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BACKGROUND: Small conductance,Ca2+-activated potassium(SK)channel,presents in various cell types and plays a crucial role in action potential profile.However,coupling and modulation of calcium and associated molecules to SK2 channel remains unclear.OBJECTIVE: To construct the recombinants of pGBAT7 and target fragments of SK2 gene,so as to observe the coupling and regulation of SK2 channel gene to calcium and other molecules.METHODS: Three pairs of primers of the target fragments of SK2 gene were designed and synthesized based on the full-length sequences of SK2.After being identified,they were individually sub-cloned into the yeast expressive plasmid pGBKT7 to construct pGBKT7-SK2 vectors.The recombinant pGBKT7-SK2 vectors were transformed into yeast AH109 by electroporation,and their activation was tested.The recombinants were extracted from yeast AH109 and verified by electrophoresis and sequencing.RESULTS AND CONCLUSION: The target fragments of SK2 gene by PCR were 411,546 and 729 bp,respectively.Three sub-clones of pGBKT7-SK2 were successfully constructed.Electrophoresis and sequencing showed that the constructed sub-clones of pGBKT7-SK2 met the expected requirements.The recombinant pGBKT7-SK2 vectors transformed into the yeast could be activated.The successful construction of the sub-clones of SK2 gene provides an important material basis for further study in the SK2 channel and function-associated molecules.
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BACKGROUND: Several studies have demonstrated that many American women who are at high risk of developing osteoporosis have higher levels of serum phosphorus. This indicates that some substances which can lower the serum level of phosphorus will supply a new and effective method to prevent and treat osteoporosis.OBJECTIVE: To observe the influences of porcine bone protein on bone mineral density (BMD) and serum levels of calcium and phosphorus in a rat model of osteoporosis.METHODS: Wistar rat models of osteoporosis were established by intramuscular injection of dexamethasone. Rat models were randomly divided into physiological saline, Jiegu Qili tablet, 50, 100, 200 mg/kg porcine bone protein groups. Rats that did not receive any treatments served as normal controls. After 12 weeks of treatment, serum was collected and serum levels of phosphorus and calcium were determined by biochemistry method. At the same time, tibia sections were made to determine tibial DMD by QDR-400 dual energy X-ray absorptiometry and to observe tibia marrow cavity by hematoxylin-eosin staining.RESULTS AND CONCLUSION: There was no significant difference in serum level of calcium among groups (P＞0.05).Compared with the physiological saline group, serum level of phosphorus in the 50, 100, 200 mg/kg porcine bone protein groups was significantly decreased (P ＜ 0.05). BMD was significantly higher in the 50, 100, 200 mg/kg porcine bone protein, Jiegu Qili tablet groups than in the physiological saline group (P ＜ 0.05). The tibia marrow cavity was smallest in the normal control group and largest in the physiological saline group. The tibia marrow cavity was larger in the 50, 100, 200 mg/kg porcine bone protein,Jiegu Qili tablet groups than in the physiological saline group. These results indicate that porcine bone protein cannot change the serum level of calcium, but it lowers serum level of phosphorus, and increases BMD, in a rat model of osteoporosis. However, the dose-dependent effect of porcine bone protein was not observed within the present experimental dosage. In addition, porcine bone protein can also reduce the marrow cavity of the tibia of rats with osteoporosis.
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BACKGROUND: Excessive nitric oxide (NO) release can cause the occurrence and development of brain injury and senile dementia due to the apoptosis induction role of NO at high concentration to nerve cells. Therefore one strategy to prevent and treat senile dementia is inhibiting the apoptosis induced by NO.OBJECTIVE: To observe whether acidic peptide will inhibit the neuron apoptosis caused by NO. DESIGN: An cell and molecule observation experiment by comparisons. SETTING: Department of Biochemistry and Molecular Biology of Basic Medical College in Zhengzhou University and the Second Laboratory of Biological Active Peptide Institute in Zhengzhou University. MATERTALS: The experiment was performed between May 2003 and May 2005, in the Second Laboratory of Biological Active Peptide Institute in Zhengzhou University and the cell culture room of Department of Biochemistry and Molecular Biology of Basic Medical College in Zhengzhou University. The newborn SD male rats within 24 hours after birth were provided by the Animal Center of Henan Province (410117).METHODS: On day 11 of primary cultures, hippocampus neurons of the newborn SD rats were pretreated with different dosages of acidic peptide for six hours. Sodium nitroprusside (SNP) of 50 μmol/L final concentration was added to the cells which were incubated for another 24 hours. Cells were collected and adopted in this experiment of five different groups, namely normal control group, group treated with SNP, group of SNP plus 0.037 5 mg/mL acidic peptide, group of SNP plus 0.075 mg/mL acidic peptide, group of SNP plus 0.15 mg/mL acidic peptide. The cell's survival rate wasmeasure by methyl thiazolyl (MTT) method; The neurofilament protein was stained with the method of immunohisto chemistry. The shape of apoptosis was display with acridine orange fluorescent stain. Then DNA ladder zone of apoptosis cells was analyzed with the method of agarose gel electrophoresis. Western Blot and absorbance scan were used to determine the expression level of Bcl-2 protein and Bax protein.MAIN OUTCOME MEASURES: ①Experimental result of cell survival rate with MTT method;②Observation results of nuclear type of apoptosis; ③DNA electrophoresis analysis of apoptosis; ④Western Blot analysis results of Bcl-2 protein and Bax protein.RESULTS: ①Neuron survival rate was 58.9％ for group treated with SNP, 70.0％ for group of SNP plus 0.037 mg/mL acidic peptide, 72.8％ for group of SNP plus 0.075 mg/mL acidic peptide, and 75.3％ for group of SNP plus 0.15 mg/mL acidic peptide. ②Observation results of nuclear type of apoptosis: Significant characteristics of apoptosis were seen in group treated with SNP. The nucleus of hippocampus neuron treated with different concentrations of acidic peptide plus SNP was similar to that of normal control group in morphology. ③The results of DNA electrophoresis analysis of apoptosis: Only the neuron DNA of group treated with SNP showed clear characteristic DNA ladder zone of apoptosis on agarose gel electrophoresis. ④Analysis results of Bcl-2 protein and Bax protein with Western Blot and absorbance scan: The expression level of Bcl-2 protein in SNP treated group was decreased while that of Bcl-2 protein was increased. Bcl2 protein levels in acidic peptide plus SNP group were increased and Bax protein levels were decreased gradually with the increasing concentrations of acidic peptide compared with SNP treated group. CONCLUSION: Acidic peptide can inhibit neuron apoptosis, increase expression level of neuron Bcl-2 protein and decrease expression level of neuron Bax protein.
