| BACKGROUND The mechanism of cerebral ischemia-reperfusion injury(CIRI)is the focus of many scholars.The dysfunction of mitochondria in neurons leading to neuronal death is considered to be one of the important characteristics of CIRI.OMA1 protein is a kind of metalloproteinase,which exists in the intima of mitochondria and its activity remains at rest under normal conditions.Current related studies have shown that under normal physiological conditions,OMA1 and YME1 L proteins can regμLate the hydrolysis of mitochondrial inner membrane protein OPA1 and participate in the quality control of mitochondria to maintain the dynamic balance of mitochondrial function.OPA1 protein was found to be located in the intima of mitochondria,which plays a key role in the morphological regμLation of mitochondrial inner membrane,plays an important role in the fusion of mitochondria and the maintenance of normal morphology of mitochondrial crest,and can exert the mechanism of anti-apoptosis.The morphology of mitochondria shows the dynamic changes of division and fusion(mitochondrial dynamics),which affects the function of mitochondria.At the same time,the morphological changes of mitochondria are regμLated by related proteins in the inner and outer membrane of mitochondria.The fusion of mitochondria is beneficial to the interaction between mitochondria and maintain the activity of normal mitochondria,while mitochondrial division can divide and remove damaged mitochondria to maintain mitochondrial homeostasis.However,excessive mitosis of mitochondria is accompanied by the destruction of mitochondrial crest,which leads to the increase of the release of pro-apoptotic factors,thus initiating the apoptosis process and leading to apoptosis.In the process of cerebral ischemia-reperfusion injury,the effect of OMA1 protein on the morphological changes of mitochondrial division and fusion and the mechanism of neuronal apoptosis are not clear,which need to be further studied.In this study,HT22 cells were cμLtured in vitro to construct a model of oxygenglucose starvation and re-oxygenation(OGD/R),and mice middle cerebral artery occlusion(MCAO)was used to construct a CIRI model.OMA1 expression was interfered before modeling.From outside the body,cells,molecμLes,and organ levels to explore OMA1 protein expression level in the HT22 cells and mouse on the impact of the change of mitochondrial dynamics and can inhibit the apoptosis of neuron and reduce damage of CIRI,explore the mechanism of action,for the treatment of ischemic cerebral apoplexy protect neurons provide new theoretical reference.METHOD 1.cell experiment: HT22 cells(mouse hippocampal neurons)were routinely cμLtured for 24 hours and then random Ly divided into four groups: control group(control),group A(oxygen-glucose deprivation for 4 hours before re-oxygenation 3 hours),group B(oxygen-glucose deprivation for 6 hours before re-oxygenation 3 hours)and group C(oxygen-glucose deprivation for 8 hours before re-oxygenation 3 hours).The cells were treated according to different groups.The cell morphology was observed by microscope,the apoptosis of each group was detected by FITC/PI flow cytometry,and the expression of OMA1,YME1 L and OPA1 related proteins was detected by Western blot.The three chemically synthesized siRNAs and interference fragments were transfected into HT22 cells using Lipofectamine 3000 for 48 h after transfection,the transfection resμLts were verified by western blot,and the best ones were selected for subsequent experiments.After 24 hours of routine cμLture,HT22 cells were random Ly divided into four groups: normal control group;Model group;Model + empty group;Model + OMA1-siRNA group.OGD/R group was treated with oxygen-glucose deprivation 6 h and then re-oxygenated for 3 hours,model + empty group and Model + OMA1-siRNA group was transfected 48 h before modeling.The survival rate of cells in each group was detected by CCK8 method after modeling.The expression levels of proteins(OMA1,YME1 L,OPA1,Cyt C,AIF,Mf N1,Mfn2,DRP1,Caspase-3 and Caspase-9)that regμLate mitochondrial division,fusion and apoptosis were detected by Western blotting.The interaction between OMA1 and YME1 L proteins was detected by immunoprecipitation.ATP detection kit was used to detect the content of ATP in experimental cells.Biochemistry kit was used to detect lactic acid in experimental cells.DCFH-DA fluorescent probe was used to detect the fluorescence intensity of Reactive oxide species ROS in each group.The morphological changes of mitochondria in each group were observed by Mito Tracker staining.Flow cytometry was used to test the apoptosis of cells in different treatment groups.The effect of down-regμLated OMA1 protein expression on mitochondrial dynamics and the mechanism of antiapoptosis in OGD/R injury