Adult human microglia secrete cytokines when exposed to neurotoxic prion protein peptide: no intermediary role for prostaglandin E2

Prion diseases are characterized by accumulation of protease resistant isoforms of prion protein (termed PrP(SC)), glial activation and neurodegeneration. The time course of PrP deposition, appearance of activated microglia, and of neuronal apoptosis in experimentally-induced prion disease suggests that microglial activation precedes the process of neuronal loss. Activated microglia and inflammatory mediators, including cytokines and prostaglandin E2 (PGE2) co-localize with PrP deposits. In vitro, mouse microglia secrete neurotoxic agents and interleukins (IL)-1 and IL-6, when exposed to synthetic peptides representing the neurotoxic fragment of PrP. In this study, adult human microglia were found to secrete IL-6 and TNF-alpha upon exposure to synthetic fibrillar PrP105-132, the putative transmembrane domain of PrP. Little cytokine release occurred following exposure of microglia to C-terminally amidated, nonfibrillar PrP105-132, suggesting that the degree of fibrillarity of PrP peptides affects their biological properties. Non-steroidal anti-inflammatory drugs (NSAIDs) are thought to exert beneficial effects in neurodegenerative disorders through suppressive effects on microglial activation and on cyclooxygenase (COX) activity. Since microglial COX-2 expression and PGE(2) synthesis are increased in human and experimental prion diseases, we investigated the effects of the NSAIDs indomethacin and BF389, an experimental COX-2 selective inhibitor, on the PrP105-132-induced microglial IL-6 and TNF-alpha synthesis in vitro. No inhibitory effects of the NSAIDs were observed. Furthermore, PrP105-132 did not stimulate microglial PGE(2) synthesis. We conclude that, unlike IL-1beta-induced IL-6 synthesis in astrocytes, the PrP-induced IL-6 synthesis in human adult microglia is not PGE2 mediated.     

羟基红花黄色素 A 对糖氧剥夺/复糖复氧后星形胶质细胞保护作用及其机制研究

目的:探讨糖氧剥夺/复糖复氧(OGD/R)状态下羟基红花黄色素A(HSYA)对星形胶质细胞(Ast)的保护作用及其机制。方法:分离小鼠原代Ast,实验设置为正常对照组、模型组和HSYA处理组。MTT、LDH检测Ast活力;免疫荧光检测GFAP蛋白表达;RT-PCR和Western blot检测C3、S100A10、PTX3极化指标;RT-PCR和ELISA法检测TNF-α、IL-1β、IL-6、IL-10炎症因子表达;氧化检测试剂盒检测CAT、SOD、GSH-Px、RNS、ROS、MDA含量;Western blot检测GFAP、p-NF-κB(p65)、p-STAT3、Nrf2、HO-1蛋白表达。结果:Ast经OGD/R处理后被激活且形态发生改变;同时A1型Ast增加,TNF-α、IL-1β、IL-6等炎症因子及RNS、ROS、MDA表达升高;A2型Ast降低,CAT、SOD、GSH-Px降低。HSYA可减轻OGD/R状态下Ast的形态异常,减少A1型Ast,同时减少炎症因子分泌以及RNS、ROS和MDA含量,降低p-NF-κB(p65)和p-STAT3表达;增加IL-10分泌和CA...     

左乙拉西坦和传统抗癫痫药体外诱导大鼠星形胶质细胞P糖蛋白表达的研究

目的研究左乙拉西坦(LEV)和传统抗癫痫药(AEDs)丙戊酸钠(VPA)、卡马西平(CBZ)对大鼠皮质星形胶质细胞P-糖蛋白(P-gp)的表达的影响。方法不同浓度(1、10、50、100μg·m L-1)的VPA、CBZ及LEV持续作用于培养的正常新生鼠大脑皮质星型胶质细胞,分别在给药后10、20和30d,用免疫细胞化学法检测P-gp的表达率。结果对照组即无药物作用的正常星形胶质细胞P-gp表达率在各时点均小于5%;CBZ组100μg·m L-1在20d,30d较对照组表达增高(P0.05),20d与30d两组间比较,P0.05,其他浓度与对照组比较P0.05;VPA组100μg·m L-1在30d时较对照组表达增高(P0.05),较20d时表达增高(P0.05),其他浓度较各时点对照组比较,P0.05。LEV组在各浓度,不同时点与对照组比较差异无统计学意义(P0.05)。结论高浓度CBZ、VPA可诱导星形胶质细胞P-gp的表达,而LEV不能诱导星形胶质细胞P-gp表达。     

