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。     

小胶质细胞在高氧暴露未成熟脑损伤中的作用

目的观察新生小鼠高氧暴露后,小胶质细胞功能变化及未成熟脑内NG2细胞的凋亡情况,以探讨小胶质细胞在高氧未成熟脑损伤中的作用。方法将新生3 d(PND3)小鼠持续90%高浓度氧暴露48 h,建立新生鼠未成熟脑高氧脑损伤模型,并腹腔注射小胶质细胞活化抑制剂米诺环素,将小鼠分为对照(A)组,空气+米诺环素(AM)组,高氧(O)组和高氧+米诺环素(OM)组,其中AM组和OM组给予45 mg/kg米诺环素。免疫组化观察小胶质细胞活化情况,real-time PCR检测TLR4(Toll样受体4,Toll-like receptor 4)、TNF-α(tumor necrosis factor-α)表达水平。免疫荧光双标检测凋亡蛋白Caspase-3表达及NG2细胞(未成熟少突胶质细胞)凋亡情况。结果与A组比较,O组激活状态的小胶质细胞明显增多,TLR4表达上调。免疫荧光结果显示O组脑组织中凋亡细胞明显增多,且免疫荧光双标可见NG2与Caspase-3共表达。而与O组相比,OM组TLR4和TNF-α的表达明显降低(P0.05),且Caspase-3表达及NG2细胞凋亡明显减少。结论围生期常压高浓度氧暴露通过激活小胶质细胞上调TLR4表达引起炎症反应致未成熟脑细胞凋亡,凋亡细胞包括少突胶质祖细胞(NG2+);抑制小胶质细胞活化可明显减轻高氧未成熟脑损伤。     

大鼠脊髓挤压伤后小胶质细胞Toll样受体4表达的变化

目的探讨脊髓挤压伤后各时间点小胶质细胞Toll样受体4(TLR4)的表达变化及其与损伤面积和免疫球蛋白G渗出范围的关系。方法 48只成年雄性SD大鼠建立T8节段脊髓挤压伤模型,随机分为6组,每组8只;假手术对照组及挤压伤组动物在手术后0h、3h、24h、72h和7d用4%多聚甲醛灌注固定,取以挤压点为中心的2cm脊髓,冷冻切片后进行HE染色和免疫荧光染色,分别观察损伤面积的变化,TLR4表达和TLR4阳性小胶质细胞的分布,以及与血浆免疫球蛋白G(IgG)渗出范围的比较,并用IPP6.0软件计算各组的阳性数目。结果脊髓损伤后TLR4主要表达在小胶质细胞,在3～24h之间开始表达,伤后在72h达到高峰,7d时已经显著下降。阳性小胶质细胞的分布与IgG渗出范围一致。结论大鼠脊髓挤压伤模型中,表达在脊髓小胶质细胞上的TLR4可在脊髓继发性损伤中发挥重要作用,并与血脊髓屏障开放有关。     

IgG刺激诱发的小胶质细胞Toll样受体4表达及细胞因子分泌

目的:了解在体外培养条件下免疫球蛋白G (IgG) 刺激对小胶质细胞表达Toll 样受体4(TLR4)和分泌细胞因子的作用.方法:用不同浓度的IgG (2 mg/L、 20 mg/L、 200 mg/L)及脂多糖(LPS)(10 mg/L)刺激原代培养的大鼠小胶质细胞,24 h后以免疫荧光染色观察TLR4的表达,ELISA法检测培养上清中肿瘤坏死因子α(TNF-α)和γ干扰素(IFN-γ)的含量.结果:IgG直接作用于体外培养的小胶质细胞后,以剂量依赖的方式引起TLR4的表达和TNF-α的分泌,但未检测到IFN-γ含量的变化.作为阳性对照的LPS引起了小胶质细胞TLR4表达,并诱导了TNF-α及少量IFN-γ的分泌.结论:同种IgG刺激可使体外培养的小胶质细胞大量表达TLR4,可能通过MyD88依赖途径导致炎性细胞因子分泌.结果提示至少在中枢神经系统的固有免疫反应中,TLR4可能发挥识别病原体之外的蛋白分子,例如IgG的作用.     

