The Effect Of Advanced Glycation End Products On APP And Aβ Metabolism In Hippocampus By Inducing The Expression Of CTGF And The Mechanism

BackgroundAlzheimer's disease is a form of neurodegenerative diseases with progressive cognitive impairment as the main manifestation. Pathological changes characteristic of AD is the formation of specific brain regions in senile plaques, neurofibrillary tangles and neuronal and synaptic loss.β-amyloid (Aβ) is the main component of senile plaques, which is the scission of amyloid precursor protein (APP). Recent studies show that, the abnormality of APP metabolic and the increase in Aβare the central link in the pathogenesis of AD. Although study on the metabolism of APP and Aβabnormal formation mechanism have achieved a breakthrough, but the specific mechanism is unclear. Therefore, the metabolism of APP and regulatory pathways are still an urgent need to address. It is important to study the the pathogenesis of AD and it is also the starting point of the project. Glycation end products are proteins of non-enzymatic glycation of terminal irreversible polymer. Under normal circumstances, the level of AGEs in vivo slowly increase with aging, while the brain is the most active organ of metabolic.It entirely dependented on aerobic energy metabolism, oxidation. The increase in brain activity can prone to sugar glycosylation then promote the produce of AGEs.Castellani [1] and other studies confirm that AGEs exist in patients with early stage AD brain neurons, with the progression of the increase in AGEs and brain Aβdeposition and senile plaque volume were positively correlated, which suggested that AGEs increased throughout the entire AD pathogenesis; Munch [2] treated AD patients with AGEs inhibitor Imatinib Division Tanzania (Tenilsetam),which showed the drug could improve cognitive function in AD patients, the study supports the AGEs are involved in AD an important factor in the occurrence and development.Connective tissue growth factor is a multi-cell expressed and secreted with the relative molecular weight of 36,000～38,000 of the mitogen-peptide. Under normal physiological conditions in vivo the expression of CTGF was low or no expression. Connective tissue growth factor (CTGF) is an mitogen-peptide with the relative molecular weight of 36,000～38,000, which is expressed and secreted by multi-cell. Under normal physiological conditions, the expression of CTGF is low or no expression in vivo. In pathological cases [3-9], CTGF over-expression is closely related to the occurrence of hyperplasia or fibrosis, for example, retinopathy, atherosclerosis, kidney fibrosis, pulmonary fibrosis, cirrhosis and chronic pancreatic fibrosis. In 2003, Ueberham [10] found that CTGF expression was significantly increased in the entorhinal area, hippocampus and temporal lobe of brain in patients with Alzheimer's disease (AD) detected by immunohistochemical techniques for the first time. CTGF expression associated with neurofibrillary tangles lesions also significantly increased in neurons near senile plaques and astrocytes. The brain neurons in the normal aging express low levels of CTGF and glial cells did not express CTGF. This means that CTGF is associated with AD in senile plaques, neurofibrillary tangles and other pathological changes.A number of studies [11,12,13] show that AGEs binding its receptor RAGE of variety of cultured cells can significantly increased CTGF expression in tissues and organs,such as, blood vessels, kidney, retina and other target organ damage. Zhao'study [14] shows that CTGF can regulate the APP metabolism and promote Aβproduction through MAPK/PI-3/AKT and it is involved in AD pathogenesis.However, CTGF expression in the brain of AD patients was not clear.The role of CTGF on APP metabolism in AD pathogenesis is also not very clear. The action of AGEs and their receptors in CTGF expression and abnormal metabolism of APP has not been reported. Therefore, it is important theoretical significance and potential applications to explore the link and possible signaling pathways between AGEs-RAGE and CTGF expression, APP metabolism and Aβgeneration and to reveal the AD pathogenesis and provide a treatment targets point.Objective1. To explore the effect of advanced glycation end products (AGEs) on the expression of connective tissue growth factor (CTGF) in rat brain.2. To explore the correlation between CTGF and the metabolism ofβ-Amyloid Precursor Protein (APP) and the possible mechanism.3. To explicit the role of AGEs-RAGE-CTGF pathway in AD pathological changes.Methods70 Wistar rats were randomly divided into 7 groups (n=10) and the hippocampus in each group was injected and produced to animal injury models by using micro-injection pump:normal control group (NC group, injected with normal saline, each side 4μl), bovine serum albumin control group, (BSA-C group, injected with bovine serum albumin, each