Early active immunization with Aβ_(3–10)-KLH vaccine reduces tau phosphorylation in the hippocampus and protects cognition of mice

Active and passive anti-Aβ immunotherapies have successfully been used for the prevention and treatment of Alzheimer's disease animal models. However, clinical use of these immunotherapies is not effective, because the vaccination is administered too late. At 1 month of age, 100 μL of Aβ3–10-KLH peptide(vaccine, 2 μg/μL) was subcutaneously injected into the neck of an amyloid precursor protein/presenilin-1/tau transgenic(3×Tg-AD) mouse model. Aβ3–10-KLH peptide was re-injected at 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5 months of age. Serum levels of Aβ antibody were detected by enzyme-linked immunosorbent assay, while spatial learning and memory ability were evaluated by Morris water maze. Immunohistochemistry was used to detect total tau with HT7 and phosphorylated tau with AT8(phosphorylation sites Ser202 and Thr205) and AT180(phosphorylation site Thr231) antibodies in the hippocampus. In addition, western blot analysis was used to quantify AT8 and AT180 expression in the hippocampus. The results showed that after vaccine injection, mice produced high levels of Aβ antibody, cognitive function was significantly improved, and total tau and phosphorylated tau levels were significantly reduced. These findings suggest that early active immunization with Aβ3–10-KLH vaccine can greatly reduce tau phosphorylation, thereby mitigating the cognitive decline of 3×Tg-AD mice. This study was approved by the Animal Ethics Committee of China Medical University, China(approval No. 103-316) on April 2, 2016.     

Effects of increased human tumor necrosis factor-like molecule 1A expression in peripheral blood of children with acute Guillain-Barre syndrome on interferon-gamma secretion

BACKGROUND： Human tumor necrosis factor-like molecule 1A （hTL1A） is a strong T helper cell type 1 （Thl） co-stimulator. Guillain-Barre syndrome （GBS） is an autoimmune disorder of the nervous system, which is mediated by Thl cells. OBJECTIVE： To determine hTL1A expression in peripheral blood T lymphocytes of acute GBS children and the effects of hTL1A on secretion of interferon-γ. DESIGN, TIME AND SETTING： A randomized, controlled, neuroimmunological in vitro study was performed at the Central Laboratory of First Hospital of Jilin University, China from November 2005 to November 2007. MATERIALS： Venous blood samples were obtained from 6 healthy donors, aged 6-12 years （all routine blood examination items were normal）, and 6 additional children with acute GBS, aged 6-12 years. The GBS children fell ill within 1 week and were not treated with hormones or immunoglobulin Purified recombinant human soluble tumor necrosis factor-like molecule 1A （rhsTL1A, 1 mg/mL, relative molecular mass 22 000, 6× His tag, soluble form） was supplied by the Central Laboratory of First Hospital of Jilin University, China. METHODS： Peripheral blood mononuclear cells were isolated from healthy donors using the standard Ficoll gradient centrifugation and were incubated in 96-well culture plates. The cells were assigned to the following groups： control （2 μg/mL phytohemagglutinin）, 2μg/mL phytohemagglutinin ＋ 25, 100 and 400 ng/mL rhsTL1A. T cell proliferation was quantified using the tritiated thymidine （3H-TdR） method. Serum interferon-γ levels in acute GBS children were detected by enzyme-linked immunosorbent assay （ELISA）. The ratio of hTL1A-positive T cells to CD3-positive T cells in peripheral blood of acute GBS children was determined using flow cytometry. Following in vitro pre-activation of peripheral blood mononuclear cells by 2 μg/mL phytohemagglutinin, the peripheral blood mononuclear cells were treated with 400 ng/mL exogenous rhsTLIA. Finally, peripheral blood mononuclear cell-secreted interferon-γlevels were measured by ELISA. MAIN OUTCOME MEASURES： The following parameters were measured： rhsTLIA stimulation index to stimulate proliferation of T cells; the serum interferon-γ levels in acute GBS children; the ratio of hTL1A-positive cells to CD3-positive cells; the levels of interferon-γ secreted by peripheral blood mononuclear cells in acute GBS children, as well as rhsTL1A-stimulated interferon-γ levels. RESULTS： T cell proliferation assay revealed that the stimulation index in each rhsTL1A group was greater than the control group. The stimulation index of the 400 ng/mL rhsTL1A group was the greatest. Serum interferon-γ levels in acute GBS children were significantly greater than the control group （P 〈 0.05）. The ratio of hTLIA＋ CD3＋ T cells to CD3＋ T cells in acute GBS children was significantly greater than the control group （P 〈 0.01 ）. Phytohemagglutinin stimulated peripheral blood mononuclear cells to a greater extent than 400 ng/mL rhsTL1A in the acute GBS group, and the secreted interferon-γ levels were significantly increased （P 〈 0.05）. CONCLUSION： In T cells pre-activated with 2 μg/mL phytohemagglutinin, proliferation was effectively increased with 400 ng/mL rhsTL1A treatment. Expression of hTLIA was increased in activated T cells from peripheral blood of acute GBS children, followed by increased interferon-γ secretion. These mechanisms are considered to be part of the pathological process that induces the secretion of inflammatory cytokines in GBS syndrome.     

