Overexpression of Human Apolipoprotein A-I Preserves Cognitive Function and Attenuates Neuroinflammation and Cerebral Amyloid Angiopathy in a Mouse Model of Alzheimer Disease

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-β (Aβ) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-β precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of Aβ in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/PS1/AI mice. In addition, Aβ-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.     

Overexpression of suppressor of cytokine signaling-5 in T cells augments innate immunity during septic peritonitis

Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT signal transduction pathway, but their role in innate immunity remains to be investigated. In the present study, we demonstrate that overexpression of SOCS5 in T cells augments innate immunity during septic peritonitis induced by cecal ligation and puncture (CLP). Mice with a cell-specific overexpression of SOCS5 in T cells (SOCS5 transgenic (Tg)) were resistant to the lethality relative to the wild-type (WT) mice. This was most likely due to the enhanced innate immunity in SOCS5Tg mice, as bacterial burden in SOCS5Tg mice was significantly lower than WT mice. Accumulation of neutrophils and macrophages was augmented in SOCS5Tg mice, an event that was accompanied by increased peritoneal levels of IL-12, IFN-gamma, and TNF-alpha. In vitro bactericidal activities of macrophages and neutrophils were enhanced in SOCS5Tg mice. Both neutrophils and macrophages from WT mice adopted enhanced bacterial killing activity when cocultured with CD4+ T cells from SOCS5Tg mice, relative to CD4+ T cells from WT mice. Adoptive transfer of SOCS5Tg-CD4+ T cells into T- and B cell-deficient RAG-2(-/-) mice resulted in augmented leukocyte infiltration and increased peritoneal levels of IL-12, IFN-gamma, and TNF-alpha after CLP, as compared with the controls. Furthermore, CLP-induced bacterial burden in RAG-2(-/-) mice harboring SOCS5Tg-CD4+ T cells was significantly reduced relative to the controls. These findings provide evidence that intervention of SOCS5 expression in T cells affects innate immunity, which highlight a novel role of T cells during sepsis.     

APP/PS1小鼠中PEG-PLA纳米粒的表面蛋白冠组成及其脑内递送的实验研究

摘要 目的：研究阿尔茨海默病（Alzhemer''s disease,AD）模型鼠中聚乙二醇聚乳酸（poly(ethylene glycol)-poly(l-lactide),PEG-PLA）纳米粒表面蛋白冠组成及其对脑内递送特性的影响。方法：制备PEG-PLA纳米粒,测定纳米粒的zeta电位及粒径,采用透射电子显微镜观察纳米粒形态。通过双光子显微镜观察APP/PS1小鼠与野生型（Wild Type,WT）小鼠脑内PEG-PLA纳米粒分布特性。采用液相色谱-质谱联用（LC-MS）技术对PEG-PLA纳米粒分别与APP/PS1小鼠和WT小鼠血浆孵育形成的两种不同蛋白冠进行蛋白组学分析。结果：制备的PEG-PLA纳米粒粒径均一,分散性较好。静脉注射PEG-PLA后,APP/PS1小鼠脑内纳米粒量明显高于WT小鼠。蛋白质组学结果显示,APP/PS1小鼠血浆孵育组PEG-PLA纳米粒表面蛋白冠中凝聚素（Clusterin）明显高于WT小鼠血浆孵育组,该蛋白与纳米粒逃避机体清除有关。此外,纳米粒蛋白冠中血管性血友病因子（Von Willebrand factor）、玻连蛋白（Vitronectin）、肌球蛋白重链-9（Myosin-9）等参与细胞粘附作用相关蛋白在APP/PS1小鼠血浆孵育组也明显多于WT小鼠血浆孵育组。结论：PEG-PLA纳米粒在AD模型小鼠中表现出的高入脑量,可能与AD疾病影响纳米粒蛋白冠组成有关。     

IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans

In the murine model of Cryptococcus neoformans infection Th1 (IL-12/IFN-gamma) and Th17 (IL-23/IL-17) responses are associated with protection, whereas an IL-4-dependent Th2 response exacerbates disease. To investigate the role of the Th2 cytokine IL-13 during pulmonary infection with C. neoformans, IL-13-overexpressing transgenic (IL-13Tg(+)), IL-13-deficient (IL-13(-/-)), and wild-type (WT) mice were infected intranasally. Susceptibility to C. neoformans infection was found when IL-13 was induced in WT mice or overproduced in IL-13Tg(+) mice. Infected IL-13Tg(+) mice had a reduced survival time and higher pulmonary fungal load as compared with WT mice. In contrast, infected IL-13(-/-) mice were resistant and 89% of these mice survived the entire period of the experiment. Ag-specific production of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with a significant type 2 cytokine shift but only minor changes in IFN-gamma production. Consistent with enhanced type 2 cytokine production, high levels of serum IgE and low ratios of serum IgG2a/IgG1 were detected in susceptible WT and IL-13Tg(+) mice. Interestingly, expression of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with reduced IL-17 production. IL-13 was found to induce formation of alternatively activated macrophages expressing arginase-1, macrophage mannose receptor (CD206), and YM1. In addition, IL-13 production led to lung eosinophilia, goblet cell metaplasia and elevated mucus production, and enhanced airway hyperreactivity. This indicates that IL-13 contributes to fatal allergic inflammation during C. neoformans infection.     

Physiologically regulated transgenic ABCA1 does not reduce amyloid burden or amyloid-beta peptide levels in vivo

ABCA1-deficient mice have low levels of poorly lipidated apolipoprotein E (apoE) and exhibit increased amyloid load. To test whether excess ABCA1 protects from amyloid deposition, we crossed APP/PS1 mice to ABCA1 bacterial artificial chromosome (BAC) transgenic mice. Compared with wild-type animals, the ABCA1 BAC led to a 50% increase in cortical ABCA1 protein and a 15% increase in apoE abundance, demonstrating that this BAC supports modest ABCA1 overexpression in brain. However, this was observed only in animals that do not deposit amyloid. Comparison of ABCA1/APP/PS1 mice with APP/PS1 controls revealed no differences in levels of brain ABCA1 protein, amyloid, Abeta, or apoE, despite clear retention of ABCA1 overexpression in the livers of these animals. To further investigate ABCA1 expression in the amyloid-containing brain, we then compared ABCA1 mRNA and protein levels in young and aged cortex and cerebellum of APP/PS1 and ABCA1/APP/PS1 animals. Compared with APP/PS1 controls, aged ABCA1/APP/PS1 mice exhibited increased ABCA1 mRNA, but not protein, selectively in cortex. Additionally, ABCA1 mRNA levels were not increased before amyloid deposition but were induced only in the presence of extensive Abeta and amyloid levels. These data suggest that an induction of ABCA1 expression may be associated with late-stage Alzheimer's neuropathology.     

Reduced retinal function in amyloid precursor protein-over-expressing transgenic mice via attenuating glutamate-N-methyl-d-aspartate receptor signaling

