| The development and regeneration of the central nervous system has always been a research in the field of Neuroscience.Recent studies have demonstrated that neural stem cells in the hippocampal dentate gyrus and subventricular zone retain the ability to proliferate and that a significant number of the daughter cells develop into neurons in adult mammalian species-neurogenesis.This is reinforced by evidence that increased survival of newborn neurons is strongly correlated with hipppcampus-dependent memory,while the inhibition of neurogenesis has adverse effects on hippocampus-depentend behaviors,thus implying that hippocampal neurogenesis contributes,at least in part,to learning and memory.Alzheimer's disease?AD?is a progressive neurodegenerative disorder that results in a gradual decline in memory and cognitive processes.The pathology of the disease is characterized by senile plaques,intracellular neurofibrillary tangles,and extensive neuronal loss.Whether newborn neurons can replace neurons in the lesion-neuroregeneration,improving cognitive impairment has become a new focus in AD research.The number of immature newborn neurons is decreased in the hippocampus of AD patients.It is also know that the overproduction and subsequent aggregation of A? limit the survival of hippocampal newborn neurons.The changes in cell cycle and proliferation related signaling pathways are the two main mechanisms inhibit cell proliferation.The aggregation of A? causes it to have a neuronal cytotoxicity that disrupts the integrity of the cell membrane and causes intracellular DNA breaks,resulting in an abnormal cell cycle.In the AD patients,the level of c AMP in hippocampus decreased,and the level of CREB phosphorylation decreased.This confirms the close relationship between CREB and AD.Steroid hormones and their metabolites within the central nervous system are commonly defined as neurosteroids.Evidence suggests progesterone exerts its neuroprotective effects through several pathways,including improving neuronal survival,and inhibiting apoptosis and inflammatory reaction.Progesterone can regulate the expression of PI3K/AKt and significantly increase the expression of BDNF.The exact mechanism by which progesterone exerts protective effects in the central nervous system remains unclear.On the one hand,progesterone binds to its classical nuclear receptors and regulates through classical gene pathways;On the other hand,progesterone binds to GABAA receptors,NMDA receptors,and PGRMC1/2 receptors on cell membranes,and it is used through non-genetic pathways.Recently,there is growing evidence that progesterone achieves neuroprotective actions by non-classic progesterone receptors mechanisms.Progesterone receptor membrane component 1,a prominent candidate mediating the nongenomic signaling events of progesterone,was suggested to be involved in neuroprotective actions.In this study,A?25-35 was used to establish AD cell model in rat hippocampal neural stem cells and APP/PS1 transgenic mice were used as AD animal models,which investigated the effect and mechanisms of progesterone on Neuroregeneration at the cellular and animal level.Part one Effect of progesterone on neuroregeneration in AD cell model Objective: To observe the effect of progesterone on the survival,proliferation,apoptosis and differentiation of neural stem cells in A?25-35-induced AD model.Methods: Issociated hippocampal neural stem cells were prepared from postnatal day 0 Sprague Dawley rats,and determined the neural stem cells purity with Nestin antibody stain,and differentiation of neuronal marker ?III-Tubulin,and astrocyte marker GFAP;The rat hippocampal neural stem cells were treated with A?25-35 to establish an AD cell model.CCK-8,Brdu Elisa,Western blot and flow cytometry were used to study the effects of progesterone on A?-induced neural stem cell survival,proliferation,apoptosis,and differentiation.Results:1.Neural stem cells purity are more than 90%,it can be induced to differentiate into neurons and astrocytes.2.Effects of progesterone on neural stem cells survival,proliferation and apoptosis after A?25–35 treatmentCCK-8 assays revealed that 20?M A?25-35 exhibited a significant decrease in cell survival?59.3±6.4?%?P<0.01?.Co-incubation with progesterone significantly improved neural stem cells survival in a concentration-dependent and time-dependent manner.Immofluorescence results showed that the Brdu antibody was located accurately in the proliferating cell nucleus.Compared with the control group,the number of Brdu positive cells were significantly decreased under A? incubation?P<0.01?.Compared with A? group,the number of Brdu positive cells in progesterone protection group increased significantly?P<0.01?.This result was consistent with Brdu elisa.Western blots results showed that cleaved-caspase 3 expression level under A? challenge was about 2.6 times relatively to normal neural stem cells.Compared with AD modle group,there was no significant difference in the expression of cleaved-caspase 3 after co-incubation of A? with PROG?P>0.05?.3.Effect of progesterone on differentiation of NSCs in AD cell modelImmofluorescence results showed that compared with the A? group,the number of DCX+ positive cells in the PROG protection group was significantly increased,and cell synapses were significantly prolonged?P<0.01?.Compared with the control group,the percentage of neural stem cells differentiated into ?III-Tubulin,GFAP positive