Multiple inflammatory pathways are involved in the development and progression of cognitive deficits in APPswe/PS1dE9 mice

Increased accumulation of amyloid-beta peptide (Aβ) and neuroinflammation is known to exist within the Alzheimer's disease (AD) brain. However, it remains unclear which form of Aβ pathologies triggers neuroinflammation and whether increased neuroinflammation contributes to cognitive deficits in AD. In the present study we found that increased inflammatory responses might occur early in preplaque APPswe/PS1dE9 mice, and were significantly enhanced in both early- and late-plaque APPswe/PS1dE9 mice. Correlational analysis revealed that multiple inflammatory indexes significantly correlated with soluble Aβ level, rather than amyloid plaque burden or insoluble Aβ level, in APPswe/PS1dE9 mice. Moreover, multiple inflammatory indexes highly correlated with the impaired spatial learning and memory in APPswe/PS1dE9 mice. Collectively, these results provide evidence that inflammatory responses might be likely triggered by soluble toxic Aβ species. Importantly, we demonstrate for the first time that multiple inflammatory pathways might be involved in the development and progression of cognitive deficits in APPswe/PS1dE9 mice, suggesting that a pharmacological approach targeting multiple inflammatory pathways may be a novel promising strategy to prevent or delay AD.     

Sorafenib inhibits nuclear factor kappa B,decreases inducible nitric oxide synthase and cyclooxygenase-2 expression,and restores working memory in APPswe mice

Alzheimer's disease (AD) is characterized by memory loss and the upregulation of pro-neuroinflammatory factors such as cRaf-1, cyclooxygenase-2 (Cox-2), and the nuclear factor kappa B (NF-κB), as well as a downregulation of protein kinase A (PKA) activity and the activation by phosphorylation of its downstream factor CREB. We investigated the effect of the anti-cancer cRaf-1 inhibitor, sorafenib tosylate (Nexavar), on the expression of these factors and on the cognitive performance of aged APPswe mice. We found that chronic treatment with sorafenib stimulated PKA and CREB phosphorylation and inhibited cRaf-1 and NF-κB in the brains of APPswe mice. NF-κB controls the expression of several genes related to AD pathology, including iNOS and Cox-²Concurrent with NF-κB inhibition, sorafenib treatment decreased the cerebral expression of Cox-2 and iNOS in APPswe mice. It has recently been observed that Cox-2 inhibition prevents cognitive impairment in a mouse model of AD and amyloid beta peptide (Aβ)–induced inhibition of long-term potentiation (LTP). Consistent with the idea that Cox-2 inhibition can improve cognitive abilities, we found that sorafenib restored working memory abilities in aged APPswe mice without reducing Aβ levels in the brain. These findings suggest that sorafenib reduced AD pathology by reducing neuroinflammation.     

Andrographolide attenuates microglia-mediated Aβ neurotoxicity partially through inhibiting NF-κB and JNK MAPK signaling pathway

Neuroinflammation plays a critical role in the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease (AD). Microglial cells after activated play critical roles in development of neuroinflammation, and may accelerate the progression of AD. Andrographolide (ANDRO), a potent naturally extracted substance, has been demonstrated to exert suppressive effects on LPS-induced inflammation by modulating macrophage and microglia overactivation. Whereas in AD, β-amyloid (Aβ) peptides have been considered as a potent activator of neuroinflammation, the effect of ANDRO on Aβ-induced neuroinflammation has not been examined. In this study, we investigated the effects of ANDRO on Aβ(1–42)-induced neuroinflammation. We found that ANDRO significantly protected neuronal cells against microglia-mediated Aβ(1–42) toxicity and attenuated the release of preinflammatory productions such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO), and prostaglandin E₂ (PGE₂). It also downregulated the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in microglial cells. Further the involved mechanism study demonstrated that ANDRO inhibited the nuclear translocation of nuclear factor-κB (NF-κB) by affecting IκB phosphorylation, and attenuated Aβ(1–42)-induced JNK–MAPK overactivation. In summary, this study, for the first time, revealed ANDRO reduced inflammation-mediated neuronal damage by blocking inflammatory responses of microglial cells to Aβ(1–42), suggesting ANDRO may be an effective agent in modulating neuroinflammatory process in AD.     

