ABSTRACT
BACKGROUND: It has been confirmed that acidic peptide has good therapeutic effect on rat models of Alzheimer disease, but the mechanism still needs further exploration.OBJECTIVE: To observe whether acidic peptide can inhibit the production of N-methyl-D-aspartate receptor (NMDAR) and beta-amyloid (β-amyloid) in brain, and accelerate the production and excretion of nerve growth factor (NGF) in rats with Alzheimer disease.DESIGN: A randomized controlled animal experiment.SETTING: Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Zhengzhou University.MATERIALS: The experiments were finished in the first laboratory of Institute of Bioactive Peptide, Zhengzhou University and the Cellular Culture Center, School of Basic Medical Sciences of Zhengzhou University from March 2005 to May 2006. Seventy 10-week-old healthy male SD rats without dementia symptoms were randomly divided into 7 groups with 10 rats in each group: normal control group, model group, saline group, glutamic acid 0.3 g/kg group, acidic peptide 15, 30 and 60 mg/kg groups.METHODS: Except the normal control group, the rats in the other 6 groups were induced into models of Alzheimer disease by damaging bilateral nucleus basalis of Meynert with ibotenic acid, and then intragastric administration of glutamic acid (0.3 g/kg) was given in the glutamic acid 0.3 g/kg group, acidic peptide of corresponding dosages in the acidic peptide 15, 30 and 60 mg/kg groups, and isovolume saline in the saline group respectively, 2 mL for each time, once a day for 20 days continuously.MAIN OUTCOME MEASURES: ① The learning ability of the rats was detected with Y-maze test immediately after the end of intragastric administration, and the times of correct responses were recorded. ② After the end of learning and memory test, the head was cut rapidly to remove brain,treated with immunohistochemical staining, and gray value was scanned with Biosens Digital Imaging System to determine the contents of NMDAR,NGF and β-amyloid in the brain of rats.RESULTS: All the 70 rats were involved in the analysis of results. ①Times of correct responses in the Y-maze test were lower in the other 6groups than in the normal control group (P ＜ 0.01), but higher in the acidic peptide 30 and 60 mg/kg groups than in the model group, saline group, glutamic acid 0,3 g/kg group and acidic peptide 15 mg/kg group (P ＜ 0.01). ②The gray values of NGF in basal forebrain in the model group, saline group,glutamic acid 0.3 g/kg group and acidic peptide 15 mg/kg group were lower than that in the normal control group (69.60±2.41, 69.62±1.46, 69.62±1.46,69.73±1.87, 80.77±2.72, P ＜ 0.01); There were no significant differences between the acidic peptide 30 and 60 mg/kg groups (79.39±2.23, 80.20±1.7, P ＞ 0.05), which were higher than the other groups. ③ The gray values of NMDAR and β-amyloid in cerebral cortex in the model group,saline group, glutamic acid 0.3 g/kg group and acidic peptide 15 mg/kg group were lower than those in the normal control group (NMDAR: 81.01±1.38, 81.31±2.06, 81.37±1.39, 79.38±1.23, 69.50±1.04; β-amyloid:74.26±1.39, 74.89±8.66, 74.88±1.46, 74.16±2.48, 67.40±3.06, P ＜ 0.01),and There were no significant differences between the acidic peptide 30and 60 mg/kg groups (P ＞ 0.05), which were lower than the other groups.CONCLUSION: Acidic peptide of 30 and 60 mg/kg can obviously ameliorate the learning and memory abilities in rat models of Alzheimer disease, which may be realized mainly through up-regulating the NGF content in basal forebrain and down-regulating the NMDAR and β-amyloid contents in cerebral cortex.