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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.
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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.
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BACKGROUND: Acidic peptide is the tripeptide composed of 3 glutamic acids, which cannot bring excitatory nerve signal transmission into playlike single glutamic acid through presynaptic release and integration withpostsynaptic NMDA receptor directly as excitable neurotransmitter. It is quite possible that acidic peptide plays its actions by integrating with multiple metabolic glutamic acidic receptors so as to promote neuron proliferation or release nerve growth factor (NGF). OBJECTIVE: To probe into whether acidic peptide induces changes in learning and memory of model rats with Alzheimer disease (AD).DESIGN: Randomized controlled single experiment was designed.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 60, 30 and 15 mg/kg, 12 rats in each group. Acidic peptide is a new small molecular peptide separated from bovine brain in this research team 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 basal ganglia 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 groups of 15, 30 and 60 mg/kg,acidic peptide of 60, 30 and 15 mg/kg was applied for gastric perfusion successively, continuously for 20 days, once per day, 2 mL/time. On the expiration of gastric perfusion, learning and memory of rats were examined with step down test in every group. The animal was placed on the safe table on step down platform to adapt to the environment for 3 minutes, afterwards, 36 V electric current was given. Error response was recorded if the animal jumped to the copper railings after electric shock and correct response was recorded if the animal jumped back the safe area. Step-up latent phase and frequency of correct response were recorded in 3 minutes.MAIN OUTCOME MEASURES: Comparison of learning and memory of rats in every group. RESULTS: Totally 84 rats were all included in the result analysis. ①Comparison of learning in every group: Compared with model group, stepup latent phase was shortened remarkably in every acidic peptide group[(102.03±5.33), (71.77±4.38), (68.28±9.53), (69.13±8.79) s, P ＜ 0.01] and the frequency of correct response was improved remarkably [(12.92±2.91),(16.17±2.79), (15.83±3.27), (16.33±2.53) times, P ＜ 0.01]. ② Comparison of memory in every group: Compared with model group, step-up latent phase was shortened remarkably in every acidic peptide group [(43.17±4.66),(29.78±4.48), (26.20±3.28), (22.09±4.43) s, P ＜ 0.01] and the frequency of correct response was improved remarkably [(15.67±2.15), (20.92±2.68),(20.83±2.29), (20.25±2.05) times, P ＜ 0.01].CONCLUSION: Acidic peptide can shorten remarkably the step-up latent phase of AD rats in step down test and improve the frequency of correct response. It is indicated that acidic peptide provides good intervention on learning and memory of rat model of Alzheimer disease.
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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.
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AIM:To investigate the protective effects of sodium ferulate(SF)on apoptosis in cultured hippocampal neurons induced by sodium nitroprusside(SNP),and the effect of SF on expression of bcl-2 and bax.METHODS:The primary cultured hippocampal neurons were exposed to 50 ?mol SNP,a nitric oxide-donor,for 24 h after pretreatment with different concentrations of SF(10-160 ?mol/mL)for 6 h.Then neuronal viability was tested by MTT assay.Fluorescent staining with Hoechst 33258 and agarose gel electrophoresis was used to analyze apoptosis.The expressions of bcl-2,bax mRNA and protein were tested by RT-PCR and Western blotting.RESULTS:Pretreatment with SF(10-160 ?mol/L)for 6 h increased the survival rate of neurons.SF prevented the neuronal nuclei from shrinkage,condensation and cleavage and blocked neuronal nuclear DNA fragmentation induced by SNP.SF also increased the expressions of bcl-2 mRNA and Bcl-2 protein and decreased the expressions of bax mRNA and Bax protein.CONCLUSION:SF prevents the cultured hippocampal neurons against SNP neurotoxicity.The mechanism of protection is related to the increase in Bcl-2 level and the decrease in Bax level.As a result,the ratio of Bcl-2/Bax is changed.