| BackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disease, characteri-zed by progressive cognitive dysfunction and memory loss in the elderly. The pathologic feature of AD is neurofibrillary tangles (NFT) and senile plaques (SP) in the brains of affected patients. Moreover, oxidative stress and endoplasmic reticulum stress may play important roles in AD. The exact mechanism of AD is still unclear and there is no effective method to cure it. Therefore, it is very necessary to explore the pathogenesis of AD and to develop new pharmaceutic method to conquer the disease.Advanced glycation end products (AGEs), formed from the non-enzymatic glycosylation of proteins, are known to be increased in diabetes patients and the elderly. AGEs are thought to play an important role in the pathogenesis of AD. Increased AGEs formation has been demonstrated in neurofibrillary tangles and senile plaques in brain. AGEs may be increased along with the disease and throughout the course of AD.There is a direct toxic effects of AGEs to promote protein crosslinking and modification and evoke oxidative and inflammatory reactions of AD. Beside the direct cytotoxicity, AGEs also interact with the receptor for AGEs (RAGE), upregulate RAGE expression and accelerate pathological process of AD through different pathway. Li discovered that AGEs bond RAGE and upregulated hyperphosphorylated tau expression, indicating that AGEs-RAGE signaling functions in NFT. AGEs-RAGE signaling may also elicited oxidative stress and endoplasmic reticulum stress to produce neuronal injury. Thus, AGEs-RAGE pathway is of great importance in AD pathogenesis. However, the precise mechanism regarding how AGEs-RAGE could affect tau phosphorylation and endoplasmic reticulum stress is not completely understood.Reducing the expression of RAGE or blocking RAGE-ligands combination might be a new therapeutic target for AD. Current anti-RAGE therapies using RAGE antibodies and soluble RAGE only block the peripheral RAGE but not the central RAGE because of the difficulty to pass the blood brain barrier (BBB). RAGE inhibitor TTP488 (also known as PF-04494700) has significant adverse events when given at high doses. A high-affinity RAGE-specific blocker, FPS-ZM1 that is able to penetrate BBB, was reported to have no side effect in physiological vital signs at a dosage that is 500-fold higher than the therapeutic dosage (1mg/kg i.p.) in AD mice. Intraperitoneal administration with FPS-ZM1 was found to improve congnitive function, inhibit Aβ production and inflammation in AD mice. Our previous research has demonstrated that FPS-ZM1 inhibited AGEs-RAGE-mediated Aβ production and inflammation. However, whether and how FPS-ZM1 could reduce AGEs induced tau phosphorylation, oxidative stress and endoplasmic reticulum stress is not clear.In the present study, a AGEs-RAGE-activated rat model by intrahippocampal injection of AGEs was established, then rat model was intraperitoneally administrated with FPS-ZM1. We aimed to characterize the neuroprotective effects of FPS-ZM1 against AGEs-induced tau phosphorylation, oxidative stress and endoplasmic reticulum stress in rat hippocampus, as well as cognitive deficits so as to provide a new insight to AD treatment.Methods1. Animals and Grouping6-8-week-old Wistar rats were fed as experimental animals and were randomly divided into four groups (ten in each group). A control group (Control) and a treatment control group (FZM1); a AGEs group (AGEs) and a AGEs treatment group (AGEs+FZM1). The bilateral hippocampi of the control and the FZM1 rats were injected with 5μL normal saline. The bilateral hippocampi of AGEs and the AGEs+FZMl rats were injected with 5μL AGE-Bovine Serum Albumin (AGE-BSA). Starting from 1 week before intrahippocampal injection, FZM1 and AGEs+FZMl rats were intraperitoneally injected with FPS-ZM1 (1 mg/kg/d at a volume of 2 mL) for 4 weeks.2. The learning and memory ability was evaluated by Morris water maze (MWM) test.3. Western blot method and immunohistochemistry method were used to analyze the expression of RAGE in rat hippocampus.4. Western blot method was used to detect the expression of Tau5 (total tau), PHF-1 (Phospho-tau at Ser396/404), PT231 (Phospho-tau at Thr231).5. Western blot method was used to detect the expression of t-Akt (total Akt), Ser473-Akt (Phospho-Akt at Ser 473), tGSK-3β (total GSK-3β),S9-GSK3β (Phospho-GSK-3β at Ser9).6. ROS was detected by 2’-7’-dichlorofluorescein-diacetate assay. The concentration of malondialdehyde (MDA), which is produced during lipid peroxidation, was determined by thiobarbituric acid reactive substances. Spectrophotometric methods were used to detect superoxide dismutase (SOD) activity,glutathione (GSH) level and glutathione peroxidase (GPx) activity.7. Western blot method was used to detect the expression of GRP78, p-PERK, p-eIF2a, CHOP, caspase-12.8. The activity of caspase-9 and caspase-3 were detected with colorimetric assay kit.Results1. Morris