Study Of Advanced Glycation Endproducts On Microglia Activation And Inflammatory Expression In Vivo And Vitro Experiments

【Background】Alzheimer's disease is a form of nervous system degenerative diseases with progressive cognitive impairment as the main manifestation. AD is the most common cause of dementia in the elderly. Three pathological changes characteristic of AD is the formation of senile plaques, neurofibrillary tangles, hippocampal pyramidal cells granulovacuolar degenerationand neuronal loss. The pathogenesis of AD is complex and not fully understood. Currently considered that chronic inflammation of the brain caused by the activation of microglia may be one of the core pathological mechanisms of AD. Advanced glycation end products are structurally diverse irreversible polymer to which the amino sugar and protein through a series of complex non-enzymatic reaction form. It has been found that AGE can induce early inflammatory reaction of AD by modifying senile plaques and activating neighboring microglia and astrocytes, which cause oxidative stress and produce inflammatory cells molecules. However, whether AGE can directly induce the activation and inflammatory reaction of microglia has not been reported. Therefore, this study explores advanced glycation end products directly or through the receptor Induce microglial activation and high expression of inflammatory cytokines in Vivo and vitro levels.【Objective】1. To explore the effect of advanced glycation end products on microglia activation and the expression of tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1β), cyclooxygenase-2(COX-2) and p-nuclear factor κB(p-NF-κB) in rats hippocampus in Vivo level.2. To explore the effect of advanced glycation end products on primary cultured microglia activation and the secretion of tumor necrosis factor alpha(TNF-α), interleukin 1 beta(IL-1β), cyclooxygenase-2(COX-2) and nitric oxide(NO) in Vivo level.3. To research the role of the activation and inflammatory reaction of microglia by induced advanced glycation end products in AD pathological changes.【Methods】Thirty Wistar rats were randomly divided into normal control group (control group), BSA group, AGE-BSA group, AGE/RAGE-Ab group and RAGE-Ab group,6 rats in each group.To establish the AD pathological model of local inflammation in rats by injecting AGE-BSA into the hippocampus. Cognitive behavioral change was detected by Morris maze test. The sections were stained by immunofluorescence to examine the expression of marker of microglia (CD11b) and astrocyte marker glial fibrillary acidic protein (GFAP) in the rats hippocampus. Immunohistochemistry and western blotting were adopted to estimate the expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), cyclooxygenase-2(COX-2) and p-nuclear factorκB (p-NF-κB) in rats hippocampus.Cultured microglial cells were intervened by AGE-BSA, then the appraisal was carried by the immunocytochemistry method and the cell morphological changes were observed. After 300μg/mL the AGE-BSA and the RAGE neutralizing antibody treated primary rat microglial cells, the levels of tumor necrosis factor alpha(TNF-α), interleukin 1 beta(IL-1β), cyclooxygenase-2(COX-2) and nitric oxide(NO) extracted from the supernatant liquid of microglia was measured by enzyme-linked immunosobent assay(ELISA).【Results】1. Compared with the control group, in AGE-BSA group,the rats cognitive function decreased, glial cell proliferation in the hippocampus was found, mainly by microglia and the expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta(IL-1β), cyclooxygenase-2(COX-2) and p-nuclear factorκB(p-NF-κB) was increased significantly (P<0.001). AGE/RAGE-Ab group microglia proliferation in the hippocampus decreased and the expression of tumor necrosis factor alpha (TNF-a), interleukin 1 beta(IL-1β), cyclooxygenase-2(COX-2) and p-nuclear factor KB(p-NF-κB)was lower than the AGE-BSA group (P<0.01), but still higher than control group. The expression of tumor necrosis factor alpha (TNF-a), interleukin 1 beta(IL-1β), cyclooxygenase-2(COX-2) and p-nuclear factorκB(p-NF-κB) in the NC group, BSA group and the RAGE-Ab group was not different (P>0.05)2. After the intervention by AGE-BSA, the cell body became bigger and the shape changed "Ameba", the levels of tumor necrosis factor alpha(TNF-a), interleukin 1 beta(IL-1β), cyclooxygenase-2(COX-2) and nitric oxide(NO) were increased significantly (P<0.001). However, comparing with the AGE-BSA group, the cells exposed to the RAGE neutralizing antibody before treated with AGE-BSA, the levels of tumor necrosis factor alpha(TNF-a), interleukin 1 beta(IL-1β), cyclooxygenase-2(COX-2) and nitric oxide(NO)were lower (P< 0.01),but still higher than the normal control group (P<0.01)【Conclusions】1. Injecting advanced glycation end products into the hippocampus could induced the proliferation and activation of microglia, promoted the product of tumor necrosis factor alpha (TNF-a), interleukin 1 beta(IL-1β), cyclooxygenase-2(COX-2) and p-nuclear factorκB(p-NF-κB) protein.2. advanced glycation end products could activate microglia and induce the time-dependent release of TNF-a., IL-1β, COX-2 and NO.3. advanced glycation end products can directly or through the receptor activate microglia-mediated immune inflammatory response, which may be another important pathogenesis of Alzheimer's disease.【Significance】In this study show that AGE-BSA can directly or through the receptor activate microglia, promote the expression of a large number of inflammatory cytokines and cause inflammation. It confirms that AGE induced microglial activation and cause inflammatory response, which has an important role in pathological changes of AD. This study suggests that AGE may be an important factor by leading to brain tissue chronic inflammation of AD, and reveal the pathogenesis of AD and provide for theoretical basis for future clinical treatment. That has important theoretical significance and potential applications.