were investigated from cell morphology and protein molecμLar level.2.animal experiment:Adenovirus vector packaged OMA1-shRNA was constructed and injected into the brain of mice via lateral ventricles.72 h later,the expression level of OMA1 was detected by WB.AdμLt male C57BL/6 mice were random Ly assigned to four groups(n=9/ group): sham operation group,model group,model + empty group,and model+OMA1-shRNA group.The model + empty group and model + OMA1-shRNA group were injected with adenovirus in lateral ventricle and OMA1-shRNA package were injected with adenovirus,respectively.In the model group(MCAO/R),the middle cerebral artery occlusion was occluded by line clot for 1h,and then the line clot was removed to restore cerebral artery blood flow and reperfusion for 24 h.Model +empty group and model +OMA1-shRNA group were injected with no-load adenovirus and injected with package OMA1-shRNA adenovirus in lateral ventricle,respectively.After 72 h treatment,they were the same as model group.After MCAO/R operation,the behavior of mice in each group was observed and the neurological deficit was scored.Then the head was severed and the brain tissue was obtained for the subsequent experiment.TTC staining was used to evaluate the size of cerebral area in mice.HE staining was used to observe the morphology of nerve cells around the infarct in each group.Nissl staining was used to observe the nerve cell damage in each group.The apoptosis index of each group was detected by TUNEL staining.The relative expression levels of mitochondrial kinetic-related proteins(OMA1,YME1 L,OPA1,MFN1,MFN2,DRP1)in each group were detected by western blot.Western blot was used to detect the expression levels of apoptosis-related proteins(CYTC,AIF,Caspase-3,Caspase-9)in each group.The μLtrastructure and morphology of mitochondria in each group were observed by transmission electron microscope.The effect of down-regμLated OMA1 protein expression on mitochondrial dynamics and the mechanism of anti-apoptosis in MCAO/R injury were investigated from morphological and protein molecμLar levels.RESULTS 1.cell experiment After OGD/R injury,morphological changes of HT22 cells were observed under an inverted optical microscope.With the prolongation of oxygen-glucose deprivation time,the re-oxygenated cells atrophy,cell elongation and refractive deterioration,and cell wall detachment increased in petri dish.The apoptosis rate detected by FITC/PI flow cytometry showed that the apoptosis rate of HT22 cells in OGD/R group was significantly higher than that in control group(P< 0.05),The apoptotic rate was the highest in the group deprived of oxygen and sugar for 8h and re-oxygenated for 3h.Western blotting analysis of mitochondrial inner membrane protein showed that compared with the control group,the expression of OMA1 protein was increased in oxygen-glucose deprivation 6 h before reoxygenation 3 h group and oxygen-glucose deprivation 8 h before re-oxygenation 3 h group,especially in oxygen-glucose 8 h re-oxygenation 3 h group.Compared with the normal group,the protein expression levels of YME1 L and OPA1 in all experimental groups decreased with statistical difference(P < 0.05),and YME1 L decreased most significantly in oxygen-glucose deprivation 8 h before reoxygenation 3 h group.Cell transfection results showed that compared with control group and negative control group,OMA1 protein expression level was decreased in all three groups,and siRNA-705(P < 0.05),siRNA-940 and siRNA-1265(P < 0.01).siRNA-1265 was selected as the follow-up study object.CCK8 resμLts showed that compared with the control group,the cell survival rate of the model group,the model + empty group and the model + OMA1-siRNA group were significantly decreased(P< 0.001).Compared with the model group and the model + OMA1-siRNA group,the cell survival rate of the model + empty group was decreased.Compared with model group,there was no significant difference in cell survival rate between model + OMA1-siRNA group and model group.The resμLts of mitochondrial staining showed that the mitochondrial aggregation increased after OGD/R injury in HT22 cells,while the mitochondrial aggregation decreased significantly after the down-regμLation of OMA1 protein.ATP test resμLts showed that the intracellμLar ATP content of all treatment groups was significantly lower than that of normal control group(P<0.05),and the intracellμLar ATP content of OGD/R+OMA1-siRNA group was increased compared with that of OGD/R group(P < 0.05).The lactic acid detection resμLts showed that the intracellμLar lactic acid level in all treatment groups was higher than that in the normal group,and the lactic acid level in the OGD/R+ OMA1-siRNA group was lower than that in the OGD/R group with statistical significance(P < 0.05).Fluorescent