water maze testFrom the third day of the test, the AGEs rats exhibited an increase in escape latency when compared to the controls (P<0.05). FPS-ZM1 treatment attenuated the effects of AGEs on escape latency (P<0.05). The AGEs rats exhibited substantially decreased number of crossings and time in target quadrant (%) when compared to the controls (P<0.01, P<0.01). FPS-ZM1 treatment rats had increased number of crossings and time in target quadrant (%) when compared to the AGEs group (P<0.01, P<0.01).2. The RAGE protein expressionWestern blot analysis showed a significant increase of RAGE in the AGEs group than in the control group (P<0.01) and a decrease of RAGE in the AGEs+FZM1 group than in the AGEs group (P<0.01).Immunohistochemistry showed a significant increase of RAGE in the AGEs group than in the control group (P<0.05) and a decrease of RAGE in the AGEs+FZM1 group than in the AGEs group (P<0.05).3. The phosphorylated tau protein and Akt/GSK-3p expressionThe expression index of PHF-1 and PT231 in the AGEs group was 0.87 and 0.79, they were 2.2 and 2.9 times than control group (P<0.01, P<0.01). The expression index of PHF-1 and PT231 in the AGEs+FZMl group was 0.38 and 0.41, and was significantly decreased than in the AGEs group (P<0.01, P<0.01).The expression index of Ser473-Akt and S9-GSK3β were 0.36 and 0.40 in the AGEs group, and were 0.72 and 0.76 in the control group (P<0.01, P<0.01). The expression index of Ser473-Akt and S9-GSK3β were 0.55 and 0.61 in the AGEs+FZM1 group and were significantly increased than in the AGEs group (P<0.01, P<0.01).No significant difference was observed between the control group and the FZM1 group (P>0.05).4. Oxidative stress detectionCompared with the control group, ROS and LPO levels significantly increased in the AGEs group (P<001, P<0.01). Reduced levels of ROS and LPO were observed in AGEs+FZM1 group compared with the AGEs group (P<0.01, P<0.05) and no significant difference was observed between the control group and the FZM1 group (p>0.05).GSH content, SOD and GPx activities in the AGEs group were significantly lower than the control group (P<0.01, P<0.01, P<0.01). However, these decreases were reversed by FPS-ZM1 treatment (P<0.05, P<0.01, P<0.01). No significant difference was observed between the control group and the FZM1 group (P>0.05).5. Expression of endoplasmic reticulum stress markersThe expression index of GRP78, p-PERK and p-eIF2a in the AGEs group were 0.66,0.69 and 0.71, they were 1.9,2.1 and 1.8 times than the control group (P<0.01, P<0.01, P<0.01). The expression index of GRP78, p-PERK and p-eIF2a in the AGEs+FZMl group was 0.44,0.43 and 0.52, and were significantly decreased than in the AGEs group (P<0.01, P<0.01, P<0.05). No significant difference was observed between the control group and the FZM1 group (P>0.05).The expression index of CHOP and caspase-12 in the AGEs group were 0.80 and 0.74, they were 2.1 and 2.2 times than the control group (P<0.01, P<0.01). The expression index of CHOP and caspase-12 in the AGEs+FZM1 group were 0.59 and 0.42, and were significantly decreased than in the AGEs group (P<0.05, P<0.01). No significant difference was observed between the control group and the FZM1 group (P>0.05).AGEs increased the activity of caspase-3 and caspase-9, they were 2.6 and 1.7 times than control group (P<0.01, P<0.01). FPS-ZM1 inhibited the activity of caspase-3 and caspase-9 than the AGEs group (P<0.01,P<0.01).Conclusion1. Intrahippocampal injection of AGEs in rat hippocampus may lead to cognitive decline through AGEs-RAGE pathway. Intraperitoneal administration with FPS-ZM1 alleviated AGEs-RAGE induced cerebral injury in rat hippocampus and improved cognitive function.2. AGEs-RAGE induced tau hyperphosphorylation. Akt/GSK-3β pathway may be involved in the process. FPS-ZM1 alleviated AGEs-RAGE-mediated tau hyperpho-sphorylation and reduced GSK-3β expression.3. FPS-ZM1 elevated the antioxidant capacity, decreased AGEs-RAGE induced oxidative damage in rat hippocampus.4. AGEs-RAGE elicited endoplasmic reticulum stress. FPS-ZM1 reduced the expression of GRP78, p-PERK and p-eIF2a, and alleviated AGEs-RAGE induced endoplasmic reticulum stress in brain.SignifanceRAGE is important in the development of AD and may be a new therapeutic target for AD. Current anti-RAGE therapies RAGE antibodies and soluble RAGE can not pass through the BBB. Therefore, there is a need to research new efficacious RAGE blockers.1. We established an animal model by intrahippocampal injection of AGEs, aimed to determine the importance of AGEs-RAGE pathway in AD pathogesis.2. We determined the protective effect of FPS-ZM1 in an AGEs-RAGE-activated animal model for the first time. Our results demonstrated that FPS-ZM1 reduces AGEs-induced tau hyperphosphorylation, oxidative stress and endoplasmic reticulum stress in rat hippocampus, and might be a potential therapeutic agent for AD. |
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