安宫牛黄丸及朱砂和雄黄对脂多糖诱导的神经损伤的作用(英文)

目的探讨朱砂和雄黄在安宫牛黄丸(AGNH)配方中对脂多糖(LPS)介导的神经损伤的保护作用及机制。方法制备大鼠中脑原代神经元-神经胶质细胞联合培养模型。实验分为正常对照组、LPS 10μg.L-1模型组、LPS +朱砂(4和40 mg.L-1)组、LPS +雄黄(4和40 mg.L-1)组和LPS +AGNH(40和400mg.L-1)组。药物与细胞作用30 min后加入LPS;7 d后进行实验指标检测。采用免疫组织化学染色法检测酪氨酸羟化酶阳性的细胞数量和形态学变化以及小神经胶质细胞的激活情况,实时RT-PCR检测细胞中肿瘤坏死因子α(TNF-α)mRNA和诱导型一氧化氮合酶(iNOS)mRNA表达水平,酶联免疫吸附试验(ELISA)与Griess试剂分别检测细胞培养上清液中TNF-α和一氧化氮(NO)的含量。结果与正常对照组比较,LPS10μg.L-1作用于细胞,多巴胺能神经元的数量明显下降了40%(P<0.05);LPS显著诱导小神经胶质细胞的激活,TNF-αmRNA和iNOS mRNA的表达分别增加了9倍和2倍(P<0.05),同时神经元-胶质细胞联合培养上清液中TNF-α和NO的含量分别增加了20倍和30倍(P<0.05)。与LPS模型组比较,AGNH400mg.L-1和雄黄40 mg.L-1能明显拮抗LPS对多巴胺能神经元的毒性作用,多巴胺神经元数量分别上升了40%和30%(P<0.05);AGNH400 mg.L-1和雄黄40 mg.L-1能抑制小神经胶质细胞的激活,小胶质神经细胞中TNF-αmRNA分别下降了61%和52%(P<0.05)和iNOS mRNA的表达分别降低了58%和51%(P<0.05),而且神经元-神经胶质细胞联合培养上清液中TNF-α的含量分别降低了55%和43%(P<0.05)以及NO的含量分别下降了53%和34%(P<0.05)。而朱砂对LPS的作用无影响。结论 AGNH能改善LPS介导的神经元损伤,雄黄是其抗炎作用的有效成分之一,朱砂未见明显的神经保护作用。     

表没食子儿茶素没食子酸酯对脂多糖诱导原代胶质细胞炎性反应的保护作用

目的:观察表没食子儿茶素没食子酸酯[(-)-Epigallocatechin-3-gallate,EGCG]对细菌脂多糖 (LPS) 所致原代神经胶质细胞炎性反应的保护作用。方法:取新生乳鼠原代神经胶质细胞培养,利用LPS引起其炎症反应。用酶联免疫吸附试验 (ELISA) 测定炎性因子肿瘤坏死因子(TNF-α)、白介素-1β(IL-1β)、白介素-1β(IL-8)表达情况和蛋白免疫印记法(Western blot) 检测炎症因子(诱导型一氧化氮合酶)iNOS蛋白含量变化。结果:LPS激活神经胶质细胞后诱导炎症因子过度表达,大幅上调TNF-α、IL-1β、IL-8炎性因子(P<0.05),iNOS蛋白质水平显著升高(P<0.05),不同浓度EGCG干预组均可明显抑制炎症因子的过度产生,一定程度上减轻LPS诱导神经胶质细胞的炎性反应。结论:一定浓度范围内,EGCG能减弱LPS引起的体外培养神经胶质细胞的炎症反应。     