高压氧对脑缺血再灌注小鼠抗氧化酶类及血脑屏障通透性的影响

目的　探讨高压氧 (HBO)在脑缺血再灌注损伤中对自由基的产生及血脑屏障 (BBB)通透性的影响。方法　昆明种小鼠 3 2 0只 ,随机分为假手术组、HBO组、脑缺血再灌注组和HBO 脑缺血再灌注组 ,每组各 80只。复制清醒脑缺血再灌注模型 ,并于术后经 0 .2 5MPaHBO治疗 5次 ,采用比色法检测各组小鼠海马组织SOD、CAT、GSH PX活性和MDA及EB的含量。取小鼠海马组织常规制片 ,透射电镜下观察并进行比较。结果　脑缺血再灌注组与假手术组比较 ,SOD、CAT及GSH PX活性明显下降 (P <0 .0 1) ,MDA含量明显升高 (P <0 .0 1) ,与脑部EB含量增高相一致。HBO 脑缺血再灌注组与脑缺血再灌注组相比 ,SOD、CAT、GSH PX活性明显升高 (P <0 .0 5 ) ,MDA含量明显下降 (P <0 .0 1) ,并与脑部EB含量减少相一致。HBO组与假手术组相比 ,GSH PX酶活性明显增高 (P <0 .0 5 )。电镜下示脑缺血再灌注组神经元坏死明显 ,星形胶质细胞胞体水肿 ,微管、微丝明显减少 ;HBO 脑缺血再灌注组神经元坏死不明显。结论　高压氧可增强脑缺血再灌小鼠脑组织抗氧化酶类活性 ,恢复星形胶质细胞功能 ,降低BBB通透性。     

RNAi抑制星形胶质细胞Cx43对缺氧/再复氧后细胞凋亡的影响

目的探讨RNA干扰技术对星形胶质细胞Cx43蛋白的抑制作用以及对细胞缺氧/再复氧后凋亡的影响。方法化学合成针对大鼠Cx43基因的小干扰RNA(Small interfering RNA,siRNA)及其阴性RNA,通过脂质体转染体外培养原代星形胶质细胞,Western blot检测Cx43蛋白表达情况,MTT法检测siRNA转染对细胞的毒性作用;通过流式细胞术观察抑制Cx43蛋白对星形胶质细胞缺氧/再复氧后细胞凋亡情况的影响。结果 Western blot结果可见,转染siRNA后12h,Cx43蛋白表达开始抑制,48h抑制最明显,MTT结果显示siRNA组和脂质体组的细胞活性明显低于未转染组(P0.05),siRNA组与脂质体组未见明显区别(P0.05);流式细胞术示缺氧12h复氧不同时间,凋亡率逐渐增加,复氧48h凋亡率最高,siRNA干扰组明显低于正常对照组(P0.05)。结论 RNAi技术是研究Cx43功能的一种有效方法,siRNA转染能有效抑制星形胶质细胞Cx43蛋白表达,siRNA转染时,细胞毒性来自脂质体;siRNA抑制Cx43蛋白能够明显减轻星形胶质细胞缺氧/再复氧后的细胞凋亡。     

Changing face of hepatic encephalopathy: Role of inflammation and oxidative stress

The face of hepatic encephalopathy(HE) is changing.This review explores how this neurocognitive disorder,which is associated with both acute and chronic liver injury,has grown to become a dynamic syndrome that spans a spectrum of neuropsychological impairment,from normal performance to coma.The central role of ammonia in the pathogenesis of HE remains incontrovertible.However,over the past 10 years,the HE community has begun to characterise the key roles of inflammation,infection,and oxidative/nitrosative s...     

刚地弓形虫速殖子与鼠星形胶质细胞体外共培养的实验观察

原代培养星形胶质细胞,待其长至培养皿的80%时,将星形胶质细胞与弓形虫速殖子(细胞与虫体的比例为1∶10)共培养0～72h,吉氏染色观察星形胶质细胞和弓形虫速殖子的变化。共培养1h,此时定为0点,再培养0～48h,单磺酰戊二胺染色在荧光显微镜下观察星形胶质细胞内自噬囊泡的变化。结果发现共培养1h时,弓形虫速殖子开始进入星形胶质细胞,并出现荧光颗粒;8h星形胶质细胞内荧光颗粒最多;12h后显著减少,星形胶质细胞开始被破坏;48h星形胶质细胞内荧光颗粒消失;72h大部分星形胶质细胞已被涨破,留下大量增殖的弓形虫速殖子。说明自噬在弓形虫速殖子体外感染星形胶质细胞的过程中是受到抑制的。     

Neuromyelitis optica and opticospinal multiple sclerosis: Mechanisms and pathogenesis

