The Study Of UCP2 Protective Role Of Neuronal Cells From Hyper-glucose Exacerbated Hypoxia Induced Injury And It’s Relationship With Mitochondria Fission And Fusion

Objective To investigate the effects of uncoupling protein 2 on nerve cells with hyperglycemia anoxic state and it’s relationship with mitochondria fission and fusion.Methods Hippocampal neurons(HT22), UCP2 transfection hippocampal neurons(UCP2) and corresponding non-transfection hippocampal neurons(HT-NC) were chosen for our experiments. HT22 cells were divided into the following groups in order to investigate the effects of hyperglycemia/acidosis.(1) The control group;(2) The hyperglycemia group: cultivated by hyperglycemia with different concentration(30,40,50 and 60mM) for 24 h. Then hypoxia 0h/4h and oxygen recovery again for different time(0,6,12 and 18h);(3)The normal glucose unite hypoxia group cultured for 24 h. Then hypoxia 0h/4h and oxygen recovery again for different time(0,6,12 and 18h);(4)The hyperglycemia unite hypoxia group: cultivated by hyperglycemia with different concentration(30,40,50 and 60mM) for 24 h. Then hypoxia 0h/4h and oxygen recovery again for different time(0,6,12 and 18h);(5)The acidosis group: cultivated by acidosis with different concentration(2.5,5,7.5 and 10mM) for 24 h. HT22 cells were divided into the following groups in order to investigate the effects of hyperglycemia on the anoxic injury of neuronal cells. In order to investigate the effects of UCP2 on hyperglycemia promoted ischemic injury, cells were divided into the following groups:(1) The HT-NC control group;(2) The HT-NC hyperglycemia accompany hypoxia; Th(3) e UCP2 control group;(4) The UCP2 grouphypoxia hyperglycemia accompany hypoxia. Cultivated with final concentration 50 mM for 24 h,then hypoxia 4h and oxygen recovery again for 12 h. CCK-8 method was used to detect the rates of cell viability. Using inverted microscope to observe the changes of cell morphology. DCFH-DA staining was used to detecte the generation of ROS. The expression of OPA1, Mfn2, Fis1, Drp1 and Cleaved Caspase-3 were measured by Western-blotting and immunohistochemistry. Apoptosis was determined via flow cytometry.ResultsPart 1:(1)The cell viability decreased after HT22 cells have been cultivated with different concentration hyperglycemia and acidosis for 24 hours. With the concentration evolution, the viability are decreased. The viability rates is decrease obviously with 50 mM hyperglycemia and 5mM acidosis(P<0.05). Compared with the control group, the cell viability rates in hypoxia-reoxygenation decreased(P<0.05);(2)The control group cells shows normal morphology; The treated groups show an increase in apoptotic cells. Apoptotic cell nuclei and membrane are incomplete;(3)After ROS-specific labeled probe DCFH-DA marking, compared with the control group, the generation of ROS is obviously increased in the different treated groups(P<0.05);(4)The results of Western-blotting shows that the expression of Cleaved Caspase-3 in he different treated groups are significantly increased comparing with control group.Part 2:(1)Compared with the UCP2 control group, the cell viability rates in UCP2 hyperglycemia unites hypoxia-reoxygenation decreased, and show significant increase than HT-NC hyperglycemia unites hypoxia-reoxygenation group. more injury was observed than the same hyperglycemia group(P<0.05). The viability rates is decrease obviously with hypoxia 4h and oxygen recovery again for 12 h group(P<0.05);(2)The control group show normal morphology; The cells in HT-NC hyperglycemia unites hypoxia-reoxygenation group show an increase in apoptotic cells than that in the UCP2 hyperglycemia unites hypoxia-reoxygenation group;(3)Compared with the HT-NC hyperglycemia unites hypoxia-reoxygenation group, the generation of ROS are obviously decreased in UCP2 hyperglycemia unites hypoxia-reoxygenation group, but still more than the control group(P<0.05);(4)Compared with control group, the expression of OPA1 and Mfn2 were significantly decreased and the expression of Fis1, Drp1 and Cleaved Caspase-3 was significantly increased in HT-NC hyperglycemia unites hypoxia-reoxygenation group(P<0.05). But that reversed comparing with that in UCP2 hyperglycemia unites hypoxia-reoxygenation group(P<0.05);(5)The result of flow cytometry show that apoptotic cells in UCP2 hyperglycemia unites hypoxia-reoxygenation group decrease than that in HT-NC hyperglycemia unites hypoxia-reoxygenationand group, but still more than the control groups.Conclusion(1)Hyper-glucose exacerbated hypoxia induced neuronal cells injury through increased the generation of ROS;(2)Acidosis can significantly increase the neuronal damage through increased the generation of ROS;(3)The increasing production of ROS is involved in the neuronal cells injury induced by hyper-glucose exacerbated hypoxia.(4)Overexpression of UCP2 can protect the neuronal cells from Hyper-glucose exacerbated hypoxia induced neuronal cells injury through decrease the generation of ROS and keeping the balance of mitochondrial fission and fusion.