吡格列酮对脂多糖诱导的星形胶质细胞炎性细胞因子释放的影响

目的研究吡格列酮对脂多糖(LPS)诱导的星形胶质细胞炎症介质释放的抑制作用及其信号传导通路。方法神经胶质酸性蛋白(glial fibrillary acid protein,GFAP)免疫荧光染色法鉴定星形胶质细胞纯度。ELISA方法检测IL-1β、IL-6和TNF-α蛋白表达量的变化。Griess法测定培养细胞上清液中一氧化氮(NO)含量。结果星形胶质细胞经GFAP免疫荧光鉴定,其阳性率可达95%以上。LPS组能明显增加星形胶质细胞分泌IL-1β、IL-6、TNF-α及NO。吡格列酮能明显抑制LPS引起的这些作用,并呈一定浓度依赖性。过氧化物酶体增殖物激活受体γ(PPARγ)的特异性阻断剂GW9662能明显对抗吡格列酮对LPS引起的IL-1β、IL-6、TNF-α及NO增加的抑制作用。与LPS组相比,JNK特异性阻断剂SP600125(5μmol·L-1)亦能有效对抗LPS诱导星形胶质细胞IL-1β、IL-6、TNF-α及NO分泌的增加;特异性iNOS抑制剂SMT可明显抑制LPS引起的NO分泌增加。结论吡格列酮能明显改善LPS诱导的大鼠皮层星形胶质细胞的损伤,这种作用可能与激活PPARγ、抑制JNK信号传导通路有关。     

异钩藤碱对脂多糖诱导的星形胶质细胞炎性介质释放的抑制作用

目的:观察异钩藤碱对LPS诱导的星形胶质细胞炎性介质释放的影响.方法:分离并鉴定新生大鼠大脑皮质星形胶质细胞,用1μg·mL<'-1>LPS激活原代星形胶质细胞,刺激其分泌IL-1β、TNF-α和NO,并大量表达iNOS mRNA.用不同浓度的异钩藤碱和LPS与星形胶质细胞共孵育.检测异钩藤碱处理后细胞培养液中这些炎性因子释放的含量.结果:异钩藤碱有效地抑制了LPS诱导的星形胶质细胞炎性介质释放,并降低了iNOS mRNA的表达水平.结论:异钩藤碱具有较好的中枢炎症抑制作用,为传统中药钩藤作为治疗缺血性脑病药物提供了科学依据.     

洛伐他汀对肾小球系膜细胞iNOS表达及氧自由基水平的影响

目的 观察洛伐他汀对脂多糖(LPS)刺激后大鼠肾小球系膜细胞诱导型一氧化氮合酶(iNOS)mRNA的表达及细胞培养上清液中羟自由基(·OH),丙二醛(MDA),总超氧化物歧化酶(tSOD)含量的影响.方法 采用逆转录-聚合酶链反应(RT-PCR)法检测LPS作用大鼠系膜细胞后,洛伐他汀在不同时间段对系膜细胞iNOS mRNA表达的影响及分别检测细胞培养上清液中的·OH、MDA、tSOD的含量.结果 系膜细胞在正常情况下iNOS mRNA无明显表达,LPS作用24h后能引起iNOS mRNA的显著表达,洛伐他汀可抑制LPS引起的系膜细胞iNOS mRNA的表达.LPS作用于大鼠系膜细胞后,可引起细胞培养上清液中·OH、MDA含量的增加及tSOD含量的减少;加入洛伐他汀后细胞培养上清液中·OH、MDA含量减少,tSOD含量没有明显的变化.结论 洛伐他汀能抑制LPS引起的系膜细胞iNOS mRNA表达的增加及下调LPS引起的细胞培养上清液中·OH、MDA含量的增加.     

绿原酸抑制巨噬细胞激活的机制研究

目的 观察绿原酸对脂多糖（LPS）致巨噬细胞激活的影响，并探讨骨髓细胞2表达的触发受体（TREM2）蛋白在其中的作用。方法 为筛选LPS造模浓度，分别以1、10、100 ng/mL的LPS培养细胞24 h，检测细胞培养上清液中白细胞介素6(IL-6)水平和细胞中诱导型一氧化氮合酶（i NOS）蛋白表达水平。为筛选绿原酸给药浓度，将细胞分为Control组、LPS处理组和3个不同浓度绿原酸（0.01、0.1、1μmol/L）干预组，检测细胞培养上清液中肿瘤坏死因子α(TNF-α)、IL-1β水平和细胞中iNOS、TREM2蛋白表达水平以及细胞活力。为观察TREM2在绿原酸抑制巨噬细胞激活中的作用，采用TREM2小干扰RNA(TREM2-siRNA)干扰细胞TREM2蛋白表达，将细胞分为Control组、LPS处理组、绿原酸+LPS组、TREM2-siRNA+绿原酸+LPS组和乱序-siRNA(SCsiRNA)+绿原酸+LPS组，在含有上述药物的培养基/空白培养基中孵育24 h后，检测细胞培养上清液中TNF-α和IL-1β水平以及细胞中TREM2、iNOS和核因子κB p65(NF-κB ...     

