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。     

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。     

蝎毒耐热多肽对脂多糖诱导的BV2细胞炎性反应的抑制作用

目的:观察蝎毒耐热多肽(SVHRP)对细菌脂多糖(LPS)诱导的BV2细胞炎性反应的影响,探讨其是否具有抑制神经炎症的作用。方法:LPS诱导BV2细胞活化,进行免疫细胞化学染色(OX-42抗体)检测细胞的形态,MTT法检测SVHRP对细胞的毒性;分别用Griess试剂法和酶联免疫吸附法(ELISA)测定细胞外液炎性介质一氧化氮(NO)和肿瘤坏死因子-α(TNF-α)的含量;Western Blot检测诱导型NO合成酶(iNOS)蛋白表达水平。结果:不同浓度的SVHRP(2~50μg/ml)对BV2细胞均无毒性。SVHRP(20μg/ml)预处理能明显抑制LPS诱导的BV2细胞的形态活化改变,减少细胞激活后产生的炎性因子NO和TNF-α,抑制iNOS的蛋白表达。结论:SVHRP明显抑制BV2小胶质细胞的炎性反应,提示SVHRP具有抗神经炎症作用。     

尼古丁对脂多糖诱导的小胶质细胞激活及活化后细胞死亡的影响

目的 观察尼古丁对脂多糖(LPS)诱导的小胶质细胞激活及活化后细胞死亡的影响. 方法 建立慢性尼古丁暴露的小鼠动物模型,腹腔注射LPS诱导小胶质细胞激活,应用免疫组织化学方法 观察皮质、海马、黑质CD-11b阳性小胶质细胞表达的变化;BV2细胞(小鼠小胶质瘤细胞系)传代培养,运用CCK-8试剂盒检测细胞活性,一氧化氮检测试剂盒检测一氧化氮(NO)释放情况,RT-PCR分析诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子(TNF-α)、白细胞介素1(IL-1β)、白细胞介素6(IL-6)、环氧化酶-2(COX-2)、干扰素调节因子1(IRF-1)、Caspase-11 mRNA的表达,免疫印迹法分析P-I-κB、Caspase-3的表达变化. 结果 尼古丁抑制LPS诱导的皮质、海马、黑质CD-11b阳性小胶质细胞的表达;尼古丁抑制LPS刺激引起的BV2细胞的死亡,NO的释放,iNOS、TNF-α、IL-1β、IL-6、COX-2、IRF-1、Caspase-11 mRNA的表达,P-I-κB、Caspase-3蛋白的表达. 结论 尼古丁可以抑制LPS诱导的小胶质细胞活化及激活诱导的细胞死亡(AICD),对脑内炎症反应具有神经保护作用.     

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。     

外源性黄体酮对Aβ_(1-42)诱导的小胶质细胞活化及其对海马神经元存活的影响

目的研究黄体酮对Aβ_(1-42)诱导的小胶质细胞激活和炎症因子的释放及其所致的海马神经元损伤的影响。方法通过Aβ_(1-42)诱导体外培养的小胶质细胞(BV2细胞)活化,并予以外源性黄体酮处理。应用Aβ_(1-42)诱导的BV2细胞的上清液处理海马神经元(HT22细胞)。应用ELISA法检测各组BV2细胞培养上清液中TNF-α、IL-1β的含量;CCK8法检测Aβ_(1-42)-BV2小胶质细胞条件培养基对HT22海马神经元细胞的存活率。结果与对照组相比,Aβ_(1-42)组的TNF-α、IL-1β的表达水平明显升高,且呈剂量依赖性。外源性黄体酮能降低Aβ_(1-42)诱导的BV2细胞IL-1β、TNF-α的释放;Aβ_(1-42)诱导BV2细胞炎症因子的释放,可导致HT22细胞死亡,而外源性黄体酮可通过抑制活化的胶质细胞炎性因子的释放而减轻神经元的死亡。结论黄体酮能够抑制Aβ_(1-42)诱导的小胶质细胞的激活并减轻其导致的神经死亡。     

