Toll样受体4、NF-κB在溃疡性结肠炎中的表达

目的:研究Toll样受体4、NF-κB在溃疡性结肠炎(UC)中的表达变化,探讨两者在UC发病机制中的作用。方法:收集UC病例及非UC对照结肠镜活检标本或手术标本。采用免疫组化和RT-PCR技术,检测非UC对照及UC患者肠黏膜TLR4、NF-κBp65的表达水平,并做统计学分析。结果:RT-PCR结果显示,TLR4、NF-κBp65在UC组织中的表达情况显著高于在非UC对照组织的表达(TLR4:143.658±33.870,30.531±8.442,t=24.253,P0.01;NF-κBp65:185.773±37.625,23.810±7.038,t=31.664,P0.01)。免疫组化染色显示TLR4、NF-κB在UC组结肠黏膜表达增强。结论:TLR4、NF-κBp65在UC中表达高度上调,推测它们可能参与了UC的发病过程。     

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...     

脂多糖诱导大鼠心肌细胞NF-κB、IκB-α的表达及意义

目的研究脂多糖(LPS)诱导大鼠心肌细胞NF-κB,IκB-α的表达及意义。方法100ng/mlLPS刺激心肌细胞0、0.5、1、2、4、6、8h和0、10、50、100ng/mlLPS刺激1h,免疫细胞化学检测NF-κBp65的表达,Westernblot检测IκB-α表达。结果LPS的刺激迅速激活心肌细胞NF-κBp65的表达,于1h时达高峰;IκB-α的表达先降低,后升高。结论LPS可诱导大鼠心肌细胞NF-κB的激活,并通过激活心肌细胞核因子-κB途径从而促进细胞损伤。     

槲皮素对脓毒症急性肺损伤大鼠肺组织NF-κB表达的影响

目的观察槲皮素对脓毒症相关急性肺损伤(ALI)大鼠的炎症信号转导通路NF-κB p65的影响。方法选用健康雄性Wistar大鼠36只,随机均分为4组:对照组、脂多糖(LPS)组、槲皮素低剂量组(30mg/kg),槲皮索高剂量组(50mg/kg),用LPS 10mg/kg溶于生理盐水2ml腹腔内注射,建立脓毒症ALI大鼠模型,对照组予等量生理盐水2ml腹腔注射,治疗组于造模前30min腹复腔注射槲皮素,所有实验动物于造模后24h应用10%水合氯醛4ml/kg腹腔注射进行麻醉,并处死。所有大鼠取肺组织进行苏木素-伊红(HE)染色,观察病理学改变并进行病理评分且对比,应用免疫组化及Western blot检测NF-κB p65在肺组织的表达。结果与对照组相比,LPS组的肺损伤明显,病理评分明显增加(P0.05);较LPS组,槲皮素治疗组肺组织HE病理染色明显改善,病理评分显著下降(P0.05),但两个治疗组之间病理评分无统计学差异。与对照组比较,LPS组的免疫组化及Western blot检测的NF-κB p65含量明显增高(P0.05),而槲皮素治疗组比LPS组NF-κB p65含量明显下降(P0.05),但两治疗组比较无明显差异。结论槲皮素对脓毒症ALI大鼠的肺脏保护作用可能与其抑制NF-κB p65的表达相关。     

戊四氮慢性致痫大鼠海马星形胶质细胞的激活

目的：研究慢性癫痫大鼠点燃时海马星形胶质细胞的激活情况。方法：采用免疫组化和双重免疫荧光标记法观察戊四氮慢性癫痫大鼠点燃后海马NF-kBp65和胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的变化。结果：癫痫发作1 h,CA1区出现NF-kBp65-IR阳性细胞,4 h胶质细胞p65-IR维持在高水平,并持续至发作后12 h;发作1 h,CA1区GFAP-IR开始增强,4～8 h观察到明显浓染和突起增多的GFAP-IR阳性细胞,并持续至24 h;GFAP/p65一IR阳性细胞发作后1 h可观察到,4 h达最高峰,24 h恢复至对照组水平。结论：戊四氮致痫大鼠点燃时,星形胶质细胞的这种早期而持续的激活提示该细胞在慢性癫痫的复发中可能起到重要作用。     

