[Effects of H102 on the memory recognition ability and AMPK-mTOR autophagy-related pathway in AD mice].

OBJECTIVE: To study the effect of ß-sheet blocking peptide H102 on the expression of AMPK-mTOR autophagy pathway-related protein in APP/PS1 double transgenic AD mice. METHODS: Thirty male APP/PS1 transgenic male AD mice of 6 months old were randomly divided into AD group and H102 intervention group, and C57BL/6J male mice of the same age were used as control group (n=15). The mice in the HF group were administered with 5 µl (5.8 mg/kg) of H102 polypeptide solution through the nasal cavity at the same time period, and the mice in the control group and the AD group were given the same amount of blank adjuvant solution daily. The memory recognition ability was tested by a new object recognition experiment 30 days after continuous administration. Immunohistochemistry and Western blot were used to detect the expressions of phosphorylated AMP-activated protein kinase(P-AMPK),phosphorylated mammalian target of rapamycin (P-mTOR) and ratio of LC32to LC31(LC3II/I )in brain tissue. RESULTS: Compared with the control group, the new object recognition index (RI) of the AD group was significantly lower (P＜0.05), and the P-AMPK and LC3II/I ratios in the brain of the mice were significantly lower (P＜0.05). The expression of P-mTOR protein was increased significantly (P＜0.05). Compared with the AD group, the RI of the H102 intervention group was increased significantly (P＜0.05), and the P-AMPK and LC3II/I ratios in the brain tissue of the mice were increased significantly (P＜0.05). The expression of P-mTOR protein was decreased significantly (P＜0.05). CONCLUSION: H102 can improve the recognition and memory ability of AD mice by activating the AMPK-mTOR autophagy-related pathway.

[Single Nucleotide Polymorphism of Mitochondrial DNA D-LOOP Region in Peripheral Blood Lymphocytes of Immuno-related Pancytopenia Patients].

OBJECTIVE: To explore the single nucleotide polymorphism(SNP) of mitochondrial DNA (mtDNA) D-LOOP region in peripheral blood lymphocytes of immuno-related pancytopenia (IRP) patients and its correlation with immune parameters. METHODS: The D-LOOP region in mitochondrial DNA of lymphocytes in peripheral blood mononuclear cells from 43 patients with untreated IRP was detected by polymerase chain reaction(PCR). The PCR products were sequenced by the pros and cons direct sequencing methods. The sequencing results were compared with the revised Cambridge reference sequence (rCRS) and the Polymorphic Sites of Human Mitochondrial Genome Database. RESULTS: Among total of 110 variant positions of D-LOOP region in 43 patients, 62 was SNP sites and 48 was mutation sites, of which 14 were the new mutation sites not yet registered in the database, 516 base variations were observed at 110 positions, the most common variations were base substitutions, among them T/C and A/G was 184/410 and 113/410 respectively. In the 110 variant positions, the high frequency variation sites were 73 and 263 for 43/43,311 for 32/43,310 and 16 224 for 27/43,16 519 for 25/43, 489 and 16 362 for 24/43. By the analysis of mitochondrial DNA D-LOOP polymorphism and related clinical immunology indicators of the patient's lymphocytes, it was found that D-loop in adult patients (age≥ 18 years old) significantly correlated with CD15 IgM, GLYCoA+ Cells IgM, CD34+ CellsIgG, CD34+ Cells IgM correlation. CONCLUSION: The high frequency of polymorphism exists in mitochondrial DNA D-loop region of lymphocytes in IRPA patients, and was significantly correlates with the autoantibodies in bone marrow mononuclear cells in adult patients, which may be associated with the IRP occurrence.

[Effect of ßsheet blocking peptide H102 on APP metabolic enzymes in hippocampal brain of double transgenic AD mice].

