The Mechanisms Of Autophagy Involved In The Effects Of Hypoxia And/or High Glucose In Neuro-2a Cells

AIMDiabetes are more likely to develop chronic cerebral hypoperfusion and disturbed function of neurons and then lead to cognitive impairment,yet the mechanisms involved is currently limited understood.Autophagy is a process by which cellular components are engulfed in autophagosome,and subsequently degraded by lysosomal proteases.It is well known that dysregulation of autophagy contributes to neurodegeneration disease such as Alzheimer's disease.To verify whether autophagy and mitophagy are complicated during diabetic chronic cerebral hypoperfusion,Neuro-2a cells were chosen to cultivated in vitro under hypoxia and/or high glucose to mimic the cerebral hypoperfusion and hyperglycemia in Diabetes.METHODS1.We divided the Neuro-2a cells into four groups according to the cultural conditions:Neuro-2a cells exposed to Normoxia?21%O₂?or Hypoxia?1%O₂?with or without High Glucose?100mM?were named as:Normoxia Control Neuro-2a cells;Normoxia High Glucose Neuro-2a cells;Hypoxia Control Neuro-2a cells;Hypoxia High Glucose Neuro-2a cells.2.Cell viability was measured by using MTS assays and the expression level of Cleaved caspase-3.3.Autophagic related proteins LC3B-II and P62 were detected by western blot.Cell Immunofluorescence?IF?and Transmission electron microscope?TEM?were used to monitor the distribution of the autophagic vesicles.4.Bafilomycin A1?BAF?was added for the final 6 h before harvesting to inhibit the autophagy degradation.5.JC-1 dye staining by flow cytometry and IF were used to analyze mitochondrial membrane potential???m?.Altered mitochondrial structure were detected by TEM.6.Mitophagic related proteins PINK1,HIF-1?,BNIP3 and TOMM20 were detected by Western blot.IF was used to access the colocalization among BNIP3,LC3B and the mitochondrial marker COX?.RESULTS1.Compared to normoxia condition,chronic hypoxia obviously decreased Neuro-2a cells viability with the expression of Cleaved caspase-3 increased,while high glucose showed a minor enhanced damage but not significantly.2.Chronic hypoxia markedly induced a upregulation of autophagic related proteins LC3B-II and P62 in Neuro-2a cells,while high glucose led to a minor decrease of LC3B-II,which had increased during hypoxia.Meanwhile we detected the accumulation of autophagy vesicles during hypoxia.3.Blocking autophagy degradation by Bafilomycin A1 obviously increased the LC3B-II and P62 proteins and reduce the cell viability in Neuro-2a cells under normoxia circumstance.However,Bafilomycin A1 neither change the expression of LC3B-II and P62 nor damage the cells further.4.Chronic hypoxia remarkably caused the loss of the mitochondrial membrane potential???m?in Neuro-2a cells.Besides the mitochondrial in Neuro-2a cells exposed to hypoxia turned rounded,giant and swollen.But high glucose did not show a significant detrimental effect.5.Chronic hypoxia dramatically led to a substantial accumulation of mitophagic related proteins HIF-1?,BNIP3 and TOMM20 in Neuro-2a cells.Meanwhile the upregulated BNIP3 showed a well colocalization with COX?,a specific mitochondrial marker.Conversely,we found that the enhanced abundance of endogenous LC3B,which accumulated in Neuro-2a cells under hypoxia condition,had less colocalization with COXIV and with BNIP3.CONCLUSIONS1.Compared to high glucose,it is chronic Hypoxia that exert a markedly detrimental effect in Neuro-2a cells which probably due to a blocked autophagy degradation.2.Chronic hypoxia mediated the damage and accumulated of mitochondrial in Neuro-2a cells,and the insufficient of Bnip3-mediated mitophagy may be involved in the process.