side 4μl), Glycosylation of bovine serum albumin group (AGE-BSA group, injected with AGE-BSA, each side 4μl), RAGE neutralizing antibody group (RAGE-Ab group, injected with RAGE monoclonal neutralizing antibody at first, each side 2μl, injected with AGE-BSA after 15 minutes, each side 4μL), RAGE neutralizing antibody control group (RAGE-Ab-C, injected with RAGE neutralizing antibodies, each side 4μl), CTGF neutralizing antibody group (CTGF-Ab group, injected with monoclonal neutralizing antibody CTGF-Ab at first, each side 2μl,15 minutes later injected with AGE-BSA for every side 4mL), CTGF neutralizing antibody control group (CTGF-Ab-C group, injected with CTGF neutralizing antibodies, each side 4μl).3 weeks later:HE staining was used to detect the pathological change in rat hippocampus; The escape latency (EL) of rats was assayed with Morris water maze test; Immunohistochemistry was employed to examine the protein expression of TGF-β1, CTGF, BACE1, PS-1, IDE, APP and Aβ42; Western blot was performed to detect the expression of RAGE, CTGF, TGF-b1, BACE1, IDE and PS-1 protein expression in rat hippocampus.Results1. The experimental results of Morris water maze test.Morris water maze test showed that the escape latency was longer in AGE-BSA group than that in NC group, BSA-C group, RAGE-Ab-C group and CTGF-Ab-C group(P<0.01); compared with NC group, the escape latency in RAGE-Ab group and CTGF-Ab group was longer(P<0.05); RAGE-Ab group and CTGF-Ab group escape latency was shorter than the AGE-BSA group (P <0.05); there was no significant difference between NC group, BSA-C group, RAGE-Ab-C group and CTGF-Ab-C group.2. The expression of TGF-β1,CTGF,BACE1,PS-1,IDE,APP and Aβ42 in rat hippocampus.There was no difference in TGF-β1,CTGF,BACE1,PS-1,APP and Aβ42 between NC group, BSA-C group, RAGE-Ab-C group and CTGF-Ab-C group. In hippocampus of AGE-BSA group, TGF-β1,CTGF,BACE1,PS-1,APP and Aβ42 were lightered brownish-yellow. The grayscales was higher than NC group. the grayscales of TGF-β1,CTGF,BACE1,PS-1,APP and Aβ42 was decreased in RAGE-Ab group and CTGF-Ab group. The expression of IDE in AGE-BSA group was deceased and the grayscales was lower than that in NC group, while, the expression of IDE in RAGE-Ab and CTGF-Ab was increased.3. The protein expression of RAGE.TGF-β1,CTGF,IDE,BACE1 and PS-1 and Semi-quantitative analysis.(1) The expression of RAGE and TGF-β1 was increased. The RAGE and TGF-β1 protein in AGE-BSA group were 1.4times,2 times than that in NC group respectively(P<0.01).Compared with AGE-BSA group, RAGE and TGF-bl protein expression in RAGE-Ab group and CTGF-Ab group significantly reduced 7% and 8% (P<0.05); there was no significant difference between NC group, BSA-C group, RAGE-Ab-C group and CTGF-Ab-C in RAGE and TGF-β1 protein expression (2) The relative index of CTGF in AGE-BSA group of protein was 0.76, which was 1.9 times than the NC group (P<0.01); compared with AGE-BSA group, CTGF protein expression in RAGE-Ab group and CTGF-Ab group was significantly reduced (P<0.05); the CTGF protein expression in NC group, BSA-C group, RAGE-Ab-C group and CTGF-Ab-C between groups was no significant difference.(3) The expression index of BACE1 and PS-1 in AGEs-BSA group were (0.76,0.74); which were 1.5 times and 1.7 times than that in NC group (0.50,0.44); and the BACE1, PS-1 protein expression in RAGE-Ab group and CTGF-Ab group were decreased compared with AGE-BSA group, the difference was statistically significant (P<0.05).(4) The expression of IDE in AGE-BSA group was lower than that in NC group (P<0.01), and IDE expression in RAGE-Ab group and CTGF-Ab group was increased (P<0.05)Conclusion(1) Glycation end products (AGEs) may promote the expression of transforming growth factorβ1 (TGF-β1) by binding with its receptor (RAGE), which leads to increase the connective tissue growth factor (CTGF) expression in hippocampal neurons of rats.(2) Connective tissue growth factor (CTGF) may play a role in the abnormal metabolism of APP and promote the generation of Aβ42 by influencing APP metabolism enzymes (β-secretase, y-secretase).(3) AGEs-RAGE-CTGF pathway that may be another important pathogenesis to Alzheimer's disease (AD) is involved in neuronal injury in hippocampus.SignificanceIn this study, three-dimensional positioning technology was used to inject AGEs into bilateral hippocampal of rats for the establishment of animal injury model. The expression of CTGF, APP expression and Aβ-generated were observed by immunohistochemistry and Western blot, and we further detected the changes of these indicators by blocking the upstream signaling molecule (RAGE) of CTGF and CTGF. This study was to explore the link between AGEs-RAGE and CTGF expression, APP metabolism and Aβgeneration. The study was to reveal the AD pathogenesis and provide a treatment targets point. That has important theoretical significance and potential applications.