The Effect Of Astragaloside In Cultured PC12 Cell With Oxygen-glucose Deprivation And Reperfusion On TLR4 Pathway

Ischemic cerebral vascular diseases in the United States and the United Kingdom have become the third killer which would endanger the human health. The incidence of ischemic stroke is 80%,and develop an annual growth of 0.2% every year, 200 million people in China suffer from it, these 70%-80% patients may result in disability. It can be seen that ischemic cerebrovascular diseases not only seriously affected people’s health, but brought great economic pressure. It has a high disability rate, mortality and morbidity characteristics. Cerebral ischemia-reperfusion injury is an important process in the ischemic cerebral vascular disease which has complex mechanisms, such as disorder of energy metabolism, cell metabolic acidosis, intracellular calcium homeostasis imbalance, excitatory amino acids, cell apoptosis and inflammatory damage and so on. More and more studies have shown that apoptosis in focal cerebral ischemia-reperfusion injury plays a key role. Study shows herbs can stimulate the regeneration of neurons, reduce the apoptosis of nerve cells. It can repair neurons in the ischemia-reperfusion injury. Astragaloside as its activity which has a protective effect on cerebral ischemia-reperfusion injury. There are varieties of mechanisms in the cell apoptosis. Recently TLR4/MyD88 pathway has been a hotspot research after cerebral ischemia-reperfusion injury in recent years. This paper studies the inhibition of Astragaloside on PC12 cells oxygen-glucose deprivation and reperfusion TLR4/MyD88 signal pathway. We bring new innovations for the future. The First Part The inhibition of cell apoptosis by Astragaloside in cultured PC12 cells with oxygen-glucose deprivation and reperfusionObjective:To observe the protective effect of Astragaloside on PC12 cells of oxygen- glucose deprivation and reperfusion, which play a role by attenuating apoptosis.Methods:PC12 cell was random divided into 6 groups: normal control group, DMSO control group, model group(oxygen-glucose deprivation and reperfusion group), Astragaloside high dose group, Astragaloside middle dose group and Astragaloside low dose group. In addition to the normal control group, other groups were made 4 hours of oxygen glucose-deprivation, then reperfusion 24 hours. Every one of the Astragaloside dose group and solvent control group were previously delivered the drug 0.5 hour before making model until the end of the experiment. We used MTT method to detect the cell viability. Apoptosis was measured by Flow Cytometry.Results:Compared with the normal group, model group cells were poor refraction, shrinkage and gathered in clumps, or even fall off. The synapses between cells disappeared and the cellular swelling was clearly. Survival rates of the cell were significantly lower and the rates of apoptosis are higher.(P<0.05).Compared with the model group, solvent control group and Astragaloside low dose group did not change significantly. There was no significant statistical difference between them in survival rate and apoptosis rate(P>0.05), but the high Astragaloside dose group and middle dose group could increase the survival rates and reduce the rate of apoptosis(P<0.05).The cellular inflation and swelling could both decreased. Compared with the Astragaloside middle and low dose group, the cell survival rate of high Astragaloside dose group of was obviously higher and the cell apoptosis rate was apparently was lower(P<0.05).Summary:1 Astragaloside could improve the survival of PC12 cell after oxygenglucose deprivation and reperfusion.2 Astragaloside could attenuate injury of PC12 cell morphology and inhibit the cell apoptosis.3 The concentration of the best effect is 100μmol/l The Second Part The effect of Astragaloside in cultured PC12 cell with oxygen glucose-deprivation and reperfusion onTLR4 pathwayObjective:To observe the effect of Astragaloside onTLR4 pathway in cultured PC12 cell with oxygen-glucose deprivation and reperfusion.Methods:To take the phase of logarithmic growth of PC12 cells. They were divided into 7 groups: normal control group, normal+artragaloside group, model group(oxygen-glucose-deprivation reperfusion), solvent group(DMSO), Astragaloside treatment group(100μmol/L),TLR4 receptor inhibition group(3μmol/L) and Astragaloside +TLR4 receptor inhibition group. The method of making model: PC12 cell was deprived oxygen and glucose for 4 hours and reintroduced them for 24 hours. Before making model each group was delivered drug for 0.5 hour, until the end of experiment. We used immunohistochemical method to detect the NF-κB and caspase-3.We use Western blot method to test the Myeloid differentiation factor88 protein and TLR4 protein.Results:Immunohistochemical results of NF-κB: Compared with the normal control group, the positive cell of NF-κB of model group increased significantly(P<0.05), but there was no difference between the normal group and the normal+Astragaloside group(P>0.05). Compared with the model group, the positive cell of NF-κB of solvent group was no difference(P>0.05), but the Astragaloside treatment group, inhibition group, the inhibition+Astragaloside group reduced significantly(P<0.05). There was no significant difference between the Astragaloside treatment group and inhibition +Astragaloside group(P>0.05). But there was significant difference between the Astragaloside treatment group and inhibition group(P<0.05). The trends of other indicators including caspase3, My D88 andTLR4 were same with NF-κB.Summary:1. Astragaloside inhibited the expression ofTLR4 and MyD88 protein.2. Astragaloside inhibited the positive cells ofcaspase-3 and NF-κB and inhibited cell apoptosis.Conclusion:Astragaloside could improve the survival rate of PC12 cells, attenuate cell morphology damage and inhibit cell apoptosis. The possible mechanism was that Astragaloside IV could inhibitTLR4/MyD88 signal pathway.