Effects of L-3-n-butylphthalide on caspase-3 and nuclear factor kappa-B expression in primary basal forebrain and hippocampal cultures after beta-amyloid peptide 1-42 treatment

BACKGROUND： L-3-n-butylphthalide （L-NBP） can inhibit phosphorylation of tau protein and reduce the neurotoxicity of beta-amyloid peptide 1-42 （Aβ1-42）. OBJECTIVE： To observe the neuroprotective effects of L-NBP on caspase-3 and nuclear factor kappa-B （NF- K B） expression in a rat model of Alzheimer＇s disease. DESIGN, TIME AND SETTING： A cell experiment was performed at the Central Laboratory of Provincial Hospital affiliated to Shandong University between January 2008 and August 2008. MATERIALS： L-NBP （purity 〉 98%） was provided by Shijiazhuang Pharma Group NBP Pharmaceutical Company Limited. Aβ1-42, 3-[4,5-dimethylthiazolo-2]-2,5 iphenyltetrazolium bromide （MTT）, and rabbit anti-Caspase-3 polyclonal antibody were provided by Cell Signaling, USA; goat anti-choactase and rabbit anti-NF- kB antibodies were provided by Santa Cruz, USA. METHODS： Primary cultures were generated from rat basal forebrain and hippocampal neurons at 17 or 19 days of gestation. The cells were assigned into five groups： the control group, the Aβ1-42 group （2 μmol/L）, the Aβ1-42 ＋ 0.1 μmol/L L-NBP group, the Aβ1-42 ＋ 1 μ mol/L L-NBP group, and the Aβ1-42 ＋ 10μmol/L L-NBP group. The neurons were treated with Aβ1-42 （2 μmol/L） alone or in combination with L-NBP （0.1, 1, 10 μmol/L） for 48 hours. Cells in the control group were incubated in PBS. MAIN OUTCOME MEASURES： Morphologic changes were evaluated using inverted microscopy, viability using the M-I-I- method, and the changes in caspase-3 and NF- k B expression using Western blot. RESULTS： Induction with Aβ1-42 for 48 hours caused cell death and soma atrophy, and increased caspase-3 and NF- K B expression （P 〈 0.05）. L-NBP blocked these changes in cell morphology, decreased caspase-3 and NF- k B expression （P 〈 0.05）, and improved cell viability, especially at the high dose （P 〈 0.05）. CONCLUSION： AI3^-42 is toxic to basal forebrain and hippocampal primary neurons; L-NBP protects against this toxicity and inhibits the induction of caspase-3 and NF- K B expression.

STUDY ON THE THERAPEUTIC EFFECTS OF GINSENOSIDE Rg-1 AND GASTRODINE ON AD MODEL RATS INDUCED BY β-AMYLOID PEPTIDE (25-35)

Objective To study the therapeutic effects of Ginsenoside Rg-1 and Gastrodine on rats model of Alzheimer's disease(AD). Methods Aggregated β-Amyloid peptide (25-35) was injected into the lateral ventricle of rats to establish AD models. Ginsenoside Rg-1, Gastrodine and Ginsenoside Rg-1+Gastrodine were intraperitoneally injected into rats of each test group(Ginsenoside Rg-1∶10mg/kg·day; Gastrodine 100mg/kg·day) for 4 weeks, the rats of control group received equal volume of saline. Passive avoidance task and Morris maze test were done to assess the ability of learning and memory. The content of superoxide dismutase (SOD), malondiadehyde (MDA), total-antioxidative capability (T-AOC), Choline acetyltransferase (ChAT) and acetylcholinesterase (AchE) in brain tissue were measured. Results Ginsenoside Rg-1 and Gastrodine significantly improved learning and memory deficits in the rats with AD induced by β-Amyloid peptide (25-35) (P<0.05). Ginsenoside Rg-1+Gastrodine group were better than Ginsenoside Rg-1 group and Gastrodine group (P<0.05). Ginsenoside Rg-1 reduced the increase of SOD, MDA, but inhibited the decrease of T-AOC, AchE and ChAT; Gastrodine reduced the increase of SOD, MDA, while inhibited the decrease of T-AOC. Gastrodine could also prevent the activity of ChAT and AchE decline in AD rats. Conclusion Both Ginsenoside Rg-1 and Gastrodine have therapeutic effects on rats with AD; Ginsenoside Rg-1 and Gastrodine injection at the same time were better than only using one of them. Their mechanisms might different. Ginsenoside Rg-1 can not only inhibit peroxidation but also increase the activity of AchE and ChAT in brain tissue, while Gastrodine can inhibit peroxidation only, but it can't prevent the decline of ChAT and AchE activity in AD rats.

