| Background and Objective:Upon the occurrence of the acut cerebral ischemic stroke, series of cascades are coming, including calcium overload, toxicity of excitatory amino acids, oxidative stress, mitochondrial dysfunction and the activation of the protease, Eventually, leading to the cell necrosis and apoptosis. It is found that oxidative stress plays a key role on the neuron damage induced by the cerebral ischemia. During the oxidative stress, free radical leads to calcium overload which activates the apoptosis program and other irreversible damage. Results found that oxidative stress can activate the autophaty-lysomomal pathway which increasing the number of the autophasome. Autophagy can digestion the impaired mitochondria and endoplasmic reticulum to control the homeostasis of the cell. However, when confronted with the ischemia--reperfusion, the response of Golgi calcium pump SPCA1and the effect of the autophagy on the function of the SPCA1are rarely involved. In this study, we will seek new ways to reduce oxidative stress injury by exploring the impact of autophagy on the Golgi calcium pump SPCA1when subjected to ischemia-reperfusion injury.Methods:1. Establish a H2O2-induced oxidative stress model in vitro. Choose different concentration of H2O2for N2a cells. The cell injury was measured by the the MTT assay.2. The test was divided into normal group, H2O2-treated group and3-MA pretreatment group; concentrations of H2O2-treated group and3-MA pretreatment group H2O2were20μM, μM,50μM;4. The activity of autophagy was measured by the MDC staining after the H2O2treatment;5. Detect the change of intracellular calcium by Fura/am;6. Using the RT-PCR to detect the SPCAlmRNA expression;7. Using the Western blot to detect the Golgi protein SPCAl and expression of the autophagy marker LC3B.Results:1. MTT results showed that with the increase of H2O2Concentration, cell damage gradually increased. H2O2damage to cells in a concentration-dependent manner, MDC staining found that following the increases the concentration of H2O2, the number of the autophagic vacuoles also increases. The concentration of H2O2increased autophagy particles. The results of western blot of LC3B also showed that autophagy activity increased with increasing concentrations of H2O2(P<0.05), while autophagy inhibitor3-MA can effectively inhibit autophagy activity.2. After H2O2treatment, Using the Fura-2/am to detect the intracellular calcium concentration found that intracellular calcium increased significantly (P<0.05), compared with the normal group. When the cells treatment by the20μM H2O2, the changes of calcium ion concentration is not obvious in H2O2treatment group and3-MA pretreatment group of (P>0.05),in addition to20μM H2O2-treated group and3-MA pretreatment group in addition to calcium ion concentration obvious external (P>0.05), the rest of the group3-MA pretreatment intracellular H2O2-treated groups compared with the calcium concentration H2O2-treated groups representing an increase of more significant (P<0.05). The rest of the3-MA pretreatment groups are experienced more obvious increase of calcium concentration than H2O2-treated groups (P<0.05).3. Compared with the normal group, H2O2-treated groups induced less expression SPCAlmRNA and SPCA1protein significantly (P<0.05),3-MA pretreatment groups expression less SPCAlmRNA and SPCA1protein than the H2O2groups (P<0.05), but20μM of H2O2concentration, SPCAlmRNA and SPCA1protein no significant change between the two groups (P>0.05).Conclusion:1. Oxidative stress injury can activate autophagy-lysosomal pathway;2. Oxidative stress can upregulate the autophagy;3. Blocking autophagy-lysosomal pathway leads to the decrease expressing of SPCA1, which aggravates calcium overload... |
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