cells in the A? group was significantly reduced?P<0.01?.Compared with the A? group,Co-incubation with progesterone the percentage of neural stem cells differentiated into ?III-Tubulin positive cells was significantly increased?P<0.01?,and the percentage of cells differentiated into GFAP positive cells was not significantly different?P>0.05?.Conclusions:1.Progesterone can increase the survival rate of neural stem cells induced by A?25-35 in a dose and time dependent manner,and promote the proliferation of NSCs.2.Progesterone promotes the regeneration of neural progenitor cells and promotes synapse growth induced by A?25-35?At the same time reversed the role of A?25-35 in inhibiting differentiation and promoting differentiation into neurons.Part two Effects of progesterone on cell cycle of neural stem cells in AD cell modelObjective: To study the effect of progesterone on the cell cycle and the expression of Cyclin D1 and PCNA of neural stem cells in AD model.Methods:Under A?25–35 induced AD cell model,the cell cycle was detected by using the flow cytometry after progesterone incubation;we examined the expression of Cyclin D1 and PCNA by Western blotting.Results:1.Effect of Progesterone on the cell cycle of NSCs in AD Cell ModelCompared with the control group,cells in the G0/G1 phase of the A? group increased significantly,S phase cells significantly decreased,and the proliferation index?PI?decreased significantly?P<0.05?.Compared with the A? group,progesterone-protected group significantly decreased G0/G1 phase cells,significantly increased S phase cells,and significantly increased PI?P<0.05?.2.Effects of progesterone on the expression of Cyclin D1 and PCNA in NSCs of AD cell modelWestern blot showed that the expression of Cyclin D1 and PCNA protein in A? group was significantly lower than that in control group?P<0.01?.Compared with A? group,the expression of Cyclin D1 and PCNA protein in progesterone protection group was significantly increased?P<0.05?.Conclusions:Progesterone promotes the proliferation of neural stem cells by reversed the number of G0/G1 phase cells block caused by A? and up-regulated the expression of Cyclin D1 and PCNA.Part three Effects of progesterone on AKt/CREB/BDNF signaling pathway of Neural Stem Cells in AD Cell ModelObjective: The contribution of AKt/CREB/BDNF signaling to the neuroprotective effect of progesterone was investigated.Methods:Western blot,Brdu incorporation,and flow cytometry were used to detect CREB/BDNF signaling?proliferation and differentiation after neural stem cells exposed to A? were pretreated with CREB activator Rolipram or treated with progesterone for 48 h in combination;Apply the above method to detect PI3K/AKt signaling?proliferation and differentiation after neural stem cells exposed to A? were pretreated with PI3K/AKt Pathway Inhibitors LY294002 or treated with progesterone.Results:1.Effects of progesterone on CREB/BDNF of neural stem cells in AD cell modelWestern-blot results showed that compared with the A? injury group,both PROG protection group and Rolipram treatment group could significantly increase the expression of p-CREB?Ser133?and BDNF protein?P<0.05?,the expression levels of T-CREB in each group had no significant difference?P>0.05?.Brdu Elisa and flow cytometry results showed that compared with the model group,PROG protection group and Rolipram group significantly promoted the proliferation of neural stem cells and differentiation into neurons?P<0.05?.2.Effects of PI3K/AKt on neural stem cells in progesterone AD cell modelWestern blot results showed that compared with PROG protected group,the expression of p-AKt protein in LY294002 treatment group was significantly decreased?P<0.05?,and the expression levels of T-AKt and T-CREB in each group had no significant difference?P>0.05?.In addition,the levels of p-CREB and BDNF in NSCs of all treatment groups were consistent with that of p-AKt.Brdu Elisa and flow cytometry results showed that compared with the model group,the progesterone group significantly promoted neural stem cell proliferation and differentiation into neurons?P<0.05?.Compared with PROG-protected group,LY294002 pretreatment group significantly inhibited the proliferation of NSCs and differentiated into neurons?P<0.05?.Conclusions:Progesterone promotes the proliferation of neural stem cells and differentiation into neurons in AD cell model by regulating the PI3K/AKt-CREB signaling pathway and promoting the expression of BDNF.Part four Study on the role of PGRMC1 progesterone promotes of neuroregeneration in AD cell modelObjective:To investigate whether PGRMC1 mediates progesteroneinduced proliferation and differentiation of neural stem cells and regulates AKt/CREB/BDNF pathways in AD cell models.Methods:The expression of PGRMC1 and classic PR in neural stem cells was detected by Immofluorescence;Western blot was used to detect the effect of A? on PGRMC1 expression in neural stem cells;PGRMC1 lentivirus was constructed and used to transfect NSCs to select the best MOI and time;PCR and Western Blot were used to detect the expression of PGRMC1 gene and protein after transfection of neural stem cells by lentivirus and the effect of lentivirus on proliferation of neural stem cells;Western Blot,Brdu Elisa and flow cytometry used to detect the effect of PGRMC si RNA on the proliferation and differentiation of neural stem cells and the expression of AKt,CREB and BDNF.Results:1.PGRMC1 are in neural stem