| BackgroundAlzheimer's disease is a common form of neurodegenerative diseases among old folks.Pathological characteristic of AD is the formation of senile plaques, neurofibrillary tangles and neuronal and synaptic loss in specific brain regions. The mechanism is very complicated,β-amyloid (Aβ) which is the principal ingredient of SP and the key aspect inducing the occurrence and development of AD is the scission of amyloid precursor protein (APP) byβ-secretion enzyme andγ-secretion enzyme. Although study on the abnormal metabolism of APP and Aβformation mechanism have achieved a breakthrough, but the specific mechanism is unclear.The Connective Tissue Growth Factor(CTGF) is one kind of secretive multi-peptides containing the cysteine richly which expresses in many kinds of cells. It includes 349 amino acids with the relative molecular weight of approximately 38000, which belongs to instantly one of early gene family CNN members. In the normal condition, CTGF does not have the expression in organism or few expresses. But the massive researches confirmed that under pathological state, CTGF can play an influential role in many kinds of fibrosis disease's occurrence and development, like skin, kidney, lung, liver, heart, retina, great vessels and so on.In recent years, some scholars studied the discovery, CTGF had a high expression in SP and NFT in AD patient brain, mainly concentrated in astrocyte and the neuron, and aggravated along with the condition increases; zhao and so on found in the AD patient brain the CTGF expression level progresses along with the condition increases, and through in vitro experiment proved that CTGF may regulate APP through the upwardγ-secretion enzyme the abnormal metabolism and promoting Aβproduction, thus explained that CTGF possibly participated in AD. But, in the AD patient brain the reason of the CTGF expression increases was not still clear, and the mechanism that the APP abnormal metabolism and Aβthe production increases was promoted by CTGF is also still not very explicitly.The advanced glycation end products(AGEs) are generated by a non-enzymatic reaction(Maillard response) of glucose and other carbohydrates with stable protein deposits, which can lead many kinds of pathology processes. In recent years the numerous researches indicated that AGEs participated in exceptional crossing linking and the deposition, the oxidized stress, the inflammatory response, the neuron loses and so on. But the mechanism of the occurrence and development of AD with AGEs and acceptor (receptor for advanced glycation end products, RAGE) has not been expounded clearly. At present the domestic and foreign researches confirmed that AGEs promoted CTGF expression in the kidney, the blood vessel and in the retina organization. Therefore we considered that whether AGEs can promote the CTGF expression to increase and affect the APP abnormal metabolism through certain circuit and Aβproduction or not in AD patients'brain, then promotes the AD progress. So we choose AGEs-RAGE-CTGF circuit to conduct the research for the first time to AD, and discusse the morbidity function and the possible mechanism of AGEs in AD in the cell level.Then we can prove that the AGEs-RAGE-CTGF circuit possibly is another mechanism about AD, thus it provides a treatment target point.Objective1. To explore the effect of AGEs with RAGE on the expression of CTGF, abnormal metabolism of APP and the produce of Aβin SH-SY5Y cells. 2. To explore the correlation between CTGF and the metabolism ofβ-Amyloid APP and the possible mechanism.3. To explicit the role of AGEs-RAGE-CTGF pathway in AD pathological changes.MethodsThe cultured SH-SY5Y cells were divided into 7 groups randomly, normal control group (NC group, injected with normal saline), bovine serum albumin control group, (BSA group, injected with bovine serum albumin,200μg/mL), Glycosylation of bovine serum albumin group (AGEs-BSA group, injected with AGEs-BSA,200μg/mL), AGEs-BSA+RAGE neutralizing antibody group (AGEs-RAGE-Ab group, injected with RAGE monoclonal neutralizing antibody at first,1:100, injected with AGE-BSA after 1 hour,200μg/mL), RAGE neutralizing antibody control group (RAGE-Ab group, injected with RAGE neutralizing antibodies,1:100), AGEs-BSA+CTGF neutralizing antibody group (AGEs-BSA+CTGF group, injected with monoclonal neutralizing antibody CTGF-Ab at first,1:100,1 hour later injected with AGEs-BSA,200μg/mL), CTGF neutralizing antibody control group (CTGF-Ab group, injected with CTGF neutralizing antibodies,1:100). To examine the cells'growth state with MTT metabolic rate and determine the best concentration and time of the AGEs-BSA。