PrP106—126诱导小胶质细胞BV-2抗氧化性能的变化

小胶质细胞活化是朊病的病理学特征之一。朊蛋白多肽PrP106—126具神经毒性,是研究异常腕蛋白（PrPSc）的理想工具。为探讨PrP106—126对小胶质细胞氧化压力的影响。本研究以小胶质细胞BV-2为细胞模型,PrP106—126作用48h,应用MTT和流式细胞仪检测细胞的活化情况,应用分子探针技术对细胞的氧化压力（reactiveoxygenspecies,ROs）进行检测,并通过荧光定量RT—PCR对与R0s相关的酶的mRNA表达进行了测定。结果表明PrPl06—126显著促进小胶质细胞BV-2的活化,并提高胞内的ROS水平;定量RT—PCR显示,PrP106—126显著降低细胞S0D-1（P〈0．01）表达水平、提高胞内Cat（P〈0．01）的表达水平;对Grx、Trx-1、和Trx-2mRNA的表达水平有升高的趋势,但未达到显著水平（P〉0．05）,对SOd-2、GPx、GR无显著性影响（P〉0．05）。从分子水平初步阐明小胶质细胞ROS升高的机理。     

Dietary curcumin supplementation attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in C57BL mice

Studies in vivo and in vitro suggest that curcumin is a neuroprotective agent. Experiments were conducted to determine whether dietary supplementation with curcumin has neuroprotective effects in a mouse model of Parkinson’s disease (PD). Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) significantly induced the loss of dopaminergic cells in the substantia nigra and deletion of dopamine in the striatum, which was attenuated by long-term (7 weeks) dietary supplementation with curcumin at a concentration of 0.5% or 2.0% (w/w). Although curcumin did not prevent the MPTP-induced apoptosis of neuroblasts in the subventricular zone (SVZ), it promoted the regeneration of neuroblasts in the anterior part of the SVZ (SVZa) at 3 days after MPTP treatment. Furthermore, curcumin enhanced the MPTP-induced activation of microglia and astrocytes in the striatum and increased the expression of glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-β1 (TGFβ1) in the striatum and SVZ. GDNF and TGFβ1 are thought to play an important role in protecting neurons from injury in the central and peripheral nervous systems. These results suggest that long-term administration of curcumin blocks the neurotoxicity of MPTP in the nigrostriatal dopaminergic system of the mouse and that the neuroprotective effect might be correlated with the increased expression of GDNF and TGFβ1. Curcumin may be effective in preventing or slowing the progression of PD.     

Vibrio vulnificus MO6-24/O Lipopolysaccharide Stimulates Superoxide Anion,Thromboxane B2, Matrix Metalloproteinase-9, Cytokine and Chemokine Release by Rat Brain Microglia in Vitro

Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus) lipopolysaccharide (LPS) has been reported to result in septic shock, its impact on the central nervous system’s innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O₂⁻), a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O₂⁻ was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), IL-6, and transforming growth factor-beta 1 (TGF-β1), chemokines macrophage inflammatory protein (MIP-1α)/chemokine (C-C motif) ligand 3 (CCL3), MIP-2/chemokine (C-X-C motif) ligand 2 (CXCL2), monocyte chemotactic protein-1 (MCP-1)/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β)/CXCL3, and brain-derived neurotrophic factor (BDNF), were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate)-stimulated O₂⁻ generation: (1) 0.1–1 ng/mL V. vulnificus LPS enhanced O₂⁻ generation significantly but with limited inflammatory mediator generation; (2) 10–100 ng/mL V. vulnificus LPS maximized O₂⁻ generation with concomitant release of thromboxane B₂ (TXB₂), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000–100,000 ng/mL V. vulnificus LPS, with the exception of TXB₂, yielded both attenuated O₂⁻ production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of neonatal brain microglia with V. vulnificus MO6-24/O LPS resulted in a significant rise in O₂⁻ production, followed by a progressive decrease in O₂⁻ release, with concomitant release of lactic dehydrogenase (LDH), and generation of TXB₂, MMP-9, cytokines and chemokines. We hypothesize that the inflammatory mediators investigated may be cytotoxic to microglia in vitro, by an as yet undetermined autocrine mechanism. Although V. vulnificus LPS was less potent than E. coli LPS in vitro, inflammatory mediator release by the former was clearly more efficacious. Finally, we hypothesize that should V. vulnificus LPS gain entry into the CNS, it would be possible that microglia might become activated, resulting in high levels of O₂⁻ as well as neuroinflammatory TXB₂, MMP-9, cytokines and chemokines.     

细胞型朊蛋白在BV2小神经胶质细胞体外激活中的作用

【目的】研究细胞型朊蛋白对小神经胶质细胞不同激活方式的影响。【方法】用IFN-γ、IL-4和IL-10分别刺激BV2细胞,RT-PCR方法检测PrPC的mRNA表达量。SiRNA干扰将PrPC沉默,用上述因子刺激细胞,用RT-PCR和Western-blot检测相关参数。【结果】用IFN-γ、IL-4和IL-10分别刺激小神经胶质细胞后可导致PrPC的mRNA表达量下降;PrPC沉默后的小神经胶质细胞对IFN-γ刺激的反应应答减弱;PrPC沉默可以显著地改变由IL-4诱导的神经胶质细胞的激活表型;但是对IL-10诱导的小神经胶质细胞激活却没有影响。【结论】PrPC既能影响小神经胶质细胞从静止状态到激活状态的转换,也在小神经胶质细胞的经典激活和替代激活途径中发挥调节作用。     

