氯喹通过抑制NF-κB和MAPK信号通路减轻脂多糖诱导的BV2小胶质细胞炎症反应

目的:探究氯喹(CQ)对脂多糖(LPS)刺激的BV2小胶质细胞活化的抑制作用及可能机制。方法:将小鼠BV2小胶质细胞分为对照组、LPS组和LPS+CQ组。LPS+CQ组预先给予CQ(10μmol/L)处理30 min再给予LPS刺激,在LPS组和LPS+CQ组中给予LPS(500μg/L)刺激后,各组细胞分别培养30 min、6 h和24 h。倒置显微镜下观察BV2细胞的形态学改变;RT-qPCR和ELISA法检测白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的mRNA和蛋白表达水平来评估BV2细胞的活化情况;用免疫荧光染色检测NF-κB蛋白核转移情况;用Western blot检测核因子κB抑制蛋白α(IκB-α)蛋白的表达情况及c-Jun氨基末端激酶(JNK)和p38蛋白的磷酸化水平。结果:LPS刺激后,BV2细胞形态由圆形或椭圆形向多极或纺锤样转变,而预先给予CQ能抑制BV2细胞的形态转变。LPS刺激后,BV2细胞中TNF-α和IL-6的mRNA水平和培养上清液中的蛋白水平明显增加,但预先给予CQ则明显抑制TNF-α和IL-6的mRNA和蛋白表达,可见CQ能减轻LPS诱导的BV2细胞炎症反应。此外,LPS刺激后,BV2细胞内IκB-α蛋白大量降解,细胞核内的NF-κB蛋白显著增多,MAPK信号通路中JNK和p38蛋白磷酸化水平明显升高;而预先给予CQ可显著减少IκB-α蛋白的降解,明显抑制NF-κB蛋白向细胞核内转移,显著降低JNK和p38蛋白的磷酸化水平,可见CQ能抑制LPS诱导下BV2细胞内NF-κB和MAPK信号通路的激活。结论:氯喹显著减轻LPS诱导的BV2细胞炎症反应,其机制与抑制NF-κB和MAPK信号通路有关。     

栀子苷下调Toll样受体4表达并拮抗脂多糖诱导的小胶质细胞炎性反应

目的:观察栀子昔对细菌脂多糖(LPS)诱导的BV2小胶质细胞炎性反应的影响并探讨其作用机制。方法:LPS诱导BV2小胶质细胞活化,CCK-8方法检测细胞存活率,Griess法测定NO释放量,ELISA测定肿瘤坏死因子-α(TNF-α)和白介素-1β(IL-1β)含量,免疫印迹检测Toll样受体4(TLR4)蛋白表达。结果:栀子苷在10～100μg/ml浓度范围内对小胶质细胞活力影响不显著,此浓度范围内,栀子苷剂量依赖性的减少LPS诱导的NO、TNF-α和IL-1β释放。此外,栀子苷还可抑制LPS诱导的BV2细胞形态活化改变,并降低LPS诱导的TLR4蛋白表达。结论:栀子苷可以拮抗LPS诱导的BV2小胶质细胞炎性反应,其机制可能与下调TLR4信号通路有关。     

