| Autophagy is a lysosomes-dependent degradation/recirculation process found in eukaryotes ranging from yeast to mammals.It is the self-protective mechanism for maintaining neuronal survival and degrading aging cell organelles and misfolded proteins under multiple stress conditions such as ischemia and hypoxia.However,excessive autophagy might induce cell death via direct autophagic cell death or indirect crosstalk with apoptosis.Mounting evidence suggests that autophagy is modulated by a variety of signal pathways.Activin A(Act A),an endogenous neuronal survival factor,elicits its neuroproteictive effects through Act A/Smads signaling pathway during cerebral ischemia.However,to date,the effect of Act A/Smads signaling pathway on autophagy activation during the process of cerebral ischemia has not been clarified.Excessive endoplasmic reticulum stress(ERS)could induce apoptosis,which is a newly discovered signal transduction pathway of apoptosis with the exception of mitochondrial-mediated apoptotic pathway and death receptor-mediated apoptotic pathway.Besides,excessive ERS may also lead to autophagic cell death.Apoptosis and autophagic cell death induced by ERS are critical components of the pathophysiology of cerebral ischemia and a variety of neurodegenerative disorders.Growing body of evidence suggests that Act A/Smads signaling exerts the neuroprotective role via suppression of mitochondrial-mediated apoptotic pathway.However,the effects of Act A/Smads signaling on ERS-mediated apoptosis and autophagic cell death remain unclear.In this study,we established the Oxygen-Glucose Deprivation(OGD)model and ERS model to examine the effects of Act A/Smads signaling on ERS-mediated apoptosis during cerebral ischemia.Besides,on the basis of OGD model of PC12 cells,we investigated the effects and mechanisms of Act A/Smads pathway on autophagy during cerebral ischemia.We further examined the effects of Act A/Smads signaling on ERS-induced autophagic cell death using ERS model of PC12 cells.1.Culture and identification of neuronal PC12 cells and establishment ofOxygen-Glucose Deprivation(OGD)model and ERS model.Objective: To establish the OGD model and ERS model of neuronal PC12 cells.Methods: NGF was used to stimulate PC12 cells into neuronal cells;Glucose free DMEM and Na S2O4 were introduced to neuronal PC12 cells to establish OGD model;Thapsigargin(TG),a representative ERS inducer,was introduced to PC12 cells to establish ERS model;Neuronal PC12 cells were identified by immunofluorescence of MAP2;CCK-8 essay and immunofluorescence staining of Hoechst33342 were used to examine the survival rates and apoptosis nuclei after OGD 0,3,6,9 and 12 h.CCK-8 essay was used to examine the survival rates after TG treatment at the concentration of 0.5μM、1μM、2μ and 5μM.Results: The DMEM complete medium with a final concentration of 100ng/ml of NGF was introduced to PC12 cells.PC12 cells showed a typical neuronal morphology with the increase in cell culture time.The immunofluorescence staining of MAP2 in the differentiated PC12 cells was strongly positive;CCK-8 assay showed that the survival rates of cells decreased gradually with extension of OGD,and Hoechst33342 staining showed that the number of dense blue nuclei increased gradually with extension of OGD;CCK-8 showed that the survival rates of cells decreased gradually with the increase of TG concentration.Conclusion: PC12 cells were differentiated into neurons after NGF stimuli;The model of OGD was established successfully;The model of ERS was established successfully.2.The effects of Act A/Smads signaling on ERS-mediated apoptosis in OGD-treated PC12 cellsObjective: To investigate the effects of Act A/Smads siganling on ERS-mediated apoptosis and unfolded protein response(UPR)signaling pathway in OGD-treated PC12 cells.Methods: On the basis of OGD model and ERS model of PC12 cells,Western blot was used to examine the protein levels of Act A and p-Smad3 to determine the effects of exogenous Act A on Act A/Smads signaling in PC12 cells subjected to OGD and ERS injury;CCK-8 assay and immunofluorescence staining of Hoechst33342 were used to detect the effects of exogenous Act A on cell death induced by OGD injury and ERS injury;Protein levels of GRP78,CHOP and cleaved-caspase12,three well-known markers of ERS,were examined by Western blot;Protein levels of ERS-associated proteins including IRE1,TRAF1,ASK1,p-PERK,e IF2α,p-e IF2α,ATF4,ATF6 and cleaved-caspase3 were examined by Western blot.Results: Western blot showed that the protein levels of Act A and p-Smad3 were up-regulated significantly when cells were subjected to OGD injury and ERS injury,and the protein levels were further enhanced by exogenous Act A in a concentrationdependent manner;CCK-8 showed that the cell survival rate was significantly reduced by OGD injury and ERS injury,which was improved by exogenous Act A;Hoechst33342 