硫辛酸抑制脂多糖诱导的星形胶质细胞细胞因子及趋化因子的表达

目的探讨硫辛酸(LA)对脂多糖(LPS)激活的星形胶质细胞分泌TNF-α、IL-1β、IL-6和IL-10及相关趋化因子的影响。方法分离并鉴定新生C57BL/6小鼠大脑皮质星形胶质细胞,1μg/mL LPS刺激第2代星形胶质细胞,100μg/mL LA进行干预,Griess法检测NO的分泌,ELISA检测TNF-α、IL-1β、IL-6和IL-10炎性因子的含量,反转录PCR检测炎症趋化因子CC亚族趋化因子配体20(CCL20),单核细胞趋化蛋白1(MCP-1)和巨噬细胞炎性蛋白1α(MIP-1α)mRNA的表达。结果与正常组比较,LPS刺激星形胶质细胞后,NO、TNF-α、IL-1β、IL-6分泌显著升高,IL-10分泌下调(P0.05);LA能抑制LPS诱导的NO、TNF-α、IL-1β、IL-6的分泌,增加IL-10的分泌,与LPS组相比差异有统计学意义(P0.05)。LA能显著下调LPS所致的CCL20、MIP-1α、MCP-1 mRNA的分泌。结论 LA能抑制LPS激活星形胶质细胞所致的炎性反应,其作用与抑制炎性因子及趋化因子的分泌有关。     

法舒地尔对LPS刺激小胶质细胞表型演变的影响

目的:利用体外培养的BV-2小胶质细胞,探讨法舒地尔(Fasudil)抑制LPS激活的炎性反应和小胶质细胞不同亚群的转换。方法:BV-2小胶质细胞进行常规培养和传代,实验分为PBS对照组、PBS联合Fasudil处理组、LPS刺激组、LPS联合Fasudil干预组。NO产生采用Griess法,TNF-α释放采用ELISA法,而M1和M2亚群分析采用流式细胞技术。结果:LPS处理导致典型的M1型小胶质细胞特征,而Fasudil处理可抑制LPS诱导的NO产生和炎性细胞因子TNF-α的释放,并且可以转化炎性M1型细胞至抗炎和修复的M2型细胞。结论:Fasudil显示了良好的抗炎效果,可能与M1小胶质细胞转化为抗炎和修复功能的M2型细胞有关。     

IL-6促进小鼠小胶质细胞系BV2坏死性凋亡

目的 探究在神经炎性反应中小胶质细胞凋亡的调控机制。方法 脂多糖(lipopolysaccharide, LPS)诱导小鼠小胶质细胞系BV2,构建神经炎性反应细胞模型,使用白介素-6(IL-6)拮抗剂塞妥昔单抗(siltuximab)处理LPS诱导的BV2细胞。CCK-8法检测细胞增殖;流式细胞测量术检测细胞凋亡;ELISA检测IL-6与TNF-α含量;RT-qPCR检测BV2细胞M1极化标志物IL-1β、IFN-γ与M2极化标志物CD206、Arg-1表达;Western blot检测JAK-STAT3信号通路关键蛋白及坏死性凋亡相关蛋白(RIP1)和RIP3表达。结果 LPS诱导后BV2细胞的增殖活力下降,凋亡增加,炎性因子IL-6与TNF-α含量增加(P<0.01)。M1极化标志物IL-1β、IFN-γ表达增加(P<0.01),M2极化标志物CD206、Arg-1表达减少(P<0.01)。JAK-STAT3通路关键蛋白磷酸化增加(P<0.01),RIP1、RIP3蛋白表达增加(P<0.01)。IL-6拮抗剂siltuximab处理细胞后,JAK-ST...     

Fractalkine对脂多糖激活的小胶质细胞炎症相关因子表达的影响

目的:探讨趋化因子Fractalkine对脂多糖(LPS)诱导的小鼠小胶质细胞(N9)激活时所分泌的TNF-α、IL-1β和一氧化氮(NO)表达的影响.方法:用Fractalkine处理经LPS激活的小鼠小胶质细胞24 h,以ELISA法检测细胞培养上清中TNF-α和IL-1β的浓度,以NO试剂盒检测培养上清中NO的浓度.结果:LPS能够激活小胶质细胞,使IL-1 β、TNF-α和NO的表达量与对照组相比明显升高;Fractalkine能够降低LPS激活的小胶质细胞IL-1β、TNF-α和NO的表达.结论:Fractalkine可能通过抑制炎症相关因子的产生而在中枢神经系统中发挥神经保护作用.     