cells body and nucleus processes,While no expression of classic PR.2.Effect of A? on expression of PGRMC1 in neural stem cellsWestern blot results showed that after 48 hours of A? treatment,PGRMC1 expression was significantly increased.Compared with control group,the relative expression of PGRMC1 in A? group increased by 67%?P<0.05?.3.Lentiviral transfection NSCs screened for optimal MOI and timeEach group of neural stem cells was added to the lentivirus in the culture medium and observed under a fluorescence microscope.After transfection of lentivirus for 48 hours,the ENi.s+Poly group had stronger green fluorescence intensity than the other groups of GFP.When the MOI value was 20,the green fluorescence of GFP was uniformly distributed and expressed in large quantities.After 48 hours,the green fluorescence of GFP was highly expressed compared with the control group.The best culture medium for NSCs transfected with lentivirus was ENi.s+Poly,the MOI value was 20 and the time was 48 hours.4.Changes of PGRMC1 gene,protein expression and the proliferation after transfection of NSCs by lentivirusCompared with the control group,the expression of gene and protein of si RNA 61634 was significantly decreased?P<0.05?.Brdu Elisa results showed that compared with the control group,each transfection group did not affect cell proliferation,the difference was not statistically significant?P>0.05?.This shows that the interference effect of si RNA 61634 is better for subsequent experiments.5.Effect of PGRMC1 si RNA on AKt/CREB/BDNF Signaling Pathway.Western blot results showed that compared with the progesterone group,the expression of p-CREB protein was significantly decreased in si RNA 61634 treated group?P<0.05?.The changes of p-AKt and BDNF in rat hippocampal neural stem cells were consistent with that of p-CREB.Brdu Elisa and flow cytometry results showed that compared with the progesterone-protected group,si RNA 61634 treated group significantly inhibited the proliferation and differentiated into neurons?P<0.05?.Conclusions:PGRMC1 may mediate the effect of progesterone on the regulation of AKt/CREB/BDNF pathway and promote the proliferation and differentiation of neural stem cells.Part five Effects of progesterone on neuroregeneration and learning and memory in APP/PS1 miceObjective:To study the effects of progesterone on the survival,differentiation of mature neurons,and learning and memory of newborn neurons in the hippocampus of APP/PS1 transgenic mice and the related mechanisms.Methods:APP/PS1 transgenic mice were selected as animal models of AD and progesterone was injected subcutaneously in the neck;The number of 28day-old Brdu+,Brdu+/Neu N+ cells in the hippocampus,and the expression of p-AKt and p-CREB proteins were detected by immofluorescence;The Elisa kit was used to detect the changes of BDNF in serum;Morris water maze was used to detect changes in learning and memory behavior in mice.Results:1.Effects of progesterone on survival and differentiation of mature neurons of newborn neurons in hippocampus of APP/PS1 transgenic miceImmofluorescence results showed that compared with APP/PS1 transgenic mice,progesterone treatment increased the number of 28day-old Brdu+ positive cells and Brdu+/Neu N+ cells in APP/PS1 mice?P<0.05?.2.Effects of progesterone on AKt/CREB/BDNF signaling pathway in hippocampus of APP/PS1 transgenic miceImmofluorescence results showed that the coloring of p-AKt and p-CREB in brain regions of APP/PS1 mice was not obvious.Compared with APP/PS1 transgenic mice,the p-AKt and p-CREB in brain regions of mice treated with progesterone was significantly increased?P<0.05?.Elisa results showed that BDNF in the blood supernatant after progesterone treatment was significantly higher than APP/PS1 transgenic mice?P<0.01?.3.Effects of progesterone on learning and memory behavior of APP/PS1 transgenic MiceMorris water maze results shows that there was no statistical difference between the average speed of the three groups of mice?P>0.05?.Compared with progesterone group,the mean escape latency in the morris water maze behavioral task was significantlyhigher in APP/PS1 group?P<0.01?.Moreover,the number of platform crossing sand the staying time ratio in platform quadrant of progesterone group was significantly increased compared with those of APP/PS1 group?P<0.05?.Conclusions:Progesterone can improve spatial learning and memory ability of APP/PS1 transgenic mice and may be related to promoting hippocampal neurogenesis,activating AKt/CREB/BDNF signaling pathway.Conclusions1.Progesterone can increase the survival rate of neural stem cells induced by A?25-35 in the concentration and time dependent manner and promote the proliferation of NSCs.And play a role in promoting the regeneration of neuronal precursor cells and their synaptic growth,and promote neuron-like differentiation.2.Progesterone can reverse A?-induced G0/G1 phase cell arrest,up-regulate the expression of Cyclin D1 and PCNA,promote the activation of PI3K/AKt-CREB signaling pathway,up-regulate the expression of BDNF and promote AD cell model in proliferation and neuron-like differentiation of neural stem cells.3.Progesterone regulates the AKt/CREB/BDNF signaling pathway through PGRMC1.4.Progesterone promoted neurogenesis in hippocampus of APP/PS1 mice,activated AKt/CREB/BDNF signaling pathway,and improved spatial learning and memory of APP/PS1 transgenic mice. |
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