壮药三七姜醇提物及含药血清对脂多糖诱导RAW264.7细胞炎症的抗炎作用及机制

目的 研究壮药三七姜醇提物及含药血清对脂多糖（LPS）诱导RAW264.7细胞炎症的抗炎作用及机制。方法 将三七姜醇提物（75.35 g/kg）或纯净水灌胃大鼠以制备含药血清或空白血清。以RAW264.7细胞为研究对象，将细胞分为正常对照组，LPS组（1μg/mL），三七姜醇提物高、中、低剂量组（50、25、12.5μg/mL）,4%或15%空白血清组，4%或15%空白血清+LPS组，4%或15%含药血清组，4%或15%含药血清+LPS组，按相应条件培养24 h后，检测各组细胞活力和细胞中一氧化氮（NO）、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6含量，以及Toll样受体4(TLR4)、核因子κB(NF-κB)mRNA表达水平，一氧化氮合酶（NOS）、环氧化酶2(COX-2)蛋白表达水平。结果 各组细胞培养24 h后，细胞活力差异无统计学意义。与正常对照组比较，LPS组细胞中NO、TNF-α、IL-1β、IL-6含量，TLR4、NF-κB mRNA表达水平和NOS、COX-2蛋白表达水平均显著升高（P<0.05）。与4%或15%空白血清组比较，4%或15...     

金钗石斛生物总碱对脂多糖激活星形胶质细胞产生炎症因子的影响

目的观察金钗石斛生物总碱对外源性内毒素脂多糖(lipopolysaccharide,LPS)激活大鼠大脑皮层星形胶质细胞(astrocyte)及诱导其产生和释放炎症介质的影响,探讨石斛生物总碱对星形胶质细胞的抗炎作用。方法通过MTS检测细胞存活率,ELISA法检测TNF-α炎性因子蛋白的表达,实时定量多聚酶链反应(real time RT-PCR)检测炎症相关基因TNF-α、IL-6 mRNA的表达。结果①LPS刺激星形胶质细胞后,MTS检测吸光度明显升高,与正常组比较差异有显著性;②金钗石斛生物总碱能够降低LPS所致的TNF-α蛋白的高表达(P<0.05);③金钗石斛生物总碱能够降低LPS诱导的星形胶质细胞吸光度的升高,同时明显抑制LPS所致的TNF-α、IL-6 mRNA的高表达。结论金钗石斛生物总碱能够拮抗LPS所引起的炎症反应,其作用与抑制星形胶质细胞的激活及其炎症因子的释放密切相关。     

去氢丹参新酮对神经炎症的抑制作用及机制研究

目的研究去氢丹参新酮对脂多糖(lipopolysaccharide,LPS)诱导小胶质细胞系BV2细胞产生炎症反应的抑制作用及其作用机制。方法不同浓度去氢丹参新酮预孵育BV2细胞后,用LPS刺激引起神经炎症相关反应。Griess试剂法检测去氢丹参新酮对活化的BV2细胞产生一氧化氮(nitric oxide,NO)的影响,ELISA检测细胞上清液中TNF-α和IL-6的释放量,Confocal观察小胶质细胞表面活化标志物MAC-1表达量的变化,Western blot检测炎症相关信号通路蛋白表达的变化。结果去氢丹参新酮可明显抑制LPS刺激BV2细胞产生的炎症因子包括NO、TNF-α和IL-6的水平,同时抑制一氧化氮合酶、环氧合酶-2等炎症相关蛋白的表达和小胶质细胞表面活化标志物MAC-1的表达。机制研究发现,去氢丹参新酮对PI3K/Akt的过度磷酸化以及NF-κB的过度活化都有明显的抑制作用。结论去氢丹参新酮具有很好的抑制神经炎症活性,其作用机制可能是通过PI3K/Akt信号通路抑制NF-κB的活化而实现的。     

金钗石斛多糖对脂多糖作用大鼠皮层胶质细胞-神经元体系的保护作用

目的观察金钗石斛多糖(NDP)对脂多糖(LPS)作用的新生大鼠大脑皮层胶质细胞-神经元混合培养体系的保护作用。方法原代制备新生大鼠大脑皮层胶质细胞-神经元混合培养体系,NDP作用于LPS刺激的胶质细胞-神经元混合培养体系,观察细胞生长情况,并采用Real time PCR法检测体系中炎症相关因子IL-1β、TNF-α、COX-2的基因表达。结果 NDP作用于LPS刺激的大鼠皮层胶质细胞-神经元混合培养体系后,胶质细胞激活减少、神经元损伤减轻,较单用LPS作用的模型组相比,炎症相关因子IL-1β、TNF-α、COX-2的基因表达明显降低。结论 NDP能抑制LPS对小胶质细胞和星形胶质细胞的激活,减少炎性因子的生成。     

