| High incidence of cerebrovascular disease seriously harm human health.Stroke, the most common life-threatening disease of the nervous system,is currently the third largest cause of death and the first cause of disability.Cerebral ischemic/hypoxic injury-induced neuronal death is the main reason leading to loss of neurological function.The mechanism of ischemic brain damage has not been clarified and there is a lack of clinically effective therapies.Therefore,the works of studying neuronal death mechanisms and exploring new therapeutic targets have important clinical significance.Evidences showed that many mechanisms were involved in ischemic neuronal death,including necrosis and apoptosis.Necrosis is a kind of non-programmed,rapid cell death,while apoptosis is a form of high-controled programmed cell death.Classic apoptosis is characteristic as caspase activation.There exists another kind of cell death distinguished form classic apoptosis after stroke.This kind of cell death is also programmed,but not dependent on caspase activation.We called it caspase-indenpendt apoptosis and the detailed mechanism is not clear.Many members of Bcl-2 family are involved in the process of cell injury.BNIP3 (Bcl-2/adenovirus E1B-19 KD interacting protein 3)is a pro-apoptotic Bcl-2 protein which is most sensitive to hypoxia.Unlike other family members,BH3 domain is not necessary for cell death and dimerization.Further studies showed that BNIP3-induced cell death is characterized by early increase of plasma membrane permeability and mitochondrial damage,but not accompanied by cytochrome c release and caspase activation.Although the mechanism of caspase-dependent cell death has a relatively clear understanding,the molecular mechanism of caspase-independent apoptosis is not clear.Because of BNIP3's sensitivity to hypoxia and its characteristic of inducing caspase independent pathway,our group intended to explore its effect in stroke and its molecular mechanism.First of all,we used oxygen glucose deprivation(OGD)/reoxygenation(R)model to mimic stroke.12 d hippocampal neurons were divided into three groups:control group,without any treatment;EBSS group,replaced with Locke's solution Earl's balanced salt solution (EBSS)medium for 4 h;OGD/R group:glucose-free EBSS medium was purged with N2/CO2/O2(94%/5%/l%)for 30 min,resulting in an oxygen content of 2-3%,then washed three times with this medium and incubated for 4 h in an oxygen-free N2/CO2/O2(94%/5%/l%)atmosphere.Thereafter,the medium was replaced by standard culture medium for 24 h.DAPI dyeing showed that the pyknotic percentage of control group and EBSS group were 10.65±2.69%and 10.16±2.94%, respectively;the pyknotic rate of OGD/R group was 37.71±6.80%.MTT results showed that the cell activities of control group and EBSS group were 100.00±6.96% and 96.31±6.91%,while the OGD/R group was 46.94±7.02%.So OGD(4 h)/R(24 h)could induce cell damage,we next observed the expression of BNIP3 at 0 h,2 h,6 h,12 h,24 h after OGD,whole cell western blot result showed that:20 KD(possible for the precursor protein)increased to 2.36±0.33 fold compared to the control group at 0 h after OGD;30 KD(monomer form)and 60 KD(dimer form)rose to 3.50± 0.46 fold and 1.84±0.31 fold at 2 h after OGD.Because previous study have reported that BNIP3 exert its function by forming a dimer and integrating into the mitochondrial outer membrane.We further observed the change of mitochondrial BNIP3,results showed that:20 KD and 60 KD bands increased to 2.14±0.23 fold and 3.42±0.31 fold at 0 h after OGD,while the 30 KD band is less than the basic observation.In order to verify whether there exists some relationship between elevated BNIP3 and cell injury,we transfected hippocampal neurons with pEGFP-C3-rBNIP3 plasmid encoding the full length of rat BNIP3 and pEGFP-C3 as control.Result showed that pEGFP-C3-rBNIP3 plasmid could induce the pyknotic