Effect And Mechanism Of PROG On Proliferation And Differentiation In Neural Stem Cells Injury By Aβ_25-35

Objective:Alzheimer’s disease（AD） is a common degenerative disease that is clinically manifested by progressive memory impairment and cognitive dysfunction within the central nervous system. In this process, β-amyloid（Aβ） deposition has been recognized as the core pathogenesis of AD. The deposition of Aβ in brain may cause a series of reactions including local inflammation of brain tissue, which will then lead to reduced synapses and neuronal apoptosis, thus eventually resulting in the initiation of AD. Neural stem cells（NSCs） are cells capable of self-renewal and multiple differentiation potential in central nervous system. Under certain conditions, NSCs can be induced for differentiation, proliferation and migration to repair deletion of nerve cells and other diseases caused by various pathological mechanisms. However, the survival and differentiation rates of NSCs in the brains of AD patients are relatively low and manifested as progressive and persistent neuron loss, suggesting that the proliferation and differentiation of NSCs are inhibited and cannot supplement the lost neurons in the brain.The local environment determines the ultimate fate of NSCs, and the neurosteroids levels are an important factor in regulating the local microenvironment. PROG plays a protective effect mainly through inhibiting nervous system damages caused by central excitability, resisting inflammation and apoptosis, and neurotrophy. Therefore, the studies on inhibition of Aβ damage in the brain of AD patients and on the drug-induced endogenous NSCs differentiation will contribute to the recovery of neurological function, which can further provide new approaches and methods for the treatment of AD. In this paper, with NSCs isolated from the hippocampus of neonatal rats as research objects, which were then treated with Aβ toxic fragment Aβ_25-35, the effect of PROG on Aβ_25-35-affected NSCs proliferation and differentiation as well as the corresponding mechanism were investigated.Methods: 1 Isolation and culture of rat neural stem cellsPrimary neural stems cell cultures were isolated from the hippocampi of 1-day neonatal Sprague-Dawley rats, which were minced and subsequently digested for 15 min at 37?C 0.125% trypsin/EDTA and mechanically dissociated to single cells. Cells were standing and then resuspended in DMEM/F12 containing 10% fetal bovine serum（FBS）. They were cultured in DMEM/F12 medium, supplemented with 2% B₂₇ and growth factor（EGF20ng/ml, b FGF20ng/ml）. Cultures were incubated at 37?C in 5% CO₂, the medium renewal every 2 days until 6 days. When neural stems cells become neurospheres, using acctuase enzyme passage. 2 Induced differentiation of neural stem cellsTake the subcultured NSCs, triturated into single cells. The cells were seeded on the culture plates precoated with poly-L-lysine in the DMEM/F12 medium containing 10% fetal bovine serum（remove b FGF and EGF） at the density of 1.0 × 10⁶ per well. Cultures were incubated at 37?C in 5% CO₂ for 6 days, followed by changing half medium every 2 days. 3 Condensed Aβ_25-35 preparationThe 2mg Aβ_25-35 is dissolved in 1000 ul sterile water and made into 2mmol/L liquid store in the refrigerator, mixing, packing. 4 Treatment and grouping 4.1 Effect of Aβ_25-35 and Aβ_25-35+PROG on proliferation in neural stem cellsThe cultures of NSCs were treated with or without Aβ_25-35（0.5 μM, 1.5μM, 5μM, 15μM, 50μM）, used to the research of MTT; Divided into 6 groups treated with Aβ_25-35 and PROG: Control, Aβ_25-35（0.5μM）, Aβ_25-35+PROG（1μM, 10μM, 100μM, 500μM） used by MTT analysis. 4.2 Effect of Aβ_25-35 and Aβ_25-35+PROG on differentiation in neural stem cellsThe induced differentiation of neural stem cells were randomly divided into Control, Aβ_25-35（0.5μM）, Aβ_25-35+PROG（10μM） used by immunocytochemistry, Real-time PCR analysis. 4.3 Mechanism of PROG on differentiation in neural stem cells injury by Aβ_25-35The induced differentiation of neural stem cells were randomly divided into Control, Aβ_25-35, Aβ_25-35+PROG, Aβ_25-35+PROG+RU-486, Aβ_25-35+PROG +AG-205 used by flow cytometry analysis.The induced differentiation of neural stem cells were randomly divided into Control, Aβ_25-35, Aβ_25-35+PROG, Aβ_25-35+ PROG+AG-490 used by western blot analysis. The concentration of Aβ_25-35 is 0.5μM and PROG, RU-486, AG-205 and AG-490 are 10μM respectively. 5 Cell viability assayThe MTT assay was used to evaluate the viability of the NSCs cultured in the glass. Briefly, 20 μL 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 μL DMSO was added to dissolve the crystals. The absorbance of the solution was read at 490 nm on a spectrophotometer and the cell viability was calculated by compare to controls. 6 Immunofluorescence of Nestin、βⅢ-Tubulin and GFAPAfter various treatments, the neural stem cells were fixed with 4% paraformaldehyde for 30 min and incubated with antibody against Nestin at 4?C overnight. After being washed, the cells were treated with the appropriate Alexa Fluor 594 secondary antibodies for 1 h at room temperature. Cell images were captured with a fluorescence microscope.After various treatments, the induced differentiation of neural stem cells were incubated with antibody against βⅢ-Tubulin and GFAP at 4?C overnight. After being washed, the cells were treated with the appropriate Alexa Fluor 488 and 594 secondary antibodies for 1 h at room temperature for double immunofluorescence experiments. 