The Mechanism And Mechanism Of Tetramethylpyrazine On The Damage Of PC12 Cells Induced By Coumaric Acid In Ischemic Model

As one of the major effective ingredients of Rhizoma Chuanxiong,ligustrazine(TMP)has been widely used in the treatment of ischemic injury.To further improve the neuroprotective property of TMP,a novel series of TMP derivatives were synthesized based on traditional Chinese medicine theory and the composition principle.Compound TMP-CA showed protective effect against CoCl2-induced neurotoxicity in differentiated PC 12 cells.Preliminary experiments confirmed that the compound TMP-CA can block the mitochondrial pathways of apoptosis pathway,inhibiting cell apoptosis and giving play to the role of the prevention and treatment of cerebral ischemia injury.(See Zhang Huazheng graduation thesis)Therefore,this study aims to further study the neuroprotective effect of compound TMP-CA and explore the compound TMP-CA nerve protective effect is involved in the death receptor pathway.At the same time,the clinical common mentioned neural protectants edaravone as positive medicine,make a preliminary evaluation on the effects of the compound TMP-CA.Objective:In vitro culture of NGF plays differentiation to ischemic injury of PC 12 cells as the object,on the basis of preliminary experiment,study compound TMP-CA and the positive medicine edaravone on PC 12 cell apoptosis and the effect of compound TMP-CA neuronal protection is involved in death receptor pathway.To observe the compound TMP-CA and the positive medicine edaravone on Bad,Bcl-xl,NF-?B/P65,Caspase-8,the effect of protein expression.The mechanism of action of compound TMP-CA on CoCl2 induced ischemic injury in PC 12 cells was investigated at the cellular and molecular level.At the same time,the clinical common mentioned neural protectant edaravone as positive medicine,and make a preliminary evaluation on the effects of the compound TMP-CA.Methods:1.Effect of compound TMP-CA and edaravone on cell viability afterCoCl2-induced neurotoxicity in differentiated PC12 cells:Differentiated PC12 cells were divided into normal group,model group,compound TMP-CA group and edaravone group.PC 12 cells were pretreated with compound TMP-CA at concentration 30?M and edaravone at various concentrations(12.5,15,20,25?M)and then exposed to CoCl2 at various concentrations(280 ?mol/L,300 ?mol/L)insult.MTT assay was used to evaluate the effect of compound TMP-CA on PC 12 cells.2.Effect of compound TMP-CA and edaravone on apoptosis after CoCl2-induced neurotoxicity in differentiated PC 12 cells:AO/EB fluorescent staining and DAPI staining were used to observe the apoptosis of PC 12 cells after CoCl2-induced neurotoxicity.3.Effect of compound TMP-CA and edaravone on morphological changes afterCoCl2-induced neurotoxicity in differentiated PC 12 cells and its mechanism:Cellular morphology was observed with hematoxylin and eosin(HE)staining.The expression of Bad and Bcl-xl was detected by immuno-histochemical analysis.4.Effect of compound TMP-CA and edaravone on the expression of apoptosis protein after CoCl2-induced neurotoxicity in differentiated PC 12 cells:Western blot was applied to evaluate the expression of NF-? B/P65?Caspase-8.Results:1.Based on the optical density(OD)values of MTT assay,compound TMP-CA could increase PC 12 cell viability after CoCl2-induced neurotoxicity(EC50=13.02?M).Edaravone can improve the differentiation after ischemia injury of PC 12 cell activity,best to determine positive drug concentrations of 20 microns.The compound TMP-CA on PC 12 cell to cell activity after ischemia injury and the effect of positive medicine according to the effect of edaravone in close,according to the edaravone EC50 value of 8.72 microns.The best concentration of building is 300 ?mol/L.2.Effect of compound TMP-CA(30?M)and edaravone(20?M)on apoptosis after CoCl2-induced neurotoxicity in differentiated PC 12 cells:?AO/EB fluorescent staining:Compared with model group,cells with the orange fluorescence decreasing in compound TMP-CA groups and positive control groups,necrosis and apoptosis of cells were decreasing.?DAPI staining:normal nucleus dyed to blue,after CoCl2 injury,cells significantly reduced,hyperchromatic cells,height of chromatin gathered,tending to edge,for the mass,or a crescent.To medicine group and control group in PC 12 quantity become more,nucleus state better,most of the oval shaped.3.Observation of morphological changes were as follows:?Cells in normal group had round cell bodies with fine dendritic networks and intact cell edges,however,apoptosis with nuclear chromatin condensation and cell shrinkage as well as the disappearance of synapse were observed in model group.Cells in compound TMP-CA groups and edaravone had long synapse and the amount of neurite-bearing cells increased compared with model group.?The immunocytochemistry results showed that compound TMP-CA and edaravone could increase the expression of Bcl-xl and decrease Bad expression compared with model group,4.Effect of compound TMP-CA(30?M)and edaravone(20pM)on the expression of apoptosis protein after CoCl2-induced neurotoxicity in differentiated PC 12 cells:Western blot results showed that the compound TMP-CA and positive medicine according to the NF-? B/P65 expression effect is not obvious,no statistically significant difference(P>0.05).In the model group the expression of Caspase-8 enhancement;Compared with model group compound TMP-CA make the expression of Caspase-8 slightly increases,but no statistically significant difference(P>0.05)?Compared with model group positive drug edaravone make the expression of Caspase-8 slightly lower,but no statistically significant difference(P>0.05).Conclusion:The protective effects of compound TMP-CA against CoCl2-induced neurotoxicity in differentiated PCl2 cells are as follows:?Increased cell activity(EC50=13.02).?Inhibition of PC12 cell apoptosis.?Increase the expression of Bcl-xl protein and down regulate the expression of Bad protein.?The expression of the NF-? B/P65 in effect is not obvious.Increase the expression of Caspase-8 but not obvious.Compound TMP-CA is not on the death receptor pathway in order to reduce cell apoptosis caused by ischemic injury.Compound TMP-CA has significant effects on reducing apoptosis and neuroprotective effect.Compound TMP-CA is close to the positive drug.