Inhibitory effects of scorpion venom heat-resistant protein on neurotoxicity of exogenous amyloid beta peptide 1-40

BACKGROUND： Studies have shown that scorpion venom heat-resistant protein （SVHRP） exhibits protective effects on primary cultured hippocampal neurons. OBJECTIVE： To determine the effects of SVHRP on astrocyte activity and synaptic density in the hippocampus induced by amyloid β peptide 1-40 （Aβ1-40） neurotoxicity. DESIGN, TIME AND SETTING： The randomized, controlled, animal experiment was performed at the Central Laboratory, the Laboratory of Human Anatomy, and the Laboratory of Physiology, in Dalian Medical University between March 2006 and June 2008. MATERIALS： Aβ1-40 was provided by Biosource, USA; SVHRP was a patented biological product of Dalian Medical University （No. ZL01 1 06166.9）. METHODS： A total of 27 healthy, 2-month-old, male SD rats were randomly assigned to 3 groups： control, Aβ, and SVHRP, with 9 rats in each group. Alzheimer＇s disease was simulated with 10 μg Aβ1-40 bilaterally injected into the hippocampus of the Aβ and SVHRP groups. The control group was injected with 2 μL 0.05% trifluoroacetic acid. One day following model establishment, the SVHRP group received an intraperitoneal injection of 2 μg/100 g SVHRP, while the control group and Aβ group received 0.5 mL/100 g tri-distilled water, once per day, for 10 consecutive days. MAIN OUTCOME MEASURES： At 16 days following model establishment, synaptophysin （p38） expression in CA1-CA4 regions of the rat hippocampus was determined by immunohistochemistry. Glial fibrillary acidic protein （GFAP） expression surrounding the hippocampal Aβ1-40 injected area was also detected. At 11 days following model establishment, escape latency, swimming time, and distance to target quadrant were measured using the Morris water maze. RESULTS： Compared with the control group, the Aβ group exhibited notably reduced p38 expression （P 〈 0.05） and notably increased GFAP expression in the rat hippocampus （P 〈 0.05）. Water maze results demonstrated that escape latency was prolonged （P 〈 0.05）, and swimming time and distance to the target quadrant were shortened in the Aβ group. Compared with the Aβ group, the SVHRP group exhibited notably increased p38 expression （P 〈 0.05） and notably decreased GFAP expression in the rat hippocampus （P 〈 0.05）. Water maze results demonstrated that escape latency was significantly reduced （P 〈 0.05）, and swimming time and distance to the target quadrant were significantly prolonged. CONCLUSION： SVHRP inhibited exogenous Aβ1-40-induced astrocyte activation and synaptic density decline in the rat hippocampus. Place navigation and spatial searching results showed that SVHRP blocked Aβ1-40-induced impaired learning and memory.

S4B-4 A Novel Allosteric Phosphodiesterase 4D Inhibitor BPN14770 Reverses Cognitive Impairment in Humanized PDE4D Mice