Here, we examined whether amyloid-beta (Abeta) protein participates in cell death and retinal function using three types of transgenic (Tg) mice in vivo [human mutant amyloid precursor protein (APP) Tg (Tg 2576) mice, mutant presenilin-1 (PS-1) knock-in mice, and APP/PS-1 double Tg mice]. ELISA revealed that the insoluble form of Abeta(1-40) was markedly accumulated in the retinas of APP and APP/PS-1, but not PS-1 Tg, mice (vs. wild-type mice). In APP Tg and APP/PS-1 Tg mice, immunostaining revealed accumulations of intracellular Abeta(1-42) in retinal ganglion cells and in the inner and outer nuclear layers. APP Tg and APP/PS-1 Tg, but not PS-1 Tg, mice had less NMDA-induced retinal damage than wild-type mice, and the reduced damage in APP/PS-1 Tg mice was diminished by the pre-treatment of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a gamma-secretase inhibitor. Furthermore, the number of TUNEL-positive cells was significantly less in ganglion cell layer of APP/PS-1 Tg mice than PS-1 Tg mice 24 h after NMDA injection. The phosphorylated form of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha), but not total CaMKIIalpha or total NMDA receptor 1 (NR1) subunit, in total retinal extracts was decreased in non-treated retinas of APP/PS-1 Tg mice (vs. wild-type mice). CaMKIIalpha and NR2B proteins, but not NR1, in retinal membrane fraction were significantly decreased in APP/PS-1 Tg mice as compared with wild-type mice. The NMDA-induced increase in p-CaMKIIalpha in the retina was also lower in APP/PS-1 Tg mice than in wild-type mice. In electroretinogram and visual-evoked potential recordings, the implicit time to each peak from a light stimulus was prolonged in APP/PS-1 mice versus wild-type mice. Hence, Abeta may impair retinal function by reducing activation of NMDA-receptor signaling pathways.     

The evolution of A beta peptide burden in the APP23 transgenic mice: implications for A beta deposition in Alzheimer disease.

BACKGROUND: High levels of A beta in the cerebral cortex distinguish demented Alzheimer's disease (AD) from nondemented elderly individuals, suggesting that decreased amyloid-beta (A beta) peptide clearance from the brain is a key precipitating factor in AD. MATERIALS AND METHODS: The levels of A beta in brain and plasma as well as apolipoprotein E (ApoE) in brain were investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting at various times during the life span of the APP23 transgenic (Tg) and control mice. Histochemistry and immunocytochemistry were used to assess the morphologic characteristics of the brain parenchymal and cerebrovascular amyloid deposits and the intracellular amyloid precursor protein (APP) deposits in the APP23 Tg mice. RESULTS: No significant differences were found in the plasma levels of A beta between the APP23 Tg and control mice from 2-20 months of age. In contrast, soluble A beta levels in the brain were continually elevated, increasing 4-fold at 2 months and 33-fold in the APP23 Tg mice at 20 months of age when compared to the control mice. Soluble A beta42 was about 60% higher than A beta40. In the APP23 Tg mice, insoluble A beta40 remained at basal levels in the brain until 9 months and then rose to 680 microg/g cortex by 20 months. Insoluble A beta40 was negligible in non-Tg mice at all ages. Insoluble A beta42 in APP23 Tg mice rose to 60 microg/g cortex at 20 months, representing 24 times the control A beta42 levels. Elevated levels of ApoE in the brain were observed in the APP23 Tg mice at 2 months of age, becoming substantially higher by 20 months. ApoE colocalized with A beta in the plaques. Beta-amyloid precursor protein (betaAPP) deposits were detected within the neuronal cytoplasm from 4 months of age onward. Amyloid angiopathy in the APP23 Tg mice increased markedly with age, being by far more severe than in the Tg2576 mice. CONCLUSIONS: We suggest that the APP23 Tg mouse may develop an earlier blockage in A beta clearance than the Tg2576 mice, resulting in a more severe accumulation of A beta in the perivascular drainage pathways and in the brain. Both Tg mice reflect decreased A beta elimination and as models for the amyloid cascade they are useful to study AD pathophysiology and therapy.     

GABA(A) receptor-mediated acceleration of aging-associated memory decline in APP/PS1 mice and its pharmacological treatment by picrotoxin