N-methyl-D-aspartate receptor subtype 3A promotes apoptosis in developing mouse brain exposed to hyperoxia

In the present study, 7 day postnatal C57/BL6 wild-type mice (hyperoxia group) and 7 day postnatal N-methyl-D-aspartate receptor subtype 3A knockout mice (NR3A KO group) were exposed to 75% oxygen and 15% nitrogen in a closed container for 5 days. Wild-type mice raised in normoxia served as controls. TdT-mediated dUTP nick end labeling (TUNEL)/neuron-specific nuclear protein (NeuN) and 5-bromo-2’-deoxyuridine (BrdU)/NeuN immunofluorescence staining showed that the number of apoptotic cells and the number of proliferative cells in the dentate subgranular zone significantly increased in the hyperoxia group compared with the control group. However, in the same hyperoxia environment, the number of apoptotic cells and the number of proliferative cells significantly decreased in the NR3A KO group compared with hyperoxia group. TUNEL+/NeuN+ and BrdU+/NeuN+ cells were observed in the NR3A KO and the hyperoxia groups. These results demonstrated that the NR3A gene can promote cell apoptosis and mediate the potential damage in the developing brain induced by exposure to non-physiologically high concentrations of oxygen.     

Effects of Acanthopanax senticosus on learning and memory in a mouse model of Alzheimer''''s disease and protection against free radical injury to brain tissue

BACKGROUND:Acanthopanax senticosus,a plant of the Araliaceae family,is used in traditional Chinese medicine.It can be used to replenish Qi,strengthen the spleen,tonify the kidney,and relieve mental strain.OBJECTIVE:To observe effects ofAcanthopanax senticosus on learning and memory in a mouse model of Alzheimer's disease and abnormal biochemical changes in the brain tissue.DESIGN:A completely randomized grouping,controlled animal experiment.SETTING:Department of Preventive Medicine,School of Basic Medical Sciences,Yanbian University.MATERIALS:A total of 50 Kunming mice,aged 1-1.5 months,equal numbers of males and females,were provided by the Laboratory Animal Center,Yanbian University Medical College.The study was performed in accordance with ethical guidelines for the use and care of animals.Acanthopanax was provided by Yanbian Chengda Pharmaceutical Co.,Ltd.Acanthopanax senticosus(0.5 kg)was soaked in water for 1 hour and transferred to 1.5 kg distilled water for extraction.It was boiled for 1 hour and extracted after 1 hour of boiling.The procedure was repeated 3 times.The extract was condensed to 500 mL and then adjusted to 500 and 1 000 g/L with water.Piracetam tablets were produced by Shandong Luoxin Pharmaceutical Corporation, China.Malonaldehyde(MDA),superoxide dismutase(SOD),and acetylcholinesterase(ACHE)kits were purchased from Nanjing Jiancheng Bioengineering Co.,Ltd.,China. METHODS:This study was performed at the Department of Preventive Medicine,School of Basic Medical Sciences,Yanbian University from January to June 2007.All mice were randomly divided into 5 groups with 10 mice in each:control group,model group,low-,and high-dose Acanthopanax senticosus-treated groups, and piracetam-treated group.All groups were administered 0.1 mL/10 g.In the control and model groups, mice were intragastrically administered saline each morning for 5 days prior to experimentation.Five days later,they were intraperitoneally perfused with saline and aluminum trichloride(100 mg/kg),respectively, every other afternoon.In the low- and high-dose Acanthopanax senticosus-treated groups,as well as the piracetam-treated groups,mice were intragastrically administered 500 and 1 000 g/L Acanthopanax senticosus and 23 g/L piracetam suspension,respectively,every morning.Five days later,they were intraperitoneally perfused with aluminum trichloride(100 mg/kg)every other afternoon,20 successive times.MAIN OUTCOME MEASURES:On days 20,23,26,and 29 after treatment,time-to-platform,number of errors,and accuracy were measured by water maze.After anesthesia,mice were euthanized and whole brain tissues were immediately resected and homogenized.MDA levels,and SOD and AChE activities,were measured using the corresponding kits. RESULTS:Fifty mice were included in the final analysis.In the model group,accuracy increased,and time-to-platform was prolonged,compared to control group(P＜0.05-0.01).In the model group,MDA levels significantly increased(P＜0.05),while SOD activity significantly decreased(P＜0.01 ),compared to control group.MDA levels were significantly lower and SOD activity was significantly higher in the low-and high-dose Acanthopanax senticosus-treated groups,compared to the model group(P＜0.05-0.01).In the model group,AChE activity significantly increased,compared to the control group(P＜0.05).AChE activity was significantly lower in the high-dose Acanthopanax senticosus-treated and the piracetam-treated groups than in the model group(P＜0.05).CONCLUSION:Acanthopanax senticosus can improve learning and memory in a mouse model of Alzheimer's disease,and concomitantly increase SOD activity,inhibit AChE activity,and decrease MDA levels.     