脂多糖通过NF-κB途径上调大鼠腹膜间皮细胞表达CD40和ICAM-1

目的 观察脂多糖(LPS)作用下大鼠腹膜间皮细胞NF-κB活性及其对CD40和细胞间黏附分子1(ICAM-1)表达的影响.方法 分离及培养大鼠腹膜间皮细胞.LPS不同浓度作用12 h 及LPS (5 μg/ml)作用不同时间点收集细胞;LPS(5 μg/ml)或BAY11-7085(一种IκBα的磷酸化抑制剂)不同浓度(1 μmol/L和5 μmol/L)预处理3 h加LPS,作用3 h后收集细胞;采用RT-PCR方法检测CD40和ICAM-1 mRNA表达.采用蛋白印迹检测NF-κB和磷酸化NF-κB(p-NF-κB)蛋白表达.结果 与常规培养基对照组相比,5 μg/ml LPS组CD40和ICAM-1 mRNA表达显著升高(P<0.05),10 μg/ml LPS组显著高于5 μg/ml LPS作用组(P<0.05).5 μg/ml LPS 作用下,ICAM-1 mRNA表达从1 h开始升高,3 h达到高峰,之后逐渐降低.CD40 mRNA表达在1 h无显著变化,3 h时迅速达到高峰,之后逐渐降低.常规培养的大鼠腹膜间皮细胞结构性表达p-NF-κB蛋白;加入LPS后,p-NF-κB蛋白表达显著增加,其中30 min～1 h表达最强,之后逐渐降低,至2 h仍显著高于常规培养组(P<0.05).加入5 μmol/L BAY11-7085后,LPS诱导的CD40和ICAM-1 mRNA表达显著降低(P<0.05),与正常对照组相比差异无统计学意义. 结论 LPS以时间依赖和浓度依赖模式上调CD40和ICAM-1的表达.NF-κB信号途径参与调节LPS诱导的大鼠腹膜间皮细胞CD40和ICAM-1的表达.     

右美托咪定调节星形胶质细胞表型改善创伤性脑损伤大鼠神经功能的机制研究

目的 探讨右美托咪定对创伤性脑损伤大鼠神经功能的保护作用及其可能的作用机制。方法30只SPF级雄性SD大鼠随机分为假手术组、模型组及右美托咪定组,每组10只,采用Feeney自由落体的方法建立脑创伤模型。NSS评分评价大鼠神经功能;免疫荧光染色检测小胶质细胞标志物Iba-1表达;ELISA检测脑组织中TNF-α、IL-1α及C1q表达情况;免疫荧光染色检测A1型星形胶质细胞标志物C3/GFAP、A2型星形胶质细胞标志物S100A10/GFAP表达情况;Western blot检测TLR4/NF-κB信号通路TLR4、NF-κB及IκB蛋白表达情况。结果 与模型组相比,右美托咪定组大鼠NSS评分明显降低,神经功能明显改善;脑内Iba-1阳性细胞表达明显减少;脑组织中TNF-α、IL-1α及C1q水平明显降低;A1型星形胶质细胞标志物C3/GFAP表达明显减少,而A2型星形胶质细胞标志物S100A10/GFAP表达明显增加;并且TLR4/NF-κB信号通路TLR4、NF-κB蛋白表达明显减少,而IκB蛋白表达明显增加。结论 右美托咪定能够改善创伤性脑损伤大鼠神经功能,其作用机制可能与抑制T...     

脂多糖对星形胶质细胞的生长具有双重性

目的:研究脂多糖(LPS)对星形胶质细胞(AC)生长的影响及其可能的机制。方法:在原代培养的AC中加入不同浓度的LPS作用不同时间,观察AC生长的情况,以及NF-κB通路抑制剂SN50对AC生长的影响。同时以不同浓度的LPS作用24h后,观察随后8dAC生长的变化。结果:在LPS作用不同时间中,只有作用24h后,低剂量的LPS可使AC的生长加快,高剂量的LPS则可抑制AC生长。以LPS作用AC24h后,短期内LPS可促进AC的生长,长期则抑制AC生长,且呈剂量依赖性。NF-κB通路抑制剂可阻断LPS对AC生长的影响。结论:低剂量的LPS短期内可促进AC生长,高剂量时则抑制AC生长,而炎症对AC的长期影响是对细胞的生长增殖起抑制作用。其可能的分子机制可能与NF-κB通路激活有关。     

葛根素通过抑制小胶质细胞TLR4/NF-κB介导的炎症反应治疗神经根性疼痛

目的:探究传统中药葛根素对腰椎间盘突出症引起的神经根性疼痛的作用及机制.方法:用自体髓核(NP)移植的方法复制大鼠腰椎间盘突出症模型,连续7天腹腔注射葛根素(100 mg·kg-1·d-1)观察镇痛效果;采用机械性撤足阈值(PWT)和热撤足潜伏期(PWL)作为痛觉阈值检测指标;Western blot方法检测脊髓组织T...     