Anti-inflammatory effects of fluoxetine in lipopolysaccharide(LPS)-stimulated microglial cells

Recent evidence has suggested that microglial activation plays an important role in the pathogenesis of depression. Activated microglia can secrete various pro-inflammatory cytokines and neurotoxic mediators, which may contribute to the development and maintenance of depression. Thus, inhibition of microglial activation may have a therapeutic benefit in the treatment of depression. In the present study, using BV2 microglial cell line and primary microglial culture, we investigated if fluoxetine, the most widely used antidepressant, can inhibit microglia activation. Our results showed that fluoxetine significantly inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α), interleukin- 6 (IL-6) and nitric oxide (NO). By RT-PCR, the mRNA level of these pro-inflammatory cytokines and iNOS was also attenuated by fluoxetine. We further investigated the intracellular signaling mechanism regulating the production of pro-inflammatory cytokines and NO from LPS-activated microglia. The results showed that fluoxetine inhibited IκB-a degradation, phosphorylation and nuclear translocation of the p65 subunit of NF-κB, and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the LPS-stimulated microglia. Taken together, our results suggest that the therapeutic effects of fluoxetine are partially mediated by modulating microglial activation.     

Ampelopsin attenuates lipopolysaccharide-induced inflammatory response through the inhibition of the NF-κB and JAK2/STAT3 signaling pathways in microglia

Increasing evidence suggests that microglia are a major cellular contributor to neuroinflammation. The present study investigated whether Ampelopsin (Amp), a type of flavanonol derivative from Ampelopsis grossedentata, may exert an anti-inflammatory effect on lipopolysaccharide (LPS)-induced BV2 and primary microglia cells. We found that pre-treatment of microglia cells with Amp before LPS with a non-cytotoxic concentration range decreased the production of nitric oxide (NO) and prostaglandin E2 (PGE2). Amp also suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the mRNA and protein levels. In addition, LPS-induced production of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) was obviously reduced by Amp. Our mechanistic study indicated that Amp suppressed LPS-induced activation of the IκB/NF-κB inflammation pathway without affecting changes in the phosphorylation levels of mitogen-activated protein kinases (MAPKs) in BV2 cells. Further studies revealed that Amp markedly reduced the phosphorylation levels of JAK2-STAT3 and STAT3 nuclear translocation. Overall, our data suggest that Amp can suppress the LPS-induced inflammatory response of microglial cells, indicating that Amp has potential for the treatment of inflammation-mediated neurodegenerative diseases.     

Effects of cobalt chloride on nitric oxide and cytokines/chemokines production in microglia

The involvement of microglial activation in metal neurotoxicity is becoming increasingly recognized. Some metal ions, such as zinc (II) and manganese (II), have been recently reported as microglial activators to induce the release of inflammatory mediators including cytokines, chemokines and nitric oxide (NO) which are involved in the pathogenesis of neurological diseases. Cobalt is essential for human life. However, excessive cobalt is cytotoxic and neurotoxic. In the present study, we determined cobalt-induced production of NO and cytokines/chemokines in N9 cells, a murine microglial cell line. High levels of cobalt significantly up-regulated iNOS mRNA and protein expression, which resulted in the release of NO. Cobalt induced the production of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in a concentration- and time-dependent manner in both N9 cells and primary mouse microglia and increased lipopolysaccharides (LPS)-induced cytokine production. Further study showed that cobalt induced cytokine production by a mechanism involving both nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The involvement of reactive oxygen species (ROS) in microglial activation was also confirmed. These findings suggested that cobalt neurotoxicity should be attributed not only directly to neuronal damage but also indirectly to microglial activation which might potentiate neuronal injury via elevation of proinflammatory mediator levels.     