核因子-κB对TNF-α诱导的大鼠气道平滑肌细胞增殖、凋亡及TGF-β1表达的影响

目的: 探讨核因子-κB(NF-κB)对TNF-α诱导的大鼠气道平滑肌细胞（ASMCs）增殖、凋亡和转化生长因子β₁（TGF-β₁）表达的影响。方法: 体外培养ASMCs，以肿瘤坏死因子-α（TNF-α）及NF-κB特异性抑制剂二硫代氨基甲酸吡咯烷(PDTC)作为工具药,将ASMCs分为对照组、TNF-α组和TNF-α+PDTC组。逆转录聚合酶链式反应(RT-PCR)检测TGF-β₁ mRNA表达，Western blotting检测NF-κB、TGF-β₁的表达，免疫细胞化学染色法检测PCNA、Bcl-2蛋白表达及定位，四甲基偶氮唑蓝(MTT)微量比色法测定ASMCs增殖，Annexin V/PI双标记流式细胞仪分析法检测细胞凋亡。结果: （1）TNF-α组ASMCs的NF-κB活性显著高于对照组(P<0.01),TNF-α+PDTC组NF-κB活性显著低于TNF-α组 (P<0.01)。（2）TNF-α组ASMCs增殖显著高于对照组(P<0.01),TNF-α＋PDTC组的增殖反应显著低于TNF-α组(P<0.01)；TNF-α+PDTC组细胞凋亡率显著高于TNF-α组及对照组(P<0.01)。（3）TNF-α组ASMCs的PCNA、Bcl-2蛋白表达量均显著高于对照组(P<0.01)，TNF-α+PDTC组的PCNA、Bcl-2蛋白表达量均显著低于TNF-α组(P<0.01)。（4）TNF-α组ASMCs的TGF-β₁的mRNA及蛋白表达水平均显著高于对照组及TNF-α＋PDTC组（P<0.01）。结论: NF-κB活化可能参与调控TNF-α诱导的大鼠气道平滑肌细胞的增殖、凋亡及TGF-β₁的表达和分泌。     

褪黑素对AD模型小鼠脑内小胶质细胞的影响

目的探讨褪黑素对APP/PS1转基因AD模型小鼠脑内小胶质细胞和炎症细胞因子COX-2的抑制效应。方法 AD转基因小鼠随机分为褪黑素处理组和对照组。14 d后,取海马通过免疫荧光化学染色检测小胶质细胞、老年斑位置情况;Western blot、ELISA方法分别检测CD11b、COX-2的变化;Western blot检测炎症信号通路TLR/NF-κB的变化。结果免疫荧光化学显示,老年斑周围有大量活化的小胶质细胞聚集;Western blot、ELISA结果显示褪黑素组小鼠脑内CD11b、COX-2表达显著减少;褪黑素组小鼠TLR/NF-κB炎症信号通路的TLR2、NF-κB-p65表达减少。结论褪黑素可能通过抑制TLR/NF-κB炎症信号通路来抑制小胶质细胞活化并抑制炎症细胞因子COX-2的分泌。     

Voluntary Running Attenuates Memory Loss,Decreases Neuropathological Changes and Induces Neurogenesis in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by loss of memory and cognitive abilities, and the appearance of amyloid plaques composed of the amyloid‐β peptide (Aβ) and neurofibrillary tangles formed of tau protein. It has been suggested that exercise might ameliorate the disease; here, we evaluated the effect of voluntary running on several aspects of AD including amyloid deposition, tau phosphorylation, inflammatory reaction, neurogenesis and spatial memory in the double transgenic APPswe/PS1ΔE9 mouse model of AD. We report that voluntary wheel running for 10 weeks decreased Aβ burden, Thioflavin‐S‐positive plaques and Aβ oligomers in the hippocampus. In addition, runner APPswe/PS1ΔE9 mice showed fewer phosphorylated tau protein and decreased astrogliosis evidenced by lower staining of GFAP. Further, runner APPswe/PS1ΔE9 mice showed increased number of neurons in the hippocampus and exhibited increased cell proliferation and generation of cells positive for the immature neuronal protein doublecortin, indicating that running increased neurogenesis. Finally, runner APPswe/PS1ΔE9 mice showed improved spatial memory performance in the Morris water maze. Altogether, our findings indicate that in APPswe/PS1ΔE9 mice, voluntary running reduced all the neuropathological hallmarks of AD studied, reduced neuronal loss, increased hippocampal neurogenesis and reduced spatial memory loss. These findings support that voluntary exercise might have therapeutic value on AD.     

Collagen gel protects L929 cells from TNFα-induced death by activating NF-κB

Purpose: Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel. Material and methods: We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy. Results: L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed. Conclusion: NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.     

