| BackgroundIschemic stroke is a neural degenerative disease with high mortality and disability, which is characterized by ischemic-hypoxic(IH) injures in neurons.Ischemic stroke is induced by a sudden reduction of blood circulation, resulting in the cutoff of oxygen and the neurological dysfunction in brain. Ischemic stroke can lead to permanent and transient ischemic–hypoxic injuries, depending on crucial reperfusion time which is only about 4-6 hours, so permanent ischemia-hypoxia is common in clinic cases.The pathological processes of IH injuries include hypoxia response, postischemic inflammatory response(PIR), cell apoptosis, periinfarct depolarization and so on.Although stroke has attracted widespread attention in the clinic, its treatment effectiveness is remarkably finite, since the mechanism of ischemic-hypoxic injuries is not understood well. Recently, some studies have revealed that autophagy plays an important role in inflammation, nutrient deprivation and even in the processes of IH.For instance, some studies have indicated that autophagy was involved in cell survival after chronic and moderate hypoxia. Considering the existence of cell death during autophagic processes, whether autophagy protects neuronal survival in IH injuries is still in debate, especially in permanent IH injuries. Autophagic flux refers to the dynamic process of autophagy, it includes the formation of autophagsomes, the engulfment of damaged organelles, autophagsomes fusion with lysosomes, and finally the degradation of autophasomes. Failure of each step can lead to imbalance of autophagy. As we know, autophagy is cascade processes in morphology and chemistrywith the participation of several autophagic proteins and signaling transduction pathways, such as LC3, Beclin-1, Atg and Notch pathway. Previous studies have showed that Wnt signaling pathway is also involved in autophagy, even though Wnt signaling pathway mainly participates in the regulation of cell proliferation and differentiation during development. Furthermore, additional studies have showed that Wnt signaling pathway probably is very important in the pathogenesis of neurodegenerative diseases, including IH, because cytokines in Wnt pathway can improve the microenvironment for neuronal repair after post-ischemic injury.However, if Wnt signaling pathway regulates autophagy in permanent IH is not yet clear, and so does autophagic flux index.Objectives1. With ischemia and hypoxia neuronal injury model in PC12 cells, the neural injuries and its mechanisms would be investigated.2. The roles of autophagy in neuronal IH injuries would be studied, through analyses of autophagy activation and autophagic flux index.3. The regulation of Wnt signaling pathway in permanent IH injuries would be analyzed, and the relationship between Wnt signaling pathway and autophagy would be discussed well.MethodsIn the present study, PC12 cells were used to establish the oxygen-glucose deprivation(OGD) model, to imitate the permanent IH injuries. Various groups were divided.(1) Control group: the cell culture in normal medium with normoxia;(2)OGD groups: the cells were cultured in OGD medium for different times from 0.5 hto 24 h. With autophagy agonist(rampycin), autophagy atagonist(3-methyl adenine,3-MA) and lysosome antagonist(MHY1485), the autophagy was intervened at OGD6 h, in order to understand the regulative mechanism of autophagy. Then, apoptotic cells were stained with Hoechst 33342 staining, meanwhile the ultrastructure of autophagic cells was observed under transmission electron microscope. In addition,the cell viability and cell apoptosis were analyzed with MTT assay, LDH test and flow cytometry as well. With immunocytochemistry and Western blotting, the expressions of some stress-related proteins, such as HIF-1α(a key regulator in hypoxia) and COX2(inflammatory indicator), were analyzed. In the meantime, the upstream proteins(Wnt1 and Wnt3a), downstream proteins(Dvl2, β-Catenin) target proteins(C-myc and Cyclin D) in Wnt/β-Catenin signaling pathway were visualized with immunocytochemistry and Western blot at various groups above.Results1. The oxidative stress response and OGD at various timesTo investigate the stress response of PC12 cells after ischemia and hypoxia, the expressions of relative stress proteins, such as HIF-1α and COX2, were investigated with immunochemistry, Western blotting, MTT, LDH test. The results showed that the