Toll样受体4在新生小鼠常压高氧脑损伤中的作用

目的探讨Toll样受体4(Toll-like receptor 4,TLR4)在新生小鼠常压高氧脑损伤中的作用。方法新生3 d(P3)的TLR4野生型(W)和TLR4突变型(M)小鼠各36只,随机分为:100%高氧暴露组(WO2组,MO2组)及空气对照组(WA组,MA组),每组18只。高氧暴露组置于100%高氧箱中2 d,以空气中常规饲养的同龄小鼠作为空气对照组。各组于P6随机取12只小鼠脑组织,用于检测TLR4 mRNA表达及石蜡切片,染色观测神经细胞密度,并检测细胞凋亡情况;其余小鼠饲养至28 d,Morris水迷宫实验评价各组小鼠学习记忆能力。结果 RT-PCR提示WO2组TLR4 mRNA表达较WA组明显增加(P<0.05);染色WO2组在前额叶皮质区平均光密度低于WA组(P<0.05)及MO2组(P<0.05),在海马CA1区小于WA组(P<0.05),在两区凋亡细胞指数均较WA组及MO2组增高(P<0.05);MO2组与MA组比较,各个脑区神经细胞密度及凋亡指数均无明显差异(P>0.05)。Morris水迷宫WO2组与WA组比较,逃避潜伏期延长(P<0.05),目标象限停留时间缩短(P<0.05),穿越虚拟平台次数减少(P<0.05)。MO2组与MA组比较,上述各项指标均无明显差异。结论 TLR4功能缺失可减少高氧导致的未成熟神经细胞凋亡,降低新生期高氧暴露对远期学习记忆能力的损害。     

小鼠脑梗死后小胶质细胞内Toll样受体9选择性上调

 目的：观察脑梗死后Toll 样受体9（TLR9）表达量及其在神经元和神经胶质细胞中的表达变化。方法：线栓法制备C57小鼠大脑中动脉闭塞（MCAO）模型,90 min后再灌注。假手术组为对照。再灌注后6 h、3 d、7 d和14 d处死动物 (n=3),制备脑冠状位冰冻切片。免疫荧光染色观察TLR9表达量及其在神经元和神经胶质细胞中的表达变化。结果：梗死灶边缘区TLR9的表达量随时间增加,始终高于对侧及假手术组。病变全程偶见神经元胞内小点状TLR9,TLR9阳性率无时间、组间差异。激活态小胶质细胞聚集于梗死灶边缘区,胞内TLR9由散在小点状变为团块状粗颗粒样。TLR9阳性率随时间先升后降,始终高于对侧及假手术组。星形细胞和少突胶质细胞未见TLR9阳性染色。结论：脑梗死后,中枢定居细胞中神经元TLR9维持固有表达,仅小胶质细胞TLR9表达选择性上调,星形细胞及少突胶质细胞无TLR9表达。     

Toll样受体2和Toll样受体4在免疫应答衣原体感染中的作用

衣原体是重要的人类病原体,其能够导致多种疾病的发生.由衣原体引起的许多人类疾病被认为是免疫病理学介导的.已经证明Toll样受体(TLRs)是多种病原体感染的主要模式识别受体( PRRs),在起始固有免疫应答,建立适应性免疫应答中发挥着重要作用.在TLR家族中,TLR2和TLR4与衣原体感染的相关性研究备受关注,在识别衣原体感染、调节宿主的早期免疫应答、炎症反应和病理形成中执行着关键性的作用.研究TLR2和TLR4在免疫应答衣原体感染中的作用可以更好地理解TLRs介导的分子免疫机制,可能有助于研发免疫治疗的分子靶标,最终有效预防、控制衣原体感染引起的疾病.     

