Autophagy/Lysosome Pathway Activation And Its Contribution To Astrocyte Death Induced By Cerebral Ischemia-Hypoxia

Aim: To investigate activation of autophagy/lysosomal pathway in astrocyte induced by cerebral ischemia-hypoxia in vivo and vitro and study the role of autophagy activation in astrocyte death.Methods: In this study, we established two cerebral ischemia-hypoxia models in vivo and vitro. Permanent middle cerebral artery occlusion (pMCAO) model was induced by using intraluminal filament technique in rats. Primary astrocyte was exposed to a paradigm of ischemic insult by using an OGD (oxygen-glucose deprivation) device. Autophagosomes in astrocyte were observed by transmission electron microscopy (TEM) both in vivo and vitro. Protein related to autophagy was determined with immunofluorescence (IF). Autophagosomes in astrocyte in vitro were observed by MDC staining. Western Blot was employed to determine alternations in LC3/Beclin 1/cathepsin B/LAMP 2/Bcl-2 levels in astrocyte at 0 h,0.5 h,1 h,3 h,6 h and 12 h after OGD, and alternations in LC3 level in OGD 3 h after 3-MA administrated. LDH showed LDH leakage alternation in astrocyte in OGD 3 h after 3-MA administrated.Results: TEM revealed that the formation of autophagosomes, enhanced presence of lysosomes, fragmented endoplasmic reticulum, swollen mitochondria in astrocyte induced by cerebral ischemia-hypoxia in vivo and vitro. Immunofluorescence demonstrated that the expression of LC3 was increased in astrocyte after pMCAO and OGD treatment, and 3-MA (10 mM) could significantly reduce the expression of LC3. MDC staining also showed that autophagosomes were appeared in astrocyte after OGD treatment. Western Blot analysis revealed that the expression of Beclin 1 was significantly up-regulated and peak at 1 h after OGD (P<0.01); the level of LC3 was increased and peak at 3 h (P<0.01); the expression of cathepsin B/LAMP 2 peak at 6 h (P<0.01); the level of Bcl-2 was decreased following OGD (P<0.01); 3-MA (10 mM) markedly decreased the level of LC3 in astrocyte (P< 0.05). LDH showed that 3-MA (5,10,20 mM) reduced LDH leakage in a partly dose-dependent manner at 3 h after OGD treatment (P< 0.05).Conclusions: 1. Autophagy/lysosome pathway was activated in astrocyte induced by cerebral ischemia-hypoxia in vivo and vitro. 2. 3-MA has protective effect on astrocyte exposured to OGD, suggesting that the excessive activation of autophagy/lysosome pathway in astrocyte injury induced by cerebral ischemia-hypoxia contributes to astrocyte cell death.