Advanced age and mutations in the genes encoding amyloid precursor protein (APP) and presenilin (PS1) are two serious risk factors for Alzheimer's disease (AD). Finding common pathogenic changes originating from these risks may lead to a new therapeutic strategy. We observed a decline in memory performance and reduction in hippocampal long-term potentiation (LTP) in both mature adult (9-15 months) transgenic APP/PS1 mice and old (19-25 months) non-transgenic (nonTg) mice. By contrast, in the presence of bicuculline, a GABA(A) receptor antagonist, LTP in adult APP/PS1 mice and old nonTg mice was larger than that in adult nonTg mice. The increased LTP levels in bicuculline-treated slices suggested that GABA(A) receptor-mediated inhibition in adult APP/PS1 and old nonTg mice was upregulated. Assuming that enhanced inhibition of LTP mediates memory decline in APP/PS1 mice, we rescued memory deficits in adult APP/PS1 mice by treating them with another GABA(A) receptor antagonist, picrotoxin (PTX), at a non-epileptic dose for 10 days. Among the saline vehicle-treated groups, substantially higher levels of synaptic proteins such as GABA(A) receptor alpha1 subunit, PSD95, and NR2B were observed in APP/PS1 mice than in nonTg control mice. This difference was insignificant among PTX-treated groups, suggesting that memory decline in APP/PS1 mice may result from changes in synaptic protein levels through homeostatic mechanisms. Several independent studies reported previously in aged rodents both an increased level of GABA(A) receptor alpha1 subunit and improvement of cognitive functions by long term GABA(A) receptor antagonist treatment. Therefore, reduced LTP linked to enhanced GABA(A) receptor-mediated inhibition may be triggered by aging and may be accelerated by familial AD-linked gene products like Abeta and mutant PS1, leading to cognitive decline that is pharmacologically treatable at least at this stage of disease progression in mice.     

Brain Prolyl Endopeptidase Expression in Aging, APP Transgenic Mice and Alzheimer’s Disease

Prolyl endopeptidase (PEP) is believed to inactivate neuropeptides that are present in the extracellular space. However, the intracellular localization of PEP suggests additional, yet unidentified physiological functions for this enzyme. Here we studied the expression, enzymatic activity and subcellular localization of PEP in adult and aged mouse brain as well as in brains of age-matched APP transgenic Tg2576 mice and in brains of Alzheimer’s disease patients. In mouse brain PEP was exclusively expressed by neurons and displayed region- and age-specific differences in expression levels, with the highest PEP activity being present in cerebellum and a significant increase in hippocampal but not cortical or cerebellar PEP activity in aged mouse brain. In brains of young APP transgenic Tg2576 mice, hippocampal PEP activity was increased compared to wild-type littermates in the pre-plaque phase but not in aged mice with β-amyloid plaque pathology. This “accelerated aging” with regard to hippocampal PEP expression in young APP transgenic mice might be one factor contributing to the observed cognitive deficits in these mice in the pre-plaque phase and could also explain in part the cognition-enhancing effects of PEP inhibitors in serveral experimental paradigms.     

IGF2 Ameliorates Amyloidosis,Increases Cholinergic Marker Expression and Raises BMP9 and Neurotrophin Levels in the Hippocampus of the APPswePS1dE9 Alzheimer’s Disease Model Mice

The development of an effective therapy for Alzheimer’s disease (AD) is a major challenge to biomedical sciences. Because much of early AD pathophysiology includes hippocampal abnormalities, a viable treatment strategy might be to use trophic factors that support hippocampal integrity and function. IGF2 is an attractive candidate as it acts in the hippocampus to enhance memory consolidation, stimulate adult neurogenesis and upregulate cholinergic marker expression and acetylcholine (ACh) release. We performed a seven-day intracerebroventricular infusion of IGF2 in transgenic APPswe.PS1dE9 AD model mice that express green fluorescent protein in cholinergic neurons (APP.PS1/CHGFP) and in wild type WT/CHGFP littermates at 6 months of age representing early AD-like disease. IGF2 reduced the number of hippocampal Aβ40- and Aβ42-positive amyloid plaques in APP.PS1/CHGFP mice. Moreover, IGF2 increased hippocampal protein levels of the ACh-synthesizing enzyme, choline acetyltransferase in both WT/CHGFP and APP.PS1/CHGFP mice. The latter effect was likely mediated by increased protein expression of the cholinergic differentiating factor, BMP9, observed in IGF2-treated mice as compared to controls. IGF2 also increased the protein levels of hippocampal NGF, BDNF, NT3 and IGF1 and of doublecortin, a marker of neurogenesis. These data show that IGF2 administration is effective in reversing and preventing several pathophysiologic processes associated with AD and suggest that IGF2 may constitute a therapeutic target for AD.     