Toll样受体2和Toll样受体4在HBV感染后的免疫作用

Toll样受体（TLR）是启动固有免疫和调节适应性免疫的重要分子,参与肝脏对病毒及细菌的免疫TLR2、TLR4过程。在HBV的慢性化进程中,TLR2、TLR4与Thl和Th2的免疫平衡及调节性T细胞（Treg）的免疫抑制相关,HBV感染后,HBcAg刺激巨噬细胞产生TNF—α的作用需要TLR2参与,HBeAg的表达与否与TLR2的表达状态有关,而TLR4通过诱导iNOS的表达和激发HBV特异性免疫在体内抗HBV过程中起重要作用：     

高氧对脂多糖诱导N9小胶质细胞促炎症作用的影响

目的:观察常压高浓度氧对脂多糖诱导N9小胶质细胞Toll受体4、TNF-α表达时序变化,初步探讨高氧对小胶质细胞促炎反应的作用及调控机制。方法:体外培养N9小胶质细胞,随机分为6组(n=3):空气组、sLPS组、hLPS组、高氧组、高氧+sLPS组、高氧+hLPS组。LPS浓度为100 ng/mL(sLPS)、1 mg/L(hLPS)。高氧(900 mL/L)暴露时间分别为2 h、6 h、12 h、16 h、24 h。RT-PCR检测TLR4的表达时序变化;Western blot检测处理12 h后各组TLR4蛋白表达;ELISA检测各组处理6 h、12 h、16 h、24 h培养上清中TNF-α的含量。结果:RT-PCR显示hLPS组在6 h后、sLPS组在16 h后TLR4 mRNA均表达上调,并随LPS浓度增加、暴露时间延长逐渐升高(P<0.05),24 h时hLPS组表达最高。6 h后在各个时间点高氧+hLPS组与hLPS比较TLR4 mRNA下调(P<0.05),在16 h、24 h最为明显(P<0.05)。Westernblot检测12 h高氧+sLPS组、高氧+hLPS组TLR4蛋白水平均低于相应浓度的LPS组(P<0.05)。ELISA结果示在各个时间点高氧+sLPS组、高氧+hLPS组与相应浓度的LPS组比较TNF-α均明显上调(P<0.05)。结论:高浓度氧暴露促进LPS诱导N9小胶质细胞的促炎症反应,TLR4可能参与此过程的负向调控。     

妊娠期脂多糖暴露通过激活子代小胶质细胞诱导大鼠孤独症样行为

目的:探讨妊娠期脂多糖(LPS)诱导的孤独症样行为仔鼠脑组织Toll样受体4(TLR4)及小胶质细胞分型的变化。方法:24只孕鼠随机平分为2组(n=12),分别于怀孕9.5 d时腹腔注射LPS(100μg/kg)或等体积磷酸盐缓冲液(PBS),所产子代大鼠即为LPS组(n=72)及PBS组(n=72)仔鼠。采用real-time PCR和Western blot技术测定仔鼠TLR4、离子钙结合衔接分子1(IBA-1)、诱导型一氧化氮合酶(iNOS)和精氨酸酶1(Arg-1)的表达水平。免疫荧光染色检测仔鼠前额叶皮质小胶质细胞的形态学改变。ELISA试剂盒测定孕鼠血清脂多糖结合蛋白(LBP)和肿瘤坏死因子α(TNF-α)及仔鼠脑组织TNF-α和白细胞介素10(IL-10)含量。三箱社交实验、嗅觉适应/去适应实验和旷场实验检测子代大鼠社交行为、探索行为及刻板行为。体外培养N9小胶质细胞,予以TLR4阻断剂瑞沙托维(TAK242)处理后,观察LPS诱导的N9细胞TLR4、iNOS及Arg-1表达水平的变化。结果:妊娠期LPS处理可诱导母鼠血清LBP及TNF-α显著升高(P<0.01或P...     

