 and Mfn2 protein were further decreased.However,there was no significant difference in the changes of the fusion factors Opa1 and Mfn2 in the normal glucose concentration without hypoxia group?NG-Con?and the high glucose culture without hypoxia group?HG-Con?.Conclusion?1?High glucose reduces the proliferation viability of astrocytes and high glucose increases astrocyte damage caused by hypoxia.?2?High glucose increase hypoxia-induced ROS production,which may be involved in the apoptosis of astrocytes induced by hypoxia.?3?Mitochondrial division/fusion imbalance may be involved in the damage of astrocytes caused by high glucose and hypoxia.