Intracellular Accumulation of Detergent-Soluble Amyloidogenic Aβ Fragment of Alzheimer's Disease Precursor Protein in the Hippocampus of Aged Transgenic Mice

To study amyloid beta-protein (A beta) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A beta were produced in brain and some peripheral tissues and A beta was produced in serum. Using antibodies specific for the A beta C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A beta42/A beta40 compared with Tg C100.WT. Approximately 30% of total brain A beta (approximately 122 ng/g of wet tissue) was water-soluble. The remaining 70% of A beta partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A beta. Immunohistochemistry with an A beta(5-8) antibody showed that A beta or A beta-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A beta levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A beta or A beta-containing fragments and to screen for compounds directed at the gamma-secretase activity.     

LiCl attenuates impaired learning and memory of APP/PS1 mice,which in mechanism involves α7 nAChRs and Wnt/β-catenin pathway

We examined the mechanism by which lithium chloride (LiCl) attenuates the impaired learning capability and memory function of dual-transgenic APP/PS1 mice. Six- or 12-month-old APP/PS1 and wild-type (WT) mice were randomized into four groups, namely WT, WT+Li (100 mg LiCl/kg body weight, gavage once daily), APP/PS1 and APP/PS1+Li. Primary rat hippocampal neurons were exposed to β-amyloid peptide oligomers (AβOs), LiCl and/or XAV939 (inhibitor of Wnt/β-catenin) or transfected with small interfering RNA against the β-catenin gene. In the cerebral zone of APP/PS1 mice, the level of Aβ was increased and those of α7 nicotinic acetylcholine receptors (nAChR), phosphor-GSK3β (ser9), β-catenin and cyclin D1 (protein and/or mRNA levels) reduced. Two-month treatment with LiCl at ages of 4 or 10 months weakened all of these effects. Similar expression variations were observed for these proteins in primary neurons exposed to AβOs, and these effects were attenuated by LiCl and aggravated by XAV939. Inhibition of β-catenin expression lowered the level of α7 nAChR protein in these cells. LiCl attenuates the impaired learning capability and memory function of APP/PS1 mice via a mechanism that might involve elevation of the level of α7 nAChR as a result of altered Wnt/β-catenin signalling.     

Promotion of β-amyloid production by C-reactive protein and its implications in the early pathogenesis of Alzheimer's disease

C-reactive protein (CRP) and β-amyloid protein (Aβ) are involved in the development of Alzheimer's disease (AD). However, the relationship between CRP and Aβ production is unclear. In vitro and in vivo experiments were performed to investigate the association of CRP with Aβ production. Using the rat adrenal pheochromocytoma cell line (PC12 cells) to mimic neurons, cytotoxicity was evaluated by cell viability and supernatant lactate dehydrogenase (LDH) activity. The levels of amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE-1), and presenilins (PS-1 and PS-2) were investigated using real-time polymerase chain reaction and Western blotting analysis. Aβ1-42 was measured by enzyme-linked immunosorbent assay. The relevance of CRP and Aβ as well as potential mechanisms were studied using APP/PS1 transgenic (Tg) mice. Treatment with 0.5-4.0 μM CRP for 48 h decreased cell viability and increased LDH leakage in PC12 cells. Incubation with CRP at a sub-toxic concentration of 0.2 μM increased the mRNA levels of APP, BACE-1, PS-1, and PS-2, as well as Aβ1-42 production. CRP inhibitor reversed the CRP-induced upregulations of the mRNA levels of APP, BACE-1, PS-1, and PS-2, and the protein levels of APP, BACE-1, PS-1, and Aβ1-42, but did not reversed Aβ1-42 cytotoxicity. The cerebral levels of CRP and Aβ1-42 in APP/PS1 Tg mice were positively correlated, accompanied with the elevated mRNA expressions of serum amyloid P component (SAP), complement component 1q (C1q), and tumor necrosis factor-α (TNF-α). These results suggest that CRP cytotoxicity is associated with Aβ formation and Aβ-related markers expressions; CRP and Aβ were relevant in early-stage AD; CRP may be an important trigger in AD pathogenesis.     