Cyclooxygenase (COX)-2 and cell cycle activity in a transgenic mouse model of Alzheimer's disease neuropathology

Prior studies have shown that cyclooxygenase (COX)-2, an enzyme involved in inflammatory mechanisms as well as neuronal activities, is up-regulated in the Alzheimer's disease (AD) brain and may represent a therapeutic target for anti-inflammatory treatments. We report the effect of neuronal overexpression of human (h)COX-2 in a murine model of AD neuropathology. Transgenic mice expressing both the human amyloid precursor protein mutation (APPswe) and the human presenilin (PS1-A246E) mutation, with resultant AD plaque pathology, were crossed with transgenic mice expressing human (h)COX-2 in neurons. At 12 months of age, the APPswe/PS1-A246E/hCOX-2 triple-transgenic mice showed an elevation in the number of phosphorylated retinoblastoma (pRb) tumor suppressor protein and active caspase-3 immunopositive neurons, compared to double APPswe/PS1-A246E or single hCOX-2 transgenic controls. No detectable influence of neuronal hCOX-2 on AD neuropathology was found in the brain of APPswe/PS1-A246E/hCOX-2 triple-transgenic mice, compared to double APPswe/PS1-A246E. In vitro studies revealed that hCOX-2 overexpression in primary cortico-hippocampal neurons derived from the hCOX-2 transgenics accelerates beta-amyloid (Abeta)(1-42)-mediated apoptotic damage which was prevented by the cell cycle dependent (CDK) inhibitor, flavoperidol. The data indicates that COX-2 overexpression causes alteration of neuronal cell cycle in a murine model of AD neuropathology, and provides a rational basis for targeting neuronal COX-2 in therapeutic research aimed at slowing the clinical progression of AD.     

Inhibitions of NF-κB and TNF-α Result in Differential Effects in Rats with Acute on Chronic Liver Failure Induced by d-Gal and LPS

In this study, we induced an acute-on-chronic liver failure (ACLF) model by human serum albumin (HSA), d-galactosamine (d-Gal) and lipopolysaccharide (LPS) in rats. Anti-TNF-α polyclonal antibody (as TNF-α inhibitor) and pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) were used to treat the liver failure animals, respectively. The results showed that TNF-α inhibition was beneficial, but NF-κB inhibition failed to protect the rats in ACLF. However, HMGB1 levels, cytokine production and activation of TLR4-NF-κB signaling pathway were all suppressed by both TNF-α and NF-κB inhibition. In order to verify the effect of PDTC on inflammatory response, we further explored its effect in vitro. Anti-inflammatory activity of PDTC was proved in U937 cell line. To conclude, both inhibitions of TNF-α and NF-κB are able to suppress the activation of TLR4 and NF-κB signaling pathway. However, NF-κB inhibition with PDTC failed to protect the rats in ACLF induced by d-Gal and LPS.     

Effects of antioxidant and NF-κB on the induction of iNOS gene in rat pulmonary microvascular endothelial cells in vitro

Effects of antioxidant and NF-κB on the induction of iNOS gene in rat pulmonary microvascular endothelial cells in vitro     

NF-κB与COX-2的正相互作用诱导子宫颈癌细胞生长及抗凋亡

目的探讨NF—κB和COX-2通路之间的相互作用对人子宫颈癌细胞株（Hela细胞）的生长及细胞凋亡的影响。方法应用细胞计数盒（CCK-8）检测细胞存活率;Hoechst33258核染色检测凋亡细胞的形态及数量的改变：Westernblot法检测Caspase-3、NF-κB和COX-2蛋白的表达。结果应用NF．KB抑制剂（PDTC）或COX-2抑制剂（NS-398）处理Hela细胞36h能明显地抑制细胞存活率,PDTC或NS-398处理Hela细胞24h能明显地促进Caspase-3表达,并增加凋亡细胞数量。PDTC处理Hela细胞能显著地抑制COX-2表达,另方面,NS-398处理Hela细胞能抑制NF-κB的表达。结论NF-κB和COX-2通路之间的正相互作用诱导Hela细胞生长及抑制细胞凋亡。     

Amyloid precursor protein increases cortical neuron size in transgenic mice

The amyloid precursor protein (APP) is the source of beta-amyloid, a pivotal peptide in the pathogenesis of Alzheimer's disease (AD). This study examines the possible effect of APP transgene expression on neuronal size by measuring the volumes of cortical neurons (microm(3)) in transgenic mouse models with familial AD Swedish mutation (APPswe), with or without mutated presenilin1 (PS1dE9), as well as in mice carrying wild-type APP (APPwt). Overexpression of APPswe and APPwt protein, but not of PS1dE9 alone, resulted in a greater percentage of medium-sized neurons and a proportionate decrease in the percentage of small-sized neurons. Our observations indicate that the overexpression of mutant (APPswe) or wild-type APP in transgenic mice is necessary and sufficient for hypertrophy of cortical neurons. This is highly suggestive of a neurotrophic effect and also raises the possibility that the lack of neuronal loss in transgenic mouse models of AD may be attributed to overexpression of APP.     