Classical inhibitors of PDE4 lack subtype selectivity due to exact amino acid sequence conservation of the catalytic site,and consequently,development of these drugs has stalled due to dose-limiting side effects of nausea and emesis.While use of subtype-selective inhibitors(i.e.,for PDE4A,B,or D)could overcome this issue,conservation of the catalytic region,to which classical inhibitors bind,limits this approach.The present study examined the effects of BPN14770,an allosteric inhibitor of PDE4D,which binds to a primate-specific,N-terminal region,conferring greater than 260-fold selectivity for PDE4D.BPN14770 was 100-fold more potent for improving memory and cognition in humanized PDE4D(hPDE4D)mice,which expressed the primate-specific binding sequence,compared to wild-type mice;meanwhile,it exhibited low potency in a mouse surrogate model for emesis.The behavioral and matching neurochemical data presented established a relationship between PDE4D target engagement and effects on cognition for BPN14770.Furthermore,BPN14770 reversed memory and cognitive deficits induced byβ-amyloid peptide 1-42(Aβ42)in Morris water maze,Y maze and novel object recognition tests in the humanized PDE4D mice.The morphological analyses suggested that the number of dendrites and the dendritic length in the CA1 of hippocampus were significantly increased after the Aβ42-treated hPDE4D mice were administered of BPN14770 for two weeks.The neurochemical and molecular biological assays suggested that neuroplasticity-related proteins and neurotrophic factor BDNF in the hippocampus of hPDE4D mice were significantly increased after the hPDE4D mice were treated with BPN14770.These findings suggest clinical potential for PDE4D selective inhibitors in disorders with cognitive deficits such as Alzheimer’s disease,which affects approximately 20 million people worldwide and nearly 5 million people in the United States.

Evaluating Anti-SmDl-amino-acid 83-119 Peptide Reactivity in Children with Systemic Lupus Erythematosus and Other Immunological Diseases

Background： SmD1-amino-acid 83-119 peptide （SmD183-119） is the major epitope of Smith （Sm） antigen, which is specific for adult systemic lupus erythematosus （SLE）. The anti-SmD183-119 antibody has exhibited higher sensitivity and specificity than anti-Sm antibody in diagnosing adult SLE. However, the utility of anti-SmD183-119 antibodies remains unclear in children with SLE （cSLE）. This study aimed to assess the characteristics of anti-StuD 183-119 antibody in the diagnosis of cSLE. Methods： Samples from 242 children with different rheumatological and immunological disorders, including autoimmune diseases （SLE [n = 46] and ankylosing spondylitis [AS, n = 11]）, nonautoimmune diseases （Henoch-Schonlein purpura [HSP, n = 60], idiopathic thrombocytopenia purpura [n = 27], hematuria [n = 59], and arthralgia [n = 39]） were collected from Shanghai Children＇s Medical Center from March 6, 2012 to February 27, 2014. Seventy age- and sex-matched patients were enrolled in this study as the negative controls. All the patients＇ sera were analyzed for the anti-SmD 183-119, anti-Sm, anti-U 1-nRNP, anti-double-stranded DNA （dsDNA）, anti-nucleosome, anti-SSA/Ro60, anti-SSA/Ro52, anti-SSB, anti-Scl-70, and anti-histone antibodies using the immunoblotting assay. The differences in sensitivity and specificity between anti-SmD183-119 and anti-Sm antibodies were compared by Chi-square test. The correlations between anti-SmD183-119 and other auto-antibodies were analyzed using the Spearman＇s correlation analysis. A value of P 〈 0.05 was considered statistically significant. Results： Thirty-six out of 46 patients with cSLE were found to be positive for anti-SmD183-119, while 12 patients from the cSLE cohort were found to be positive for anti-Sm. Compared to cSLE, it has been shown that anti-SmD183-119 was only detected in 27.3% of patients with AS and 16.7% of patients with HSP. In comparison with anti-Sm, it has been demonstrated that anti-StuD 183-119 had a higher sensitivity （78.3% vs. 26.1%, 2＇2 = 25.1, P 〈 0.05） and a lower specificity （90.8% vs. 100%, x^2 = 13.6, P 〈 0.05） in the diagnosis of cSLE. Further analysis revealed that anti-StuD 183-119 antibodies were positively correlated with anti-dsDNA, anti-nucleosome, and anti-histone antibodies in cSLE. Moreover, it has been clearly shown that anti-SmD183-119was more sensitive than anti-Sm in discriminating autoimmune diseases from nonautoimmune disorders in patients with arthralgia or hematuria. Conclusions： Measurement of anti-SmD183-119 in patients with cSLE has a higher sensitivity and a marginally lower specificity than anti-Sin. It has been suggested that inclusion of anti-SmD183-119 testing in the integrated laboratory diagnosis ofcSLE may significantly improve the overall sensitivity in child populations.

