Tetramethylpyrazine Protects Rat Astrocytes Against H₂O₂-induced Oxidative Stress Injury

ObjectiveIn this study,rat astrocytes were isolated and cultured in vitro,tetramethylpyrazine（TMP）-containing medium was introduced as an intervention and protective factor,and hydrogen peroxide was used to construct astrocyte oxidative damage model to observe the correlation of cell oxidative damage Detect the expression levels of apoptotic proteins and key upstream and downstream proteins of the PI3K/AKT pathway,explore the protective effect of TMP on oxidative stress damage of astrocytes and the relationship between this effect and the PI3K/AKT signaling pathway.MethodsAstrocytes were isolated from SD newborn rats（24～48 h old）and cultured,and then the cells were randomly divided into control group（cultured with normal culture medium）,model group(50、100、200、300μmol·L^-1 H₂O₂ for 24 h),TMP groups(pretreatment of 25,50,100μmol·L^-1 TMP for 48 h,and then H₂O₂ was added for 24 h).CCK-8 assay was used to detect cell survival rate,GFAP staining to observe cell morphology,flow cytometry to detect cell apoptosis,corresponding reagent test kits to detect the lactate dehydrogenase（LDH）content and superoxide dismutase（SOD）activity,real-time fluorescent quantitative PCR to measure the detect the m RNA expression of PI3K,AKT,Bcl-2,Bax and Caspase-3,and Western blotting to test the protein expression levels of PI3K,AKT,p-AKT,Bcl-2,Bax and Caspase-3 in each group of cells.ResultsThe model group had significantly reduced cell survival rate（P<0.05）,the apoptosis rate of 100μmol/L is about 50%,and this concentration will be used in the following experiments,increased apoptotic rate（P<0.05）,elevated LDH content（P<0.05）,decreased SOD activity（P<0.05）;down-regulated PI3K,AKT,Bcl-2 m RNA and protein levels,and up-regulated the expression levels of Bax and Caspase-3m RNA and protein levels（P<0.05）.Compared with the model group,TMP pretreatment resulted in significantly increased cell survival rate（P<0.05）[with the dose of 50μmol/L highest（P<0.05）],lowest apoptotic rate（P<0.05）and LDH content（P<0.05）,enhanced SOD activity（P<0.05）,up-regulation of PI3K,AKT,and Bcl-2 at m RNA and protein levels,and down-regulation of Bax and Caspase-3 at m RNA and protein levels（P<0.05）.ConclusionTMP exerts protective effects on oxidative stress injury of rat astrocytes induced by H₂O₂,and improves cell morphology and inhibits cell apoptosis after injury,and this effect may be related to the regulation of PI3K and p-AKT activation.