脐带间充质干细胞对小胶质细胞活化表型的调节

目的:探讨脐带间充质干细胞(hUC-MSCs)是否以外分泌的方式调节小胶质细胞M1/M2极化表型,并且探讨其机制。方法:提取原代脐带间充质干细胞,并制备含有hUC-MSCs分泌的全部营养因子的条件培养基(hUC-MSCs-CM)。实验分为:空白对照组,单纯LPS刺激组,单纯hUC-MSCs-CM刺激组,LPS、hUC-MSCs-CM共刺激组,刺激24 h后收集上清和蛋白。ELISA方法检测上清中TNF-α、IL-6浓度,Western blot检测CD86、精氨酸酶1(Arg1)、PI3K的蛋白表达量。结果:①LPS诱导BV2细胞分泌TNF-α、IL-6因子增多,LPS、hUC-MSCs-CM共刺激组与LPS组相比,TNF-α、IL-6表达明显降低(P 0. 05);②LPS刺激组BV2细胞M1型标志物CD86表达明显增多,hUC-MSCs-CM共刺激组与LPS组相比CD86表达降低(P 0. 05)。LPS、hUC-MSCs-CM共刺激组M2型标志物Arg1表达增多(P 0. 05);③LPS、hUC-MSCs-CM共刺激组上调PI3K磷酸化水平(P 0. 05)。结论:①hUC-MSCs以外分泌的方式抑制小胶质细胞炎性因子的分泌;②hUC-MSCs促进活化的小胶质细胞由M1型向M2型转化,其机制可能与激活PI3K/AKT通路相关。     

羟基积雪草苷对LPS诱导小胶质细胞增殖的抑制作用及机制研究

目的:研究羟基积雪草苷对LPS刺激的小胶质细胞增殖的抑制作用及机制。方法:取SD大鼠的新生小鼠,进行小胶质细胞的原代培养,分离纯化小胶质细胞;MTT法筛选LPS刺激小胶质细胞增殖的最佳浓度,观察不同浓度羟基积雪草苷对LPS刺激小胶质细胞后的作用。ELISA检测细胞上清液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)的含量,流式细胞术检测细胞周期及细胞凋亡,Western blotting法检测Toll样受体4(TLR4)蛋白的表达,RT-PCR检测NF-κB mRNA的表达。结果:LPS可以明显诱导体外培养小胶质细胞的增殖和炎症因子释放。与LPS组比较,羟基积雪草苷对LPS诱导的小胶质细胞增殖具有显著抑制作用,且具有剂量依赖性,羟基积雪草苷处理小胶质细胞48 h的IC50为10.97 nmol/L。同时羟基积雪草苷使小胶质细胞TNF-α和IL-6的释放显著降低(P0.05);羟基积雪草苷使小胶质细胞的G2期细胞与细胞凋亡率增加,并降低小胶质细胞TLR4和NF-κB的表达。结论:羟基积雪草苷对LPS刺激的小胶质细胞的增殖和炎症因子的生成具有抑制作用,其作用机制可能与抑制TLR-4和NF-κB表达、改变细胞周期并诱导细胞凋亡有关。     

磷脂酰肌醇3-激酶通路对脂多糖预激动小胶质细胞高迁移率族蛋白1表达的影响

目的 探讨磷酸肌醇-3激酶/蛋白激酶B(phosphoinositide 3-kinase/protein kinaseB,P13K/Akt)通路对脂多糖 (lipopolyssacride,LPS)预处理体外培养大脑皮质小胶质细胞高迁移率族蛋白1(high mobility group box-1protein 1,HMGB-1)表达的影响.方法 体外分离纯化小鼠大脑皮质小胶质细胞,纯度鉴定后按未刺激对照组、0.01μg/mL LPS单次刺激组(18 h)、1μg/mL LPS刺激组(24 h)、预处理组、PI3K/AKT通路抑制组(2 h)分别进行处理.收获各组细胞与培养上清,采用酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)分泌水平,免疫荧光法检测HMGB-1在细胞中的定位情况,Western blot 检测 HMGB-1和磷酸化Akt的表达水平.结果 PI3K/Akt通路抑制后,LPS预刺激小胶质细胞产生的TNF-α水平较1μg/mL单次刺激组、预刺激组水平降低(P=0.043;P=0.046);磷酸化Akt表达降低,小胶质细胞HMGB-1表达以及向胞浆中转位呈现降低趋势.结论 PI3K/Akt通路在LPS预激诱导的HMGB-1活化中起正性调节作用,这些分子及调节通路的变化参与LPS耐受诱导的重新调配过程,从而共同影响小胶质细胞的最终效应.     

