The Study Of The Protective Effect Of Phenols Of Gastrodia Elata Blume. On Oxidative Damage Of PC12Cells Induced By H₂O₂

Objective: In a previous study, our research group found that the effectivecomponents of Gastrodia elata Blume. had effects on resisting brain ischemiareperfusion injury and the mechanism was associated with resisting to oxidativedamage. Its activity was related to a group of fat-soluble phenolic compounds. Wechosed PC12cells oxidative damage model induced by H₂O₂as the oxidative stressmodel to screen the protective activities of3extracts and15monomer components ofGastrodia elata Blume.and investigated their possible mechanisms against oxidativestress injury.Methods:1、We chosed RPMI-1640culture medium containing10%FBS and0.4%doubleantibody to culture, passage and frozen cells. The cells growth curve was drown byMTT, cell counting and trypan bule staining method.We controled preliminary thegrowth characteristics of PC12cell in vitro.2、The cell morphology observation and MTT assay were used to explore theappropriate concentration of H₂O₂induced PC12cell oxidative damage model.3、The cell morphology observation and MTT assay were used to explore the effectiveconcentration of edaravone in H₂O₂induced PC12cell oxidative damage model.4、The cell morphology observation and MTT assay were used to evaluate theprotective effects of3extracts and15monomer components of Gastrodia elata Blume. in H₂O₂induced PC12cell oxidative damage model.5、The DCFH-DA fluorescence probe, LDH ELISA， TBA method and T-SODhydroxylamine method were used to measure the intracellular ROS level,extracelluarLDH activity，LDH leakage rate, intracellular MDA content and T-SOD activity toevaluate the effects of Gastrodia elata Blume.active ingredient by H₂O₂inducedPC12cell oxidative damage model.Results:1、In our lab cultivation conditions in96hole plate，the growth curve drawn by MTTassay showed the PC12cells cultured6d by100cells/mL didn’t reach the plateauwhile cultured3d by100000cells/mL reached. The appropriate inoculationconcentrations were6000，8000and10000cells/mL. The8000cells/mL was culturedin25cm2culture flask and the growth curve was drawn by the cell counting andtrypan blue staining counting method，which showed the PC12cells cultured4dreached about90%coverage rate and cultured5d was full. Cells showed contactand density inhibition with the gradual increase in the number of cells and the growthspace reduction.2、Injured by40μM-80μM H₂O₂the number of cells was decreased gradually，mostcells synapses retracted，cells body rounded，part of round cells grouped，cells bodyedge changed blurry，diopter decreased and adherent ability reduced；compared withthe normal group，the cell suvival rate was at range of18.6%-66.0%（P<0.01），therefore, the optimal H₂O₂damage concentration was40μM-80μM.3、Edaravone under the0.918mmol·L^-1(160μg·mL^-1) dose improved the damagedcells morphology significantly in50μM H₂O₂damage model. Cell viability assayshowed that compared with the normal group，the survival rate of model cells injuredby50μM H₂O₂for2h was26.9%（P<0.01）；compared with the model group，thecells survival rate of edaravone under the0.918mmol·L^-1dose was increased10.9%（P<0.01）.4、In3extracts and15monomer components of Gastrodia elata Blume.,the model cells morphology of EtOAc (20μg·mL^-1) and8#(80μg·mL^-1) groups were improved.Cell viabilities assay showed that compared with the model group，the survival ratesof EtOAc (20μg·mL^-1) and8#(80μg·mL^-1) groups were increased14.7%and9.9%respectively （P<0.01）. Survival rate of1#and6#were also enhanced significantly（P<0.05or P<0.01）， but the results of the same concentration had a poorrepeatability.5、The results of intracellular ROS level, extracelluar LDH activity， LDH leakagerate, intracellular MDA content and T-SOD activity on H₂O₂induced PC12celloxidative damage model showed that compared with the model group，fluorescenceintensity of EtOAc (80μg·mL^-1) and8#(40，80μg·mL^-1) groups were reducedsignificantly （P<0.05or P<0.01）；extracelluar LDH activity and LDH leakage rate ofEtOAc (20，40and80μg·mL^-1) groups were reduced significantly （P<0.05orP<0.01），extracelluar LDH activity and LDH leakage rate of8#(40，80μg·mL^-1)groups were increased significantly （P<0.05or P<0.01）；intracellular MDA content ofEtOAc (40μg·mL^-1) group was reduced significantly （P<0.05）；intracellular T-SODactivity of EtOAc (20，40μg·mL^-1) and8#(20,40μg·mL^-1) groups were increasedsignificantly （P<0.01）； intracellular T-SOD activity of EtOAc (20，40μg·mL^-1) and8#(20，40μg·mL^-1) groups were reduced significantly （P<0.01）.Conclusions:1、Cultured in25cm2culture bottle using8000cells/mL （1600cells/cm2） for4d wasthe optimal condition for passag, inoculation and frozen storage of PC12cells, whichpopulation doubling time （PDT） was between12h to24h.2、The PC12cell oxidative stress damage model could been copied by using40μM to80μM H₂O₂induced PC12cell for2h.3、The optimal concentration of edaravone protecting the oxidative damage modelinjured by50μM H₂O₂for2h was0.918mmol·L^-1(160μg·mL^-1).4、Phenols of8#、1#、6#and EtOAc could improve the morphology and increasesurvival rate of the oxidative damage PC12cells. 5、The EtOAc of Gastrodia elata Blume.had that the antioxidant effects in vitro byprotecting the intrgity of cell membrane afer H₂O₂injury, decreasing the intracellularROS level, raising intracellular T-SOD and inhibiting LPO.8#phenolic compoundof Gastrodia elata Blume.had the antioxidant effects by decreasing the intracellularROS level and raising intracellular T-SOD activity afer H₂O₂injury，which under useddoses had no effect on protecting the intrgity of cell membrane and inhibiting LPO.