Apigenin exhibits anti-inflammatory effects in LPS-stimulated BV2 microglia through activating GSK3β/Nrf2 signaling pathway

Abstract

Objective: Apigenin is a natural flavonoid compound extracted from Matricaria chamomilla. We evaluated the anti-inflammatory effects of apigenin in this study using the Lipopolysaccharide (LPS)-stimulated BV2 microglia.

Methods: BV2 cells were treated with apigenin for 1?h and then treated with LPS. The inflammatory cytokine productions were tested by qRT-PCR and ELISA. The expression of GSK3β, Nrf2, and NF-κB signaling pathways were measured by western blot analysis.

Results: Apigenin significantly attenuated LPS-induced TNF-α, IL-1β, and IL-6 production. Apigenin suppressed LPS-induced NF-κB activation. Furthermore, GSK3β, Nrf2, and HO-1 were concentration-dependently increased by apigenin. The suppression of apigenin on LPS-induced inflammatory response and NF-κB activation were prevented when Nrf2 was knocked out or by GSK3β inhibitor.

Conclusions: Collectively, apigenin suppressed LPS-induced microglia activation via activating GSK3β/Nrf2 signaling pathway.     

黄芪总皂苷对脂多糖诱导的BV2小胶质细胞的抗炎机制

目的:探讨黄芪总皂苷(TAS)对脂多糖(LPS)诱导的BV2小胶质细胞炎症损伤的抗炎作用机制.方法:用CCK-8法筛选出对细胞活力无抑制的药物浓度;用浓度为1 mg/L的LPS刺激BV2细胞24 h,建立细胞炎症模型;实验分为正常组、LPS组、高剂量(75 mg/L)TAS组和低剂量(50 mg/L)TAS组;应用流式...     

视黄酸通过TLR4/NF-κB通路对脂多糖诱导血管炎症与氧化应激的影响

目的 研究视黄酸(RA)对脂多糖(LPS)诱导的血管炎症与氧化应激的保护作用并探讨其作用机制。 方法 SD大鼠给予RA和TLR4抑制剂连续口腔灌胃2周后,除对照组外,LPS组、RA 3 mg/kg组、RA 15 mg/kg组和TLR4抑制剂组（TAK-242, 3 mg/kg）,通过腹腔注射LPS (10 mg/kg)以建立血管炎症模型。血管张力测定系统检测血管舒张功能,硝酸还原酶法检测大鼠血清中一氧化氮（NO）的含量,ELISA法检测血清中IL-18、IL-1β、TNF-α、IL-6和GSH-px的水平,WST-1法和TBA法分别检测血清中超氧化物歧化酶（SOD）活力、丙二醛（MDA）,DHE荧光探针检测血管活性氧(ROS)的水平,免疫组化法检测血管 NF-κB p65的表达,免疫印迹法检测血管TLR4、eNOS和p-eNOS的表达。 结果 与LPS组相比,RA能够改善血管舒张功能,增加p-eNOS和NO的水平,减少血清炎症因子IL-18、IL-1β、TNF-α、IL-6的含量,降低血清中MDA和ROS的生成,升高血清中SOD的生成和GSH-px的释放量,并且下调血管TLR4和NF-κB p65的表达水平。另外,RA对LPS诱导的血管炎症与氧化应激的影响与TLR4抑制剂作用相似。 结论 RA对LPS诱导的血管炎症与氧化应激具有抑制作用,其可能通过TLR4/NF-κB p65信号通路发挥作用。     

