| Objective:Alzheimer's disease?AD?is a progressive neurodegenerative disorder that results in a gradual decline in cognitive processes.The deposition of amyloid-??A??has been considered an extremely critical factor for AD development.Endoplasmic reticulum?ER?as an important organelle,its changes of function have a profound impact on cells.The endoplasmic reticulum stress?ERS?can promote conformation dysfunction of protein and lead to the formation of excess A?,thus triggering a series of pathological changes.Therefore endoplasmic reticulum stress plays an important influence on the onset of Alzheimer's disease.The unfolded protein response?UPR?is normally a protective cellular response that protects against endoplasmic reticulum stress,which occurs with the build up of misfolded proteins.Excessive ERS or ERS mechanism abnormalities are related to AD nervous system damage.ER stress is involved in neurodegenerative diseases including AD.Astrocytes play important role on AD and are largest number of cells in nervous tissue.Inhabiting the astrocytes abnormal changes is good for the treatment of neurodegenerative diseases.In recent years,a large number of studies have found that progesterone?PROG?,as a neuroactive steroid,has neuroprotective effects,but its specific mechanism of endoplasmic reticulum stress is unclear in Alzheimer's disease.There is growing evidence that progesterone achieves neuroprotective actions by progesterone receptor membrane component 1?PGRMC1?.In this study,we using A?1-42 treated the rat cortical astrocytes,investigate the expression of endoplasmic reticulum stress-related proteins,eg,glucose regulated protein78?GRP78?,PKR-like ER kinase?PERK?,eukaryotic initiation factor 2 kinase?eIF2??,investigate the effect of PROG and PGRMC1 on A?1-42-triggered endoplasmic reticulum stress and detail the involvement of the ER PI3K/Akt/GSK3? pathways in AD.Methods:1 Cell cultures1.1 Primary cultures of rat cortical astrocytesPrimary astroglial cell cultures were obtained from cerebral cortices of 1-day neonatal Sprague-Dawley rats,which were minced and subsequently digested for 15 min at 37? 0.125 % trypsin and mechanically dissociated to single cells.Cells were standing and then resuspended in DMEM containing 10 % fetal bovine serum?FBS?.Cultures were incubated at 37? in a 5% CO2 incubator,first renewal the medium at 24 h,then medium renewaled every 2-3 days until confluence.Cultures were purified from contaminating microglia and oligodendrocytes by gentle shaking,200 rpm.The astrocyte purity was over 95 %,determined by GFAP-staining.2 A?1-42 oligomers preparationThe 1mg A?1-42 is dissolved in hexafluoroisopropanol,mixing,packing.To obtain beta peptide,in a hood HFIP were evaporated to dryness,stored at-20?.Before using the DMSO solution,dilutions were further performed in DMEM medium to obtain appropriate concentrations.The peptides were oligomerized at 4? for 24 h to use.3 The GFAP immunofluorescence observe the morphology change of astrocytes.Immunofluorescent analysis of GFAP?an astrocyte-specific intermediate filament component?was performed to observe the morphological activation of astrocytes.After various treatments,astrocytes were fixed with 4 % paraformaldehyde for 30 min and incubated with a rabbit antibody against GFAP?1: 1000?at 4? overnight.After being washed,the cells were treated with the appropriate secondary antibodies for 1 h at room temperature.Cell images were captured with a fluorescence microscope.Astrocytes were treated with A?1-42?1 ?M?for different time,and were randomly divided into Control,A??2 h?,A??4 h?,A??8 h?,and Western blot was performed to detect the changes of GRP78 protein level.4.2 Effect of PROG on the proliferation of astrocytesAstrocytes were randomly divided into Control,PROG?1 ?M?,A?1-42?1 ?M?and A?1-42+PROG used by MTT analysis.4.3 Effect of PROG on the endoplasmic reticulum stress of astrocytesAstrocytes were randomly divided into Control,PROG,A?1-42,A?1-42+PROG,used by western blot analysis.The concentration of A?1-42 or PROG is1 ?M.4.4 The mechanism of A?1-42 induced endoplasmic reticulum stress in astrocytes4.4.1 Astrocytes were randomly divided into Control,A?1-42,A?1-42+PROG,A?1-42+ LiCl?GSK3? inhibitor?used by western blot analysis.The concentration of A?1-42 or PROG is 1 ?M and LiCl is 10 mM.4.4.2 Astrocytes were randomly divided into Control,A?1-42,A?1-42+PROG,A?1-42+PROG +LY294002?PI3K/Akt pathways inhibitor?used by western blot analysis.The concentration of A?1-42 or PROG is 1 ?M and LY294002 is 10 ?M.4.4.3 Astrocytes were randomly divided into Control,A?1-42,A?1-42+PROG,A?1-42+PROG+AG205?PGRMC1 inhibitor?used by western blot analysis.The concentration of A?1-42 or PROG is 1 ?M and AG205 is 10 ?M.5 Cell viability assayThe MTT assay was used to evaluate the viability of the astrocytes cultured in the glass.Briefly,20 ?L MTT was added to the culture media with a final concentration of 3.6 mmol?L-1.After 4 hours of culture,the culture media was removed and 200 ?L DMSO was added to dissolve the crystals.The absorbance of the solution was read at 570 nm on a spectrophotometer and the cell viability was calculated by compare to controls.4 Treatment and grouping4.1 Effect of A?1-42 on endoplasmic reticulum stress in astrocytes6 The relative amounts of GRP78,p-PERK,p-eIf2?,p-Akt,p-GSK3? protein levels were detected by Western blot analysis.Equal amounts of protein?20 ?g?were separated by 10% SDS-PAGE,and electrotransferred to a PVDF membrane.Membranes were blocked at room temperature,and incubated with the primary antibodies,1: 1 000 rabbit anti-GRP78,p-PERK,p-eIf2?,p-Akt,p-GSK3? and rabbit anti-?