Role of Toll-like receptor 4 in hyperoxia-induced lung inflammation in mice

Objective: Prolonged exposure to hyperoxia causes lung inflammation, but the role of Toll-like receptor 4 (TLR4) in hyperoxia-induced signal transduction remains unclear. Material or subjects: We evaluated neutrophil accumulation, signal transduction and cytokine production during hyperoxia, comparing TLR4 mutant (C3H/HeJ) and wild type (C3H/HeN) mice. Methods: The mice were exposed to 80% oxygen in a hyperoxic chamber for 0 (control), 48, or 96 h. After the exposure, bronchoalveolar lavage (BAL) was performed for differential cell counting and cytokine measurement. In lung homogenate, activation of NF-κB and STAT1 was also examined. Results: In C3H/HeJ mice, hyperoxia-induced neutrophil accumulation in BAL fluid was significantly decreased compared with C3H/HeN. Hyperoxia for 96 h caused NF-κB translocation in C3H/HeN mice, which was significantly attenuated in C3H/HeJ mice (p < 0.05). In contrast, STAT1 activation occurred as early as after 48 h of oxygen exposure, which did not differ between the two strains. The levels of TNF-α, IL-6, and KC in BAL fluid were increased after oxygen exposure, which was suppressed by the lack of TLR4 signaling. Conclusion: These results suggest that TLR4-dependent NF-kB activation may be an important process of the upregulation of proinflammatory mediators and subsequent neutrophil accumulation into the lung during hyperoxia. Received 21 March 2007; accepted without revision by G. Wallace 11 April 2007     

Notch通路在大鼠肾脏缺血再灌注损伤TLR4介导的炎症反应中的作用

 目的: 探讨Notch通路对大鼠肾脏缺血再灌注损伤(IRI)中Toll样受体4(TLR4)介导的炎症反应的作用。方法: 雄性SD大鼠75只随机分为假手术组(sham组)、缺血再灌注组(IRI组)和γ-分泌酶抑制剂DAPT干预组(DAPT组)。分别于再灌注6 h、12 h、24 h、48 h、72 h时点观察各组肾脏病理改变,检测血尿素氮(BUN)和血清肌酐(Scr)水平,ELISA检测血清肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平,免疫组化和Western blot分别检测大鼠肾脏Notch1、TLR4和NF-κB p65蛋白表达水平。结果: 在IRI组,呈现不同程度的以肾小管上皮细胞和间质损伤为主的肾脏病理改变,BUN、Scr及血清炎性因子TNF-α、IL-6含量在各时点均显著高于sham组(P<0.05),而在DAPT干预组,在各时点肾脏病理损伤明显减轻,BUN、Scr和血清TNF-α、IL-6水平均显著低于IRI组(P<0.05)。Notch1、TLR4和NF-κB p65主要表达于肾小管上皮细胞胞质中,在sham组仅有微量表达,在IRI组则有高表达,在各时点表达与sham组相比均显著增强(P<0.05),而在DAPT组,各因子的表达水平在各时点均较IRI组显著降低(P<0.05)。结论: 大鼠肾脏IRI出现显著的肾功能及肾脏病理改变,血清炎性因子TNF-α和IL-6水平升高,Notch1、TLR4及NF-κB p65在肾组织中表达增强;而DAPT可通过抑制Notch1活化和TLR4/NF-κB通路,抑制TLR4所介导的炎症反应,从而发挥肾脏保护作用。     

Effect of Toll-like receptor 4 inhibitor on LPS-induced lung injury

Objective and design

Toll-like receptor 4 (TLR4) plays important roles in the recognition of lipopolysaccharide (LPS) and the activation of inflammatory cascade. In this study, we evaluated the effect of TAK-242, a selective TLR4 signal transduction inhibitor, on acute lung injury (ALI).

Materials and methods

C57BL/6J mice were intravenously treated with TAK-242 15 min before the intratracheal administration of LPS or Pam3CSK4, a synthetic lipopeptide. Six hours after the challenge, bronchoalveolar lavage fluid was obtained for a differential cell count and the measurement of cytokine and myeloperoxidase levels. Lung permeability and nuclear factor-κB (NF-κB) DNA binding activity were also evaluated.

Results

TAK-242 effectively attenuated the neutrophil accumulation and activation in the lungs, the increase in lung permeability, production of inflammatory mediators, and NF-κB DNA-binding activity induced by the LPS challenge. In contrast, TAK-242 did not suppress inflammatory changes induced by Pam3CSK4.