staining showed that the number of apoptotic cells with dense blue nuclei increased after OGD injury and ERS injury,which was significantly decreased by exogenous Act A;Western blot showed that the protein levels of CHOP,GRP78,cleaved-caspase12 and cleaved-caspase3 were increased after OGD injury and ERS injury,which were reduced by exogenous Act A;Western blot showed that ERSassociated proteins including IRE1,TRAF1,ASK1,p-PERK,e IF2α,p-e IF2α,ATF4 and ATF6 were all upregulated after OGD injury and ERS injury,while exogenous Act A inhibited the changes of IRE1,TRAF1,ASK1,p-PERK,p-e IF2α and ATF4.The levels of e IF2α and ATF6 showed no significant changes after Act A treatment.Conclusion: Act A/Smads signaling could reduce the ERS-mediated apoptosis during cerebral ischemia,which might be associated with the suppression of IER1-TRAF2-ASK1 signaling and PERK-e IF2α-ATF4 signaling.3.The effects of Act A/Smads signaling on OGD-induced autophagy in neuronalPC12 cellsObjective: To investigate the effects of Act A/Smads signaling on the regulation of autophagy during cerebral ischemia.Methods: The dynamic changes in protein levels of LC3 and Beclin1,two wellknown markers of autophagy,during OGD injury were examined by Western blot;Exogenous Act A and Act RIIA-Ab were used to enhance and inhibit the strength of Act A/Smads signaling,respectively.JNK1 inhibitor SP600125 was used to block JNK1 signaling.P38 inhibitor SB203580 was used to block p38 signaling.The effects of Act A、Act RIIA-Ab、SP600125 and SB203580 on OGD-induced autophagy in neuronal PC12 cells were examined by Western blot and Immunofluorescence staining;The effects of Act A and Act RIIA-Ab on the OGD-induced activation of JNK1 and p38 were examined by Western blot.Results: Western blot showed that LC3 and Beclin1 were both progressively upregulated with the extension of OGD injury;The OGD-induced expression of LC3 IIand Beclin1 were both suppressed by treatment with exogenous Act A in a concentration-dependent manner and upregulated significantly by Act RIIA-Ab.These results were further confirmed by Monodansylcadaverine(MDC)and Acrodine orange(AO)staining;Western blot showed that either SB203580 or SP600125 markedly attenuated OGD-induced elevation in the protein levels of Beclin1 and LC3II;The OGD-induced elevation in the protein levels of JNK1,p-JNK1,p38 and p-p38 was inhibited by exogenous Act A and upregulated by Act RIIA-Ab significantly.Conclusion: Prolonged OGD injury resulted in a significant increase in the autophagy activation in a time-dependent fashion in PC12 cells;Act A/Smads signaling pathway negatively regulated the OGD-induced autophagy in PC12 cells;JNK1 and p38 pathways were involved in the OGD-induced autophagy in PC12 cells;JNK1 and p38 were the downstream factors of IRE1-TRAF2-ASK1 signaling.Combined with the 2nd part of experiment,the negative effects of Act A/Smads on OGD-induced autophagy might be associated with the suppression of IRE1-TRAF2-ASK1-JNK1/p38 pathway.4.The effects of Act A/Smads signaling on ERS-induced autophagic cell death inPC12 cells.Objective: To investigate the effects of Act A/Smads signaling on ERS-induced autophagic cell death in PC12 cells.Methods: On the basis of ERS model,3-methyladenine(3-MA),a specific inhibitor of autophagy,was used to inhibit the activation of autophagy.JNK1 inhibitor SP600125 was used to block JNK1 signaling.P38 inhibitor SB203580 was used to block p38 signaling;Western blot was used to examine the protein levels of LC3 and Beclin1.AO staining was used to detect the strength of autophagy;Western blot was used to examine the protein expression of phosphorylation of JNK1 and p38.Results: Compared with DMSO group,the expression of LC3 IIand Belcin1 were all upregulated in TG group,which were decreased in TG+Act A group and TG+3-MA group;AO staining showed that TG treatment induced far more red spots of AO in the cytoplasm of PC12 cells.However,the alteration was suppressed by treatment with Act A and 3-MA;CCK-8 showed that the cell survival rate was significantly decreased in TG group compared with DMSO group,which was increased in TG+Act A group and TG+3-MA group;Western blot showed that either SB203580 or SP600125 markedly attenuated ERS-induced elevation in the protein levels of Beclin1 and LC3 II.The ERSinduced elevation in the protein levels of JNK1,p-JNK1,p38 and p-p38 was inhibited by exogenous Act A.Conclusion: Autophagy was involved in the cell death induced by ERS;Act A/Smads signaling could reduce the ERS-induced autophagic cell death in PC12 cells;JNK1 and p38 pathways were involved in the ERS-induced autophagy in PC12 cells;Combined with results of the 2nd part of experiment,the inhibitory effects of Act A/Smads signaling on ERS-induced autophagic cell death might be associated with the suppression of IER1-TRAF2-ASK1-JNK1/p38 signaling and PERK-e IF2α-ATF4 signaling. |
