Inhibition Of Autophagy Stabilizes Lysosomal Membrane Via Up-regulation Of Lysosomal Heat Shock Protein 70.1B In Ischemic Astrocytes

Aim: According to our previous findings,inhibition of autophagy has a protective effect on cerebral ischemia.But its mechanism is unknown.Therefore,this study is aim to further explore the mechanism of it.Methods: In vivo,the permanent middle cerebral artery occlusion(pMCAO)model was performed 2,3,5-triphenyltetrazolium chloride(TTC)staining was used to detect the cerebral infarction volume.In vitro,primary astrocytes were cultured,and oxygen and glucose deprivation(OGD)was performed.Lactate dehydrogenase(LDH)assay was used to detect the injury of astrocytes induced by OGD.3-MA and Wort were used to inhibit autophagy pharmacologically,and knockdown of Atg5 was used to inhibit autophagy genetically.And then western blotting and immunofluorescence were used to detect the expression of cathepsin B,cathepsin L and Hsp70.1B in astrocytes induced by OGD.AO staining and Lyso-Tracker Red staining was used to detect the lysosomal membrane permeability in astrocytes induced by OGD.In astrocytes induced by OGD,shRNA Hsp70.1B was used to knockdown Hsp70.1B,and then AO staining was used to detect the lysosomal membrane permeability.Results: In a rat model of permanent middle cerebral artery occlusion(pMCAO),we found that the inhibition of autophagy reduced the cerebral infarction volume.Consistent with the vivo model,inhibition of autophagy deceased LDH leakage.These results suggest that inhibition of autophagy has a protective effect on cerebral ischemia.Furthermore,Western blotting showed pharmacological inhibition of autophagy and knockdown of Atg5 blocked OGD-induced activation of cathepsin B and cathepsin L in astrocytes.Immunofluorescence showed the inhibition of autophagy stabilized lysosomal membrane and blocked them from the lysosome into the cytoplasm.AO staining and Lyso-Tracker Red staining showed that inhibition of autophagy decreased OGD-induced lysosomal membrane permeability in astrocytes.There results suggest that the inhibition of autophagy decreases OGD-induced lysosomal membrane permeability in astrocytes.Moreover,Western blotting and immunofluorescence showed inhibition of autophagy further increased OGD-induced up-regulation of Hsp70.1B in astrocytes.And AO staining suggested that knockdown of Hsp70.1B further enhanced OGD-induced lysosomal membrane permeability and blocks autophagy inhibition–induced lysosomal membrane stability in OGD-treated astrocytes.Conclusion: There results suggest that inhibition of autophagy has a protective effect on cerebral ischemia and OGD-induced astrocytes.And its mechanism is that inhibition of autophagy decreases OGD-induced lysosomal membrane permeability via up-regulation of lysosomal heat shock protein 70.1B in astrocytes.