The Role Of Antioxidant Gene In The Death Of PC12Cell Induced By Glucose Deprivation

Objective:The earliest pathological change of cerebral ischemia is energy metabolicdisorders. Cells rely on glucose metabolism to provide energy. Glucose isbroken down by oxidation mainly through glycolysis and oxidativephosphorylation in the body. Mitochondrial oxidative phosphorylation disorderdecrease ATP in cerebral ischemia and produce a lot of reactive oxygenspecies at the same time. Therefore, this research adopts the EBSS,glycolysis blocking agent2-DG, oxidative phosphorylation blocker oligomycinto explore the influence of different energy metabolic pathways in cells surviveand the role of antioxidant gene in the cell death induced by the absence ofglucose.Methods:1)PC12cell culture glucose depletion model, the cell can be divided into fivegroup namely Con, EBSS,E+2-DG,E+Oligo and E+2+O for30minutes.MTTmethod was used to detect cell viability. Inverted microscope was used toobserve the cell morphology. Then we use the Flow cytometry to test the formof cell death in PC12cells.2)Western Blot detected expression changes in apoptosis-related proteincleaved Caspase-3.3)By Hoechst33342staining, the nucleus morphology was observed underconfocal microscopy in PC12cells.4)RT-PCR and real-time PCR detected expression changes in antioxidantgene SOD2,HO-1,NQO-1,so did the Western Blotting.5)PC12cell culture glucose depletion model, the cell can be divided into fivegroup namely Con,EBSS,E+2-DG,E+Oligo and E+2+O. Then, join the NACpretreatment of half an hour, dosing half an hour. MTT method was used todetect cell viability. Inverted microscope was used to observe the cellmorphology.Results:1)Under the condition of the lack of glucose inhibition of different metabolicpathways and a half hours, different groups of PC12cells survival rate haveobvious change, single plus2-DG group found that cell survival rate is very low, other groups are low, too. And E+2+O group is the lowest, but nostatistical significance; PC12cells form clearly, it become tentacles retractionand cell density decreased; The results of Flow cytometry are similar to theabove trend.2) WB result also shows the same trend, caspase-3has the larger set ofcracking degree in single plus2-DG group. But in E+2+O group, caspase-3has the smallest set of cracking degree accidentally at this time. And wespeculates that may be due to low energy which leads to cell death.3) With Hoechst33342staining, we observed pycnosis, fragmentation andenhanced staining in the nucleus of PC12cells, which were further enhancedby co-function of2-DG and Oligo.4) PCR results show that reducing intracellular antioxidant capacity, SOD2,NQO-1, HO-1at the transcription level is reduced, also similar trends in thelevel of translation.5) After adding exogenous antioxidants NAC, the survival rate of PC12cells ispicking up. Cell morphology is recovered under inverted microscope. Cellsdensity increase a lot compared the none-NAC.Conclusion:1. Interference glycolysis and aerobic oxidation can promote apoptosis ofPC12cells2. Antioxidant capacity is reduced in PC12cells by interference glycolysis andaerobic oxidation.3. Oxidation reaction is regulated by the glycolysis and aerobic oxidation.4. PC12cells rely mainly on glycolysis to maintain cells survive under thecondition of lack of nutrition.