Protective Effects Of Ethyl Acetate Extract Of Polygonum Cuspidatum On Hydrogen Peroxide-induced Injury In PC12 Cells

ObjectiveA variety of pathological mechanisms are evidently associated with humanneurodegenerative disorders,with no particular mechanism emerging as a major contributor.Apparently the outcomes of any effective neuroprotective strategy,targeting specificdisease components,will remain uncertain until validation in human subjects.The research about the protective of ethyl acetate extract of Polygonum cuspidatumcan provide a chance for treating the ROS-induced neurodegenerative diseases.MethodsIn this study,we used cell viability assay,morphological assay,flow cytometrydetection of cell death,mitochondrial membrane potential assay to investigate whetherethyl acetate extract of Polygonum cuspidatum exerted neuroprotective effects on H₂O₂induced injury in PC12 cells and to study the possible mechanisms.1.Establish H₂O₂ induced PC12 injury model.2.Apply MTT asssy to detect the cell viability of different concentrations of ethyl acetate extract of Polygonum cuspidatum on PC12.3.The effect of ethyl acetate extract of Polygonum cuspidatum on H₂O₂ induced PC12injury by LDH,MTT assay. 4.Effect of ethyl acetate extract of Polygonum cuspidatum on H₂O₂ induced changes inmitochondrial membrane potential.5.Adopt Annxin V/PI assay to detect the apoptosis and necrosis rate of ethyl acetateextract of Potygonum cuspidatum on on H₂O₂-induced injury.6.Nuclear staining for assessment of apoptosis were observed using the chromatin dyeHoechst 33324.7.Effect of ethyl acetate extract of Potygonum cuspidatum on the cell cycle by H₂O₂induced injury in PC12 cells.8.The research about the other compounds protective effect on H₂O₂-induced injury inPC12 cells from ethyl acetate extract of Potygonum cuspidatum.Results1.Estabilsh 880μM H₂O₂ induced PC12 injury model.2.The results showed that the concentration of EAPC from 0.15 to 5μg/ml has nocytotoxicity.3.Following exposure of the cells to 880μM H₂O₂ for 24h,the significant reduction incell survival and activities lactate dehydrogenase (LDH)release.The cell survival wereincreased and the LDH release is reduced by EAPC (0.31-5μg/ml)plus 880μM H₂O₂.4.EAPC (3,1,0.3μg/ml)can reduce 880μM H₂O₂-induced mitochondrial membranepotential change of JC-1 stained mitochondria in PC12 cells at 2H.5.The apoptotic nature of H₂O-induced cell death was further confirmed by annexin V-FITC labeling of PS exposed on the plasma membrane.EAPC(3,1,0.3μg/ml)can inhibitthe apoptosis and necrosis.6.Further evidence for the apoptotic nature of the observed cell death was provided bythe nuclear morphology by using Hoechst 33324 under a microscope.H₂O₂ clearly inducednuclear fragmentation.7.Since apoptosis and the cell cycle are closely linked,EAPC changed the cell cycleexposure to H₂O₂ for 24h.EAPC (3μg/ml,1μg/ml,0.3μg/ml)inhibit the S stage.8.To screening the compounds from EAPC by MTT,LDH assay.The results showed thatpolydatin,2-Methoxy-6-acetyl-7-methyljuglone,Anthraglycoside A and Anthraglycoside Bhave protective effects.ConclusionThe concentrations of EAPC(3μg/ml,1μg/ml,0.3μg/ml)can increase the depolarization of mitochondrial membrane potential(MPP),reduce the apoptosis and necrosis rate andnuclear fragmentation.Taken together,these results suggest that EAPC shows protectiveagainst H₂O₂-induced cell injury.The finding is of a higher value in searching for newtherapeutic agent for treating oxidative damage-derived neurodegenerative disorders.