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) while neuromyelitis optica (NMO) is an inflammatory disease of the CNS that selectively affects the optic nerves and spinal cord. In Asians, MS is rare; however, when it appears, the selective and severe involvement of the optic nerves and spinal cord is characteristic. This form, termed opticospinal MS (OSMS), has similar features to the relapsing form of NMO in Western populations. Recently, a specific IgG against NMO, designated NMO-IgG, was discovered, and the relevant antigen was found to be aquaporin-4 (AQP4), one of the major water channel proteins in the CNS. Because NMO-IgG has been reported to be present in 30-60% of OSMS patients, OSMS in Asians has been suggested to be the same entity as NMO.The sensitivity of NMO-IgG/anti-AQP4 antibody for NMO varies from 30% to 80%, while the specificity is 90-100%. Pathological studies on NMO have revealed perivascular immune complex (IgM, IgG and C9neo) deposition and extensive loss of AQP4 in active lesions, where myelin basic protein (MBP) staining was relatively preserved. IgG from NMO-IgG-seropositive NMO patients induces astrocyte death in culture in the presence of complements, and reproduces astrocyte loss in vivo when MBP-specific T cells are co-transferred to cause experimental autoimmune encephalomyelitis. It is thus postulated that the complement-activating anti-AQP4 antibody plays a pivotal role in the development of NMO lesions through astrocyte necrosis, and that demyelination is a secondary event.However, in autopsied cases of NMO, we and others found that some demonstrated selective AQP4 loss while others showed preservation of AQP4, even in the acute lesions. We also found that, in some MS lesions, AQP4 was lost extensively far beyond the areas of myelin loss. In the CSF, proinflammatory cytokines such as IL-17, IL-8, IFNγ, and G-CSF are markedly elevated in OSMS patients, irrespective of the presence or absence of anti-AQP4 antibody. In OSMS and NMO patients, T cells reactive to myelin proteins show intra- and inter-molecular epitope spreading, suggesting that T cells are already stimulated with myelin antigens in vivo. These findings suggest that mechanism of NMO and OSMS in Asians is heterogeneous, anti-AQP4 antibody-related and -unrelated, and that not only anti-AQP4 antibody but also myelin-autoreactive Th17 or Th1 cells may also play a role in triggering CNS inflammation. Possible mechanisms for NMO and OSMS are discussed in this review.     

Retinal astrocyte differentiation mediated by leukemia inhibitory factor in cooperation with bone morphogenetic protein 2

Retinal astrocytes and their precursor cells migrate from the optic nerve. Interleukin 6 family cytokines, whose signal transduction requires gp130, promote astrocyte differentiation in the optic nerve, though the mechanism of astrocyte differentiation in the retina has not been clarified. We found that GFAP-positive astrocytes were significantly decreased in number but that a considerable number of astrocytes were still present in gp130-deficient mouse retina. These findings suggest that gp130-dependent signaling pathways play essential roles in retinal astrocyte differentiation and that retinal astrocyte differentiation can also be promoted by other signaling pathways. We found that leukemia inhibitory factor, bone morphogenetic proteins, and their receptors are expressed in P0 retina. In addition, leukemia inhibitory factor and bone morphogenetic protein 2 synergistically promote astrocyte differentiation of retinal precursor cells isolated from P0 mouse retina. These observations demonstrated that not only gp130-dependent signaling but also bone morphogenetic proteins play essential roles in retinal astrocyte differentiation.     

Diclofenac enhances proinflammatory cytokine-induced nitric oxide production through NF-κB signaling in cultured astrocytes

Recently, the number of reports of encephalitis/encephalopathy associated with influenza virus has increased. In addition, the use of a non-steroidal anti-inflammatory drug, diclofenac sodium (DCF), is associated with a significant increase in the mortality rate of influenza-associated encephalopathy. Activated astrocytes are a source of nitric oxide (NO), which is largely produced by inducible NO synthase (iNOS) in response to proinflammatory cytokines. Therefore, we investigated whether DCF enhances nitric oxide production in astrocytes stimulated with proinflammatory cytokines. We stimulated cultured rat astrocytes with three cytokines, interleukin-1β, tumor necrosis factor-α and interferon-γ, and then treated the astrocytes with DCF or acetaminophen (N-acetyl-p-aminophenol: APAP). iNOS and NO production in astrocyte cultures were induced by proinflammatory cytokines. The addition of DCF augmented NO production, but the addition of APAP did not. NF-κB inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-κB p65 Stealth small interfering RNA suppressed iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. LDH activity and DAPI staining showed that DCF induces cell damage in cytokine-stimulated astrocytes. An iNOS inhibitor, l-NMMA, inhibited the cytokine- and DCF-induced cell damage. In conclusion, this study demonstrates that iNOS and NO are induced in astrocyte cultures by proinflammatory cytokines. Addition of DCF further augments NO production. This effect is mediated via NF-κB signaling and leads to cell damage. The enhancement of DCF on NO production may explain the significant increase in the mortality rate of influenza-associated encephalopathy in patients treated with DCF.