血管紧张素Ⅱ诱导RAW264.7细胞Toll样受体4表达及髓过氧化物酶活性的机制研究

目的:探讨血管紧张素Ⅱ(AngⅡ)对RAW264.7细胞Toll样受体4(TLR4)mRNA、蛋白表达及髓过氧化物酶(MPO)活性的影响及其致炎致动脉粥样硬化(AS)机制.方法:体外培养RAW264.7细胞, 用不同浓度的AngⅡ(1、 10、 100、 1000 nmol/L)及100 μg/L脂多糖(LPS)刺激RAW264.7细胞24 h后, RT-PCR法测RAW264.7细胞TLR4 mRNA水平, Western blot检测RAW264.7细胞TLR4蛋白表达, 比色法测细胞培养上清中MPO活性.同时, 预先加入不同剂量的TLR4阻断剂(1 mg/L、 5 mg/L)后, 再用AngⅡ(100 nmol/L)刺激RAW264.7细胞24 h, 检测细胞培养上清中MPO活性.结果:AngⅡ浓度依赖性地增加RAW264.7细胞TLR4 mRNA及蛋白表达(P<0.01), LPS也可诱导RAW264.7细胞TLR4 mRNA及蛋白表达(P<0.01); AngⅡ浓度依赖性地升高RAW264.7细胞MPO活性(P<0.01), LPS也可升高RAW264.7细胞MPO活性(P<0.01), 而TLR4阻断剂明显抑制AngⅡ这一效应(P<0.01).结论:AngⅡ上调RAW264.7细胞TLR4表达, 通过跨膜受体TLR4诱导MPO分泌, 加重炎症反应, 促进AS的形成和发展.     

The role of toll-like receptors in systemic lupus erythematosus

Systemic lupus erythematosus is an autoimmune disease characterized by the production of autoantibodies against a relatively limited range of nuclear antigens. These autoantibodies result in the formation of immune complexes that deposit in tissues and induce inflammation, thereby contributing to disease pathology. Growing evidence suggests that recognition of nucleic acid motifs by Toll-like receptors may play a role in both the activation of antinuclear B cells and in the subsequent disease progression after immune complex formation. The endosomal localization of the nucleic acid-sensing Toll-like receptors (TLRs), TLR3, 7, and 9, is believed to contribute to the distinction between endogenous nucleic acids and those of foreign origin. In this article we review recent work that suggests a role for the B-cell receptor and Fcγ receptors in delivering nuclear antigens to intracellular compartments allowing TLR activation by endogenous nucleic acids. A number of in vitro studies have presented evidence supporting a role for TLRs in SLE pathology. However, recent studies that have examined the contributions of individual TLRs to SLE by using TLR-deficient mice suggest that the situation is far more complicated in vivo. These studies show that under different circumstances TLR signaling may either exacerbate or protect against SLE-associated pathology. Further understanding of the role of TLRs in pathological autoreactivity of the adaptive immune system will likely lead to important insights into the etiopathogenesis of SLE and potential targets for novel therapies.     

Rotavirus and coxsackievirus infection activated different profiles of toll-like receptors and chemokines in intestinal epithelial cells

Objective  To understand the inflammatory-immune response in intestinal epithelial cells after infection of rotavirus and coxsackievirus B3. Methods  We examined by quantitative PCR the expression profiles of genes encoding five toll-like receptors (TLR) and levels of three chemokines in response to rotavirus and coxsackievirus B3 infection in a human intestinal epithelial cell line (HT-29 cells). Results  We demonstrated that rotavirus induced significantly increased levels of mRNA expression for TLR2, TLR3, TLR7 and TLR8 in HT-29 cells in a time-dependent manner. In contrast, coxsackievirus B3 did not stimulate mRNA expression for TLR3. Rotavirus and coxsackievirus B3 also induced higher levels of mRNA expression for RANTES, IP-10 and IL-8 during the period of infection in a different manner. Finally, significantly elevated levels of RANTES, IP-10 and IL-8 were detected by ELISA in rotavirus-infected cells from 24 to 48 h. Conclusion  Our findings suggest that different patterns of TLRs and chemokines were induced in the initiation and modulation of immune response to rotavirus and coxsackievirus B3 infection.     