Conclusion

TAK-242 may be a promising therapeutic agent for ALI, especially injuries associated with pneumonia caused by Gram-negative bacteria.     

Role of microglia and toll-like receptor 4 in the pathophysiology of delirium

Delirium is a serious medical condition that commonly afflicts elderly inpatients. This is especially common in the post-operative setting where it increases mortality, length of hospital stay and health care costs. The exact mechanisms involved in its pathogenesis remain uncertain and there is currently no effective pharmacological therapy for treatment or prevention of delirium. We hypothesize that microglia-mediated neuroinflammation via toll-like receptor 4 signalling is a significant contributor to post-operative delirium. Based on our proposed mechanism, three novel pharmacological therapies have been suggested to be effective to prevent or treat delirium. Curcumin, ibudilast and minocycline have been shown to interfere with various steps in the proinflammatory microglial activation intracellular signalling pathway, disrupting the subsequent neuroinflammatory cascade. We hypothesize that these drugs could be a novel pharmacotherapy that could significantly improve the outcome of post-operative delirious patients.     

Role of microglia and toll-like receptor 4 in the pathophysiology of delirium

Delirium is a serious medical condition that commonly afflicts elderly inpatients. This is especially common in the post-operative setting where it increases mortality, length of hospital stay and health care costs. The exact mechanisms involved in its pathogenesis remain uncertain and there is currently no effective pharmacological therapy for treatment or prevention of delirium. We hypothesize that microglia-mediated neuroinflammation via toll-like receptor 4 signalling is a significant contributor to post-operative delirium. Based on our proposed mechanism, three novel pharmacological therapies have been suggested to be effective to prevent or treat delirium. Curcumin, ibudilast and minocycline have been shown to interfere with various steps in the proinflammatory microglial activation intracellular signalling pathway, disrupting the subsequent neuroinflammatory cascade. We hypothesize that these drugs could be a novel pharmacotherapy that could significantly improve the outcome of post-operative delirious patients.     

Role of Toll-like receptor 4 in gram-positive and gram-negative pneumonia in mice

To determine the role of Toll-like receptor 4 (TLR4) in the immune response to pneumonia, C3H/HeJ mice (which display a mutant nonfunctional TLR4) and C3H/HeN wild-type mice were intranasally infected with either Streptococcus pneumoniae (a common gram-positive respiratory pathogen) or Klebsiella pneumoniae (a common gram-negative respiratory pathogen). In cases of pneumococcal pneumonia, TLR4 mutant mice showed a reduced survival only after infection with low-level bacterial doses, which was associated with a higher bacterial burden in their lungs 48 h postinfection. In Klebsiella pneumonia, TLR4 mutant mice demonstrated a shortened survival after infection with either a low- or a high-level bacterial dose together with an enhanced bacterial outgrowth in their lungs. These data suggest that TLR4 contributes to a protective immune response in both pneumococcal and Klebsiella pneumonia and that its role is more important in respiratory tract infection caused by the latter (gram-negative) pathogen.     

Toll-like receptor 1 inhibits Toll-like receptor 4 signaling in endothelial cells

Toll-like receptor 4 (TLR4) is the signal-transducing component of the LPS recognition complex and is essential for LPS-induced septic shock. Here we demonstrate that TLR1 has the capacity to abrogate TLR4 signaling. Human microvascular endothelial cells express TLR4 but not TLR1 and respond to LPS through TLR4. The ability of these cells to respond to LPS was lost, however, when they were transfected with TLR1. Inhibition was specific for TLR1 because TL5 failed to block TLR4 function. Moreover, TLR1 had no effect upon TNF-alpha signaling, indicating that TLR1 operated at a step upstream of the convergence between the two pathways. Inhibition of TLR4 signaling was mediated by the extracellular, but not cytoplasmic domain of TLR1. In addition, TLR1 physically associated with TLR4 in co-precipitation experiments. These findings suggest that TLR1 might restrain potentially dangerous innate response to LPS by binding to TLR4 and preventing the formation of active signaling complexes.