CysLT2受体拮抗剂HAMI3379抑制LPS诱导小鼠小胶质瘤细胞系的炎性反应

目的探究半胱氨酰白三烯2(CysLT2)受体拮抗剂HAMI3379对LPS诱导小鼠小胶质细胞(BV-2)炎性反应的调控作用及其可能的作用机制。方法体外培养BV-2,将BV-2分为对照组、LPS(100 ng/mL)组、HAMI3379(0.01、0.1和1μmol/mL)组和LPS+HAMI3379组。CCK-8法检测BV-2细胞的增殖;ELISA检测细胞上清液中炎性因子IL-1β、TNF-α、IL-10的含量;Western-blot检测PKCα、IKBα、NF-κB p50和p65蛋白的表达。结果LPS能够激活BV-2细胞,促进其细胞的增殖(P<0.05);显著增加细胞上清液中炎性因子IL-1β、TNF-α的分泌,减少IL-10的分泌(P<0.05);且显著上调PKCα、IKBα、p65蛋白的表达水平(P<0.05)。CysLT2受体拮抗剂HAMI3379能够显著减轻上述变化(P<0.05)。结论CysLT2受体拮抗剂HAMI3379能够抑制LPS激活BV-2细胞,抑制炎性反应,其作用机制可能与抑制PKCα/NF-κB信号通路有关。     

天抗(TK)对脂多糖诱导小鼠炎症模型的抗炎作用及其机制

目的研究天抗(TK)对脂多糖(LPS)诱导的小鼠炎症模型的抗炎作用及机制研究。方法将42只昆明小鼠随机分为正常对照(NC)组、模型对照(LPS)组、地塞米松(DXM)组、天抗低(TK-L)、中(TK-M)和高(TK-H)剂量组(0.2,0.8和3.2 g/kg)。各组分别灌胃给药7 d后,腹腔注射30 mg/kg的LPS诱导小鼠急性炎性模型,6 h后处死小鼠,检测小鼠脾脏指数,ELISA测定小鼠血清中IL-1β、IL-6和TNF-α的表达水平;生化法检测小鼠血清中SOD和MDA的表达;qRT-PCR检测小鼠脾脏TLR4、MyD88、TRAF6、p65、IL-1β、IL-6和TNF-αmRNA的表达水平;Western blot检测小鼠脾脏TLR4、MyD88、TRAF6、p-p65和p65蛋白表达水平。结果与LPS组相比,TK组小鼠的脾脏指数明显降低,血清和脾脏组织中IL-1β、IL-6、TNF-α和MDA水平显著下降,SOD水平明显升高,小鼠脾脏组织的TLR4、MyD88、TRAF6和p-p65等蛋白及mRNA表达水平均明显降低。结论天抗对LPS诱导的小鼠急性炎症模型具有抗炎作用,其作用机制可能是通过TLR4/MyD88/NF-κB(p-65)信号通路抑制炎症因子的释放。     

Artesunate Protects LPS-Induced Acute Lung Injury by Inhibiting TLR4 Expression and Inducing Nrf2 Activation

Artesunate, a derivative of artemisinin, has been reported to have anti-inflammatory property. However, few studies showed the protective effects of artesunate on lung injury. In this study, we aimed to investigate the effects of artesunate on LPS-induced lung injury in mice. The mice were treated with artesunate 1 h before or after LPS treatment. The effects of artesunate on lung MPO activity and malondialdehyde (MDA) content were detected. The lung wet/dry radio and the numbers of inflammatory cells in BALF were also measured. ELISA was used to evaluate the levels of TNF-α, IL-1β, and IL-6 in BALF. Western blot analysis was adapted to detect TLR4 and Nrf2 signaling pathways. The results showed that artesunate protected against LPS-induced ALI by decreasing the numbers of inflammatory cells, lung edema, MPO activity, and MDA content. Furthermore, artesunate significantly inhibited the levels of TNF-α, IL-1β, and IL-6. Artesunate also inhibited LPS-induced IL-6 and IL-8 production in the A549 cells. In addition, artesunate dose-dependently suppressed LPS-induced TLR4 expression and NF-κB activation. The expression of Nrf2 and HO-1 were also up-regulated by artesunate. The data suggest that artesunate possesses anti-inflammatory and anti-oxidant properties against LPS-induced ALI via inhibiting TLR4 signaling pathway and activating Nrf2 signaling pathway.     

