| Objective：Investigate the role and mechanisms of autophagy-lysosomal pathway on thecell damage of PC12induced by corticosterone.Methods：1. PC12cells were randomly grouped: corticosterone group (set up three dosegroups:1×10-7M;1×10-6M and1×10-5M) and vehicle (DMSO)in the control group. The two groups were experimentally studied afterincubation for24hours. To investigate the effects of corticosterone on thePC12cell damage and the activity of autophagy-lysosomal pathway, the PC12cells activity and apoptosis were detected by MTT assay and flow cytometerwith Annexin-V/PI double stain after treatment with corticosterone,respectively. Western blotting method was used to measure the ratio ofLC3-II/LC3-I, p62and Caspase-3protein levels. Furthermore, to evaluate theextent of autophagosome and autolysosome accumulation, the PC12cells weretransfected with a marker protein, mCherry-GFP-LC3tandem taggedfluorescent protein (tfLC3) and analyzed them by confocal microscopy.2. PC12cells in experimental groups:(1) corticosterone (1×10-5M) group (2)corticosterone+chloroquine (10μ M) group (chloroquine group)(3)corticosterone+rapamycin (20μM) group (rapamycin)(4) solvent (DMSO)control group. These groups were experimentally studied after incubation for24hours. To investigate the role and mechanisms of autophagy-lysosomalpathway in corticosterone-induced damage of PC12cell, cultured PC12cellwere randomly divided into four groups: the control, corticosterone, corticosterone+10μM chloroqunie and corticosterone+20μM rapamycin.The proliferation and apoptosis of cells were detected by MTT and flowcytometry methods, respectively. The ratio of LC3-II/LC3-I, the expression ofp62and Caspase-3protein were measured by Western blotting. Furthermore,the LC3conjugation and the autophagosome maturation process of themRFP-GFP-LC3reporter protein were also characterized by confocalmicroscope analysis.Results：1. MTT results indicated that high-level corticosterone (10-6,10-5M) couldsignificantly reduce activity of PC12cells after treatment of24h. The flowcytometry results demonstrated that high-level corticosterone would markedlyincrease the apoptotic rate of PC12cells. Western blotting results showed thathigh-level corticosterone up-regulate the ratio of LC3-II/LC3-I and theexpression of p62and Caspase-3proteins. Confocal microscopy resultsshowed that high-level corticosterone treatment resulted in the accumulationof yellow autophagosomes (vesicles that appear yellow in the merged images)but lead to a decrease red-only autolysosomes. This profile could be due? todamaged autogaphagic flux.2. The autophagy-lysosomal pathway inhibitor chloroqunie rapidly increased theapoptotic rate, the ratio of LC3-II/LC3-I, as well as and up-regulated theexpressions of p62and Caspase-3proteins in PC12cells treated with1×10-5M corticosterone. Confocal microscopy results indicated that cells treatedwith cloroqunie had a larger increase in autophagosomes, but fewerautolysosomes, compared with untreated cells. Conversely, theautophagy-lysosomal pathway rapamycin decreased the increases theapoptotic rate induced by high level corticosterone, increased the ratio ofLC3-II/LC3-I, while decreased p62and Caspase-3proteins levels. In addition,the number of red puncta was significantly greater after rapamycin treatment,and the yellow puncta were also significantly increased after in the merged images. These results imply that autophagic flux was enhanced in response torapamycin in corticosterone-treated PC12cells.Conclusion:Corticosterone treatment not only damage autophagy-lysosomal pathway, butalso cell activity, thus exerting a harmful effect on the survival of PC12cells.Corticosterone induced PC12cells injury may be through a mechanism ofautophagy-lysosomal pathway.x... |
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