Mechanism Of Protective Effect Of Histone Deacetylase Inhibitor SAHA On Cerebral Ischemia

PART ? The molecular mechanism underlying the neuroprotective effects of deacetylase inhibitors SAHA on ischemic injuriesAims:To investigate the protective effects and the underlying mechanism of histone deacetylase inhibitor(HDACis)Suberoylanilide hydroxamic acid(SAHA)on ischemic stroke.Methods:Healthy male BALB/c mice were randomly divided into four groups:Sham operation group(Sham),cerebral ischemia/reperfusion(I/R)group,SAHA group and vehicle group(DMSO).I/R models with 60minute ischemia and reperfusion using Zea-Longa suture method.SAHA group was divided into three subgroups:tail vein injection of SAHA10O?l immediately after reperfusion(SAHA1 with 12.5mg/Kg,SAHA2 with 25mg/Kg and SAHA3 with 50mg/Kg).DMSO group:with tail vein injection of 25%DMSO100?l immediately after reperfusion.24h after reperfusion,the neurological deficit score were evaluated,and then mice were sacrificed.The infarct volume were measured by TTC staining,the levels of acetylation in brain tissue were detected by western blot.Results:24h after reperfusion,there were some neurological deficits in SAHA group and I/R group,the scores of neurological deficit were form 1 to 3,and Sham group had no neurological deficit.Compared with DMSO group,neurological deficit score and infarct volume was significantly reduced in SAHA25mg/Kg and 50mg/Kg groups,compared with I/R group(P<0.01.The expression of acetylated protein declined significantly in the left side of I/R mice.SAHA can upregulate the protein acetylation level in normal mouse.Treated with SAHA(25mg/Kg and 50mg/Kg)after reperfusion robustly increase the acetylate level of protein(P<0.05).SAHA prevented against ischemia-induced protein hypoacetylation.Conclusion:Treated with SAHA immediately after cerebral ischemia/reperfusion,neurological deficit score and infarct volume was significantly reduced.In addition,SAHA attenuated the decline of acetylation level as well.These indicates that SAHA SAHA can upregulate acetylation expression and protect the brain from ischemia reperfusion injury.PART ? The quantitative proteomic study on the protective effects of SAHA on the cerebral damage induced by transient focal cerebral ischemia in miceAims:According to our previous study of the protective effects of SAHA on the brain damage induced by transient focal cerebral ischemia in mice,an quantitative proteomic strategy based on isobaric tag for relative and absolute quantitation(iTRAQ)labeling was adopted to detect the differentially expressed proteins and investigate the molecular mechanism underlying its protection of I/R injury.Methods:A transient occlusion of middle cerebral artery(tMCAO)model was established in BALB/c by using suture method.iTRAQ labeling coupled with online two dimensional LC/MS/MS technology was adopted to detect the differentially expressed proteins;the biological function of those differentially expressed proteins are categorized by Gene ontology analysis and IPA(Ingenuity Pathway Analysis)and some of them are further validated by Western blot or ICC.Results:There are four groups in the present study,Sham,Sham with SAHA treatment,MCAO and MCAO with SHAH treatment.From two independent biological experiments,1579 proteins were detected with FDR less than 1%.However,to obtain a more reliable result,a more stringent criteria was adopted,only proteins with two or more peptides were used for subsequent data analysis,whereby 1117 proteins were included,and among which 1113 proteins with quantitative information.Compared the MCAO group with Sham group,the expression of 68 proteins was changed significantly(1.5 fold),33 were up regulated and 35 were down regulated.The up-regulated proteins included the components of blood(HEMO,HPT and FIBG etc.),proteins specifically expressed in astrocyte and oligodendrocytes(GFAP,EAA2 and MAG etc.).Cytoskeleton(Tau),proteins involved in protein synthesis and post-transcriptional modification(EF1A1,ALG2),and transportation(CAMKV,S12A5,VPS52)are down regulated.The most important finding in the present study is SAHA treatment partially diverts the changing tendency of those proteins caused by ischemic injury.In the 33 up-regulated and 33 down-regulated,expression of 14 and 19 are suppressed and raised respectively after SAHA treatment,and 4 of them(GFAP,EAA2,Tau and GSK3?)were validated by WB.Conclusion:For the first time,our study demonstrated the multiple protective effects of HDACi on the I/R injury from a proteomic level,including BBB permeability,reduce glia activation,stabilize tissue and cellular structure and improve energy metabolism.These data not only confirm previous studies,more importantly,SAHA treatment could partially divert the changing tendency of proteins caused by I/R injury.This may be the reason for the multiple protective effects of SAHA.Beside,astrocytes also involve in the cerebral protection of SAHA.