二氢杨梅素对APP/PS1转基因小鼠抗炎的作用及其机制初探

目的研究二氢杨梅素(Dihydromyricetin,DHM)对阿尔茨海默病(Alzheimer’s disease,AD)模型小鼠脑内肿瘤坏死因子α(TNF-α)及小胶质细胞M1、M2表型转化的影响。方法在体外实验中,以20μg/ml Aβ_(1-42)刺激BV2细胞24 h诱导其活化,通过2.5μg/ml DHM及等质量浓度的DMSO稀释液干预BV2细胞24 h,分别作为治疗组和对照组,采用免疫印迹法(Western blot)检测炎症因子TNF-α、小胶质细胞M1和M2表型的表达水平。在动物实验中,取4月龄的APP/PS1双转基因模型小鼠10只,按随机数字表法平均分为DHM治疗组和对照组。DHM治疗组小鼠腹腔注射DHM 1 mg/(kg·d),对照组小鼠腹腔注射等量经生理盐水稀释的DMSO溶液,连续治疗2周。采用Western blot检测2组小鼠脑内炎症因子TNF-α的表达水平,用免疫荧光染色观察2组小鼠脑内M1、M2型小胶质细胞表型的表达情况。结果与对照组比较,DHM治疗抑制BV2细胞释放TNF-α,减少APP/PS1小鼠脑内炎症因子TNF-α的表达,可明显减少诱导型一氧化氮合酶(i NOS)的表达水平,增加精氨酸酶1(Arginase-1)的表达。结论 DHM可有效抑制APP/PS1转基因小鼠脑内的炎症反应,其机制可能与DHM促进M1型小胶质细胞向M2型转化有关。     

PYNOD对LPS活化的BV2小胶质细胞炎症因子释放的影响

 目的:观察PYNOD对LPS活化的BV2小胶质细胞炎症因子释放的影响。方法: 将表达PYNOD的重组质粒pEGFP-C2-PYNOD瞬时转染BV2细胞后,加入LPS作用24 h,Griess 法检测一氧化氮（nitric oxide, NO）的释放,实时荧光定量PCR（real-time PCR）检测诱导型一氧化氮合酶（inducible NO synthase, iNOS）和白细胞介素-1β（interleukin-1β,IL-1β）mRNA的表达,此外Western blotting和ELISA法检测iNOS和IL-1β的蛋白表达。结果: 转染PYNOD重组质粒能显著抑制LPS诱导的BV2小胶质细胞炎症因子NO的释放（P＜0.05）。Real-time PCR证实PYNOD可抑制iNOS和 IL-1β 的mRNA表达,差异有统计学意义（P＜0.05）。ELISA和Western blotting证实PYNOD可下调iNOS和 IL-1β 蛋白的表达（P＜0.05）。结论: PYNOD蛋白可以在转录水平和翻译水平显著抑制LPS刺激的BV2小胶质细胞活化产生的炎症反应。     

法舒地尔(Fasudil)抑制脂多糖诱导的小鼠星形胶质细胞活化和炎性反应及其机制

目的探讨法舒地尔(Fasudil)对脂多糖(LPS)诱导的星形胶质细胞活化和炎症反应及Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路的影响。方法体外培养新生C57BL/6小鼠大脑皮质星形胶质细胞,细胞分为PBS对照组、1μg/m L LPS刺激组、1μg/m L LPS联合15μg/m L盐酸法舒地尔处理组,Griess法检测培养细胞上清液一氧化氮(NO)的水平,ELISA检测肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-10和IL-4的水平,免疫荧光细胞化学染色检测星形胶质细胞胶质原纤维酸性蛋白(GFAP)及TLR4的表达,Western blot法检测GFAP、TLR4和磷酸化的NF-κBp65(p-NF-κBp65)蛋白水平。结果与PBS组比较,LPS组NO、TNF-α和IL-6水平显著升高,IL-10和IL-4水平降低;法舒地尔能抑制LPS诱导的NO、TNF-α和IL-6的分泌,增加IL-10和IL-4的分泌。法舒地尔处理组星形胶质细胞GFAP表达显著降低,同时TLR4和NF-κB蛋白的水平也降低。结论法舒地尔阻断TLR4/NF-κB信号通路抑制LPS诱导的星形胶质细胞活化及炎性反应。     

