Role Of Autophagy In Neuronal SH-SY5Y Cell Injury Induced By Ischemia And Hypoxia

Background Cerebrovascular disease is a serious threat to human health,of which stroke is the most common cerebrovascular disease.At present,the death caused by stroke is the firstcause of death in our country.With the increasing aging of population,stroke will become the main killer threatening the health of the elderly.The majority of stroke patients suffer from ischemic stroke.When ischemic stroke occurs,the ischemic core of brain tissue will be irreversible infarcted quickly due to ischemia and hypoxia while the ischemic penumbra area around the ischemic core is only partial ischemia and hypoxia.The ischemic penumbra area is just in the early stage of pathological damage for a certain period of time.The neurons that show hydropic degeneration in this area not only have a tendency to aggravate mortality but a possibility of reversing to normal.Therefore,it is an urgent practical problem to find the mechanism of nerve cell self-protection,prolong the survival time of penumbra cells,and properly expand the therapeutic window so as to gain time for the follow-up treatment of patients.In recent years,many studies have shown that autophagy,as a self-rescue mechanism of cells,plays an important role in physiological and stress conditions.However,the mechanism of autophagy during ischemia is not clear.Whether autophagy can promote survival or aggravate injury is controversial.Neuroblastoma cells SH-SY5 Y as an in vitro cell model to study neuronal injury is recognized by academia.Therefore,by observing the changes of microfilaments,mitochondria,cell apoptosis,necrosis and autophagy,the damage changes of neural cells were comprehensively reflected.The SH-SY5 Y cells are used as a vitro cell model to simulate the environment of ischemic penumbra where the hypoxic and glucose are deficient to reveal the mechanism of neuron self-salvation,and provide experimental basis for further expanding the therapeutic window and promoting rehabilitation of ischemic stroke in clinic.Objective The aims of this study were to investigate the damage of SH-SY5 Y cells under hypoxic and ischemic conditions,the effect of Beclin1 protein on neuron autophagy and anti-injury,the relationship between mitochondrial changes and neuron autophagy,and the role of microfilament in SH-SY5 Y neuron injury and anti-injury under the ischemic conditions.In order to provide evidence for the protection of penumbra neurons,the factors affecting the self-rescue of nerve cells were explored.Methods SH-SY5 Y cells was cultured.Oxygen glucose deprivation(OGD)was used to mimic the penumbra environment.RNA interference(RNAi)technique was used to construct Beclin1-siRNA interference model by which the transcription and translation level of Beclin1 was down-regulated.To observe the effects of ischemia and hypoxia on SH-SY5 Y injury,the experiment was divided into three groups,control group,OGD 6 hours group and OGD 24 hours group.To investigate the role of autophagy in SH-SY5 Y injury induced by ischemia and hypoxia,the experiment was divided into four groups,control group,RNAi group,OGD 6 hours group and RNAi+OGD 6 hours group.RT-PCR and Western Blot analysis were used to identify Beclin1-siRNA interference model.CCK-8 was used to detect cell viability.Microplate method was used to detect lactate dehydrogenase(LDH)content in supernatant of cell culture.Specific fluorescence probe was used to label cell microfilaments and mitochondria.Changes of microfilament cortex and stress fibers,mitochondrial distribution,quantity and morphology were observed under fluorescence microscope.Reactive oxygen species(ROS)were detected by DCFH-DA fluorescence probe under the flow cytometry and fluorescence microscopy.Indirect immunofluorescence staining labeled LC3 protein.Western Blot was used to detect Beclin1,LC3 and P62 to evaluate the level of autophagy.Cell necrosis was tested by Hoechst33258/PI staining.DAPI and Annexin V-FITC/PI staining were used to investigate the cyteblasts morphology and the apoptotic level of cells respectively.The expression of Bcl-2,Bax were tested by Western Blot.Results 1.The viability of SH-SY5 Y cells under ischemic conditions decreased with the prolongation of OGD time and the leaked LDH increased.The cortex of microfilaments became thinner and the stress fibers decreased in OGD 6 hours group than in control group,even agglutinated at 24 hours after OGD.The number of mitochondria reduced with the decrease of tubular mitochondria and augment of punctate mitochondria.Both the results of flow cytometry and fluorescence microscopy showed the level of ROS increased gradually with the prolongation of OGD time.After cells treated with OGD,expression of Beclin1 and LC3-II/LC3-I increased relatively while the P62 decreased.Immunofluorescence staining also showed that bright spots of LC3-II increased,level of autophagy increased and autophagy flux was smooth after OGD.Compared with the control group,the cells after OGD showed bright blue and red staining,indicating the increase of necrotic cells.As for cells apoptosis,the chromatin agglutination appeared in the OGD6 h group,the morphological changes after 24 hours OGD were more obvious after DAPI staining.All detections showed that the prolongation of OGD time gradually increased the apoptosis.2.The results of Beclin1-siRNA transfection test showed that the level of Beclin1 gene and protein after Beclin1-siRNA interference decreased significantly and the interference was successful.3.By pretreatment of RNAi,the expression of LC3-II/LC3-I decreased,P62 increased and autophagy downregulated in ischemic conditions,but the autophagy level after treated with RNAi and OGD was still higher than that in control.Compared with the group of OGD,the cell viability,the number of mitochondria and stress fibers decreased and microfilament cortex became thinner after treated by RNAi and OGD.Meanwhile the expression of Bax and Bcl-2 changed.The levels of necrosis and apoptosis further increased.Conclusions Ischemia-hypoxia impairs SH-SY5 Y cells.Beclin1-dependent autophagy contributes to protect effects from ischemia-hypoxia in SH-SY5 Y cells.