OBJECTIVE: To investigate the effect of ß-sheet breaker peptide H102 on APP associated secretase in the hippocampus brain regions of APP/PS1 double transgenic mice(AD mice). METHODS: Thirty 6-month-old APP/PS1 double transgenic mice were randomly divided into AD group and H102 group, a group of C57BL/6J mice with the same age, number and background was set as controls(n=15). H102 (5.8 mg/kg) 5 µl was infused by intranasal administration to mice in H102 treatment group, and equal volume of blank solution of H102 was given to mice in control group and AD group. The ability of spatial reference memory was tested by Morris water maze after 30 days of treatment. And then immunohistochemistry tests and Western blot were used to detect the content of α-secretase (ADAM10, ADAM17), ß-secretase (BACE1), γ-secretase (PS1, APH1a, PEN2) in the hippocampus brain regions. RESULTS: Compared with the control group, the expression of BACE1, PS1, PEN-2 and APH1-a protein in the hippocampus of AD group were significantly increased, ADAM10, ADAM17 protein expression were significantly reduced (P<0.05); Compared with the model group, H102 could significantly improve the spatial learning and memory ability of AD mice, significantly decreased the expression of BACE1, PS1, PEN-2 and APH1-a protein in the hippocampus, significantly increased the expression of ADAM10 and ADAM17 protein(P<0.05). CONCLUSIONS: ß sheet peptides blocked H102 can reduce the formation of Aß in the hippocampus brain area, improve the activity of α-secretase in the hippocampus brain region, decrease the activity of ß-and γ-secretase, improve the learning and memory ability of AD mice.

[The effects of H102 on NF-κB signal pathway in brain of transgenic AD mice].

OBJECTIVE: To investigate the effects of ß-sheet breaker peptide H102 on NF-κB signal pathway in brain of APP/PS1 double transgenic mice. METHODS: Thirty 8-week-old APP/PS1 double transgenic mice were randomly divided into model group and treatment group. A group of C57BL/6J mice with the same age and background were served as controls (n=15). H102 5 µl(5.8 mg/kg) was infused by intranasal administration to mice in H102 treatment group, and equal volume of blank solution of H102 (chitosan, BSA) was given to mice in control group and model group. After 16 weeks, the ability of spatial reference memory was tested by Morris Water Maze. Then immunohistochemistry tests and Western blot technique were used to detect the content of amyloid beta peptide 1-42(Aß1-42), nuclear factor-kappa B (NF-κB), inhibitor of NF-κB (IκB), IκB kinase (IKK), the corresponding phosphorylated proteins (p-NF-κB、p-IκB、p-IKK), inducible nitric oxide synthase (iNOS) and cleaved Caspase 3 proteins in mice brain. RESULTS: ①The ability of learning and memory was significantly lowered in model group than that in control group. And the ability of learning and memory was significantly improved in treatment group than that in model group (P<0.05). ②The contents of Aß1-42, p-IKK, p-NF-κB, p-IκB, intranuclear NF-κB,iNOS and cleaved Caspase 3 in mouse brain were significantly increased in model group than those of control group, and these protein expressions were significantly lowered in treatment group than those in model group (P<0.05). CONCLUSIONS: H102 can inhibit NF-κB signal pathway in brain of APP/PS1 double transgenic mice, reduce the levels of inflammation and apoptosis in nerve cells, and improve the ability of learning and memory in transgenic AD mice.

[Effects of ß-sheet breaker peptide H102 on synaptic plasticity associated proteins in double transgenic AD mice].