1-(3-Aminopropyl)-3-butylimidazolium bromide for carboxyl group derivatization:potential applications in high sensitivity peptide identification by mass spectrometry

The cationic reagent 1-(3-aminopropyl)-3-butylimidazolium bromide(BAPI) was exploited for the derivatization of carboxyl groups on peptides.Nearly 100% derivatization efficiency was achieved with the synthetic peptide RVYVHPI(RI-7).Furthermore,the peptide derivative was stable in a 0.1% TFA/water solution or a 0.1%(v/v) TFA/acetonitrile/water solution for at least one week.The effect of BAPI derivatization on the ionization of the peptide RI-7 was further investigated,and the detection sensitivity was improved >42-fold via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS),thus outperforming the commercial piperazine derivatization approach.Moreover,the charge states of the peptide were largely increased via BAPI derivatization by electrospray ionization(ESI) MS.The results indicate the potential merits of BAPI derivatization for high sensitivity peptide analysis by MS.

Clinical Study on Treatment of Advanced Non-small Cell Lung Cancer by Arsenious Acid Combined with Tα-1 Thymus Peptide and Megestrol Acetate

Objective: To observe the therape utic effect of arsenious acid combined with Tα-1 thymus peptide and megestro l acetate on advanced non-small cell lung cancer.Methods: Nintey-two patients were divided randomly into the tr eated group( n =45) and the control group( n =47). The treated group were tr eated with arsenious acid combined with Tα-1 thymus peptide and megestrol ac etate, and the control group were treated with chemotherapy in the NP protocol .Results: (1) Therapeutic effect :In the 36 patients of the treated group, 2 were evaluated as CR, 8 as PR, 9 as MR, 8 as SD and 9 as PD, the CR+PR rate being 27.7% (10/36), while in the 40 patients of the control group, the co rresponding numbers were 3, 10, 11, 9, 7 and 32.5% (13/40). Comparison between t he CR+PR rate between the two groups showed insignificant difference ( P >0.05 ). (2)Clinical benefit rate: The positive rate of Karnofsky scores in the treate d group and the control group was 44.4% and 20.0% respectively; and the positive rate of body weight in the two groups was 33.3% and 12.5% respectively, the dif ference between the two groups was significant ( all P <0.05). (3)Changes of T- cell subsets and NK cell activity: CD4 and CD4/CD8 ratio after treatment i n the treated group increased significantly ( P <0.05), while in the control group, CD3, CD4, CD4/CD8 ratio and NK cell activity all lowered significantly ( P <0.01). Comparison between the two groups after treatment showed significa nt difference in CD4, CD4/CD8 ratio and NK cell activity, with those in the trea ted group all higher than those in the control group ( P <0.01). (4)Adeverse -reaction: No serious adverse reaction was found in both two groups. (5)Media n survival period:The treated group was 30 weeks and that in the control group w as 28.5 weeks, and the difference between the two groups was insignificant ( P >0.05).Conclusion: Arsenious acid combined with Tα-1 thymus peptide and megestrol acetate was a relatively effective scheme with low toxicity in tre ating advanced NSCLC.

Supramolecular assembly of Cp1-11 peptide and insulin for rapid-acting formulation

In order to improve the life quality of diabetic patients,it is very important to develop rapid-acting insulin formulations that can mimic the physiological meal-time secretion profile of insulin in healthy people.Although several insulin analogues have been designed to provide postprandial glycemic control,still there are some serious disadvantages.A supramolecular strategy is presented here to inhibit insulin aggregation and improve its bioactivity by using Cp1-11 peptide.As a fragment of C-peptide in proinsulin,Cp1-11 peptide was found to influence insulin oligomerization by supramolecular interactions.This work demonstrates that the Cp1-11 peptide can interact with oligomeric insulin and facilitate its disaggregation into the physiologically active monomeric form.Computer simulation indicates that Cp1-11 can insert into the space between the C-terminal tail and the N-terminal helix of the B-chain of insulin,causing dissociation of the insulin dimer.The supramolecular assembly of Cp1-11 and insulin can improve the bioavailability and therapeutic effect of insulin on the control of in vivo blood glucose levels.These results suggest that Cp1-11 peptide can modulate the intermolecular interaction of aggregated insulin and prevent the transition from monomeric to multimeric states,and shows great potential for the development of an effective rapid-acting strategy to treat diabetes.

