The Investigation Of Anti-neuroinflammation Mechanism Of Granulocyte Colony-stimulating Factor In Interference Of Alzheimer's Disese

BackgroundAlzheimer'S disease named as a German psychiatrist, is a neurodegenerative disorders correlated wim age, approximately 60 percent in dementia. More and more olds are bothered by AD, and AD has been the fourth cause of old people's death next to cardiovascular diseases,cancer,stroke. This disease not only cause the decline of living levels of old people but also bring the burden of enconomy and mentality to the society and family. So, it is most improtant for us to find the effective treatment of AD.Accroding to the current research of cytology and microbiology,β-amyloid protein is the main reason of the death of neurons.β-amyloid protein is composed of neurofibrillary tangles and senile plaques. Aβcombined with receptors on microglia can active the microgalia and cause the secrete of pro-inflammatory factors, such as, IL-1, TNF-α, IL-6. They will cause the function and behavioral disturbance. Latest proofs indicated that mediators of inflammation will aggravate the inflammation processing through up-regulate theβ-secretase.Many reports indicated that anti-inflammatory agents may degrade the risk of suffer AD. To conclusion, chronic inflammation is the main pathology of AD. Anti-inflammation should be the potential treatment of AD.Granulocyte colony-stimulating factor (G-CSF), a hematopoietic factor, is a stimulator of neutrophil differentiation, proliferation and function. It is used clinically to treat neutropenia. Recently, reports described G-CSF with anti-apoptosis, anti-inflammation, neurotrophy, and angiogenesis roles related to neuroprotection. G-CSF decreases the levels of interleukin-1β(IL-1β), IL-6, and IL-8 in several conditions. G-CSF administration can rescue cognitive abilities through anti-inflammation in AD and in mice models of cerebral ischemia, but the precise mechanism of G-CSF reducing the secretion of proinflammatory cytokines remain unknown.Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels widely expressed in mammalian cells. They are composed of a diverse pentameric assembly ofα-andβ-subunits. The subtype a7 nAChR is one of the predominant nAChRs in the brain. It is extensively expressed in microglia, astrocytes and neurons.α7 nAChR plays a crucial role in modulating postsynaptic transmission. Moreover,α7 nAChR decreases the level of inflammatory mediators, including TNF-a, IL-1β, IL-6, and IL-18. As a regulator of neuroinflammation, the increased expression of a7 nAChR can attenuate the expression of pro-inflammation. Up-regulate the expression ofα7 nAChR could improve the cognitive ability of AD patients.G-CSF can cure AD via anti-inflammatory. As the new strategy of AD treatment, we will investigate its ability of anti-inflammatory and possiable mechanisms of anti-inflammation. These will provide evidents for the cure of AD. There are four parts of my research: Part 1 Influence of G-CSF on the cognitive function of the Alzheimer's disease mice modelObjective:To investigate ethology and pathological changes of Alzheimers disease mice model and wild mice, Morris water maze was used to evaluate their abilities of spatial learning and memory. This research discussed the influence of the G-CSF upon the cognition function of Alzheimers disease mice model and wild mice.Methods:1. Alzheimers disease mice model and wild mice were divided into G-CSF group, control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.2. The mice were given Morris water maze examination before and after G-CSF administration.3. Mice were perfused with 4% paraformaldehyde and the brains were used to made pathological sections. Hippocampal pathological changes were observed with haematoxylin & eosin (HE) and Nissl staining.4. To observe the influence of the G-CSF upon the cognition function of Alzheimers disease mice model and wild mice, statistics software was used to analyse the experimental statements of Morris water maze examination before and after G-CSF administration.Results1.Morris water maze:Spatial acquisition:In the spatial acquisition phase Of testing, Control group had significantly longer swim time and swim distant than wild type group(P<0.01). Compare to Control group, G-CSF group sigficantly reduced swim time and swim distant, which was close to swim time and swim distant of wild type group. Moreover, there Was a significantly decreased trend for swim time and swim distant in G-CSF group(P＜0.01). There Was no effect of block or treatment on swim speed, and no treatment by block interaction. In the Spatial retention trial, both quadrant time and platform crossings,There was no statistical difference between wild type group and G-CSF group(P<0.05) (Figurel-4).2. HE and Nissl staining show a clear structure of hippocampus neurons.The cell nucleus structure is normal.The Nissl's bodies in the intracytoplasm were abundant.The chromatins of cell nucleus were distributed equally. Hippocampus neurons of Alzheimer's disease mice model were injuried.Swelled neurons and broken neurons were observed and they ranked chaos in the sections.The Nissl's bodies in the intracytoplasm and survival neurons were reduced compared with control groups (P<0.05).The structure of hippocampus neurons were similar as the wild type groups (Figure5,6,Tablel,2)Conclusions1. The cognition function of Alzheimer's disease mice model was decreased.2. The cognition function of Alzheimer's disease mice model was improved after the administration of G-CSF.3. G-CSF administration was benefited for the cognitive function of Alzheimer's disease mice model.Part 2 Influence of G-CSF on the neuroinflammation in the brain of Alzheimer's disease mice modelObjectiveInvestigate the influence of G-CSF on the neuroinflammation in the brain of Alzheimer's disease mice model.Methods:1. Alzheimers disease mice model and wild mice were divided into G-CSF group,control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of IL-1βand TNF-a in mice's frontal lobe cortex was detected by immunofluorescence, and observe the impact of G-CSF on their expression.Results1. Analysis result of image showed that the relative immunofluorescence intensity levels of IL-1βin the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P＜0.05) (Figurel,2). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figurel,.2). There is significate difference bwteen three groups (P<0.05).2. Analysis result of image showed that the relative immunofluorescence intensity levels of TNF-a in the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P<0.05) (Figure3,4). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figure3,4). There is significate difference bwteen three groups (P<0.05).Conclusions1. It was confirmed in my research that neuroinflammation exist in the brain of Alzheimer's mice model.2. MDL-28170 protects hippocampal neuron after pilocarpine-induced seizure against PCD through the inhibited effect onμ-calpain.3. The administration of G-CSF attenuate neuroinflammtion in Alzheimer's mice model through repress the expression of inflammatory factors IL-1βand TNF-a. It protected the cortical neurons from neuroinflammation of Alzheimer's disease mice model. Part 3 Influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice modelObjectiveInvestigate the influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice model. Discuss the critical effect of a7 nAChR on the cholinergic anti-inflammatory pathway.Methods:1. Alzheimers disease mice model and wild mice were divided into G-CSF group,control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration.2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of a7 nAChR in mice's frontal lobe cortex was detected by immunohistochemistry (IHC), and observed the impact of G-CSF on their expression.3. Mice were sacrificed at different time-points. Their brains were obtained and stored at-80℃.4. The proteins expression of a7 nAChR was detected by Western blotting and the effects of G-CSF on their expression were observed.Results1.Analysis result of image showed that density values ofα7nAChR in G-CSF day 7 group, G-CSF day 14 group, G-CSF day 28 group, G-CSF day 56 group were higher statistically than that of control group and wild type group(P<0.01 or 0.05). There is significant difference between the three groups (P<0.05) (Figurel,2).2.Comparison of protein levels ofα7nAChR:The protein levels of a7nAChR protein in G-CSF group increased significantly compared to that in control group(P<0.01).There is significant difference between the three groups (P<0.05) (Figure3,4).Conclusion1.α7 nAChR expressed generally on the front lobe cortex of mice's brain.2. The expression of a7 nAChR on the front lobe cortex of Alzheimer's mice model's brain was significately depressed.3. The administration of G-CSF could increase the expression of a7 nAChR on the brain of Alzheimer's mice model in different time points. It has the function of neuroprotection.Part 4 Influence of G-CSF on the expression NF-kB in the brain of Alzheimer's disease mice model and study of inflammation mechanismsObjectiveInvestigate the influence of G-CSF on the expression a7 nAChR in the brain of Alzheimer's disease mice model. Discuss the critical effect of a7 nAChR on the cholinergic anti-inflammatory pathway.Methods1. Alzheimers disease mice model and wild mice were divided into G-CSF group,control group and wild type group. Both of the G-CSF group and the control group are APP V717I transgenic mice. Mice in the G-CSF group were subcutaneously injected with G-CSF for 7 days. The control animals and the wild type animals were injected with PBS in parallel. The mice of each group were divided into three sub-groups as 7,14 and 28 days after the administration. 2. Mice at different time-points were perfused with 4% paraformaldehyde in phosphate-buffered saline respectively and the brains were used to make frozen sections. The expression of NF-κB in mice's frontal lobe cortex was detected by immunofluorescence, and observed the impact of G-CSF on their expression.3. Spearman correlation analysis was used to analysis the relationship of the expression ofα7 nAChR and NF-κB at different time points.Results1. Analysis result of image showed that the relative immunofluorescence intensity levels of NF-κB in the APP V717I transgenic mouse treated by G-CSF were significant lower than those in the control group (P<0.05) (Figurel,.2). The relative immunofluorescence intensity levels of control groups was the highest group at any time points compare with the other two groups (Figurel,.2). There is significate difference bwteen three groups (P<0.05).2. Result of Spearman correlations analysis showed that there was significant negative correlations betweenα7 nAChR and NF-κB expressions (7d:rho=-0.73, p=0.007; 14d:rho=-0.83, p=0.008; 28d:rho=-0.89, p=0.02).Conclusion there is an inverse correlation between the expression ofα7 nAChR and NF-κB in the brain of Alzheimer's disease mice model after the treatment of G-CSF. This finding suggests that G-CSF significantly attenuates the expression of proinflammatory cytokines by increasing the expression ofα7 nAChR in the brain of APP transgenic mice.