Inhibitory Effects of Lycopene on the Induction of NO,Cytokines, and Mitogen-Activated Protein Kinase Expression by Lipopolysaccharide in Primary Cultured Microglia

Abstract

Microglia are activated in response to brain injury and release neurotoxic factors including nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Lycopene, a potent antioxidant, is known to inhibit brain injury. In this study, we found that lycopene (5–20 μ M) significantly inhibited lipopolysaccharide (LPS)-induced NO release in primary cultured microglia. Lycopene (5–20 μM) also concentration-dependently diminished the LPS-induced production of proinflammatory cytokines such as TNF-α and IL-1β in microglia. Further study of the molecular mechanisms revealed that lycopene markedly inhibited extracellular signal-regulated kinase (ERK1/2) but not c-Jun N-terminal kinase (JNK1/2) or p38 mitogen-activated protein kinase (MAPK) phosphorylation stimulated by LPS in microglia. These results suggest that microglial inactivation by lycopene is at least partially due to activation of ERK1/2 phosphorylation Therefore, inhibition of NO and proinflammatory cytokine production in activated microglia by lycopene may represent a powerful and potential therapeutic strategy for various neurodegenerative diseases including ischemia-reperfusion cerebral infarction.     

6-Shogaol, a ginger product, modulates neuroinflammation: a new approach to neuroprotection

Inflammatory processes in the central nervous system play an important role in a number of neurodegenerative diseases mediated by microglial activation, which results in neuronal cell death. Microglia act in immune surveillance and host defense while resting. When activated, they can be deleterious to neurons, even resulting in neurodegeneration. Therefore, the inhibition of microglial activation is considered a useful strategy in searching for neuroprotective agents. In this study, we investigated the effects of 6-shogaol, a pungent agent from Zingiber officinale Roscoe, on microglia activation in BV-2 and primary microglial cell cultures. 6-Shogaol significantly inhibited the release of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS). The effect was better than that of 6-gingerol, wogonin, or N-monomethyl-l-arginine, agents previously reported to inhibit nitric oxide. 6-Shogaol exerted its anti-inflammatory effects by inhibiting the production of prostaglandin E(2) (PGE(2)) and proinflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and by downregulating cyclooxygenase-2 (COX-2), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB) expression. In addition, 6-shogaol suppressed the microglial activation induced by LPS both in primary cortical neuron-glia culture and in an in vivo neuroinflammatory model. Moreover, 6-shogaol showed significant neuroprotective effects in vivo in transient global ischemia via the inhibition of microglia. These results suggest that 6-shogaol is an effective therapeutic agent for treating neurodegenerative diseases.     

Inhibitory effect of fucoidan on nitric oxide production in lipopolysaccharide‐activated primary microglia

1. Microglial activation plays an important role in the pathogenesis of neurodegenerative diseases by producing various pro‐inflammatory cytokines. Microglia‐derived nitric oxide (NO) is critical for the lipopolysaccharide (LPS)‐induced selective loss of dopaminergic neurons. 2. Fucoidan is a sulphated polysaccharide extracted from brown seaweeds. It has a variety of biological actions, including anticoagulant, antiviral and anti‐inflammatory effects. The aim of the present study was to investigate the effects of fucoidan on LPS‐induced cellular activation in microglia and to evaluate the inhibitory mechanisms involved. 3. To investigate the effects of fucoidan on LPS‐induced cellular activation in microglia, primary microglial cells were preincubated with fucoidan (31.25, 62.5 and 125 μg/mL) for 10 min, followed by stimulation with LPS (0.01 μg/mL). Then, cell shape and NO production were determined 24 h after LPS stimulation, whereas inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined at 6 and 18 h after LPS stimulation, respectively. To evaluate the inhibitory mechanisms involved, mitogen‐activated protein kinase (MAPK) activation was also evaluated. 4. Lipopolysaccharide transformed cells into an amoeboid shape, whereas 62.5 μg/mL fucoidan inhibited this activation. Moreover, 125 μg/mL fucoidan significantly inhibited microglial NO production to 75% of that in LPS‐treated group and also significantly diminished the expression of iNOS mRNA and protein by nearly 50%. Fucoidan (125 μg/mL) also suppressed phosphorylation of p38 and extracellular signal‐regulated kinase (ERK) by approximately 50%, but not that of c‐Jun N‐terminal kinase. 5. The results provide the first evidence that fucoidan has a potent inhibitory effect against LPS‐induced NO production by microglia. The results also suggest that this inhibitory action of fucoidan involves suppression of p38 and ERK phosphorylation.