Effect of propofol on glutamate-induced activation and elated inflammatory cytokines of astrocytes from spinal cord dorsal horn

BACKGROUND: Astrocytes participate in central nervous system-mediated physiological or pathological processes, such as pain. Activated dorsal horn astrocytes from the spinal cord produce nerve active substances and proinflammatory cytokines, such as interleukin-I beta (IL-1 β ), IL-6, and tumor necrosis factor-a (TNF-a ), which play important roles in pain transduction and regulation. OBJECTIVE: To investigate the effects of different doses of propofol on activation of cultured spinal cord dorsal horn astrocytes induced by glutamate, as well as changes in IL-1 β, IL-6, and TNF-a, and IL-10 (anti-inflammatory cytokine) expression in rats, and to explore the dose relationship of propofnl. DESIGN, TIME AND SETTING: The cellular and molecular biology experiment was performed at the Central Laboratory of Yunyang Medical College between March 2006 and December 2007. MATERIALS: Forty healthy, Wistar rats, aged 2-3 days, were selected. Propofol was provided by Zeneca, UK; glutamate by Sigma, USA; EPICS XL flow cytometry by Beckman culture, USA; rabbit-anti-mouse glial fibrillary acidic protein (GFAP) antibody kit and inflammatory cytokine detection kit were provided by Zhongshan Biotechnology Company Ltd., Beijing; multimedia color pathologic image analysis system was a product of Nikon, Japan. METHODS: Astrocytes were harvested from T11-L6spinal cord dorsal horn of Wistar rats and incubated for 3 weeks. The cells were divided into seven groups, according to various treatment conditions: control group was cells cultured in Hank's buffered saline solution; intralipid group was cells cultured in intralipid (0.2 mL/L); glutamate group was cells cultured with 100 μ mol/L glutamate; propofol group was cells cultured with 250 μ mol/L propofol; three glutamate plus propofol groups were cultured in 100 μ mol/L of glutamate, followed by 5, 25, and 250 μ mol/L of prnpofol 10 minutes later. MAIN OUTCOME MEASURES: GFAP-labeled astrocytes were analyzed using a multimedia pathology imaging analysis system to detect area density (AD) and average optical density (AOD) of positive cells. The supernatant concentrations of IL-1 β, TNF- a, IL-6, and IL-10 were determined using radioimmune assays. RESULTS: Compared with the control group, cells in the glutamate plus low-dose propofol group were activated and hypertrophic, and AD and AOD were significantly increased (P < 0.01 ). Concentrations of IL-1 β, TNF- a, and IL-6 were also significantly increased (P < 0.01 ), while IL-10 levels remained unchanged (P > 0.05), but still higher than the control and glutamate groups (P>0.05). Compared with the glutamate group, astrocyte activation was inhibited by moderate and high-dose propofol. In addition, with moderate and high-dose propofol, AD, AOD, IL-1 β, TNF-a, and IL-6 concentrations were significantly decreased (P < 0.05-0.01 ), and IL-10 levels were increased (P < 0.01 ). CONCLUSION: Propofol can effectively inhibit glutamate-induced astrocyte activation in the spinal cord dorsal horn, significantly inhibit production of IL-1β, TNF-a, and IL-6, and increase IL- 10 synthesis and release in a dose-dependent manner.     