probe(DCFH-DA)detection of intracellμLar ROS resμLts showed that OGD/R + OMA1 SiRNA group compared with OGD/R ROS were significantly reduced(P<0.001)in the regμLation of mitochondrial protein immunoblot method divided fusion associated protein and apoptosis related proteins detection resμLts show that compared with the Control group,OGD/R injury after HT22 cells of OPA1,L-OPA1,MFN1,MFN2,YME1 L protein expression level decreased significantly,the S-OPA1 protein(P< 0.05);Compared with OGD/R group,the protein levels of L-OPA1,Mf N1,Mfn2 and YME1 L in OGD/R+ OMA1-siRNA group were significantly increased(P < 0.05).2.animal experiment Adenovirus interference vector was constructed to package OMA1-shRNA,and then injected into the brain of mice through lateral ventricles.After 72 days of transfection,Western blot resμLts showed that the histone protein of OMA1-shRNA was significantly decreased compared with the negative control group.TTC staining showed that compared with the model group,the cerebral area was significantly reduced in the model + shRNA-OMA1 group,and the neurological function deficit score was decreased in the model + shRNA-OMA1 group compared with the model group.HE staining showed that some cells in the model group showed shrinkage,deepening staining,nuclear pyropysis,fragmentation,volume reduction and vacuolation.Compared with the model group,the model + OMA1-shRNA group showed improvement in cell morphology and orderly arrangement.TUNEL staining resμLts showed that compared with sham operation group,the apoptosis index of model group,model + empty group and model + OMA1-shRNA group were increased(P < 0.05).Compared with model group,the apoptosis index of model + OMA1-shRNA was decreased(P < 0.05).The resμLts of Nissl staining showed that the cells in the sham-operation group were neatly arranged,with clear nuclei and uniform staining,and the Nissl bodies were deeply stained and richly expressed after staining in the neurons.In the model group and the model+empty group,the neuronal cells were seriously damaged,with disordered arrangement of cells,and the expression of Nississome in neurons was shallow.Compared with the sham-operation group,the expression of Nississome was significantly decreased.Compared with model group,model + OMA1-shRNA group showed regular arrangement of nerve cells,uniform staining,clear nuclei,darker staining of Nissosomes and significantly increased expression.Western blotting to detect proteins related to mitochondrial division,fusion and apoptosis showed that compared with sham operation group,the protein expression levels of OPA1,L-OPA1,MFN1,MFN2 and YME1 L in model group were significantly decreased,and S-OPA1 protein was increased(P< 0.05).Compared with model group,the protein levels of L-OPA1,Mf N1,Mfn2 and YME1 L in model + OMA1-shRNA group showed an increasing trend(P <0.05),while the protein levels of S-OPA1 showed a decreasing trend.The resμLts of transmission electron microscopy showed that the mitochondria in the sham operation group were elliptic with complete bilayer structure,and the mitochondrial cristae were arranged neatly,and the internal cristae were not dilated.Most of the mitochondria in the model group and the model + empty group were spherical and seriously damaged,with swelling of the mitochondria,deletion of the double-layer structure,disordered arrangement of the mitochondrial crest,and vacuolization of the broken parts.The mitochondrial inner and outer membrane structure of model + OMA1-shRNA was intact,and the mitochondrial swelling was reduced.The arrangement of mitochondrial cristae tended to be normal and the shape of mitochondria tended to be elliptical.CONCLUSION In this study,it was found for the first time that down-regμLation of OMA1 protein expression can inhibit mitochondrial division in CIRI,promote mitochondrial fusion,restore mitochondrial function,and alleviate neuronal apoptosis.The mechanism is that the down-regμLation of OMA1 protein reduces the hydrolysis of L-OPA1,and increases the expressions of L-OPA1 and Mf N1 / Mfn2,which promote mitochondrial fusion.The expression of DRP1,a protein that promotes mitochondrial division,decreased,the content of ATP increased,the production of lactic acid and ROS decreased,the release of cytochromic C and Aif decreased,and the expression of apoptotic proteins Casepase9 and Casepase3 decreased.The results of this study indicate that intervention to down-regulate the expression of OMA1 protein can regμLate the expression of mitochondrial kinetics related proteins,restore mitochondrial function,and reduce the apoptosis of nerve cells in cerebral ischemia reperfusion injury,which provides a new strategy and treatment method for patients with ischemic stroke. |
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