| Aim: To study the role of autophagy and endoplasmic reticulum stress (ER stress) in primary cultured cortical neurons oxygen and glucose deprivation (OGD) preconditioning.Methods: An oxygen/glucose deprivation (OGD) preconditioning model in primary cultured murine cortical neurons was established in vitro. For ischemic preconditioning (IPC), the cortical neurons were deprived of oxygen and glucose for 30 min. 24h after the preconditioning stimulus, the cultures were again deprived of oxygen and glucose for 3hr to cause lethal OGD. 3-MA was added 30min before IPC. The activation of autophagy in neurons after OGD was observed by using MDC staining. Protein level of LC3 was determined by immunofluorescence. The autophagy related protein LC3,Beclin1 and p62 as well as ER stress related protions HSP70,GRP78,CHOP,caspase-12 were assessed by Western Blot analysis.Results: The expression of protein LC3 and Beclin-1, which were the markers for autophagy, was significantly increased after IPC. LDH results and microscopic observations indicated that IPC induced neuroprotection, while 3-MA (10,20mM), significantly increased the cell damage, suggesting that autophagy inhibitors abolished the IPC-induced preconditioning neuroprotection. MDC staining and immunofluorecsence revealed that autophagy was activated after IPC and inhibited by 3-MA treatment. Western Blot analysis showed that LC3,Beclin1,HSP70,GRP78 rapidly up-regulated after IPC and was attenuated by 3-MA treatment. However, 3-MA treatment increased the expression of p62,caspase-12 and CHOP.Conclusions: 1. IPC induced autophagy activation in the primary cultured cortical neurons. 2. 3-MA completely suppressed the neuroproteciton induced by IPC and reduced the expression of LC3 and Beclin1. 3. IPC induced ER stress and HSP70,GRP78 upregulation, while inhibition of autophagy may cause excessive ER stress, and increases CHOP, caspase-12 expression, leading to neuronal death. |
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