-actin overnight,respectively,and then with secondary antibody for 2 h.ECL luminescenced and exposure.7 Statistical analysisAll experiments were repeated three times independently.Results are given as the mean±SD.Statistical analysis of data were performed by One-Way ANOVA followed by Dunnett t test with SPSS 16.0.P<0.05 was considered statistical significance.Results:1 A?1-42 induced ER stress in the astrocytes1.1 GFAP staining revealed that astrocytes were flat,full and cytoplasm rich1.2 Western Blot showed that: compared with the control group,the relative expression of GRP78 was significantly increased by A?1-42?1 ?M?treatment for 4 h?P<0.05?.2 Effect of progesterone on astrocytes survival rateMTT results showed that compared with the control group,1 ?M A?1-42 treatment astrocytes for 4 h,cell survival rate is?78.9±2.64?%?P<0.05?.After the united to join A?1-42 and progesterone,compared with the A?1-42 treatment group,cell survival rate is?120.0±1.73?%?P<0.05?.3 Effect of progesterone on A?1-42-induced ER stress of astrocytesWestern Blot showed that: compared with control group,the relative expression of GRP78,p-PERK,p-e If2? was significantly increased by A?1-42 treatment?P<0.05?.Compared with control group,GRP78,p-PERK,p-eIf2? proteins ratio did not show significant difference with the addition of progesterone under basal condition?P>0.05?.Compared with injury group,the relative expression of GRP78,p-PERK,p-eIf2? was significantly decreased?P<0.05?by A?1-42 plus PROG treatment.4 Glycogen synthase kinase3??GSK3??mediates A?1-42-induced ER stress.Western Blot showed that: compared with control group,the relative expression of GRP78,p-GSK3? was significantly increased?P<0.05?by A?1-42 treatment.Compared with injury group,the relative expression of GRP78,p-GSK3? was significantly decreased?P<0.05?by A?1-42 plus PROG treatment;the relative expression of GRP78,p-GSK3? was significantly decreased?P<0.05?by A?1-42 plus LiCl treatment.5 Progesterone down-regulates of GSK3? activation via PI3K/Akt pathway in astrocytes.Western Blot showed that: compared with control group,the relative expression of GRP78,p-GSK3? was significantly increased and p-Akt was significantly decreased by A?1-42 treatment?P<0.05?;Compared with injury group,the relative expression of GRP78,p-GSK3? was significantly decreased and p-Akt was significantly increased?P<0.05?by A?1-42 plus PROG treatment;Compared with A?1-42 plus PROG group,the relative expression of GRP78,p-GSK3? was significantly increased?P<0.05?and p-Akt was significantly decreased?P<0.05?by A?1-42 plus PROG plus LY294002 treatment.6 Involvement of PGRMC1 in progesterone regulating A?-induced endoplasmic reticulum stress.Western Blot showed that: compared with control group,the relative expression of GRP78,p-PERK,p-eIf2? was significantly increased?P<0.05?by A?1-42 treatment.Compared with injury group,the relative expression of GRP78,p-PERK,p-eIf2? was significantly decreased?P<0.05?by A?1-42 plus PROG treatment.Compared with A?1-42 plus PROG group,the relative expression of GRP78,p-PERK,p-eIf2? was significantly increased?P<0.05?by A?1-42 plus PROG plus AG205 treatment.7 Involvement of PGRMC1 in progesterone regulating PI3K/ AKt/GSK3? signaling pathwayWestern Blot showed that: compared with control group,the relative expression of p-AKt was significantly decreased?P <0.05?and p-GSK3? was significantly increased?P <0.05?by A?1-42 treatment.Compared with injury group,the relative expression of p-AKt was significantly increased?P <0.05?and p-GSK3? was significantly decreased?P <0.05?by A?1-42 plus PROG treatment;Compared with A?1-42 plus PROG group,the relative expression of p-AKt was significantly decreased?P <0.05?and p-GSK3? was significantly increased?P <0.05?by A?1-42 plus PROG plus AG205 treatment.Conclusions:1 A?1-42 can induce ER stress in the astrocytes.2 The protective effect of progesterone was through the expression of GRP78,p-PERK,p-e If2? to inhibit A?1-42 induced ER stress.3 PROG may regulate PI3K/Akt/GSK3? signaling pathway that inhibiting A?1-42-triggered ER stress in astrocytes4 The protective effect of progesterone was partially through PGRMC1 to regulate the expression of astrocytes associated proteins inhibiting A?1-42-triggered ER stress. |
PDF Full Text Request