Age-related changes of brain iron load changes in the frontal cortex in APPswe/PS1ΔE9 transgenic mouse model of Alzheimer's disease

Alzheimer's disease (AD) as a neurodegenerative brain disorder is a devastating pathology leading to disastrous cognitive impairments and dementia, associated with major social and economic costs to society. Iron can catalyze damaging free radical reactions. With age, iron accumulates in brain frontal cortex regions and may contribute to the risk of AD. In this communication, we investigated the age-related brain iron load changes in the frontal cortex of 6- and 12-month-old C57BL/6J (C57) and APP_swe/PS1_ΔE9 (APP/PS1) double transgenic mouse by using graphite furnace atomic absorption spectrometry (GFAAS) and Perls’ reaction. In the present study, we also evaluated the age-related changes of DMT1 and FPN1 by using Western blot and qPCR. We found that compared with 6-month-old APP/PS1 mice and the 12-month-old C57 mice, the 12-month-old APP/PS1 mice had increased iron load in the frontal cortex. The levels of DMT1 were significantly increased and the FPN1 were significantly reduced in the frontal cortex of the 12-month-old APP/PS1 mice than that in the 6-month-old APP/PS1 mice and 12-month-old C57 mice. We conclude that in AD damage occurs in conjunction with iron accumulation, and the brain iron load associated with loss control of the brain iron metabolism related protein DMT1 and FPN1 expressions.     

apoE/LDLR双基因缺失幼龄小鼠主动脉中动脉粥样硬化相关基因的表达

利用RT-PCR以及实时定量RT-PCR检测11个动脉粥样硬化(atherosclerosis,AS)相关基因在1、2和3月龄的载脂蛋白E(apolipoproteinE,aopE)/低密度脂蛋白受体(low-density lipoprotein receptor,LDLR)双基因缺失(apoE-/-/LDLR-/-)小鼠主动脉中的表达变化,同时应用血生化指标和病理形态学观察AS早期病变特点,探讨apoE和LDLR基因联合缺失引发的血脂代谢紊乱和血管炎症损伤的关系以及AS的炎症反应机制.结果显示,apoE-/-/LDLR-/-小鼠IL-18、TLR2、MCP-1、ICAM-1、VCAM-1、GM-CSF、CD36和ET-1表达在1月龄时较同龄野生型(wild type,WT)小鼠显著上调(P<0.05,P<0.01),PDGF-α和TNF-α表达在2月龄时较同龄WT小鼠显著上调,除ET-1表达在2月龄时以及了LR2、VCAM-1和ICAM-1表达在3月龄时降至WT小鼠水平以外,其余各基因表达随年龄增长继续升高(P<0.05,P<0.01),其中MCP-1表达在2月龄时达到峰值.NF-kB在各年龄段apoE-/-/LDLR-/-小鼠中的表达与同龄WT小鼠相比均无显著差异.各年龄段apoE-/-/LDLR-/-/小鼠血清了C、TG、LDL、HDL、TNF-α、IL-1β和ox-LDL含量均显著高于同龄WT小鼠(P<0.05,P<0.01),并随年龄增长逐渐升高.apoE-/-/LDLR-/-小鼠1月龄时主动脉内膜出现少量的散在的脂质沉积,随着年龄增长病变区域增多,脂质沉积增厚.上述结果提示:apoE和LDLR双基因缺失形成的高脂血症可能通过刺激主动脉中炎症基因时序表达,起始并扩大病变部位的炎症反应,共同促进AS的发生发展.     

Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice

Background

Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration.

Methods

We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes.

Results

At 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment.

Conclusion

IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.     

Mutual modulation between interleukin-10 and interleukin-6 induced by Rhodococcus aurantiacus infection in mice

The interaction between interleukin-10 (IL-10) and interleukin-6 (IL-6) was investigated in the inflammatory response to Rhodococcus aurantiacus (R. aurantiacus) infection, in which both cytokines act as anti-inflammatory cytokines. Compared with wild-type (WT) counterparts, IL-6 gene-deficient (IL-6(-)/(-)) mice mounted a more robust production of IL-10 and tumor necrosis factor-alpha (TNF-alpha) during the initial phase of infection. Administration of anti-IL-10 antibody resulted in all the mice dying within 3 days post-infection as well as a further elevated TNF-alpha release. In vitro challenge of the macrophages from IL-6(-)/(-) and WT mice with heat-killed R. aurantiacus also showed similar results. Addition of exogenous IL-6 depressed IL-10 and TNF-alpha production by either IL-6(-)/(-) mice or IL-6(-)/(-) mouse macrophages. Likewise, WT mouse macrophages pretreated with anti-IL-10 or anti-IL-6 antibody exhibited increased production of TNF-alpha and IL-6 or IL-10 respectively. Moreover, neutralization of both IL-10 and IL-6 induced a further increase in TNF-alpha production by WT mouse cells. Overall, we conclude that IL-10 is a key element in protecting mice against mortality, and that IL-10 and IL-6 production are negatively regulated by each other although they are additive in suppressing TNF-alpha release in R. aurantiacus-infected mouse model.     

Rho‐associated coiled‐coil kinase 1 activation mediates amyloid precursor protein site‐specific Ser655 phosphorylation and triggers amyloid pathology

Rho‐associated coiled‐coil kinase 1 (ROCK1) is proposed to be implicated in Aβ suppression; however, the role for ROCK1 in amyloidogenic metabolism of amyloid precursor protein (APP) to produce Aβ was unknown. In the present study, we showed that ROCK1 kinase activity and its APP binding were enhanced in AD brain, resulting in increased β‐secretase cleavage of APP. Furthermore, we firstly confirmed that APP served as a substrate for ROCK1 and its major phosphorylation site was located at Ser655. The increased level of APP Ser655 phosphorylation was observed in the brain of APP/PS1 mice and AD patients compared to controls. Moreover, blockade of APP Ser655 phosphorylation, or inhibition of ROCK1 activity with either shRNA knockdown or Y‐27632, ameliorated amyloid pathology and improved learning and memory in APP/PS1 mice. These findings suggest that activated ROCK1 targets APP Ser655 phosphorylation, which promotes amyloid processing and pathology. Inhibition of ROCK1 could be a potential therapeutic approach for AD.     

APPswe/PS1dE9转基因小鼠小脑内突触素及BDNF/Trk-B蛋白表达的变化

目的：观察APP/PS1转基因小鼠小脑突触素及BDNF/Trk-B蛋白表达变化。方法：选用9月龄APP/PS1雄鼠（n1）和同窝对照野生型WT雄鼠（n2）。采用Western blot （n1=6;n2=6）、免疫组化（n1=4;n2=4）两种方式定量、定位测定小脑组织功能活性依赖蛋白突触素、脑源性神经营养因子（BDNF）和其高亲和力受体（Trk-B）的蛋白表达。用透射电镜观察小脑皮质突触超微结构变化（n1=2;n2=2）。结果：与WT组相比,APP/PS1组小脑皮质内突触素、BDNF/Trk-B表达明显减少;突触间隙增宽,突触后致密区变薄,密度降低。结论：APP/PS1小鼠小脑皮质中突触素、BDNF/Trk-B蛋白含量均明显降低,突触超微结构也发生明显改变,提示AD小脑突触数量及形态变化可能与BDNF合成及释放减少有关。