Study On Phenolic Composition C Extract From Gastrodia Elata Protect PC12 Cells Against Antioxidant Injury

Objective:Ischemic cerebrovascular disease is one of the main causes of human death at present. It’s important for the treatment to open clogged blood vessels and restore blood flow of diseased region in time. However, restoration of blood flow beyond the time window of reperfusion will result in more severe reperfusion injury, namely ischemic reperfusion injury(IR). During cerebral ischemia-reperfusion, plenty of reactive oxygen species(ROS) are released by mitochondria and accumulate in the cells. This will cause toxic reaction of cells and lead to tissue injury finally. ROS plays a regulating role on apoptosis mainly through its mediation on the mitochondrial apoptosis pathway, thus leading to oxidative tissue damage. On the one hand, ROS can directly or indirectly cause damages to the mitochondrial membrane, resulting in the decline of mitochondrial membrane potential(MMP), release of cytochrome C into the cytoplasm, the Caspase cascade reaction and apoptosis eventually. On the other hand, ROS can activate the signaling pathway of mitogen activated protein kinase(MAPK) to participate in the regulation of apoptosis. And studies have shown that MAPK can be effectively activated during cerebral ischemia reperfusion, which is a specific pathway in the process of cerebral ischemia reperfusion. Therefore,researches on effects of drug in MAPK signal pathway an important significance on the drug research and development for the prevention and treatment of ICVD.Our earlier studies indicated that Gastrodia elata showed a strong resistance against cerebral ischemic reperfusion injury in rats, whose mechanism was related to anti-free radical injury and anti-apoptosis. The active ingredients were mostly in theethyl acetate extract. A group of lipid soluble phenols was separated from the ethyl acetate extract of Gastrodia elata, and its activity was screened with oxidative damaged PC12 cell models induced by H2O2. The results showed that the phenolic composition C extract from Gastrodia elata had a strong activity of anti-oxidative injury in vitro.To further discuss the mechanism of the anti-oxidative activity of the phenolic composition C extract from Gastrodia elata, H2O2 induced oxidative damaged PC12 cell models was used to evaluate the activity of the phenolic composition C with the indicators such as ROS level in cells, T-SOD activities, and GSH content. The effect of the composition C on apoptosis of oxidative damaged PC12 cell was studied according to MMP variation and expressions of Caspase-3 and Caspase-9. The expression of proteins related to the MAPK signaling pathway were measured to see whether the composition C performed its anti-oxidative activity through regulating MAPK signaling pathway.Methods:1. According to preliminary grope for preparing PC12 cells oxidative damage model induced by H2O2, detect cell survival rate by MTT to evaluate the effect on PC12 cells induced by H2O2 oxidation damage model of phenolic composition C extract fron Gastrodia elata, and detect intracellular ROS level, T- SOD activities and GSH content.2. To detect Caspase 3 activity and MMP variation by kits, and detect Caspase-3,Caspase-9 protein expression by western-blot in PC12 cells, for expore the effect of phenolic composition C extract from Gastrodia elata on cell apoptosis.3. Before establishing the oxidative damage model, use the inhibitor PD98059,Results:1. Compared with control, model group cell survival rate was lower significantly(P < 0.01), remarkable increase of intracellular ROS level(P < 0.01), T-SOD activity and GSH content were significantly decreased(P<0.05 or P < 0.01); Compared with model, use edaravone and phenolic composition C extract from Gastrodia elata preconditioning cells 24 h, the cell survival rate was increased significantly(P < 0.01),intracellular ROS level was decreased(P < 0.01), T-SOD activities and GSH content was increased significantly(P < 0.01),2. Compared with control, Caspase-3 was activated(P < 0.01), MMP was decreased, expression of Caspase-3, Caspase-9 protein were increased in model group(P < 0.05 or P < 0.01); Compared with model, in edaravone group, Caspase-3activation degree was lower(P < 0.01), MMP was to rise, and inhibited the protein expression of Caspase-3 and Caspase-9(P < 0.05); Phenolic composition C extract from Gastrodia elata could inhibited the Caspase-3 activation degree and protein expression of Caspase-3 and Caspase-9, and increased MMP(P < 0.05 or P < 0.01).3. Compared with drug group, pretreated with ERK1/2 inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580, cell survival rate was decreased significantly(P < 0.01); Compared with control, P-JNK and P-p38 protein expression of model group were increased(P < 0.01); Compared with model, edaravone could inhibit P-JNK protein expression(P < 0.01); Phenolic composition C extract from Gastrodia elata could inhibit P-JNK protein expression(P < 0.01), after pretreatment PD98059, SP600125 and SB203580, the protein expression of P-ERK1/2, P-JNK and P-p38 were significantly inhibited(P < 0.01 or P < 0.05); ERK1/2, JNK and p38 protein expression of each goup were not statistically difference.Conclusions:1.Phenolic composition C extract from Gastrodia elata showed a significant protective effect on PC12 cells oxidative damage induced by H2O2.2. Phenolic composition C extract from Gastrodia elata protected the oxidative damage of PC12 cells by regulate JNK signaling pathways.