Retinal ganglion cell death in a DBA/2J mouse model of glaucoma Microglial activation and intraocular pressure

BACKGROUND: Retinal microglia has been shown to reactivate in a murine model of pigmentary glaucoma. However, the relationship between microglial activation and intraocular pressure (IOP) elevation and retinal ganglion cell (RGC) death is still unclear. OBJECTIVE: To verify that microglial activation and tumor necrosis factor alpha (TNF-α) expression is involved in RGC death with elevated IOP and prolonged time of glaucomatous optic nerve lesion in a DBA/2J mouse model of glaucoma. DESIGN, TIME AND SETTING: This randomized, controlled, animal experiment was performed at the Peking University Third Hospital, Peking University Eye Center, China between December 2006 and May 2008. MATERIALS: DBA/2J mice and C57BL/6J mice (Jackson Laboratory, USA), rat anti-mouse CD11b monoclonal antibody (Serotec, UK), and goat anti-TNF-α polyclonal antibody (Sigma, USA) were used in this study. METHODS: A total of 100 female, DBA/2J mice at 3, 6, 9,12, and 14 months of age (20 mice per age group) were used for the glaucoma model, and 18 C57BLV6J mice at 3, 9,14 months of age (6 mice per age group) were used as normal controls. The anterior segment of the eye was observed using a slit-lamp biomicroscope. IOP was measured using a microneedle system. Morphology and number of retinal microglia were observed using immunohistochemistry. RGCs were quantified using Nissl staining. Co-localization of TNF-a and microglia was observed using double-labeling immunofluorescence. Excavation of the optic nerve head was observed utilizing hematoxylin-eosin staining. MAIN OUTCOME MEASURES: The following parameters were measured: IOP levels, numbers of RGCs and activated microglia, and TNF-α expression. RESULTS: In 6-month-old DBA/2J mice, dispersed pigment was observed, and some mice developed increased IOP. At 9 months of age, IOP levels reached a peak. In 3-month-old DBA/2J mice, microglia were activated. In 6-month-old DBA/2J mice, the number of activated microglia was significantly increased and migrated to the outer retinal layer. In 9-month-old mice, TNF-α expression was co-localized with microglia. Significant RGC loss occurred in mice aged 9 to 14 months, with the presence of optic nerve fiber loss and optical nerve head excavation. IOP returned to normal levels at 12 months of age, but microglia remained activated, which was consistent with RGC loss. CONCLUSION: Retinal microglial activation was partially attributed to increased IOP. Activated microglia might be mainly responsible for RGC loss. TNF-α expression was evident in the inner retinal layer. However, the relationship between TNF-α and RGC loss remains poorly understood.     

Hyperoside protects the blood-brain barrier from neurotoxicity of amyloid beta 1–42

Mounting evidence indicates that amyloid β protein(Aβ) exerts neurotoxicity by disrupting the blood-brain barrier(BBB) in Alzheimer's disease. Hyperoside has neuroprotective effects both in vitro and in vivo against Aβ. Our previous study found that hyperoside suppressed Aβ_(1–42)-induced leakage of the BBB, however, the mechanism remains unclear. In this study, bEnd.3 cells were pretreated with 50, 200, or 500 μM hyperoside for 2 hours, and then exposed to Aβ_(1–42) for 24 hours. Cell viability was determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Flow cytometry and terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay were used to analyze cell apoptosis. Western blot assay was carried out to analyze expression levels of Bax, Bcl-2, cytochrome c, caspase-3, caspse-8, caspase-9, caspase-12, occludin, claudin-5, zonula occludens-1, matrix metalloproteinase-2(MMP-2), and MMP-9. Exposure to Aβ_(1–42) alone remarkably induced bEnd.3 cell apoptosis; increased ratios of cleaved caspase-9/caspase-9, Bax/Bcl-2, cleav ed caspase-8/caspase-8, and cleaved caspase-12/caspase-12; increased expression of cytochrome c and activity of caspase-3; diminished levels of zonula occludens-1, claudin-5, and occludin; and increased levels of MMP-2 and MMP-9. However, hyperoside pretreatment reversed these changes in a dose-dependent manner. Our findings confirm that hyperoside alleviates fibrillar Aβ_(1–42)-induced BBB disruption, thus offering a feasible therapeutic application in Alzheimer's disease.     