胰高血糖素样肽-1在消化系统中的病理生理作用

胰高血糖素样肽（glucagon-like peptide，GLP）-1是一种主要由肠道L细胞分泌的胃肠肽类激素，在消化系统和中枢神经系统中均有发现。GLP-1可作用于胰岛细胞并发挥多种功能，包括刺激胰岛素的生物合成和分泌，抑制胰高血糖素的分泌。GLP-1还可作用于下丘脑和胃肠道，发挥抑制食欲、延缓胃排空及抑制肠道运动等作用。

黑逍遥散对AD模型小鼠海马区Aβ_(1-42),GSK-3β,NEP,IDE表达的影响

目的:研究黑逍遥散对阿尔茨海默症(Alzheimer disease,AD)小鼠海马区β淀粉样多肽1-42(β-amyloid 1-42peptide,Aβ1-42),糖原合成酶激酶-3(glycogen synthase kinase-3β,GSK-3β),脑啡肽酶(neprilysin,NEP),胰岛素抵抗酶(insulindegrading enzyme,IDE)表达的影响。方法:42只APP/PSI双转基因小鼠称体质量后,按随机原则分为4组,分别为模型组,阳性药组(盐酸多奈哌齐,3. 25 mg·kg-1),黑逍遥散高、低剂量组(6,3 g·kg-1)。10只同月龄、同种系的野生型C57BL/6J小鼠为正常组。连续灌胃12周后,Morris水迷宫予以行为学检测,苏木素-伊红(HE)染色观察海马神经元的形态改变,采用免疫组化技术分别检测小鼠海马区Aβ1-42,GSK-3β,NEP,IDE蛋白的表达。结果:治疗3个月后,与正常组比较,AD模型组小鼠,逃避潜伏期均延长,跨原平台次数减少(P <0. 01),小鼠海马区Aβ1-42,GSK-3β蛋白阳性表达显著增强,NEP与IDE蛋白阳性表达显著减弱(P <0. 01),HE染色显示AD模型小鼠海马神经细胞损伤严重;与模型组比较,用药各组小鼠的逃避潜伏期均显著缩短、跨原平台次数均显著增加(P <0. 05,P <0. 01),小鼠海马区Aβ1-42,GSK-3β蛋白阳性表达显著减弱,NEP与IDE蛋白阳性表达显著增强(P <0. 05,P <0. 01),HE染色显示各治疗组小鼠海马神经细胞损伤减轻。结论:黑逍遥散能够显著改善AD小鼠的学习记忆能力,可能与调节Aβ在海马区的异常沉积和降解作用等方面来减轻AD小鼠认知能力损伤有关。

The ubiquitin ligase Nedd4-2 mediates the regulation of PepT2 by mTORC1 in bovine mammary epithelial cells

Peptide transporter 2(PepT2)transports short peptides from the blood into bovine mammary epithelial cells(BMEC)to stimulate milk protein synthesis.Despite the fact that the effect of PepT2 is acknowledged in BMEC,little is known about its regulation.This study was completed to investigate the role of mammalian target of the rapamycin(mTOR)signaling in regulating the expression and function of PepT2 in BMEC.The regulation of PepT2 by mTOR in BMEC was studied in vitro using peptide transport assay,gene silencing,Western blot.The membrane expression of PepT2 and the uptake of b-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid(b-Ala-Lys-AMCA),a model dipeptide,in BMEC were reduced by rapamycin(a mTOR inhibitor)and silencing of either mTOR complex 1(mTORC1)or mTOR complex 2(mTORC2),stimulated by DEP domain-containing mTOR-interacting protein(DEPTOR,endogenous inhibitor of mTORC1 and mTORC2)silencing.The trafficking of PepT2 to the membrane and the uptake of b-Ala-Lys-AMCA was promoted by neuronal precursor cell-expressed developmentally down-regulated 4 isoform 2(Nedd4-2)silencing.The effects of knockdown of mTORC1,but not mTORC2,on cell membrane expression and transport activity of PepT2 was abolished by Nedd4-2 silencing.With immunofluorescence staining,PepT2 was identified to be interacting with Nedd4-2.The Nedd4-2 expression and the interaction between PepT2 and Nedd4-2 was increased through mTORC1 knockdown,indicating an increased ubiquitination of PepT2.The results revealed that mTORC1 can regulate the expression and function of PepT2 through Nedd4-2 in BMEC.