Caspase-1 inhibition attenuates activation of BV2 microglia induced by LPS-treated RAW264.7 macrophages

Neuroinflammation has been recognized as a factor in the pathogenesis of neurodegenerative diseases. Emerging evidence suggests that peripheral inflammation, besides neuroinflammation, functions as a modulator of disease progression and neuropathology in several neurodegenerative diseases. However, detailed correlations among peripheral inflammation, neuroinflammation and neurodegeneration remain unknown. In the present study, we prepared a peripheral inflammation model with lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages to explore its activation on BV2 microglia. We found that LPS induced the production of IL-1β, IL-6 and TNF-α in the culture medium of RAW264.7 macrophages. We further showed that LPS plus ATP activated inflammasome, evidenced by the upregulation of caspase-1 and IL-1β, which was suppressed by ZYVAD, a caspase-1 inhibitor. Furthermore, the conditioned medium obtained from LPS-treated RAW264.7 macrophages activated BV2 microglia, stimulating the release of IL-1β, IL-6 and TNF-α from BV2 cells. ZYVAD pretreatment markedly suppressed BV2 microglia activation induced by RAW264.7 cells conditioned medium. Taken together, our study indicates that macrophage-mediated peripheral inflammation subsequently evokes neuroinflammation and may aggravate neural damage. Inflammasome and caspase-1 may be potential targets for modulating systemic inflammatory responses in neurodegenerative diseases.     

Sphingosine kinase 1 regulates the expression of proinflammatory cytokines and nitric oxide in activated microglia

Microglial activation has been implicated as one of the causative factors for neuroinflammation in various neurodegenerative diseases. The sphingolipid metabolic pathway plays an important role in inflammation, cell proliferation, survival, chemotaxis, and immunity in peripheral macrophages. In this study, we demonstrate that sphingosine kinase1 (SphK1), a key enzyme of the sphingolipid metabolic pathway, and its receptors are expressed in the mouse BV2 microglial cells and SphK1 alters the expression and production of proinflammatory cytokines and nitric oxide in microglia treated with lipopolysaccharide (LPS). LPS treatment increased the SphK1 mRNA and protein expression in microglia as revealed by the RT–PCR, Western blot and immunofluorescence. Suppression of SphK1 by its inhibitor, N, N Dimethylsphingosine (DMS), or siRNA resulted in decreased mRNA expression of TNF-α, IL-1β, and iNOS and release of TNF-α and nitric oxide (NO) in LPS-activated microglia. Moreover, addition of sphingosine 1 phosphate (S1P), a breakdown product of sphingolipid metabolism, increased the expression levels of TNF-α, IL-1β and iNOS and production of TNF-α and NO in activated microglia. Hence to summarize, suppression of SphK1 in activated microglia inhibits the production of proinflammatory cytokines and NO and the addition of exogenous S1P to activated microglia enhances their inflammatory responses. Since the chronic proinflammatory cytokine production by microglia has been implicated in neuroinflammation, modulation of SphK1 and S1P in microglia could be looked upon as a future potential therapeutic method in the control of neuroinflammation in neurodegenerative diseases.     

黄连乙醇提取物与盐酸小檗碱体外抗炎对比研究

目的:比较黄连乙醇提取物与盐酸小檗碱体外抗炎活性,探索体外抗炎机制。方法:通过脂多糖体外刺激小鼠单核巨噬细胞建立细胞炎症模型,给药干预后,LPS 长时间刺激RAW264.7 细胞,MTT 比色法分析黄连乙醇提取物及盐酸小檗碱对RAW264.7 细胞生长活性的影响。酶联免疫吸附法检测细胞上清液中IL-β、IL-6、TNF-α、NO、前列腺素E2 (PGE2)含量。实时荧光定量RT-PCR 法检测iNos、HO-1、TNF-αmRNA 表达。结果:在5 ~80 mg/ L 范围内,黄连乙醇提取物及盐酸小檗碱对RAW264.7 细胞无抑制作用;各浓度给药组IL-6、IL-1β、TNF-α、NO、前列腺素E2 (PGE2 )含量与LPS 刺激模型组比较均有显著性(P<0.01),且浓度与剂量无效应相关。实时荧光定量RT-PCR 结果显示,各浓度给药组均明显降低iNos、HO-1、TNF-αmRNA 表达(P<0.05,P<0.05,P<0.01),且与浓度不呈效应关系。结论:黄连乙醇提取物具有体外抗炎作用,抗炎活性优于盐酸小檗碱,其作用机制可能与抑制TNF-α、NO 等炎症因子的活化,进而影响花生四烯酸(AA)代谢有关。