Absence of ephrin-A2/A3 increases retinal regenerative potential for Müller cells in Rhodopsin knockout mice

Müller cells(MC) are considered dormant retinal progenitor cells in mammals.Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain.It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC.Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells.In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC.The level of ephrin As/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC.Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2~(–/–) A3~(–/–) mice by 5-ethynyl-2′-deoxyuridine(EdU) incorporation.We detected a significant increase of EdU~+ cells in MC derived from A2~(–/–) A3~(–/–) mice.Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout(Rho~(–/–)) mice.To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2~(–/–) A3~(–/–), Rho~(–/–) and Rho~(–/–) A2~(–/–) A3~(–/–) mice and the numbers of EdU~+ cells distributed among different layers of the retina.Ephrin As/EphA4 expression was upregulated in the retina of Rho~(–/–) mice compared to the wild-type mice.In addition, cultured MC derived from ephrin-A2~(–/–) A3~(–/–) mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice.Interestingly, we detected a significant increase of EdU~+ cells in the retinas of adult ephrin-A2~(–/–) A3~(–/–) mice mainly in the inner nuclear layer; and these EdU~+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC.In Rhodopsin knockout mice(Rho~(–/–) A2~(–/–) A3~(–/–) mice), a significantly greater amount of EdU~+ cells were located in the ciliary body, retina and RPE than that of Rho~(–/–) mice.Comparing between 6 and 12 weeks old Rho~(–/–) A2~(–/–) A3~(–/–) mice, we recorded more EdU~+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration.Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC.Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration.All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA(approval No.S-353-0715) on October 24, 2012.     

Ratanasampil reduced levels of Aβ1-40 and Aβ1-42 and Aβ42/Aβ40 ratio in brains of Tg2576 mouse model of Alzheimer's disease

Objective To investigate the effect of ratanasampil (RNSP) which is traditional Chinese Tibet-Medicines on the β-amyloid peptide as a therapeutic target in Alzheimer's disease.Methods Thirteen female mice of 13-14 months old with Tg2576 transgene and ten BL6 × SJL mice as control were used in drug test All mice sacrificed after 8 weeks of RNSP treatment at 15-16 months of age.Open field activity and Y-maze tests were performed for animal behavioral change and memory ability.The β-amyloid peptides (Aβ1-40 and Aβ1-42) and β-amyloid precursor protein (APP) in Tg2576 mice brain were measured with western blot and enzyme linked immunosorbent assay (ELISA).Immunostaining with 1 E8 ( 1: 25 ) was carried out on brain sections of RNSP and vehicle-treated mice.Results The training times of Y-maze test decreased in RNSP-treated Tg2576 mice (P ＜0.01 ).After 2 months of RNSP treatment, a decrease in open field behavior was seen in Tg2576 mice ( P ＜ 0.05 ).The level of Aβ1-40 and Aβ1-42 in the brain was significantly decreased after RNSP treatment ( P ＜ 0.05 ).The Aβ42/Aβ40 ratio was significantly decreased after RNSP treatment as compared to vehicle-treated mice ( P ＜ 0.05 ) .But levels of APP in brain unchanged.Preliminary plaque counting of the sections showed reduced plaque numbers in the drug-treated mice in brain.Conclusions These results suggest that ratanasampil may reduce β-amyloid peptide production in brain to improve the learning and memory ability of Tg2576 mice of Alzheimer's disease.     

Ratanasampil reduced levels of Aβ1-40 and Aβ1-42 and Aβ42/Aβ40 ratio in brains of Tg2576 mouse model of Alzheimer's disease

Objective To investigate the effect of ratanasampil (RNSP) which is traditional Chinese Tibet-Medicines on the β-amyloid peptide as a therapeutic target in Alzheimer's disease.Methods Thirteen female mice of 13-14 months old with Tg2576 transgene and ten BL6 × SJL mice as control were used in drug test All mice sacrificed after 8 weeks of RNSP treatment at 15-16 months of age.Open field activity and Y-maze tests were performed for animal behavioral change and memory ability.The β-amyloid peptides (Aβ1-40 and Aβ1-42) and β-amyloid precursor protein (APP) in Tg2576 mice brain were measured with western blot and enzyme linked immunosorbent assay (ELISA).Immunostaining with 1 E8 ( 1: 25 ) was carried out on brain sections of RNSP and vehicle-treated mice.Results The training times of Y-maze test decreased in RNSP-treated Tg2576 mice (P ＜0.01 ).After 2 months of RNSP treatment, a decrease in open field behavior was seen in Tg2576 mice ( P ＜ 0.05 ).The level of Aβ1-40 and Aβ1-42 in the brain was significantly decreased after RNSP treatment ( P ＜ 0.05 ).The Aβ42/Aβ40 ratio was significantly decreased after RNSP treatment as compared to vehicle-treated mice ( P ＜ 0.05 ) .But levels of APP in brain unchanged.Preliminary plaque counting of the sections showed reduced plaque numbers in the drug-treated mice in brain.Conclusions These results suggest that ratanasampil may reduce β-amyloid peptide production in brain to improve the learning and memory ability of Tg2576 mice of Alzheimer's disease.     

