| ObjectiveTo observe the effects of homocysteine(Hcy) on apoptosis of neural cells and proliferation, differentiation of endogenous neural stem cells(NSCs) in the ischemic brain by establishing rat model of middle cerebral artery occlusion(MCAO). To investigate the possible mechanism of DNA methylation during the changes in the proliferation and differentiation of endogenous NSCs caused by Hcy and Azacitidine(Azac, a DNA methylation blocking agent) intervention.MethodsOne hundred and twenty adult male SD rats were randomly divided into sham operation group(SO group), middle cerebral artery occlusion group(MCAO group), Hcy+middle cerebral artery occlusion group(Hcy+MCAO group), Azacitidine+ middle cerebral artery occlusion group(Azac+MCAO group) and Hcy+ Azacitidine +middle cerebral artery occlusion group(Hcy+Azac+MCAO group). There were twenty-four rats in each group. The rats in SO and MCAO group were intraperitoneally injected with saline at 5 m L/(kg·d). The rats in Hcy+MCAO and Azac+MCAO group were intraperitoneally injected with 2% Hcy and 0.05% Azac solution at 5 m L/(kg·d) respectively. 2% Hcy and 0.05% Azac mixed solution at 5 m L/(kg·d) was injected in Hcy+Azac+MCAO group. Middle cerebral artery occlusion was induced by intraluminal filament method after 7 d intervention. The brain infarct volume were measured by triphenyltetrazolium chloride(TTC)staining. Apoptosis rate of neural cells was tested by TUNEL assay. The HE stain was used for the histological analysis. The concentrations of Hcy, SAM and SAH in plasma of rats were examined by HPLC method. Immunofluorescence labeling was used to detect Hcy distribution in brain tissue. Methyflash TM methylated DNA Quantification kit was used to detect global DNA methylation levels. Active Motif's DNA Methyltransferase Activity/Inhibition Assay kit was used to measure DNMTs activity. Endogenous NSCs proliferation(7 d), differentiation(14 d) and the level of 5m C in DCX+ cells and Neu N+ cells were detected by double immunofluorescence techniques. ResultsAfter the intervention of Hcy, Azac, and establishment of rats MCAO model, the result of TTC stain indicated that percentage of the cerebral infarction volume of rats in Hcy+MCAO, Azac+MCAO and Hcy+Azac+MCAO significantly increased compared with MCAO group(P<0.05). The apoptosis rate of neural cells in Hcy+MCAO, Azac+MCAO and Hcy+Azac+MCAO group was higher than MCAO group(P<0.05). The total methylation level was significantly decreased in Azac+ MCAO, Hcy+Azac+MCAO and Hcy+MCAO groups versus MCAO group(P<0.05). The DNMTs activity in Hcy+MCAO, Azac+MCAO and Hcy+Azac+MCAO groups was significantly decreased, compared with MCAO group(P<0.05). Immunofluorescence results showed that Hcy positive cells can be observed in cortex and hippocampus of rats that in Hcy+MCAO and Hcy+Azac+MCAO group. HPLC test indicated that the serum Hcy and SAH concentration of rats in Hcy+MCAO, Hcy+Azac+MCAO group was significantly higher, and the SAM/SAH lower than that in other three groups(P<0.05). Immunofluorescence detection results showed that the number of DCX+/Brd U+, Neu N+/Brd U+, DCX+/5m C+ and Neu N+/5m C+ cells was significantly increased in MCAO group versus SO group(P<0.05), while DCX+/Brd U+, Neu N+/Brd U+, DCX+/5m C+, Neu N+/5m C+ cells in Hcy+MCAO, Azac+MCAO and Hcy+Azac+MCAO groups was decreased, compared with MCAO group(P<0.05). ConclusionsThe rat cerebral artery occlusion model was successfully established and imitated clinical pathology of cerebral ischemia. The results indicated that Hcy could induce the brain damage of rats with cerebral infarction, increase the apoptosis of neural cells. Hcy could also inhibit the proliferation, migration of endogenous NSCs, and the differentiation of NSCs into neurons after focal cerebral infarction. The possible mechanisms for the inhibitory effect of Hcy on the proliferation and differentiation of endogenous NSCs are as follows: Hcy could inhibit the activity of DNMTs, increase the concentration of SAH and decrease the ratio of SAM/SAH, DCX+ cells and Neu N+ cells. |
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