Decrease in toll-like receptors 2 and 4 in the spleen of mouse with endotoxic tolerance

OBJECTIVE: Down-regulation of toll-like receptor (TLRs) is present in animals with endotoxin tolerance. The spleen is the largest peripheral lymphatic organ in vivo, and it plays an important role in inflammatory reactivity. In this study we investigated TLR2 and TLR4 proteins in the spleen of endotoxic tolerant mice. METHODS: The TLR2 and TLR4 proteins were quantitatively detected by Western blotting, and their locations in the spleen were observed by immunohistochemistry. IL1-beta mRNA in the spleen was analyzed by real-time quantitative PCR. RESULTS: The mice pretreated with 0.5 mg/kg/day of LPS for 14 days exhibited a decrease of TLR2 and TLR4, and presented endotoxin tolerance. In addition, a bolus injection of 10 mg/kg LPS for mice with endotoxic tolerance caused no alteration of TLR2 and TLR4 proteins and no change of IL1-beta mRNA expression in the spleen. CONCLUSION: TLR4 is the target of endotoxic tolerance induction. The induction of cross-tolerance of TLR2 following LPS challenge is present in vivo with endotoxin tolerance.     

Expolysaccharides from Bifidobacterium animalis RH activates RAW 264.7 macrophages through toll-like receptor 4

A water-soluble expolysaccharide (EPS) was prepared from Bifidobacterium animalis RH by enzymatic hydrolysis, ethanol precipitation and Sepharose CL-6B column chromatography. Its immunomodulating activity was evaluated using murine macrophage cell line RAW 264.7. The data obtained showed that the EPS promoted proliferation and phagocytosis activity of RAW 264.7 cells. In addition, the EPS promoted RAW 264.7 cells to produce NO, IL-6, TNF-α, MCP-1 and MIP-1α in a dose-dependent manner. Further work revealed that the EPS activated RAW 264.7 cells majorly through TLR4.     

Roles of toll-like receptors in natural interferon-producing cells as sensors in immune surveillance

Natural IFN-alpha/beta producing cells (IPCs) play a central role in innate immunity against microbial infections. In primary immune responses, toll-like receptors (TLRs), as major pattern-recognition receptors, are essential for IPCs as well as other antigen presenting cell (APC) subsets to recognize microbes. IPCs unequivocally express TLR7 and TLR9, and can respond to the respective ligand to produce IFN-alpha/beta and to rapidly differentiate into dendritic cells (DCs). Thereby, IPCs can not only activate innate immune system but also provoke T cell responses. Thus, IPCs link innate and adaptive immunity through TLR system. In addition, recent work has revealed the regulatory system of DC subsets in response to microbial invasion. In this context, by the different but complementary expression profile of TLRs, IPCs together with myeloid APC subsets constitute a rational system of immune surveillance that can cover a wide variety of pathogens and enlarge immune adjuvant effects.     

Rapid translocation of intracellular toll-like receptors depends on endosomal NADPH oxidase

Endosomal toll-like receptors (TLRs) must be translocated from the endoplasmic reticulum (ER) to the endosome and proteolytically cleaved within the endosome before they can induce cellular signals. As ligands for these TLRs are also liberated from apoptotic or necrotic cells, this process is controlled by several mechanisms which shall ensure that there is no inadvertent activation. We have shown previously that antiphospholipid antibodies induce endosomal NADPH-oxidase (NOX) followed by the translocation of TLR7/8 to the endosome. We show now that endosomal NOX is required for the rapid translocation of TLR3, TLR7/8, and TLR9. Deficiency of gp91phox, the catalytic subunit of NOX2, or inhibition of endosomal NOX by the chloride channel blocker niflumic acid both prevent immediate (i.e., within 30 min) translocation of these TLRs as shown by confocal laser scanning microscopy. Under these conditions, the induction of mRNA synthesis for TNF-α and secretion of TNF-α is delayed by approx. 6–9 h. However, maximal expression of TNF-α mRNA or secretion of TNF-α is not significantly reduced. In conclusion, these data add NOX2 as another component involved in the orchestration of cellular responses to ligands of endosomal TLRs.     

TLR4胞内区的同源模建

目的 为进一步研究新发现的TLR4蛋白在LPS对机体细胞产生作用的机制，分析其Q结构上的功能区域。方法 同源模建，通过计算机模拟从理论上对TLR4胞内区的功能区域进行探讨。结果 Pro712His突变体溶液可及表面的亲疏水性质有明显变化，且相应位置所带的电荷从中性变为正电荷。结论 TLR4胞内区在发生作用时，起主要作用的是疏水性作用，下一个接头蛋白MyD88与TLR4胞内区相互作用的部位应当是一个有较强疏水作用的区域。这样当Pro712His突变体出现时，由于电荷的排斥，疏水作用无法达到点突变以前的强度，2个蛋白的结合不能得以实现，信号无法继续传递。从而出现对LPS的耐受现象。