Hydrogen-rich saline reduces oxidative stress and inflammation by inhibit of JNK and NF-κB activation in a rat model of amyloid-beta-induced Alzheimer's disease

This study is to examine if hydrogen-rich saline reduced amyloid-beta (Aβ) induced neural inflammation and oxidative stress in a rat model by attenuation of activation of JNK and NF-κB. Sprague–Dawley male rats (n = 18, 280–330 g) were divided into three groups, sham operated, Aβ1–42 injected and Aβ1–42 plus hydrogen-rich saline treated animals. Hydrogen-rich saline (5 ml/kg, i.p., daily) was injected for 10 days after intraventricular injection of Aβ1–42. The levels of IL-1β were assessed by ELISA analysis, 8-OH-dG by immunohistochemistry in the brain slides, and JNK and NF-κB by immunohistochemistry and western blotting. After Aβ1–42 injection, the level of IL-1β, 8-OH-dG, JNK and NF-κB all increased in brain tissues, while hydrogen-rich saline treatment decreased the level of IL-1β, 8-OH-dG and the activation of JNK and NF-κB. In conclusion, hydrogen-rich saline prevented Aβ-induced neuroinflammation and oxidative stress, possibly by attenuatation of activation of c-Jun NH₂-terminal kinase (JNK) and nuclear factor-κB (NF-κB) in this rat model.     

显性失活IκBα质粒对胰腺癌PC-3细胞株核因子-κB和环氧合酶-2表达的影响

目的：观察显性失活IκBα质粒转染胰腺癌PC-3细胞株后，对细胞核因子-κB（NF-κB）和环氧合酶-2（COX-2）表达的影响。 方法： 免疫组织化学证实NF-κB和COX-2在胰腺癌PC-3细胞株中的表达，逆转录-聚合酶链反应（RT-PCR）和蛋白免疫印迹（Western blotting）检测PC-3细胞转染显性失活IκBα质粒后，细胞中NF-κB和COX-2表达的变化。 结果： 胰腺癌PC-3细胞株中存在NF-κB和COX-2的表达，转染显性失活IκBα质粒后，细胞中NF-κB和COX-2表达均下调，且体现出一定的时间依赖性关系。 结论： 胰腺癌PC-3细胞株中存在NF-κB和COX-2的阳性表达。显性失活的IκBα质粒可抑制细胞中NF-κB和COX-2的表达。     

Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues

Objective: Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues.

Methods: Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10?ng/ml) treatment for 4?h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay.

Results: Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB.

Conclusions: The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.     

Inhibition of LPS-induced inflammatory biomarkers by ethyl acetate fraction of Patrinia scabiosaefolia through suppression of NF-κB activation in RAW 264.7 cells

Patrinia scabiosaefolia (PS) has been used for curing various types of inflammatory-related disorders. However, the precise mechanism of the anti-inflammatory activity of PS remains unclear. Here, we investigated the anti-inflammatory effects of several fractions isolated from the PS in RAW 264.7 macrophages. The results indicated that the ethyl acetate fraction of PS (EAPS) concentration highly suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) and IL-6 productions without a cytotoxic effect on RAW 264.7 cells. EAPS inhibited the expressions of LPS-induced iNOS and COX-2 protein and their mRNA in a dose-dependent manner. Particularly, EAPS suppressed the level of nuclear factor-κB (NF-κB) activity, which was linked with the suppression of LPS-induced phosphorylation of p65 at serine 276 and p65 translocation into nuclei, but not MAPK signaling. In addition, treatment with EAPS inhibited the production of TNF-α in LPS-injected mice and suppressed the production of IL-6 and TNF-α in LPS-stimulated splenocytes from BALB/c mice. Therefore, we demonstrate here that Patrinia scabiosaefolia potentially inhibits the biomarkers related to inflammation through the blocking of NF-κB p65 activation, and it may be a potential therapeutic candidate for the treatment of inflammatory diseases.     

抗氧化剂和NF-κB对大肠癌细胞IL-8表达的作用及机制

目的：探讨NF-κB在大肠癌细胞IL-8诱导表达中的作用及抗氧化剂对大肠癌细胞IL-8诱导表达的影响及其机制。 方法： IL-8 mRNA表达采用逆转录/聚合酶链反应(RT/PCR)检测，培养上清IL-8蛋白含量用ELISA检测，EMSA法测定细胞核内NF-κB结合活性。 结果： 抗氧化剂可阻断大肠癌细胞培养体系TNF-α诱导的IL-8生成及IL-8 mRNA的表达, TNF-α可诱导大肠癌细胞NF-κB的激活并被抗氧化剂阻断。 结论： TNF-α诱导的大肠癌细胞IL-8基因和蛋白表达依赖于NF-κB的激活；抗氧化剂可通过抑制NF-κB的活化而阻断IL-8基因和蛋白的诱导表达。