| BackgroundHypoxic-ischemic brain damage(HIBD)represents a major reason of acute death and chronic neurological damages in neonates.There are several severe neurological sequelae associated with HIBD,such as cerebral palsy,seizures,cognitive impairment,etc,making drastic effects on the children,families and society.Clinically,the mortality caused by HIBD has significantly reduced,but the occurrence of disability exhibits increasing trend.Therefore,in order to reduce the disability in children and improve the population quality,it is pivotal to explore novel and effective therapeutic strategies for HIBD.Until now,specific treatments for HIBD are limited to alleviating symptoms,and the neurological sequela rate remains high.Thus,it is necessary to investigate the mechanisms underlying neuronal damage/protection after HIBD which may provide a new insight into the treatments of HIBD.Hypoxia inducible factor-1 alpha(HIF-la)is an important transcriptional regulator of cellular response to hypoxia.Under normal physiological condition,HIF-1? is degraded via Ubiquitin-protease pathway.But when the cells in hypoxia,the degradation of HIF-1? is inhibited,and the accumulated HIF-1? may play critical roles in maintaining the stable of cell cycle and energy metabolism through regulating its targeted genes expression.Autophagy is a specific life phenomenon in eukaryotic cells,and in nature autophagy is an essential cellular process in which cytoplasmic components are degraded by lysosomes.The main function of autophagy is to maintain normal metabolic balance and cellular environment homeostasis.Under starvation,autophagy is responsible for providing amino acids and small molecules to support cell survival by degradation of cytoplasmic material.Recently,growing studies have demonstrated that in addition to maintain the normal cellular metabolism,authophagy also takes part in disease process,like cancer.Autophagy is highly conserved in evoluation from yeast to mammals.Beclin-1 is a homologous gene of yeast autophagy related genes in mammals which is a direct executor of autophagy.Beclin-1 is involved in formation of autophagosome,and its high expression represented the activation of autophagy.Microtubule-associated protein 1 light chain 3(LC3)is located at the cytomembrane surface of autophagic vacuole,and its expression levels are positively correlated with numbers of autophagic vacuoles.Therefore,the expression of Beclin-1 and LC3 protein can be applied to evaluate the degree of cell autophagy.Recent investigations have reported that the autophagy induced by hypoxia may be attributed to the up-regulation of HIF-1 a.In hypoxia,the elevated expression of HIF-1 a can enhance the transcription and protein expression of BCL-2/interacting protein3(BNIP3),and the over-expression f BNIP3 may lead to elevated level of Beclin 1 through competitive binding bcl-2.The dissociative Beclin 1 can activate autophagy.Therefore,over-expression HIF-1 a can drive autophagy via enhancing the expression of its targeted gene BNIP3.Currently,the association of HIF-1 a with autophagy was widely researched in tumor cells,but its function in neurons after HIBD remained unclear.The purpose of the study was to investigate the functionof autophagy in HIBD,as well as the underlying regulation mechanisms.The current study may provide a theoretical basis for the prevention and treatment of HIBD.Objectives1.To investigative the function of autophagy in cortical neurons oxygen glucose deprivation model,as well as its effects on cell survival.2.To detect the expression pattern of HIF-1? in neurons in hypoxia.In addition,we investigated the association between HIF-1? expression and autophagy.3.To verify whether HIF-la regulated autophagy via HIF-1?/BNIP3/Beclinl pathway in cortical neurons after oxygen glucose deprivation.4.To verify the function and mechanisms of autophagy in HIBD via animal models that might provide a novel approach for clinical treatments.Methods1.The cortical neurons were isolated from neonatal SD rats within 24h.DMEM was selected as culture medium,and the neurons were cultured in 37?,5%C02(volume fraction)and saturated humidity incubator.Then neuron specific enolase(NSE)was used for identification of cortical neurons.2.Cell hypoxia model was performed for the logarithmic growth phase of neurons through oxygen glucose deprivation(OGD)method.The culture medium was replaced by a sugar free DMEM medium.The glucose-free median was filled with oxygen-free mixture for 30 min in advance.Then the cells were cultured for 1.5h in a saturated humidity incubator under 37?.PI and MTT assays were performed to detect neurons survival at Oh,6h,12h,24h and 48h.The expression profiles of HIF-1?,BNIP3,Beclin 1 and LC3 proteins were estimated by western blot method.We also investigated the neurons survival,the numbers of autophagic vacuole(MDC fluorescence staining assay),neurons autophagy(laser scanning confocal microscope),and the challenges of cell structure relating to autophagy(transmission electron microscope)after suppressing the expression of HIF-la via adding its inhibitor 2ME2 and siRNA technology.3.The cultured neurons was treated with 10 mmol/1 2ME2 for 30min.Then the cells were used for construction of OGD model.12h after,western blot was applied to evaluate the expression of BNIP3 and Beclin 1.In addition,BNIP3 siRNA vectors were transfected to the neurons cells before construction of OGD.12h later,western blot was performed to investigate the expression pattern of HIF-1? and LC3.4.A total of 60 7-day-old SD rats were randomly divided into Sham group and HIBD group,30 in each group.The rats were killed on 6h,12h,24h,48h,and 72h after model establishment.QRT-PCR and western blot were used to investigate the mRNA and protein levels of HIF-la,BNIP3 and Beclin 1,separately.5.54 SD rats with 7 days were randomly grouped into Sham,HIBD,and HIBD+3MA groups.At 24h after HIBD modeling,the rats were killed and their brain were detected by immunofluorescence,western blot,encephaledema and apoptosis assays.Results1.At the beginning of in vitro culture,the primary neurons cells displayed adhering to the wall,circular or oval shape,small size,and strong sense of three dimensional.After cultured for 2 days,almost all the cells had a processes,and their volumes began to increase.The cells were circular or oval shape,with obvious halo.On the third day of culture,the cells continued to increase,and the processes extended.The cell volumes of the neurons significantly increased after culture for 5days.The cells showed round\oval or cone shape,and bipolar and three pole type were the prevalent type,occasionally the unipolar type.2.In OGD models,neurons survival increased,and their survival decreased with time.Moreover,the expression of HIF-1? exhibited increasing trend.Inhibition of HIF-1? expression would reduce neurons apoptosis and enhance survival.3.The expression of LC3 was increased with time in OGD models.At 12h in OGD models,a large number of green spots were observed under confocal laser scanning microscopy,plenty of double vesicles were found by transmission electron microscopy,and the positive cells labeled by MDC was obviously increased.4.3-MA,the inhibitor of autophagy,could protect neurons survival in OGD models,while the promote of autophagy,Rapa,led to decreased cell survival.5.In OGD models,knockdown the expression of Beclin 1 might enhance the survival and reduce apoptosis of neurons.6.In HIBD rats models,the activation of autophagy in neuronal cells in ischemic area suggested high death rate of.neuronal cells and brain edema.The application of 3-MA,an inhibitor of autophagy,could improve neurons autophagy and apoptosis,as well as brain edema.Conclusions1.Hypoxia-ischemia may induce abnormal activation of autophagy in neurons,thus leading to cell apoptosis and brain damage.2.In OGD model,the up-regulation of HIF-1? may contribute to activation of autophagy via HIF-1?/BNIP3/Beclin 1 signal pathway,thus promoting cell apoptosis and enhance neurological impairment.3.Suppression of autophagy can improve the survival of neurons that may be a potential therapeutic target for HIBD in clinic. |
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