乌司他丁对首次单纯二尖瓣机械瓣置换患者术后早期认知功能的影响

目的 探讨乌司他丁对首次单纯二尖瓣人工机械瓣置换患者术后早期认知功能的影响.方法 将40例老年单纯二尖瓣人工机械瓣置换患者随机分为对照组和实验组.实验组在麻醉诱导后开始恒速静脉输注乌司他丁6000U/kg~(-1)(30min内输完),然后以1000U/kg~(-1)/h~(-1)的速率持续静脉输注至手术结束,对照组则不使用乌司他丁.于CPB前(T1)、转流后60min(T2)、停机后60min(T3)、停机后360min(T4)抽取动脉血,测定血浆肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)浓度.分别在术前1d和术后第10天对患者进行神经精神功能9项测验.结果 两组患者血浆TNF-α、IL-6和IL-10的浓度,在12～T4各时点与体外循环前比较均明显增加(P<0.01);T2～T4时点实验组血浆TNF-α和IL-6浓度明显低于对照组(P<0.01),IL-10浓度明显高于对照组(P<0.05).术后认知功能障碍发生率对照组为40%(8例)、实验组为15%(3例),实验组组明显低于对照组(P<0.05).结论 乌司他丁可以降低首次单纯二尖瓣人工机械瓣置换患者术后早期认知障碍的发生率,这可能与其抑制体外循环过程中促炎细胞因子TNF-α和IL-6的释放、促进抗炎细胞因子IL-10的释放有关.

Abstract：

Objective To determine the effect of ulinastatin on early postoperative cognitive function to primary simple mitral valve replacement patients.Methods Forty adult patients suffered from primary simple mitral replacement were randomly divided into control group and study group.In study group,ulinastatin 6000U/kg in 50ml of normal saline was in fused intravenously over 30 min after induction of anesthesia followed by continuous infusion of ulinastatin at 1000 U/kg~(-1)/h~(-1) until the end of the operation.In control group.was not.The blood samples were collected at the same time point of preoperation(T1),60 min of cardiopulmonary bypass(T2),and 1h(T3),6h(T4)after eardiopulmonary bypass.Plasma concentration of IL-6,IL-10 and TNF-α were detected with ELISA.A battery of 9 neuropsychologieal tests Was performed and scored pre-operation and 10d after surgery.Results The plasma levels of IL-6,IL-10 and TNF-α in each group were significantly increased at T2,T3,T4.The plasma levels of IL-6 and TNF-α in study group were significantly lower than control group at T2,T3,T4.The plasma levels of IL-10 in study group were significantly higher than the levels in control group at T2,T3,T4.The incidence of postoperative cognitive dysfunction Was significantly lower in study group(40%)compared with the control group(15%)(P<0.05).Conclusions This study indicated that ulinastatin could reduce the incidence of postoperative cognitive dysfunction through the following mechanism through inducing the increased amplitude of plasma levels of TNF-α and IL-6,whereas enhancing the increased amplitude of the plasma IL-10 levels that resulted from CPB.     

Protective effects of Dendrobium nobile Lindl. alkaloids on amyloid beta(25–35)-induced neuronal injury

Dendrobium nobile Lindl.alkaloids(DNLA),the active ingredients of a traditional Chinese medicine Dendrobium,have been shown to have anti-oxidative effects,anti-inflammatory action,and protective effect on neurons against oxygen-glucose deprivation.However,it is not clear whether DNLA reduces amyloid-beta(Aβ)-induced neuronal injury.In this study,cortical neurons were treated with DNLA at different concentrations(0.025,0.25,and 2.5 mg/L)for 24 hours,followed by administration of Aβ_(25–35)(10μM).Aβ_(25–35) treatments increased cell injury as determined by the leakage of lactate dehydrogenase,which was accompanied by chromatin condensation and mitochondrial tumefaction.The damage caused by Aβ_(25–35) on these cellular properties was markedly attenuated when cells were pretreated with DNLA.Treatment with Aβ_(25–35)down-regulated the expressions of postsynaptic density-95 mRNA and decreased the protein expression of synaptophysin and postsynaptic density-95,all changes were significantly reduced by pretreatment of cells with DNLA.These findings suggest that DNLA reduces the cytotoxicity induced by Aβ_(25–35) in rat primary cultured neurons.The protective mechanism that DNLA confers on the synaptic integrity of cultured neurons might be mediated,at least in part,through the upregulation of neurogenesis related proteins synaptophysin and postsynaptic density-95.     