OBJECTIVE: To investigate the effects ofß-sheet breaker peptide H102 on expression of synaptic plasticity associated proteins and learning and memory functions in double transgenic Alzheimer's disease(AD) mice,and to discuss its mechanisms. METHODS: Thirty APP-swe/PS1dE9 double transgenic male mice of 8 weeks were randomly divided into model group and H102 treatment group (15 mice per group). In addition,a group of C57BL/6J mice with the same age and background was set as normal. H102 (5.8 mg/kg) 5 µl was infused by in-tranasal administration to mice in H102 treatment group,and equal volume of blank solution of H102 (chitosan,BSA) was given to mice in con-trol group and model group. The ability of spatial reference memory was tested by Morris Water Maze after 16 weeks treatment,then immunohis-tochemistry tests and Western blot technique were used to detect the content ofß-amyloid peptide(Aß1-42) protein and phospho protein kinase C α、ß2、γ(p-PKCα, p-PKCß2, p-PKCγ), phospho-N-methyl-D-aspartate receptor1(p-NMDAR1), phospho-Calcium/Calmodulin dependent pro-tein kinaseⅡα(p-CaMKⅡα) and phospho-cAMP response element binding protein(p-CREB) of synaptic plasticity associated proteins in mice brain. RESULTS: The ability of learning and memory was significantly improved in H102 treatment group than that in model group by the test of Morris Water Maze. The contents of Aß1-42 proteins and p-PKCα, p-PKCß2, p-PKCγ, p-NMDAR1, p-CaMKⅡαand p-CREB of synaptic plas-ticity associated proteins in mice brain were improved significantly in H102 treatment group than those in model group by the test of immunohis-tochemistry tests and Western blot technique. CONCLUSIONS: ß-sheet breaker peptide H102 can significantly improve synaptic plasticity and the ability of learning and memory in double transgenic AD mice.

Feasibility of ß-sheet breaker peptide-H102 treatment for Alzheimer's disease based on ß-amyloid hypothesis.

ß-amyloid hypothesis is the predominant hypothesis in the study of pathogenesis of Alzheimer's disease. This hypothesis claims that aggregation and neurotoxic effects of amyloid ß (Aß) is the common pathway in a variety of etiological factors for Alzheimer's disease. Aß peptide derives from amyloid precursor protein (APP). ß-sheet breaker peptides can directly prevent and reverse protein misfolding and aggregation in conformational disorders. Based on the stereochemical structure of Aß1-42 and aggregation character, we had designed a series of ß-sheet breaker peptides in our previous work and screened out a 10-residue peptide ß-sheet breaker peptide, H102. We evaluated the effects of H102 on expression of P-tau, several associated proteins, inflammatory factors and apoptosis factors, and examined the cognitive ability of APP transgenic mice by behavioral test. This study aims to validate the ß-amyloid hypothesis and provide an experimental evidence for the feasibility of H102 treatment for Alzheimer's disease.

[The combined effects of beta-sheet breaker and hUCMSC on APP transgenic mice].

OBJECTIVE: To study the effect of combining the injection of beta-sheet breaker H102 with human umbilical cord mesenchymal stem cell (hUCMSC) on APP transgenic mice behavior, P-tau, apoptosis and the expression of relevant enzymes in the brain. METHODS: APP transgenic mice were randomly divided into model group, hUCMSC group, H102 group, H102 with hUCMSC group and a group of C57BL/6J mice with the same age and background was set as normal. After two weeks and four weeks, the ability of spatial reference memory was tested by Morris Water Maze. After four weeks, immunohistochemical stain and Western blot were done to detect the content of Bad, Bax, Bcl-2, Bcl-xl, P-tau, GSK-3beta, PP-2A and PP-1 in mice brain. RESULTS: The ability of memory of hUCMSC in 2 weeks group was slightly improved than that in the model group. hUCMSC in four weeks group, H102 group and H102 with hUCMSC group significantly improved the ability of and memory, and reduced the phosphorylation of tau and brain cell's apoptosis of the Alzheimer disease (AD) mice. CONCLUSION: Beta-sheet breaker H102 together with transplanting hUCMSC is an effective therapeutic strategy for AD.

[Glycyrrhetinic acid induces apoptosis and alters survivin gene expression in human myeloma cell line U266].