ABSTRACT
BACKGROUND: Nerve growth factor (NGF) and brain-derived neurotrophic factor(BDNF) are very important to the survival and proliferation of nerve cells. In the patients with Alzheimer disease (AD), the levels of NGF and BDNF are low.OBJECTIVE: To investigate whether acidic peptide can stimulate rat astrocytes to secrete NGF and BDNF.DESIGN: A randomized control animal experiment.MATERLALS: The experiment was finished in the First Laboratory of Institute of Biopeptide, Zhengzhou University; Cellular Culture Center,School of Basic Medical Sciences of Zhe ngzhou University from September 2003 to May 2005. Fifteen neonatal SD rats within 2 days after birth were selected.METHODS: ① The cerebral cortex of the neonatal SD rats was removed under sterile condition, the astrocytes were isolated and cultured, and then identified with the glial fibriliary acidic protein immunohistochemical staining. ② The cultured astrocytes were randomly divided into six group:blank control group, serum control group, positive control group and acidic peptide treated groups. No treatment was given in the blank control group,serum of 0.2 in volume fraction and 1 000 U/mL interferon were added in the serum control group and positive control.group, 37.5, 75 and 150 mg/L acidic peptides were added in the acidic peptide treated groups respectively. ③ The astrocytes of the 2nd generation, which covered the whole bottom of bottle, were digested to single cell suspension, and then inoculated to three 12-well plates equally at 5×105 /mL. The survival rate and the contents of NGF and BDNF in the supernatant of each group were determined at 24, 48 and 72 hours respectively.MAIN OUTCOME MEASURES: ① Cell numbers and survival rates at different culture time-points; ② Effect of acidic peptide on the proliferation of astrocytes in rats; ③ Changes of NGF and BDNF in the supernatant of astrocytes at different culture time-points.RESULTS: ① As compared with the blank control group, the cell numbers and survival rates at 24, 48 and 72 hours were obviously increased in the acidic peptide groups treated with 75 and 150 mg/L (P＜0.05, 0.01,0.001), but not obviously increased in the acidic peptide group treated with 37.5 mg/L. ② As compared with the blank control group, the rates of proliferation in the acidic peptide groups treated with 37.5, 75 and 150 mg/L were all significantly increased (17.5%, 45.5%, 72.5%, P＜0.001). ③ As compared with the blank control group, the absorbance (A) values of NGF in the supernatant at 24, 48 and 72 hours were all markedly increased in the acidic peptide groups treated with 37.5, 75 and 150 mg/L (P＜0.001),and the A values of BDGF in the supernatant at 48 and 72 hours were significantly increased (P＜0.05, 0.01).CONCLUSION: Acidic peptide can increase the secretions of NGF and BDNF of rat astrocytes to different extent.
ABSTRACT
BACKGROUND: It is pointed in some experiment that acidic peptide improves learning and memory of model rat with Alzheimer disease (AD) by inhibiting the synthesis of toxic compounds of nitric oxide (NO).OBJECTIVE: Animal model with Alzheimer disease was established to observe the changes in the levels of NO, nitric oxide synthase (NOS) and acetylcholinesterase (AChE) treated with acidic peptide of various dose concentration.DESIGN: Randomized control and single experiment.SETTING: Teaching-Research Room of Biochemistry and Molecular Biology of Basic Medical College of Zhengzhou University.MATERIALS: The experiment was performed in 2nd Research Room and Experimental Animal Room of Teaching-Research Room of Biochemistry and Molecular Biology of Basic Medical College of Zhengzhou University.Totally 100 SD male rats were selected and some of them were excluded due to retarded response in step down test. Totally 84 rats were included in the experiment and randomized into 7 groups, named normal control,model group, physiological saline group (PS group), Piracetam group, acidic peptide groups of 15, 30 and 60 mg/kg, 12 rats in each group. Acidic peptide was a new small molecular peptide separated from bovine brain and is tripeptide composed of three glutamic acids.METHODS: Except normal control, in the rest groups, after 1 week routine breeding, cerebral stereotactic microinjection was used to inject 5 μg ibotenic acid in hippocampus of rats to destroy bilateral Meynert's nucleus basalis to establish AD model. In normal control and model group, no medication was applied. In PS group, physiological saline was used for gastric perfusion. In piracetam group, piracetam of 0.3 g/kg was used for gastric perfusion and in acidic peptide groupsof 15, 30 and 60 mg/kg,acidic peptide of 15, 30 and 60 mg/kg was applied for gastric perfusion successively, continuously for 20 days, once per day, 2 mL/time. On the expiration of gastric perfusion, the rats were sacrificed after anesthetized and the brain was collected on ice plate to prepare tissue homogenate. After centrifugated at 1 000 r/minute, 4℃ for 10 minutes, the supernatant was collected to assay the levels of NO, NOS and AChE with NO, NOS and AChE kits successively.MAIN OUTCOME MEASURES: Levels of NO, NOS and AChE in brain of rat in each groupRESULTS: Totally 84 rats were employed in the experiment and all entered result analysis. Comparison of levels of NO, NOS and AChE in rat brain of each group: compared with model group, NO levels in acidic peptide groups of 15, 30 and 60 mg/kg were reduced remarkably[(1.95±0.20), (1.39±0.10), (1.25±0.07), (1.00±0.04) mmoL/kg, P ＜ 0.05],NOS levels were reduced remarkably [(4.53±0.18), (3.39±0.09), (3.10±0.06),(2.97±0.06) μmol/kg, P ＜ 0.05] and AChE did not change remarkably[(0.67±0.12), (0.71±0.11), (0.72±0.08), (0.72±0.07) mmol/L, P ＞ 0.05].CONCLUSION: Acidic peptide reduces significantly the synthesis of NO and NOS in brain of AD rat, but it dose not affect AChE activity remarkably. It is suggested that acidic peptide improves learning and memory of rat with Alzheimer disease probably by inhibiting the synthesis of toxic compound of NO or its toxicity.