Protective effects of components of the Chinese herb grassleaf sweetflag rhizome on PC12 cells incubated with amyloid-beta42

The major ingredients of grassleaf sweetflag rhizome are β-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms of β-asarone and eugenol, components of the Chinese herb grassleaf sweetflag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations(between 1 × 10–10 M and 1 × 10–5 M) of β-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions(1 × 10–6 M β-asarone and eugenol). The survival rates of PC12 cells significantly increased, while expression levels of the m RNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl m RNA increased. In addition, the combination of β-asarone with eugenol achieved better results than either component alone. Our experimental findings indicate that both β-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.     

Neurotoxic role of interleukin-17 in neural stem cell differentiation after intracerebral hemorrhage

Interleukin 17(IL-17)and its main producer,T cell receptorγδcells,have neurotoxic effects in the pathogenesis of intracerebral hemorrhage(ICH),aggravating brain injuries.To investigate the correlation between IL-17 and ICH,we dynamically screened serum IL-17 concentrations using enzyme-linked immunosorbent assay and explored the clinical values of IL-17 in ICH patients.There was a significant negative correlation between serum IL-17 level and neurological recovery status in ICH patients(r=–0.498,P<0.01).To study the neurotoxic role of IL-17,C57 BL/6 mice were used to establish an ICH model by injecting autologous blood into the caudate nucleus.Subsequently,the mice were treated with mouse neural stem cells(NSCs)and/or IL-17 neutralizing antibody for 72 hours.Flow cytometry,brain water content detection,Nissl staining,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling results indicated that NSC transplantation significantly reduced IL-17 expression in peri-hematoma tissue,but there was no difference in T cell receptorγδcells.Compared with the ICH group,there were fewer apoptotic bodies and more Nissl bodies in the ICH+NSC group and the ICH+NSC+IL-17 group.To investigate the potential effect of IL-17 on directional differentiation of NSCs,we cultured mouse NSCs(NE-4 C)alone or co-cultured them with T cell receptorγδcells,which were isolated from mouse peripheral blood mononuclear cells,for 7 days.The results of western blot assays revealed that IL-17 secreted by T cell receptorγδcells reduced the differentiation of NSCs into astrocytes and neurons,while IL-17 neutralization relieved the inhibition of directional differentiation into astrocytes rather than neurons.In conclusion,serum IL-17 levels were elevated in the early stage of ICH and were negatively correlated with outcome in ICH patients.Animal experiments and cytological investigations therefore demonstrated that IL-17 probably has neurotoxic roles in ICH because of its inhibitory effects on the directional differentiation of NSCs.The application of IL-17 neutralizing antibody may promote the directional differentiation of NSCs into astrocytes.This study was approved by the Clinical Research Ethics Committee of Anhui Medical University of China(For human study:Approval No.20170135)in December 2016.All animal handling and experimentation were reviewed and approved by the Institutional Animal Care and Use Committee of Anhui Medical University(approval No.20180248)in December 2017.     

缺陷腺病毒载体表达重复10次的Aβ3-10基因疫苗鼻黏膜免疫APPswe, PSEN1dE9转基因小鼠可诱导出Th2型免疫反应

Immunotherapy for Alzheimer’s disease (AD) is effective in improving cognitive function in transgenic mouse models of AD. Because the AN1792 [beta-amyloid (Aβ) 1-42] vaccine was halted because of T cell mediated meningoencephalitis, many scientists are searching for a novel vaccine to avoid the T cell mediated immune response caused by the Aβ1-42. Importantly, the time when the immunization is begun can influence the immune effect. In this study, an adenovirus vaccine was constructed containing 10 × Aβ3-10 repeats and gene adjuvant CpG DNA. Transgenic AD mice were immunized intranasally for 3 months. After 10 × Aβ3-10 vaccine immunization, high titers of anti-Aβ42 IgG1 predominant antibodies were induced. In spatial learning ability and probe tests, the 10 × Aβ3-10 immunized mice showed significantly improved memories compared to control mice. The 10 × Aβ3-10 vaccine resulted in a robust Th2 dominant humoral immune response and reduced learning deficits in AD mice. In addition, the 10 × Aβ3-10 vaccine might be more efficient if administered before Aβ aggregation at an early stage in the AD mouse brain. Thus, the adenovirus vector encoding 10 × Aβ3-10 is a promising vaccine for AD.     

