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.

Objective To investigate the effect of RBP on 3T3-L1 adipocyte differentiation,lipid metabolism and glucose transporter 4(GLUT-4)gene expression.Methods We constructed an expression vector for rat resistin gene and transfected it into 3T3-L1 adipocytes.RBP was added to the medium of 3T3-L1 adipocytes or resistin-overexpressing adipocytes on day 0 of differentiation.Cell differentiation and lipid accumulation were determined by oil red O staining.The mRNA expressions of differentiation marker genes(pref-1,C/EBP?,FAS)and GLUT-4 gene were evaluated by RT-PCR.Triglyceride(TG)and free fatty acids(FFAs)in adipocytes were measured by colorimetric kit.Results(1)When 10-12mol/L RBP was applied,the percent of living cells was high and the shape was unchanged.(2)RBP had no effect on the differentiation of normal adipocytes,but significantly decreased the number of lipid droplets in resistin-overexpressing adipocytes without affecting the lipid droplets-presenting day.(3)C/EBP? and FAS expressions in resistin-overexpressing adipocytes were down-regulated after RBP was applied,without changing their expressions in normal adipocytes.(4)RBP had no effect on the cellular TG and FFAs levels in normal cells,whereas it can significantly decrease the levels in resistin-overexpressing adipocytes.(5)There was no difference in the expression of GLUT-4 gene between 3T3-L1 adipocytes and RBP-applied cells.Conclusions(1)RBP has no effect on the cell differentiation and lipid metabolism in normal 3T3-L1 adipocytes.(2)RBP can inhibit the cell differentiation and lipid metabolism of resistin-overexpressing 3T3-L1 cells.(3)RBP has no effect on the expression of GLUT-4 gene.

AIM　To explore the effect of berberine in inhibition of proliferation and induction of apoptosis of gastric cancer cell MGC-803.METHODS　The MGC-803 cell morphology and cell counting were studied by Trypan blue, MTT, Methyl green pyronin stain assay, the MGC-803 cell DNA changes were investigated by flow cytometry and agarose gel electrophoresis.RESULTS　Berberine could significantly inhibit the growth of MGC-803. After 48 hours of treatment with different concentration berberine (8, 4, 2, 1 mg*L-1), the inhibitive rate of MGC-803 were 86.7%, 65.9%, 20.0%, 0%, respectively. Meanwhile, MGC-803 showed typical apoptosis features: cell shrinkage, nuclear condensation, DNA fragmentation and formation of apoptotic bodies. A typical subdiploid peak before G0/G1 phase was observed by flow cytometry. The Trypan blue stain achromatophilia rates of death cells, which with well membranes, were between 60%～82%. Agarose gel electrophoresis analysis, which showed that the chromosomes of MGC-803 were broken by berberine before the structures of membrane were damaged, while DNA broke into long fragments rather than small fragments, supported that the activation effect of berberine on nuclease was mild.CONCLUSION　Berberine could significantly inhibit proliferation of gastric cancer cell MGC-803 and induced apoptosis, and the cytotoxicity of berberine on MGC-803 was weak.

Objective To explore the diagnostic value of the combined detection of serum Dickkopf-1 (DKK1 )and EB viral capsid antigen immunoglobulin A (VCA-IgA)in patients with nasopharyngeal carcinoma (NPC).Methods Serum levels of DKK1 and VCA-IgA were measured by enzyme-linked immunosorbent assay (ELISA)for the 80 patients with NPC and 65 normal controls.Receiver operating characteristic (ROC) curve was used to calculate the diagnostic value.Results The serum levels [M(QR )]of DKK1 in patients with NPC were significantly higher than those in normal controls [580.773 (429.1 46 )pg/ml vs.31 6.1 74 (252.965)pg/ml],with a significant difference (Z=4.846,P<0.000 1 ).ROC curves showed that the opti-mum diagnostic cutoff for serum DKK1 was 611.981 pg/ml,with an area under curve (AUC)of 0.734 (95%CI:0.654-0.81 5,50.0% sensitivity,96.9% specificity).Measurement of VCA-IgA demonstrated an AUC of 0.71 4 (95%CI:0.631-0.798,47.5% sensitivity,95.4% specificity).The combined detection of DKK1 and VCA-IgA demonstrated an AUC of 0.849 (95%CI:0.783-0.91 4,76.3%sensitivity,95.4%spe-cificity).For patients with early-stage NPC,the detection effect of combined detection of DKK1 and VCA-IgA was much better than that in normal controls,with a significant difference (χ2 =23.784,P <0.001 ). Conclusion Serum DKK1 has potential diagnostic value for NPC.Combined detection of DKK1 and VCA-IgA may aid the early diagnosis of NPC.