expression of HIF-1α and COX2 were up-regulation in OGD groups with time prolongation, and significant difference appeared after 12 hour’s OGD(p < 0.05). The cell viability decreased in OGD groups with time prolongation. LDH release rate showed positive correlation with OGD time prolongation, and significant difference appeared after 12 hour’s OGD(p < 0.05). OGD could induce cells apoptosis with time dependency, and the significant difference started after 6h OGD(p < 0.05).Autophagy activation was investigated with transmission electron microscopy(TEM) and immunocytochemistry for some autophagy related proteins. The study showed that numerous LC3 positive puncta were found in OGD groups with time dependency, and the LC3 positive puncta were demonstrated as autophagasomes under TEM. Western blot results showed that the expression of autophagy related proteins LC3 and Beclin-1 were up-regulation in OGD groups with statistical difference. LC3 Ⅱ / Ⅰ ratio, a criterion for the quantity of autophagic flux, was enhanced in OGD groups with time-dependency as well, suggesting that autophagy was activated. Protein P62, the autophasosomes degradation related protein decreased in OGD groups with time-dependency, indicating autophagosomes degradation enhanced. MHY1485, the lysosome antagonist, was also treated in OGD groups to examine the regulation of LC3Ⅱ/Ⅰratio as well, the golden criterion for autophagic flux. We found that LC3 Ⅱ / Ⅰ ratio increased significantly in all OGD groups,comparing with control group, suggesting autophagic flux was up-regulated.3. Permanent ischemia hypoxia and autophagy’s neuroprotectionTo investigate the autophagy’s neuroprotection in permanent IH, autophagy agonist and antagonist were administrated to OGD 6 h groups. In OGD 6 h group,after the administration of Rampycin(autophagy agonist), the autophagy was activated with LC3 expression up-regulation. Meanwhile, cell viability was enhanced,but LDH was declined, and the expressions of hypoxia and inflammation related proteins, such as HIF-1α and COX2, decreased as well. After administrations of 3-MA and MHY(autophagy antagonist), autophagy was inhibited with LC3down-expression, cells viability decline, but HIF-1α and COX2 over-expression,indicating autophagy play a protective role.2. Autophagy and OGD at various times4. Autophagy and the regulation of Wnt/β-Catenin signaling pathway in ischemia and hypoxiaSome proteins in Wnt/β-Catenin pathway were tested in the study after OGD treatment for examination the Wnt/β-Catenin pathway involvement in IH. The upstream proteins(Wnt1 and Wnt3a) expressions were up-regulation in OGD groups with time prolongation, the significant difference appeared after 12h(p < 0.05).However, the expressions of downstream proteins(Dvl2, β-Catenin) and target proteins(C-myc and Cyclin D) decreased in OGD groups with time dependency. The data suggested that Wnt/β-Catenin pathway was involved in permanent ischemia and hypoxia.To understand the relationship between autophagy and Wnt/β-Catenin signaling pathway, autophagic agonist and antagonist were administrated in OGD 6 h group.After administration of Rapamycin, autophagy was enhanced, and the upstream proteins(Wnt1 and Wnt3a) expressions were up-regulation. By the contrast, the expressions of downstream proteins(Dvl2, β-Catenin) and target proteins(C-myc,and Cyclin D) decreased. After the administration of 3-MA and MHY(autophagy antagonist), autophagy was inhibited, with upstream proteins(Wnt1 and Wnt3a)expressions down-regulation and downstream and target proteins up-regulation. The results indicated Wnt/β-Catenin pathway was regulated by autophagy negatively.Conclusion:1. OGD can lead to cellular stress response, including inflammatory response,neuronal apoptosis, neuronal necrosis, with time dependency.2. OGD can induce autophagy in OGD groups with time dependency.3. Autophagy is activated and up-regulated through autophagy flux index in permanent IH. Autophagy plays neuroprotection in the process of IH.4. Wnt/β-Catenin signaling pathway is involved in the regulation of permanent IH neuronal injuries. The upstream proteins are up-regulation, but the downstream proteins are down-regulation.5. Autophagy regulates Wnt/β-Catenin signaling pathway negatively. Autophagy plays a protective role in the permanent IH, due to the regulation of Wnt/β-Catenin signaling pathway. |
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