Fasudil通过TLR4通路抑制脂多糖诱导的小鼠BV-2小胶质细胞TNF-α和IL-1β的表达

目的探讨Fasudil对脂多糖(LPS)诱导BV-2小胶质细胞系促炎细胞因子表达中的作用。方法体外培养BV-2小胶质细胞系,实验分为PBS对照组、LPS刺激组、LPS联合Fasudil干预组,ELISA检测细胞TNF-α、IL-1β的释放,Griess法检测NO释放水平,流式细胞术检测Toll样受体4(TLR4)、TLR2蛋白表达。结果 LPS刺激BV-2细胞可导致TNF-α、IL-1β和NO的释放明显增加,还可导致炎性信号通路中的受体TLR4表达明显增加。Fasudil能明显抑制炎性因子的释放和TLR4的表达。结论 Fasudil可抑制LPS诱导的小胶质细胞NO、TNF-α和IL-1β释放,其作用机制可能与Fasudil下调TLR4通路有关。     

二肽基肽酶抑制剂类似物对LPS诱导的小胶质细胞炎症反应的抑制作用

目的探讨二肽基肽酶抑制剂类似物对脂多糖(LPS)诱导的小胶质细胞炎症反应的抑制作用及机制。方法取新生SD大鼠进行小胶质细胞的原代培养并分离纯化。本研究分为空白组、阴性对照组、LPS组、药物组(每组平行测定3次),药物提前48 h预处理。使用MTT法筛选LPS诱导小胶质细胞增殖的最佳浓度,观察不同浓度下二肽基肽酶抑制剂类似物对LPS刺激小胶质细胞的作用。使用ELISA检测细胞上清液中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)的含量,Western blotting法检测Toll样受体4(TLR-4)蛋白的表达,RT-PCR法检测NF-κB mRNA的表达。结果经LPS刺激后,SD大鼠小胶质细胞生长迅速,并可检测到大量炎性因子释放。与空白组相比,加入二肽基肽酶抑制剂类似物后,LPS诱导的小胶质细胞增殖被明显抑制,处理48 h后对小胶质细胞的IC50为1.014×10-2μmol/L。二肽基肽酶抑制剂类似物能明显抑制小胶质细胞TNF-α和IL-1β的释放(P0.01),并降低小胶质细胞TLR-4和NF-κB的表达。结论二肽基肽酶抑制剂类似物对LPS刺激的小胶质细胞增殖及炎症反应具有抑制作用,可能与抑制小胶质细胞TLR-4、NF-κB表达有关。     