PrP106-126对BV-2小胶质细胞NO含量影响及作用机制

朊蛋白多肽PrP106-126可激活小胶质细胞并产生活性物质。探讨PrP106-126作用于BV-2小胶质细胞对NO生成状况影响,从酶学角度检测其作用机制。在细胞培养液中加入50μmol.L-1PrP106-126,培养48 h后检测培养液NO含量,采用实时定量RT-PCR检测细胞内iNOs和nNOs在mRNA表达水平。结果表明,PrP106-126显著提高细胞培养液中NO含量(9.34倍,P<0.01),且iNOs和nNOs表达水平均极显著提高(11.60倍和4.36倍,P<0.01),从而为解释朊病发生机制提供基础数据。     

Increased phosphorylation of caveolin-1 in the spinal cord of irradiated rats

Phosphorylation of caveolin-1 occurs during cell activation by various stimuli. In this study, the involvement of caveolin-1 in an irradiation injured spinal cord was examined by analyzing the phosphorylation of caveolin-1 in the spinal cord of rats after irradiation with a single dose of 15 Gray from a ⁶⁰Co γ-ray source at 24 h post-irradiation (PI). A Western blot analysis showed that the phosphorylated form of caveolin-1 (p-caveolin-1) was expressed constitutively in the normal spinal cords and was significantly higher in the spinal cord of irradiated rats at 24 h PI. The increased expression of ED1, which is a marker of activated microglia/macrophages, was matched with that of p-caveolin-1. In the irradiated spinal cords, there was a higher level of p-caveolin-1 immunoreactivity in the isolectin B4-positive microglial, ependymal, and vascular endothelial cells, in which p-caveolin-1 was weakly and constitutively expressed in the normal control spinal cords. These results suggest that total body irradiation induces activation of microglial cells in the spinal cord through the phosphorylation of caveolin-1.     

Diterpenoids from the Brown Alga Rugulopteryx okamurae and Their Anti-Inflammatory Activity

Brown algae of the Family Dictyotaceae produce an array of structurally diverse terpenoids, whose biomedical potential in the anti-inflammatory area has been scarcely explored. Herein, the chemical study of the alga Rugulopteryx okamurae has led to the isolation of ten new diterpenoids: rugukadiol A (1), rugukamurals A–C (2–4), and ruguloptones A–F (6–10). The structures of the new compounds were established by spectroscopic means. Compound 1 exhibits an unprecedented diterpenoid skeleton featuring a bridged tricyclic undecane system. Compounds 2–10 belong to the secospatane class of diterpenoids and differ by the oxygenated functions that they contain. In anti-inflammatory assays, the new diterpenoid 1 and the secospatanes 5 and 10 significantly inhibited the production of the inflammatory mediator NO in LPS-stimulated microglial cells Bv.2 and macrophage cells RAW 264.7. Moreover, compounds 1 and 5 were found to strongly inhibit the expression of Nos2 and the pro-inflammatory cytokine Il1b in both immune cell lines.     

褪黑素对LPS致大鼠海马炎性损伤的保护作用

本研究旨在探究褪黑素（MT）对脂多糖（LPS）致大鼠海马炎性损伤的保护作用。选取40只4周龄健康雄性SD大鼠,随机分为4组：空白组（CON组）、模型组（LPS组）、褪黑素干预组（LPS+MT组）及褪黑素组（MT组）。通过腹腔注射的方式给予大鼠10 mg·kg^-1MT和/或10 mg·kg^-1LPS,4 h后,采用旷场试验对各组大鼠进行行为学测试;试验结束称大鼠体重,解剖取海马称重并计算海马体系数;苏木精-伊红（HE）染色观察脑切片中海马区域病理变化;RT-PCR技术检测海马中小胶质细胞激活标记物Iba-1和CD11b mRNA表达;Western blot法检测海马中炎性因子IL-1β、TNF-α、IL-6、IL-10及TGF-β蛋白表达。结果表明,与CON组相比,LPS组大鼠自主探索行为减少、运动能力下降,海马组织神经细胞排列松散、细胞间隙增大、胞质固缩深染、胶质细胞浸润,小胶质细胞激活标志物Iba-1和CD11b mRNA表达极显著升高（P<0.01）,促炎因子IL-1β、TNF-α及IL-6蛋白表达极显著升高（P<0.01）,抗炎因子IL-10和TGF-β蛋白表达极显著降低（P<0.01）。而与LPS组相比,LPS+MT组大鼠自主探索行为增加、运动能力增强,海马组织神经细胞排列紧密,未见明显病变,小胶质细胞激活标记物Iba-1和CD11b mRNA表达极显著降低（P<0.01）,促炎因子IL-1β、TNF-α及IL-6蛋白表达极显著降低（P<0.01）,抗炎因子IL-10和TGF-β蛋白表达极显著增加（P<0.01）。此外,MT组与CON组相比,所有指标差异均不显著（P>0.05）。结果提示,MT可抑制小胶质细胞激活,减轻海马炎症反应,从而改善LPS造成的大鼠海马炎性损伤。     

Differential effects of domoic acid and E. coli lipopolysaccharide on tumor necrosis factor-alpha, transforming growth factor-beta1 and matrix metalloproteinase-9 release by rat neonatal microglia: evaluation of the direct activation hypothesis

The excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).