The Role Of RIP1 Kinase Inhibition In The Stabilization Of Lysosomal Membrane In Ischemic Astrocytes And Its Hsp70.1B Dependent Mechanisms

Aim: According to our previous reports,inhibition of RIP1 kinase has a protective effect on cerebral ischemia,and its mechanism is associated with inhibiting the activation of cathepsins and its release from lysosome to cytoplasm,suggesting that inhibition of RIP1 kinase contributes to the stabilization of lysosomal membrane.Therefore,this study is aimed to further explore the role of RIP1 kinase inhibition by RIP1 K knockdown in the stabilization of lysosomal membrane in ischemic astrocytes and its mechanism.Methods: Inhibition of RIP1 K was mediated by pharmacological(Nec-1)or genetic(shRNA RIP1K)technics in vivo and in vitro.The permanent middle cerebral artery occlusion(pMCAO)model was established in vivo.The oxygen and glucose deprivation(OGD)was performed in the primary cultured astrocytes in vitro.The morphology of astrocytes and its lysosome were examined with transmission electron microscopy(TEM)in ischemic astrocytes;Annexin-V-FITC and propidium iodide(PI)staining were used to detect astrocytic apoptotic and necrotic death,and AO staining was used to detect the lysosomal membrane permeability.Genechip,western blotting,immunohistochemistry,and immunofluorescence assays were used to analyse the expression and location of Hsp70.1B.Lentivirally-delivered shRNA against Hsp70.1B(shRNA Hsp70.1B)was used to observe the role of Hsp70.1B in the infarction volume,neurological functional behavioral score and lysosomal membrane permeability.Western blotting,immunohistochemistry and immunofluorescence,and Co-Immunoprecipitation assays were used to analyse the expression and location of proteins that related to the regulating proteins for Hsp70.1B,such as heat shock protein 90(Hsp90),heat shock factor 1(Hsf1)and μ-calpain.Results:(1)Western blotting analysis showed that the time-course changes of RIP1 K,Hsf1 and Hsp70.1B were consistent that these proteins were immediately up-regulated after ischemic injury,and reached their peak value 6 h after ischemic injury,and decreased 12 h later,but still kept in high level.The time-course changes of Hsp90 were significant different from these of RIP1 K,Hsf1 and Hsp70.1B that Hsp90 levels kept in high level in normal condition,decreased significantly 6 h later and further decreased 12 h later.(2)We found that knockdown of RIP1 K increased the integrity of the cell structure and the lysosomal membrane in ischemic astrocyteswith TEM.Annexin-V-FITC and PI staining showed that knockdown of RIP1 K decreased necrosis and apoptosis of ischemic astrocytes.AO staining showed that integrity of the lysosomal membrane was increased after RIP1 K knockdown.These findings suggested that knockdown of RIP1 K increases the integrity of the lysosomal membrane in ischemic astrocytes.(3)Genechip showed the up-regulation of Hsp70.1B after RIP1 K knockdown.Western blotting,immunohistochemistry and immunofluorescence analysis further showed an increase in the expression of lysosomal Hsp70.1B after RIP1 K knockdown in ischemic astrocytes.Furthermore,Hsp70.1B knockdown further increased the cerebral infarction volume,aggravated behavioral disorder and decreased the integrity of the lysosomal membrane.AO staining showed that the integrity of the lysosomal membrane was decreased after Hsp70.1B knockdown.These findings suggested that RIP1 K knockdown increases the levels of lysosomal Hsp70.1B and has protective effect on lysosomal membrane.(4)The regulation proteins of Hsp70.1B such as Hsp90,Hsf1 and μ-Calpain were analyzed by western blotting,immunohistochemistry and immunofluorescence,and Co-Immunoprecipitation assays,these results suggested that knockdown of RIP1 K could further decrease the expression of Hsp90 and its combination with Hsf1 in ischemic astrocytes cytoplasm and further increase the translocation of Hsf1 to the nucleus.Conclusion: Knockdown of RIP1 K confers an increase in the integrity of the lysosomal membrane in ischemic astrocytes in a lysosomal Hsp70.1B dependent manner;and its mechanisms may be involved in further decreasing the expression of Hsp90 in the cytoplasm of ischemic astrocytes and the combination of Hsp90 with Hsf1 and then promoting the translocation of Hsf1 to the nucleus.