Identification of a New Member of the Ep-CAM (17-1A) Tumor-Associated Antigen Family

The tumor-associated antigen Ep-CAM (17-1A antigen), defined by the murine monoclonal antibody (mAb) 17-1A, has been identified as a 42-kD glycoprotein. The mAb 17-1A has been used for immunotherapy of colorectal cancer. We obtained mAb 19F4 using a synthetic peptide containing antigen determinants of 17-1A antigen. The mAb 19F4 can bind the corresponding dominants of the 17-1A antigen in ELISA. Western-blot analysis demonstrated that mAb 19F4 recognized a 50-kD protein from cell lysates of MCF-7 (breast cancer cell line). Both mAb 19F4 and 17-1A detected a 42-kD protein in the cell lysates of HT-29 (colorectal cancer cell line). The results suggest that new members of the tumor-associated antigen family 17-1A may exist.

Relationship between Growth Hormone and Cytokines in Short Children Undergoing Growth Hormone Stimulation Testing

Background: The relationship between growth hormone (GH) and cytokines remains unclear. Several studies have suggested that GH increases tumor necrosis factor (TNF)-α production in both children and adults. However, a number of studies have demonstrated a negative correlation between GH and TNF α. The aim of this study is to explore the relationship between endogenous GH secretion and certain pro and anti-inflammatory cytokines in short children undergoing GH stimulation testing for evaluation for GH deficiency. Methods: Plasma growth hormone, TNF α, CRP, IL-6, IL1-β, IL-4 and IL-10 levels are obtained at baseline and every 30 minutes for 150 minutes following two provocative agents (clonidine, and either arginine or glucagon). Results: Among the 23 children, 7 are found to be GH deficient. No significant differences in baseline TNF α levels are found between GH deficient and GH sufficient children. No correlation is identified between TNF α levels and GH levels during stimulation testing. Furthermore, no relationship is found between GH and pro-inflammatory cytokines or GH and anti-inflammatory cytokines. Conclusion: Our results do not demonstrate an acute relationship between endogenous GH secretion and the cytokines examined.

Protective effect of Xuebijing injection on myocardial injury in patients with sepsis: a randomized clinical trial

OBJECTIVE: To investigate the protective effect and possible mechanism of Xuebijing Injection on myocardial injury in patients with sepsis, and to evaluate its prognostic implications.METHODS: Patients with septic myocardial injury were recruited, and were randomly divided into two groups: treatment group and control group. All patients in two groups received conventional cluster treatment, the patients in treatment group additional received Xuebijing injection dissolved in0.9% sodium chloride injection, and the patients in control group received the same amount of 0.9%sodium chloride injection. At the beginning of treatment and 3, 7 and 10-day after treatment, lab-oratory indicators of cardiac troponin Ⅰ(cTnI),N-terminal pro B-type natriuretic peptide(NT-pro BNP) and procalcitonin(PCT) were respectively tested in venous blood. The patient's length of stay in Intensive Care Unit(ICU) and the mortality in 28 days were recorded.RESULTS: At 3, 7 and 10-day after treatment, the improvements of c Tn I, NT-pro BNP and PCT in treatment group were better than those in control group, and the differences were statistically significant(P < 0.05). The mortality of treatment group in28 days was not significantly different from that of control group(P > 0.05). The ICU length of stay of treatment group was shorter than that of control group(P > 0.05).CONCLUSION: Xuebijing injection could improve the levels of c Tn I, NT-pro BNP and PCT in patients with septic myocardial injury.and it had a protective effect on myocardial injury.