AIM To explore the effect of berberine in inhibition of proliferation and induction of apoptosis of gastric cancer cell MGC 803.METHODS The MGC 803 cell morphology and cell counting were studied by Trypan blue, MTT, Methyl green pyronin stain assay, the MGC 803 cell DNA changes were investigated by flow cytometry and agarose gel electrophoresis.RESULTS Berberine could significantly inhibit the growth of MGC 803. After 48 hours of treatment with different concentration berberine (8, 4, 2, 1 mg?L -1 ), the inhibitive rate of MGC 803 were 86 7%, 65 9%, 20 0%, 0%, respectively. Meanwhile, MGC 803 showed typical apoptosis features: cell shrinkage, nuclear condensation, DNA fragmentation and formation of apoptotic bodies. A typical subdiploid peak before G 0/G 1 phase was observed by flow cytometry. The Trypan blue stain achromatophilia rates of death cells, which with well membranes, were between 60%～82%. Agarose gel electrophoresis analysis, which showed that the chromosomes of MGC 803 were broken by berberine before the structures of membrane were damaged, while DNA broke into long fragments rather than small fragments, supported that the activation effect of berberine on nuclease was mild.CONCLUSION Berberine could significantly inhibit proliferation of gastric cancer cell MGC 803 and induced apoptosis, and the cytotoxicity of berberine on MGC 803 was weak.

Objective To explore the rule of traditional Chinese medicine in treating tinnitus based on literature and Logistic multiple regression analysis. Methods Articles about tinnitus treatment using traditional Chinese medicine were searched in several databases, i.e. CNKI (1984-2013), VIP (1989-2013), CBM (1990-2013), and PubMed (1984-2013), and medication frequency was analyzed. Then, models for tinnitus medication were established through metrological method and Logistic multiple regression. Results The common prescriptions with highest frequency usage were Liuwei Dihuang Pill, Longdan Xiegan Decoction, and Erlong Zuoci Pill. Corni Fructus, Psoraleae Fructus, and Lycii Fructus were commonly used for kidney-deficiency type of tinnitus;Astragali Radix, Atractylodis Macrocephalae Rhizoma, and Codonopsis Radix were commonly used for the spleen-qi-deficiency type of tinnitus;Gardeniae Fructus, Gentianae Radix et Rhizoma, and Cyperi Rhizoma were commonly used for the liver-qi-dysfunction type of tinnitus;Pinelliae Rhizoma, Aurantii Fructus Immaturus, and Stephaniae Tetrandrae Radix were commonly used for the phlegm-fire disturbance type of tinnitus;Forsythiae Fructus, Menthae Haplocalycis Herba, and Mori Cortex were commonly used for the wind-fire-invasion type of tinnitus. Conclusion Analysis of medication frequency and Logistic multiple regression analysis can provide evidence and reference for the treatment of tinnitus and syndrome differentiation.

Objective To predict the biological process and signaling pathways in which hsa-miR-1908 might be in-volved by a series of bioinformatics analysis, so as to lay foundations and provide theoretical basis for the further studies of hsa-miR-1908 biological function in human preadipocytes. Methods The sequence of hsa-miR-1908 was acquired from miR-Base database, and target genes of hsa-miR-1908 were predicted by miRanda, and then the intersection of the results and the results of gene-chip as gene set were further analyzed by gene ontology and pathway enrichment. Results The hsa-miR-1908 had some conserved property among different species. The functions of the target genes were enriched in Wnt receptor signal-ing pathway through beta-catenin, cell cycle, cell apeptosis and other biological processes. The GnRH signaling, MAPK sig-naling, insulin signaling, cell cycle signal transduction pathways and signaling pathway in pancreatic cancer were signiifcantly enriched. Conclusions The target genes set of hsa-miR-1908 were enriched in multiple biological process which are related with the obesity. This study provides guidance for the further study in human preadipocytes.

Objective To compare the pharmacological actions of Liandai Tablet(LT) and its main active components on apoptosis of gastric cancer cells and DNA damage. Methods Effects of Liandai Tablet(LT) and its main active components,berberine and indirubin,on growth and apoptosis of gastric cancer cell strain MGC_803 were explored and their effects on DNA damage were also studied. Results LT serum in high and low dosages and berberine could inhibit the growth of MGC_803 as compared with the control group,and typical morphological features of apoptosis were found in the MGC_803 by methyl green pyronin stain assay.But indirubin at various concentrations showed no obvious inhibitory effects. Agarose gel electrophoresis assay revealed that the MGC_803 cell DNA was split into large fragments when treated with berberine. Conclusion LT serum exerts a similar inhibitory effect on the growth and apoptosis of gastric cancer cells as compared with berberine.The effects of LT at various serum concentrations on MGC_803 DNA was less than that of berberine,and indirubin at the given concentration had no this effect.

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.

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.