| ObjectiveBased on the oxygen-glucose deprivation(OGD)model of primary cultured fetal rat hippocampal neurons,we use the agonists Resveratrol(RSV)and inhibitor EX527 of silent mating type information regulation 2 homolog-1(Sirtl),to observe changes of it in mRNA and protein levels,the apoptosis of cells and changes in oxidative stress indicators after OGD,and changes in Peroxisome proliferator activator receptor gamma coactivator 1α(PGC-1α),in order to explore the regulatory effect of Sirtl on oxidative stress of hippocampal neurons after OGD and the possible regulatory pathway.MethodsIn this project,primary cultured fetal rat hippocampal neurons were used as experimental subjects.The purity of the cultured neurons was identified by immunofluorescence technology labeled Neuronal Nuclei(NeuN).Based on the OGD model,cells were treated with RSV and EX527,to dectet the expression levels of Sirtl in mRNA and protein level,and to measure cell death,cell viability,SOD and MDA contents and the expression of PGC-1α in different groups.The data of different groups were compared by independent sample T-test,and the data were expressed by mean ± SD.Inspection level was α=0.05,and P<0.05 for the difference is statistically significant.Results1.Culture and identification of primary hippocampal neurons and establishment of oxygen and glucose deprivation modelsThe cultured neuron cells grew well.After seven days of inoculation,dendrites and axons were intertwined into a network,and the neurons matured.Immunofluorescence technology showed that the purity of neurons was>95%,and subsequent experiments can be performed.12 hours after the model we observed cell shrinkage,nuclear fragmentation,and synaptic rupture.The cell viability of control group and OGD12h group measured by MTT was 100.000 ± 4.526%and 63.247 ±1.930%,respectively.The cell viability of OGD group was significantly lower than that of control group(P<0.01,n=3),so the model was made successfully.2.Changes in mRNA and protein expression levels of Sirtl after OGDCompared to control group,the relative expression of Sirtl in OGD group reduced in both mRNA and protein levels(P<0.01,n=3);(P<0.05,n=3).3.Effects of EX527 and RSV on Sirtl expression in hippocampal neurons after OGD1)EX527 treatment:Compared to OGD group,the relative expression of Sirtl in OGD+EX527 group reduced in both mRNA and protein levels(P<0.05,n=3);(P<0.05,n=3).Compared to OGD+EX527 group,the relative expression of Sirtl in OGD+EX527+RSV group increased in both mRNA and protein levels(P<0.05,n=3);(P<0.05,n=3).2)RSV treatment:Compared to OGD group,the relative expression of Sirtl in the OGD+RSV group increased in both mRNA and protein levels(P<0.01,n=3);(P<0.05,n=3).4.Effects of EX527 and RSV on hippocampal neural cell viability,apoptotic rate,and MDA and SOD content after OGD1)EX527 treatment:Compared to OGD group,the cell viability of OGD+EX527 group decreased significantly(P<0.01,n=6),the SOD content per unit protein also decreased(P<0.01,n=6),and the MDA content per unit proteinincreased(P<0.01,n=6),the apoptosis rate alsoincreased(P<0.01,n=6),but the expression of Caspase3 did not increase significantly(P>0.05,n=3).Compared with OGD+EX527 group,the cell viability of OGD+EX527+RSV group increased(P<0.01,n=6),the SOD content per unit protein also increased(P<0.01,n=6),and the MDA content per unit protein decreased.(P<0.01,n=6),the apoptosis rate also decreased(P<0.01,n=6),and the expression of Caspase3 decreased(P<0.01,n=3).2)RSV treatment:Compared to OGD group,the cell viability of OGD+RSV group increased(P<0.01,n=6),the SOD content per unit protein also increased(P<0.01,n=6),and the MDA content per unit protein decreased(P<0.01,n=6),the apoptosis rate decreased(P<0.01,n=6),and the expression of Caspase3 also decreased(P<0.01,n=3).3)RSV pretreatment:Compared to OGD group,the cell viability of OGD+RSV pretreated group increased(P<0.01,n=6),the apoptosis rate decreased(P<0.01,n=6),but the expression of Caspase3 did not decrease significantly(P>0.05,n=3).5.Regulation of Sirtl on PGC-1a1)EX527 treatment:Compared to OGD group,the relative expression of PGC-1a in OGD+EX527 group were reduced in both mRNA and protein levels(P<0.05,n=3);(P<0.05,n=3).Compared to OGD+EX527 group,the relative expression of PGC-1a in OGD+EX527+RSV group increased in mRNA level(P<0.05,n=3),and the relative expression did not increase significantly in protein level(P>0.05,n=3).2)RSV treatment:Compared to OGD group,the relative expression of PGC-1αin OGD+RSV group increased in both mRNA and protein levels(P<0.01,n=3);(P<0.01,n=3).3)Relationship between expression changes of Sirtl and PGC-1α after EX527 and RSV treatment:①EX527 treatment group:Compared to OGD group,the relative expressionof Sirtl and PGC-1α in OGD+EX527 group decreased in mRNA and protein levels.Compared to OGD+EX527 group,the relative expression levels of Sirtl and PGC-1α in OGD+EX527+RSV group were both increased in mRNA level;the relative expression level of Sirtl in protein level increased,but PGC-1α did not increasesignificantly.② RSV treatment group:Compared to OGD group,the relative expressionof Sirtl and PGC-1α in the OGD+RSV group increased in mRNA and protein levels.ConclusionIn oxygen-glucose deprived hippocampal neuron cells,Sirtl may exert its neuroprotective effect against oxidative stress by regulating PGC-1α. |
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