This study was aimed to investigate the effects of glycyrrhetinic acid (GA) on proliferation, apoptosis and survivin mRNA expression in human myeloma cell line U266 in vitro. Cell proliferation was assayed by MTT method. Both cell apoptosis and cell distribution in cell cycle were analyzed by using flow cytometry. Scanning electron microscopy was used to observe the morphological changes in U266 cells induced by GA. Expression of survivin mRNA was detected by quantitative real-time reverse transcription-polymerase chain reaction. The results showed that GA inhibited proliferation of U266 cells in a time- and dose-dependent manners in vitro. GA presented apoptosis induction potency to U266 cells, obvious changes in morphology of U266 cells was observed under scanning electron microscope. The cells were arrested at the G(0)/G(1) phase, showing the accumulation in G(0)/G(1) phase, reduction of cells in G(2)/M phase and S phase. GA could down-regulate the expression of survivin gene in U266 cells in a dose-dependent manner. It is concluded that GA can inhibit proliferation of U266 cells in a time- and dose-dependent manners and induce apoptosis of this cell line in vitro through arresting G(0)/G(1) phase and down-regulating expression of survivin.

[Influence of H102 on the expression of amyloid protein and amyloid precursor protein in the hippocampus of APP695 transgenic mice].

OBJECTIVE: To observe the influence of H102 on the expression of amyloid protein and amyloid precursor protein in the hippocampus of APP695 transgenic mice. METHODS: The 9-month-old APP695 transgenic mice were randomly divided into the model group and the H102 group; C57BL/6J mice were adopted as normal control group. The H102 group were injected with H102 in a dose of 3 microl/per mouse in lateral ventricle, once a day, for ten days; while the model group and the control group were injected with saline. The hippocampus and temporal cortex of the brain sections from transgenic mice and wild type female mice were subjected to immunohistochemistry and Congo red histological staining, and observed the difference of the protein expression under microscope. The expression of the APP protein was detected by Western blot. RESULTS: Abeta and APP immunohistochemistry showed density of positive cell in the CA1 region of hippocampus of control group were less than model group. H102 peptide reduced the area, and density of positive cells. Congo red staining showed there were lots of amyloid plagues in the brains of model mice but not in the brains of normal control. And the Western blot showed the content of the APP protein of the model group was much higher than the H102 group. H102 significantly decreased the amyloid plagues. CONCLUSION: The expression of APP, Abeta are increased in APP695 transgenic mice, and H102 can decrease the level of APP, Abeta in transgenic mice.

[Damage effects of sleep deprivation on myocardium and its antioxygen index in rats].

AIM: To explore the effects of sleep deprivation (SD) on myocardium and its antioxygen index. METHODS: 35 Sprague Dawley rats were randomly divided into five groups: Cage control, Tank control, SD 2 d, SD 4 d and SD 6 d. The "flower pot" technique was used to establish rats sleep deprivation model followed by record of surface electrocardiogram, detection of myocardium morphology changes under microscope and transmission electron microscope and investigation of MDA content and SOD activity of myocardial mitochondria. RESULTS: After sleep deprivation, heart rate increased and ECG showed ischemia of myocardium; subcellular organelles such as chromosome, endoplasmic reticulum, mitochondria, intercalated disk impaired, myofibril lysis or necrosis, and lipid peroxidation reaction effects spread widely; edema, bleeding of the microvessels and invasion of the monocytes could be seen in the lumen. The MDA level increased and SOD activity increased followed by a decreased trend. CONCLUSION: Sleep deprivation can induce damage on myocardium, and the stress especially oxygen stress caused by SD may be the possible mechanism.

[Modulation with bupleurum root on brain electrical activities in epileptic model].

AIM: To study the effect of bupleurum root on epileptic seizure. METHODS: The rabbits and rats were injected by pilocarpine as epileptic models, and observed the effect of bupleurum on the electroencephalogram (EEG) and hippocampal slice by electroencephalograph and glass microelectrode extracellularly. RESULTS: The seizure time and duration of each major seizure of epilepsy were significantly shortened and the interval of seizure significantly prolonged (P < 0.05) after intraabdominal injection of bupleurum root. After instilling the injection of bupleurum root onto the slices could reduce the amplitude of evoked field potential in epileptic hippocampal slices remarkably, the average of fall is 20.41%, and restore in 6.86 minutes on average (P < 0.001). CONCLUSION: Bupleurum root can inhibit the brain electrical activities in epileptic model, it is suggest that bupleurum has the distinct effect of antiepilepsy.