Study Of Effects Of Continuous Hypoxia At Different Time On Autophagy

OBJECTIVETo evaluate the autophagy activity of 83213 pancreatic islet ? cells at different continuous hypoxic duration by monitoring the changes of autophagy-related proteins LC3 BII,Beclin1 and P62.METHODS1.83213 islet cells were divided into two groups: control group and hypoxia group.The control group was normoxia?21% oxygen?and no rapamycin treatment,hypoxia group was treated with continuous hypoxia?1% O₂,5%CO₂?at different duration for 2 h,3 h,6 h,12 h,24 h,48 h.The total protein was extracted and its concentration was measured.Then extract the total protein of two groups and measure its concentration.2.The expression of hypoxia-inducible factor-1?HIF-1a?was detected by Western Blot in order to reflect the dynamics of HIF-1? at different time points after hypoxia.3.The expression of Beclin1,LC3B? and P62 in the normoxia group and hypoxia group was detected by Western Blot to reflect the dynamic changes of autophagy level at each time point after hypoxia treatment.RESULTS1.Compared with the normoxia group,the expression of HIF-1? protein was increased at 2 h,3 h,6 h,12 h,24 h and 48 h,and reached the peak at 24 h,and the difference was statistically significant?P <0.05?2.Compared with the normoxia group,the level of Beclin1 in pancreatic islets increased with hypoaxia treatment for 2h and 3h,and decreased at 6h after hypoxia,but still higher than that in normoxia group,while the level of Beclin1 decreased with hypoxia for 12 h,24h,48 h,and the difference was statistically significant?P<0.05?.Compared with normoxia group,the level of autophagy-related protein LC3B? increased at 2h,3h and 6h after hypoxia?P <0.05?,and decreased gradually at 12 h,24h and 48 h after hypoxia?P <0.05?.Compared with the normoxia group,the P62 protein decreased at 2h and 3h after hypoxia,while the difference has statistically significance at 3h only;The hypoxia group has been significantly higher than that in the normoxia group since 6h after hypoxia?P <0.05?,and reached the peak at hypoxia 24 h.CONCLUTION1.The expression of HIF-1? was increased in hypoxic condition.2.During sometime of hypoxia,the level of autophagy was increased with the higher autophagy-associated protein,Beclin1 and LC3?.However,with time goes by,autophagy is no longer enhanced,but weakened with the lower Beclin1 and LC3?.So,The survival state of the cells is related to the duration and intensity of hypoxia.In the short-term hypoxia conditions,autophagy may be involved in the protection of islet ? cells.And the dynamic changes of another autophagy-associated protein,P62,were opposite to that of Beclin1 and LC3 BII.