Protective Effects Of Histone Deacetylase Inhibitor (HDACi) On Brain Ischemia And Its Mechanism

Objective: To establish a mice model of focal cerebral ischemia/reperfusion (I/R) and a model ofoxygen-glucose deprivation (OGD) in vitro, and investigate the protective effects and theunderlying mechanism of histone deacetylase inhibitor (HDACi) on ischemic stroke, whereby toprovide experimental basis for clinical application of HDACi treatment of cerebral ischemicdiseases.Methods:1、Healthy male Balb/c mice were randomly divided into2groups: Sham operationgroup (Sham), I/R group. Injury models with30-minute ischemia and reperfusion of left middlecerebral artery were prepared in I/R group mice following Zea-Longa suture method. Vascularwas isolated and MCAO monofilament was not put into middle cerebral artery in Sham groupmice. I/R group was divided into three subgroups: I/R24h group、I/R48h group、I/R72h group.24h after reperfusion, the neurological deficit score were evaluated, and then mice weresacrificed to measure infarct volume by TTC staining.2、Healthy male Balb/c mice were randomly divided into4groups: Sham group, I/R group,SAHA group and DMSO group. The Zea-Longa suture method was used to block the middlecerebral artery for30min, and then did a reperfusion for48hours. SAHA group was divided intothree subgroups: SAHA12.5mg/Kg preconditioning group (SAHA1group), SAHA25mg/Kgpreconditioning group (SAHA2group), SAHA50mg/Kg preconditioning group (SAHA3group).SAHA and DMSO were injected through abdominal cavity1hour before cerebral ischemia.24hafter reperfusion, the neurological deficit score were evaluated, and then48h after reperfusion,mice were sacrificed to measure infarct volume by TTC staining, the levels of protein acetylationin brain tissues were detected by Western blot.3、SH-SY5Y cells were cultured with DMEM medium, OGD model was established andevaluated based on cell viability. Afterwards, the influences of HDACi treatment on the neuroninjury induced by OGD were analyzed, morphological changes were observed with Invertedmicroscope, cell viability was measured by methyl thiazoly tetrazolium (MTT) assay, cellapoptosis and necrosis were detected by PI and Hoechst staining, the cells injury was detected bythe lactate dehydrogenase (LDH) kit, the levels of intracellular reactive oxygen species (ROS)and potential of mitochondrial membrane (MMP) were evaluated by Fluorescence microscope or flow cytometry, the levels of acetylation were evaluated by Western blot.Results:1、Neurological deficit score showed that, compared with Sham group, there weredifferent levels of neurological defects in I/R groups, the neurological deficit score was（2.60±0.15）points, and Sham group had no neurological deficit. TTC staining showed that mice in I/Rgroup had clear infarction when compare with the lack of brain infarction in Sham control.2、Western blot showed that, compared with Sham group, acetylated protein levels droppedsignificantly in the left middle cerebral artery territory of I/R mice. By intraperitoneal injectionof SAHA can significantly increase the normal mice protein acetylation levels at6h (P<0.05),there was no difference after24h.25mg/Kg and50mg/Kg SAHA prevent ischemia-inducedholoprotein hypoacetylation. Neurological deficit score and TTC staining showed that:Compared with Sham group, there were different levels of neurological defects in SAHA groupand I/R group, the neurological deficit scores were between1and3points, and Sham group hadno neurological deficit. Compared with I/R group, neurological deficit score was no significantimproved in SAHA12.5mg/Kg group. Although infarct volume was decreased in SAHA12.5mg/Kg group (11.45%decrease compared with I/R group), but the difference was notstatistically significant. Neurological deficit score and infarct volume was significantly reducedin SAHA25mg/Kg and50mg/Kg groups, compared with I/R group, the infarct volume wasdecreased by42.92%and56.53%respectively.3、Oxygen-glucose deprivation for2hours significantly suppressed the cell viability，holoproteinacetylation and MMP of SH-SY5Y, the intracellular levels of ROS increased obviously.Moreover, the number of apoptotic and necrosis cells increased as the extending ofoxygen-glucose deprivation. Compared with OGD group, TSA or SAHA significantly improvedcellular viability, and they also ameliorated the neuronal morphological injury. In addition,SAHA pretreatment was shown to reduce the number of necrosis and apoptosis cells, prevent theOGD-induced holoprotein deacetylation, decrease cytotoxicity, lactate dehydrogenase (LDH)release in SH-SY5Y cells. Moreover, SAHA reversed the mitochondria dysfunction after OGD,as evidenced by decreasing mitochondria ROS levels, elevating the levels of mitochondrialmembrane potential.Conclusion:1、Zea-Longa suture method is a simple and reliable production of mice transientMCAO model approach, MCAO30min/reperfusion48h can cause stability infarction, suitable for study of brain ischemia-reperfusion injury.2、Intrapertioneal injection a dose of SAHA before1hour on focal cerebral ischemia-reperfusioninjury exerted significant brain protective effect, but the specific mechanism remains to befurther research.3、 HDAC inhibitor protect SH-SY5Y cells from OGD induced injury, the underlyingmechanisms may partially due to the effects of HDACi on an anti-oxidative stress, andmaintaining of mitochondria function.