Role of Notch-1 signaling pathway in PC12 cell apoptosis induced by amyloid beta-peptide(25–35)

Recent studies have demonstrated that Notch-1 expression is increased in the hippocampus of Alzheimer's disease patients. We speculate that Notch-1 signaling may be involved in PC12 cell apoptosis induced by amyloid beta-peptide(25–35)(Aβ25–35). In the present study, PC12 cells were cultured with different doses(0, 0.1, 1.0, 10 and 100 nmol/L) of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a Notch-1 signaling pathway inhibitor, for 30 minutes. Then cultured cells were induced with Aβ25–35 for 48 hours. Pretreatment of PC12 cells with high doses of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester( 10 nmol/L) prolonged the survival of PC12 cells after Aβ25–35 induction, decreased the expression of apoptosis-related proteins caspase-3,-8,-9, increased the activity of oxidative stress-related superoxide dismutase and catalase, inhibited the production of active oxygen, and reduced nuclear factor kappa B expression. This study indicates that the Notch-1 signaling pathway plays a pivotal role in Aβ25–35-induced PC12 apoptosis.     

Altered serous levels of monoamine neurotransmitter metabolites in patients with refractory and non-refractory depression

The study examined plasma metabolite changes of monoamine neurotransmitters in patients with treatment-resistant depression (TRD) and non-TRD before and after therapy. All 30 TRD and 30 non-TRD patients met the diagnostic criteria for a depressive episode in accordance with the International Classification of Diseases, Tenth Revision. Before treatment, and at 4, 6, and 8 weeks after treatment, the plasma metabolite products of monoamine neurotransmitters in TRD group, including 5-hydroxyindoleacetic acid, 3-methoxy-4-hydroxyphenyl ethylene glycol and homovanillic acid, were significantly lower than those in the non-TRD group. After two types of anti-depressive therapy with 5-serotonin and norepinephrine reuptake inhibitor, combined with psychotherapy, the Hamilton Depression Rating Scale scores were significantly reduced in both groups of patients, and the serous levels of 5-hydroxyindoleacetic acid and 3-methoxy-4-hydroxyphenyl ethylene glycol were significantly increased. In contrast, the homovanillic acid level exhibited no significant change. The levels of plasma metabolite products of peripheral monoamine neurotransmitters in depressive patients may predict the degree of depression and the therapeutic effects of treatment.     

T cells promote the regeneration of neural precursor cells in the hippocampus of Alzheimer＇s disease mice

Alzheimer's disease is closely associated with disorders of neurogenesis in the brain, and growing evidence supports the involvement of immunological mechanisms in the development of the disease. However, at present, the role of T cells in neuronal regeneration in the brain is unknown. We injected amyloid-beta 1–42 peptide into the hippocampus of six BALB/c wild-type mice and six BALB/c-nude mice with T-cell immunodeficiency to establish an animal model of Alzheimer's disease. A further six mice of each genotype were injected with same volume of normal saline. Immunohistochemistry revealed that the number of regenerated neural progenitor cells in the hippocampus of BALB/c wild-type mice was significantly higher than that in BALB/c-nude mice. Quantitative fluorescence PCR assay showed that the expression levels of peripheral T cell-associated cytokines(interleukin-2, interferon-γ) and hippocampal microglia-related cytokines(interleukin-1β, tumor necrosis factor-α) correlated with the number of regenerated neural progenitor cells in the hippocampus. These results indicate that T cells promote hippocampal neurogenesis in Alzheimer's disease and T-cell immunodeficiency restricts neuronal regeneration in the hippocampus. The mechanism underlying the promotion of neuronal regeneration by T cells is mediated by an increased expression of peripheral T cells and central microglial cytokines in Alzheimer's disease mice. Our findings provide an experimental basis for understanding the role of T cells in Alzheimer's disease.     

Intranasal insulin ameliorates neurological impairment after intracerebral hemorrhage in mice

In Alzheimer’s disease and ischemic stroke,intranasal insulin can act as a neuroprotective agent.However,whether intranasal insulin has a neuroprotective effect in intracerebral hemorrhage and its potential mechanisms remain poorly understood.In this study,a mouse model of autologous blood-induced intracerebral hemorrhage was treated with 0.5,1,or 2 IU insulin via intranasal delivery,twice per day,until 24 or 72 hours after surgery.Compared with saline treatment,1 IU intranasal insulin treatment significantly reduced hematoma volume and brain edema after cerebral hemorrhage,decreased blood-brain barrier permeability and neuronal degeneration damage,reduced neurobehavioral deficits,and improved the survival rate of mice.Expression levels of p-AKT and p-GSK3βwere significantly increased in the perihematoma tissues after intranasal insulin therapy.Our findings suggest that intranasal insulin therapy can protect the neurological function of mice after intracerebral hemorrhage through the AKT/GSK3βsignaling pathway.The study was approved by the Ethics Committee of the North Sichuan Medical College of China(approval No.NSMC(A)2019(01))on January 7,2019.