ELISA was employed to examine the protein expression of Aβ1-40 and Aβ1-42. Immunohistochemistry was employed to examine the protein expression of APP, CTGF, RAGE, Aβ1-40 and Aβ1-42; Western blotting was performed to detect the expression of APP,RAGE, CTGF, BACEland PS-1 protein expression in SH-SY5Y cells.Results1. The influence of different protein density of BSA/AGEs-BSA to SH-SY5Y cells growth conditionThis experimental results showed that AGEs-BSA(200μg/mL) caused the SH-SY5Y cells vigor to drop to about 50% comparing to normal control group 48 hours later, the same density of BSA did not have the obvious influence to the SH-SY5Y cells vigor. Therefore we selectd 200μg/mL of AGEs-BSA intervention SH-SY5Y cells for 48 hours, compared with other groups, and observed the expression and production of APP, RAGE, CTGF, BACE, PS-1,Aβ1-40 and Aβ1-42.2. The expression of Aβ1-40 and Aβ1-42 secreted by SH-SY5Y cellsThere was no difference in Aβ1-40 and Aβ1-42 between NC group, BSA group, RAGE-Ab group and CTGF-Ab group. In SH-SY5Y cells of AGEs-BSA group, Aβ1-40 and Aβ1-42 were more increased than NC group. And the expressions of Aβ1-40,Aβ1-42 were decreased in AGEs-RAGE-Ab group and AGEs-CTGF-Ab group.3. The expression of Aβ1-40,Aβ1-42,APP,CTGF,RAGEIn SH-SY5Y cells of AGEs-BSA group, Aβ1-40, Aβ1-42, APP, CTGF, RAGE were brownish-yellow. There was no difference in Aβ1-40, Aβ1-42, APP, CTGF and RAGE between NC group, BSA group, RAGE-Ab group and CTGF-Ab group, and the cells were lighter brownish-yellow. Compared to AGEs-BSA group, the grayscales of Aβ1-40, Aβ1-42, APP, CTGF was decreased in AGEs-RAGE-Ab group; the grayscales of Aβ1-40, Aβ1-42, APP was decreased and AGEs-CTGF-Ab group,but the expression of RAGE did not significantly changed, and all of these indexes were higher than NC group4. The protein expression of APP,RAGE,CTGF,BACE1 and PS-1 and Semi-quantitative analysis.(1) There was no significant difference between NC group, BSA group, RAGE-Ab group and CTGF-Ab group in APP, BACE1 and PS-1 protein expression. In SH-SY5Y cells of AGEs-BSA group, the expression of APP, BACE1 and PS-1 protein were 1.49 times,1.76 times and 2.03 times than that in NC group respectively(P<0.05). Compared with AGEs-BSA group, APP, BACE1 and PS-1 protein expression in AGEs-RAGE-Ab group significantly reduced 18.26%,14.88% and 29.51%(P<0.05) but were 1.22 times,1.51times and 1.43 times than that in NC group respectively(P<0.05). Compared with AGEs-BSA group, APP, BACE1 and PS-1 protein expression in AGEs-CTGF-Ab group significantly reduced 14.05%,10.69% and 31.03% (P<0.05) but were 1.45 times,1.60 times and 1.21 times than that in NC group respectively(P<0.05).(2) There was no significant difference between NC group and BSA group in RAGE protein expression. In SH-SY5Y cells of AGEs-BSA group and AGEs-CTGF-Ab group, the expression of RAGE protein were 1.42 times and 1.44 times than that in NC group respectively(P<0.05), and there was no significant difference between AGEs-BSA group and AGEs-CTGF-Ab group in RAGE protein expression.(3) There was no significant difference between NC group, BSA group and RAGE-Ab group in CTGF protein expression. In SH-SY5Y cells of AGEs-BSA group, the expression of CTGF protein were 1.98 times than that in NC group (P<0.05). Compared with AGEs-BSA group, CTGF protein expression in AGEs-RAGE-Ab group significantly reduced 30.29%(P<0.05) but were 1.38 times than that in NC group (P<0.05).Conclusion(1) In the cells level, AGEs promoted the expression of RAGE,and also induced the expression of CTGF, abnormal metabolism of APP and Aβformation by binding with its receptor (RAGE)in SH-SY5Y cells.(2) CTGF played a role in the abnormal metabolism of APP and promoted the generation of Aβ1-40 and Aβ1-42 by influencing APP metabolism enzymes (β-secretase,γ-secretase).(3) AGEs-RAGE-CTGF pathway that may be another important pathogenesis to Alzheimer's disease (AD) is involved in neuronal injury in SH-SY5 Y cells.SignificanceIn this study, we injected AGEs into SH-SY5Y cells for the establishment of cells injury model. The expression of RAGE, CTGF, APP, BACE and PS-1 expression and Aβ-generated were observed by Elisa,immunohistochemistry and Western blotting, and we further detected the changes of these indicators by blocking the RAGE and CTGF. This study was to explore the mechanism of AGEs-RAGE-CTGF in AD. Thus we provide one new method for the early diagnosis, preventing and treating AD. |
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