7 Real-time PCR analysis the relative expression of βⅢ-Tubulin and GFAP m RNAWe used Trizol reagent kit for total RNA isolation. c DNA synthesis and PCR amplification was carried out using two-step RT-PCR kit with SYBR green. RT-PCR and melting curve analysis were performed in multiplicate in optimized conditions as described by manufacturer. The cycle number（CT） at which the signals crossed a threshold set within the logarithmic phase was recorded and the differences in cycle threshold（△△Ct） were used to analyze the gene expression in the experimental group and control group :experimental group /control group = 2_{-△△Ct}. 8 Percentage of βⅢ-Tubulin and GFAP positive cells by flow cytometry analysisAfter various treatments, the induced differentiation of neural stem cells are mixed and drilled. Adding βⅢ-Tubulin monoclonal antibody, then PE labeled secondary antibodies, avoid light 30 minutes flow cytometry analysis. GFAP uses the straight standard method, GFAP monoclonal antibody with PE conjugated 30 minutes were detected, avoid light, flow cytometry analysis. 9 The relative amounts of t-JAK2, t-STAT3, p-JAK2, p-STAT3 protein levels were detected by Western blot analysis.Equal amounts of protein（20μg） were separated by 10% SDS-PAGE, and electrotransferred to a PVDF membrane. Membranes were blocked with 5 % BSA for 2 h at room temperature, and incubated with the primary antibodies, 1: 1000 rabbit anti- JAK2, STAT3, p-JAK2 and p-STAT3, and rabbit anti β-actin overnight, respectively, and then with secondary antibody for 2 h. ECL luminescenced and exposure.10 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 Culture and identification of neural stem cellsIsolated from the hippocampus of newborn rats neural stem cells, grown in serum-free medium after 1day the majority of cells death, a small part of cells aggregates formed in 2⁴ cells group. After 7days become dozens to hundreds of cells colony, they have clear boundary, strongrefraction. Nestin immunofluorescence staining showed that NSCs of red fluorescence.2 Induced differentiation of neural stem cellsUsing the DMEM/F12 medium containing 10% fetal bovine serum（remove b FGF and EGF） induced differentiation of neural stem cells. After 12 h see more cells crawl from single cells. When cultured 24 h, a few cells differentiate into cells which have synaptics. Immunofluorescence staining showed that the terminal cells, neural stem cells can differentiate into various corresponding specific marker expression: expression of βⅢ-Tubulin is neurons and expression of GFAP is astrocytes.3 Effect of Aβ_25-35 and Aβ_25-35+ PROG on proliferation in neural stem cellsMTT results showed that compared with the control group, 0.5μM Aβ_25-35 can obviously reduce the formation of neural stem cell spheres, cell survival rate decreased by 32.5%（P<0.05）. After the United to join Aβ_25-35 and different concentrations of progesterone, compared with the injury group, 10μM PROG makes cell survival rate decreased by 15%（P<0.05）.4 Effect of Aβ_25-35 and Aβ_25-35+PROG on differentiation in neural stem cellsImmunofluorescence showed that: Aβ_25-35 can significantly decrease the expressions of βⅢ-Tubulin and GFAP compared with the control group（P<0.05）; Compared with injury group, the expressions of βⅢ-Tubulin and GFAP increased more significantly by Aβ_25-35 plus PROG treatment.Real-time PCR showed that: compared with the control group, the relative expression of βⅢ-Tubulin and GFAP decreased 50%, 70% respectively treated with Aβ_25-35（P<0.01）; compared with injury group, the m RNA of βⅢ-Tubulin and GFAP significantly increased by PROG treatment（P<0.001）.5 Study on the mechanism of progesterone promotes differentiation of stem cells injury by Aβ_25-35Flow cytometry analysis showed that: compared with the control group, percentage of βⅢ-Tubulin positive cells and GFAP positive cells are significantly decreased by Aβ_25-35 treatment（P<0.01）. Compared with injury group, percentage of βⅢ-Tubulin positive cells and GFAP positive cells are significantly increased by PROG treatment（P<0.01）. Compared with Aβ_25-35 plus progesterone group, percentage of βⅢ-Tubulin positive cells and GFAP positive cells are significantly decreased by AG-205 treatment group（P<0.001）; However, percentage of βⅢ-Tubulin positive cells and GFAP positive cells by RU-486 treatment group has not statistically significant difference（P>0.05）.Western Blot showed that: compared with the control group, the relative expression of p-JAK2, p-STAT3 was significantly increased by Aβ_25-35 treatment（P<0.05）. Compared with injury group, the relative expression of p-JAK2, p-STAT3 was significantly decreased by PROG treatment（P<0.05）. Compared with Aβ_25-35 plus PROG group, expression of p-JAK2, p-STAT3 was significantly decreased by AG-490 treatment（P<0.05）.Conclusions:1 In vitro conditions,neural stem cells can isolated from the hippocampi of neonatal rats and cultured in serum-free medium. The removal of b FGF and EGF in medium adding 10%FBS, NSCs can be induced for differentiate into neurons, astrocytes.2 Aβ_25-35 can does dependently decrease the viability of NSCs and inhibit differentiation of neural stem cells.3 PROG can decrease the proliferation and promote differentiate into neurons and astrocytes of NSCs injury by Aβ_25-35.4 PROG may inhibit JAK2/STAT3 signaling pathway that promoting NSCs differentiation injury by Aβ_25-35, the underlying mechanism involves the progesterone receptor membrane components1.