Effects Of Homocysteine On Autophagy Level Of Neural Stem Cells In Vitro And Cerebral Cortex In Focal Cerebral Artery Occlusion-reperfusion Rats

ObjectiveTo observe the effects of homocysteine(Hcy)on damage and apoptosis of neural cells in the ischemic brain by establishing rat model of middle cerebral artery occlusion-reperfusion(MCAO)and investigate the possible mechanism of damage of neural cells caused by Hcy through Hcy and 3-methyladenine(3MA,an autophagy blocking agent)intervention;To observe the effects of Hcy on proliferation,differentiation of neural stem cells(NSCs)in vitro and investigate the possible mechanism by Hcy intervention and gene expression microarray.The purpose of our study is to investigate the mechanisms of hyperhomocysteinemiae(HHcy)on damage of the ischemic brain and provide new ideas for the prevention and treatment of cerebral infarction.Methods1.Eighty adult male SD rats were randomly divided into sham operation group(SHAM group),MCAO group,MCAO+Hcy group and MCAO+Hcy+3MA group.There were twenty rats in each group.By tail vein the rats in MCAO+Hcy and MCAO+Hcy+3MA group were injected with 0.8 mg/mL Hcy at 4 m L/kg every other day.Rats were weighed once a week and intervention doses were adjusted according to the weight.Middle cerebral artery occlusion-reperfusion was induced by intraluminal filament method after 21 d Hcy intervention.The rats in MCAO+Hcy+3MA group were injected 3MA(5 mmol/L,4 mL/kg·d)by tail vein for five days before the MCAO surgery.The pathological changes of brain were measured by HE staining.Apoptosis rate of neural cells was tested by TUNEL assay.The concentrations of Hcy in plasma of rats were examined by a cycling enzymatic method.Cell morphology and the level of autophagy were detected by transmission electron microscopy.Western Blot(WB)was used to detect the expression of LC3 and Beclin-1,the markers of autophagy.Immunofluorescence labeling(IF)was used to detect the expression and distribution of LC3 and Beclin-1.2.NSCs were isolated from 24 h newborn SD rat brain and cultured in vitro.NSCs were randomly divided into Control group,Hcy intervention group(Hcy 150 μmol/L).WB was used to detect the expression of autophagy-relative proteins,such as Beclin-1,LC3,mTOR and p70S6 K.Cell morphology and level of autophagy were detected by transmission electron microscopy.Gene expression microarray [Agilent SurePrint G3 Rat GE(8*60K,Design ID:028279)] and WB were used to detect the gene expression of NSCs Results1.The result of HE staining indicated that the neuronal cells in the SHAM group were arranged regularly.After MCAO,most cells were arranged disorderly.The morphology changes in the MCAO+HCY group were more severe than in the MCAO group.Compared with the MCAO+HCY group,less cellular damage was observed in the MCAO+HCY+3MA group.Compared with the SHAM group,the apoptosis rate in the brain was significantly increased in the MCAO group(P<0.05).The apoptosis rate in MCAO+Hcy group was higher than MCAO group(P<0.05).Compared with the MCAO+HCY group,the apoptosis rate of neural cells was significantly decreased in the MCAO+HCY+3MA group(P<0.05).To show whether autophagy is involved in cell death in Hcy-treated MCAO rats,transmission electron microscope(TEM)was used to observe the formation of autophagosomes.In the MCAO group,some double membrane-bound compartments which contained cytoplasmic material(autophagosomes)were visible.Autophagosomes were frequently observed in the MCAO+HCY group than in the MCAO group.Western Blot results showed that ischemia injury resulted in a significant increase in LC3 B and Beclin-1 expression compared with the SHAM group(P<0.05).Compared to the MCAO group,the protein levels of LC3 B and Beclin-1 were increased in the MCAO+HCY group(P<0.05).The level of LC3B/Beclin-1 in MCAO+HCY+3MA group was significantly decreased,compared with the MCAO+HCY group(P<0.05).Immunofluorescent co-staining of LC3B/Beclin-1 and NeuN/GFAP showed that LC3B/Beclin-1 was mostly present in neurons of the ischemic penumbra of cortex and LC3B/Beclin-1 and GFAP were not expressed in the same cell.Immunofluorescence of 8-OHdG showed that number of 8-OHdG positive cells was significantly higher in the MCAO+HCY group,compared to the MCAO group.However,autophagy inhibitor 3-MA treatment reduced 8-OHdG expression.2.After the intervention of Hcy on NSCs,results of Western Blot showed that Hcy intervention resulted in a significant increase in LC3-II/I and Beclin-1 protein expression compared with the Control group(P<0.05).Transmission electron microscope results showed that neuronal damage was more pronounced and more autophagosomes were observed after Hcy intervention.Gene expression microarray and WB results showed that Hcy intervention down-regulated mTOR and p70S6 K which were the key proteins of mTOR pathway.ConclusionsThe rat cerebral artery occlusion-reperfusion model was successfully established and imitated clinical pathology of cerebral ischemia.The results indicated that Hcy could enhance autophagy and aggravate neuronal cell injury after MCAO with the generation of autophagosomes and the upregulation of LC3B/Beclin-1 protein expression in neurons.Conversely,the autophagic inhibitor 3MA could inhibit autophagy activation and ameliorate the ischemic injury caused by Hcy intervention.Immunofluorescence co-staining of LC3B/Beclin-1 and NeuN/GFAP indicated that their expression occurred mainly in neurons and hardly in astrocytes.8-OHdG expression was increased after Hcy treatment.The oxidative stress might serve as a possible link between the overactivation of autophagy and the elevated Hcy level after ischemic stroke.We successfully isolated and cultured NSCs from neonatal rats.Hcy could induce autophagy and increase NSCs damage.Gene expression microarray and WB results showed that Hcy intervention down-regulated genes of mTOR pathway.Therefore we made a conclusion that Hcy treatment enhanced the autophagy level of NSCs by mTOR pathway and enhanced the autophagy level of neuron by ROS pathway,which contributes to brain damage.