黄芪多糖抑制小鼠EAE 及调控BV-2 神经小胶质细胞活化的实验研究

目的:研究黄芪多糖(APS)对实验性自身免疫性脑脊髓膜炎(EAE)的治疗作用以及对参与EAE 发病机制的神经小胶质细胞活化的调控作用及其可能的作用机制。方法:动物实验:MOG35-55 诱导C57BL/6 小鼠建立EAE 模型,予APS 给药干预,通过5 级临床症状评分观察APS 对小鼠EAE 的治疗作用。细胞实验:MTT 法检测脂多糖(LPS)对于BV-2 神经小胶质细胞的增殖抑制作用,筛选合适的LPS 刺激浓度活化神经小胶质细胞,构建BV-2 神经小胶质细胞活化的模型;倒置显微镜观察BV-2 神经小胶质细胞形态学的改变;ELISA 法检测BV-2 神经小胶质细胞IFN-酌、TNF-α的分泌水平变化;观察不同浓度的APS 对BV-2 神经小胶质细胞活化的调控作用;APS 干预后,Western blot、Real-time PCR 方法分别检测BV-2 神经小胶质细胞PD-L1 蛋白和mRNA 表达水平的变化。结果:APS 能够有效治疗小鼠EAE 的临床症状,成功建立了体外BV-2 神经小胶质细胞活化模型,一定浓度的APS 能够抑制BV-2 神经小胶质细胞的活化,提高活化的BV-2 细胞的生存活性,降低IFN-酌、TNF-α的分泌水平,促进活化的BV-2 神经小胶质细胞PD-L1 基因及蛋白表达上调。结论:APS 对小鼠EAE 具有明显的治疗作用,其发挥作用的机制可能是APS 能够有效抑制神经小胶质细胞的活化,降低炎性细胞因子IFN-酌、TNF-α的分泌,对神经小胶质细胞有抗炎保护作用,PD-1/ PD-L1 通路可能是APS 发挥抗炎作用的重要途径。     

Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain

Our previous studies have shown that intracerebral administration of endotoxin, lipopolysaccharide (LPS), induces selective white matter injury and hypomyelination in the neonatal rat brain and that the LPS-induced brain injury is associated with activation of microglia. To test the hypothesis that inhibition of microglial activation may protect against LPS-induced white matter injury, we examined roles of minocycline, a putative suppressor of microglial activation, on LPS-induced brain injury in the neonatal rat. A stereotactic intracerebral injection of LPS (1 mg/kg) was performed in postnatal day 5 Sprague-Dawley rats and control rats were injected with sterile saline. Minocycline (45 mg/kg) was administered intraperitoneally 12 h before and immediately after LPS injection and then every 24 h for 3 days. Inflammatory responses, activation of microglia and brain injury were examined 1 and 3 days after LPS injection. LPS injection resulted in brain injury in selective brain areas, including bilateral ventricular enlargement, cell death at the sub- and periventricular areas, loss of O4+ and O1+ oligodendrocyte (OL) immunoreactivity and hypomyelination, as indicated by decreased myelin basic protein immunostaining, in the neonatal rat brain. Minocycline administration significantly attenuated LPS-induced brain injury in these rat brains. The protective effect of minocycline was associated with suppressed microglial activation as indicated by the decreased number of activated microglial cells following LPS stimulation and with consequently decreased elevation of interleukin 1beta and tumor necrosis factor-alpha concentrations induced by LPS and a reduced number of inducible nitric oxide synthase expressing cells. Protection of minocycline was also linked with the reduction in LPS-induced oxidative stress, as indicated by 4-hydroxynonenal positive OLs. The overall results suggest that reduction in microglial activation may protect the neonatal brain from LPS-induced white matter injury and inhibition of microglial activation might be an effective approach for the therapeutic treatment of infection-induced white matter injury.     

The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.     

The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.     

米诺环素调控P2X7受体抑制BV-2细胞活化的研究*

 目的：探索P2X7受体在米诺环素(Mino)抑制脂多糖（LPS）刺激的BV-2细胞活化中的作用。方法：将体外培养的BV-2细胞分为5组：空白对照组、LPS处理组、LPS+ 0.1 μmol/L Mino组、LPS+ 1 μmol/L Mino组和LPS+ 10 μmol/L Mino组。分组处理8 h后行real-time PCR检测P2X7受体mRNA表达;处理24 h后观察各组细胞形态学变化,Western blotting检测P2X7受体蛋白表达,取细胞培养液上清行ELISA检测TNF-α和IL-1β的分泌情况。结果：经LPS处理后,BV-2细胞P2X7受体mRNA及蛋白的表达均升高,细胞培养液上清的TNF-α和IL-1β表达升高,同时伴有形态学的改变,而0.1～10 μmol/L Mino能抑制这一趋势,差异有统计学意义（P<0.01）。 结论： Mino抑制BV-2细胞活化的机制可能与抑制P2X7受体的活性相关。