percentage increased from 22.86±6.82%to 62.46±9.62%.What's more,BNIP3 siRNA could reduce the cell death from 32.87±1.80%to 18.07±1.46%after OGD/R 12 h,could reduce the cell death from 37.25±2.15%to 16.02±1.21%after OGD/R 24 h.So,BNIP3 is involved in OGD/R induced cell death.However,the mechanism of BNIP3-mediated caspase-independent apoptosis is not clear.Studies have shown that endonuclease G(Endo G)and apoptosis-inducing factor(AIF),as mitochondrial dysfunction downstream molecules,were involved in caspase-independent cell death pathway.Endo G and AIF both are mitochondrial proteins.Endo G is a mitochondrial DNA enzyme which translocates to nucleus after releasing from mitochondrion and cuts chromatin DNA into nucleosome fragments in a caspase-independent manner.AIF is a mitochondrial flavoprotein which combines with Endo G after releasing from mitochondrion to carry DNA degradation.This is the only currently identified DNA degradation pathway of caspase-independent.So we presume that BNIP3 maybe have some relationship with Endo G and AIF during OGD/R.First,we observed the change of AIF and Endo G during OGD/R process. Western Blot showed that whole cell AIF and Endo G increased to 1.84±0.18 fold and 1.54±0.18 fold at 6 h after OGD.Previous studies proved that AIF and Endo G release from mitochondrion and translocate to nucleus after injury,we further observed the change of AIF and Endo G in mitochondrial and nuclear fractions. Results showed that mitochondrial AIF and Endo G reduced to 0.63±0.12 fold and 0.58±0.80 fold at 2 h after OGD and the corresponding nuclear AIF and Endo G increased to 1.60±0.11 fold and 3.62±0.41 fold.This suggested that AIF and Endo G translocate from mitochondrion to nucleus in OGD/R.To verify the relation between AIF and Endo G nuclear translocation and cell death,we further observed the percentage of AIF and Endo G nuclear translocation and cell death at 0 h,2 h,6 h, 12 h,24 h after OGD.Nucleus shrinkage rate increased significantly at 12 h after OGD,while AIF and Endo G nuclear translocation rate showed a significant difference at 2 h after OGD,which preceded the nuclear pyknosis,suggesting that AIF and Endo G nuclear translocation occurred before cell damage,which may be a causative agent for cell death but not a accompanying phenomenon.Secondly,in order to verify the relationship of BNIP3 upregualtion and nuclear AIF/Endo G translocation,we observed the effect of exogenous BNIP3 on AIF and Endo G nuclear translocation.pEGFP-C3-rBNIP3 plasmid could cause the percentage of nuclear AIF increase from 30.44±5.57%to 70.12±7.51%and nuclear Endo G from 25.92±3.99%to 60.01±8.51%.So,exogenous BNIP3 could induce AIF and Endo G nuclear translocation.At the same time,BNIP3 siRNA could reduce the rate of AIF nuclear transfection from 24.58±3.39%to 12.5±3.66%after OGD/R 2 h, could reduce the rate from 51.67±5.60%to 29.29±6.74%after OGD/R 6 h, suggesting that BNIP3-siRNA could delay the AIF nuclear transfection.Once again,we used AIF mutant C57 mouse(Hq)cells to prove above mentioned results again.This kind of mouse showed an 80%reduction of AIF expression.pEGFP-C3-rBNIP3 plasmid could induce shrinkage rate increased from 22.13±5.32%to 58.86±6.50%in wild-type C57 hippocampal neurons,but only caused shrinkage rate increased from 23.25±4.95%increased to 35.38±6.19%in Hq hippocampal neurons.Furthermore,AIF gene mutation does not affect BNIP3 expression after OGD/R.Nuclear staining and MTT results showed that AIF gene mutation can block OGD/R-induced pyknosis and cell activity change.So,AIF is a very important downstream regulatory factor in OGD/R.To sum up,OGD/R could induce BNIP3 upregulation and AIF and Endo G nuclear translocation,there may be exist some close relation between these three proteins.This research could provide a molecular theory for preventing the neuronal damage after stroke.
|
PDF Full Text Request