TLR4基因沉默下调TLR2信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用

目的 研究单核巨噬细胞RAW264.7受烟曲霉孢子刺激时,TLR2和TLR4信号通路发挥的作用,以及沉默TLR4基因后,对TLR2信号通路的影响.方法 利用RNAi技术将TLR4-siRNA转染RAW264.7细胞24h后给予烟曲霉孢子刺激12h,将细胞随即分为正常组(N组)、正常+烟曲霉孢子刺激组(N+Af组)、正常+TLR4-siRNA组[TLR4(RNAi)组]、正常+TLR4-siRNA+烟曲霉孢子刺激组[ TLR4(RNAi) +Af组],RT-PCR和Western blot法检测细胞受烟曲霉孢子刺激后TLR2、TLR4、MyD88 mRNA及TNF-α蛋白的表达变化.结果 (1)TLR4基因沉默前:与N组比较,N+Af组TLR2、TLR4、MyD88 mRNA及TNF-α蛋白表达量均显著升高(P＜0.05).(2)TLR4-siRNA( 100nmoL/L)转染RAW264.7细胞,沉默效率达83％.(3)TLR4基因沉默后:与N组比较,TLR4( RNAi)组TLR2、MyD88 mRNA的表达量均显著降低(P＜0.05);与N+Af组比较,TLR4( RNAi)+Af组的TLR2、MyD88 mRNA和TNF-α蛋白的表达量均显著降低(P＜0.05);与TLR4( RNAi)组比较,TLR4(RNAi)+ Af组MyD88 mRNA表达量显著升高(P＜0.05),而TLR2 mRNA及TNF-α蛋白表达量却无显著变化(P＞0.05).结论 RAW264.7细胞受烟曲霉孢子刺激时,TLR2和TLR4信号通路被激活,通过释放促炎细胞因子TNF-α发挥抗烟曲霉孢子刺激作用;当沉默TLR4基因后,TLR2信号通路不能被很好地激活来抵抗烟曲霉孢子对细胞的刺激作用,沉默TLR4基因下调了TLR2信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用,可能TLR4较TLR2在抵抗烟曲霉孢子刺激时发挥更重要的作用.     

青蒿琥酯对小鼠巨噬细胞RAW264.7中TLR4介导的炎症通路的抑制作用

目的观察青蒿琥酯(artesunate,AS)对脂多糖(lipopolysaccharide,LPS)刺激小鼠RAW264.7细胞Toll样受体4(Toll-like receptor 4,TLR4)介导炎症通路活化的影响,以探讨AS的抗炎作用机制。方法采用免疫荧光观察胞内TLR4表达和分布;免疫印迹检测TLR4及下游炎症通路关键分子的表达和活化;酶联免疫吸附法检测细胞培养上清中TNF-α、IL-6浓度。结果 AS对LPS诱导的TLR4表达及其在细胞中的聚集均有抑制作用;AS同时抑制TLR4衔接蛋白髓分化因子88和含TIR结构域干扰素诱导衔接蛋白表达,也抑制依赖于二者活化的肿瘤坏死因子受体相关因子6表达;对下游MAPK通路,AS抑制p38表达和磷酸化、JNK磷酸化,但对ERK1/2无显著影响;对下游NF-κB通路,AS下调抑制性-κBα(inhibitory-κBα,IκBα)的磷酸化,进而减少NF-κB亚基p50和p65活化入核的数量;最后,AS能够抑制LPS诱导的TNF-α和IL-6释放。结论 AS通过抑制LPS诱导的TLR4通路活化,减少致炎细胞因子的释放,从而发挥其抗炎作用。     

云芝糖肽对人PBMC中TLR5信号通路调控机制的研究

目的:研究云芝糖肽(PSP)对人外周血单个核细胞(PBMC)中TLR5信号通路的调控作用,探索PSP对Toll样通路的免疫调节机制。方法:体外分离、培养人PBMC,分为对照组和PSP处理组,采用实时荧光定量PCR(Q-PCR)技术对PSP处理人PBMC前后,PBMC中的TLR5信号通路中相关基因的表达差异情况进行定量分析。结果:PSP处理的实验组与对照组相比较,TLR5信号通路中的TLR5、IRAK4、TRAF6、IRF5和NF-κB基因的表达水平均显著增高(P<0.05),但AP-1基因的表达降低(P<0.05)。结论:PSP对PBMC中TLR通路的调控可能主要通过NF-κB和IRF5调控终端效应因子来发挥免疫调节作用,这些为进一步探讨PSP的免疫调节作用机理提供了实验依据。     

TLR信号通路研究进展

TLR在识别病原体相关分子中起到关键作用,提示其与免疫炎症相关.TLR在天然免疫反应中不仅可以清除病原体,而且可通过识别病原体相关分子过程中有效建立获得性免疫体系.在TLR介导的细胞信号通路中,接头分子TLR/IL-1R结构域对免疫炎症反应起到了重要的作用,它可产生一些前炎症细胞因子如NO、1型干扰素等,并上调共刺激分子表达.现就TLR与其相关配体作用关系及TLR介导的天然免疫激活进行综述.     

三百棒通过调控TLR4/NF-κB信号通路对脂多糖诱导的RAW264.7细胞极化的影响

目的：探讨三百棒醇提物(TAAE)对脂多糖(LPS)诱导的小鼠单核-巨噬细胞RAW264.7极化趋向以及其对炎症反应的影响。方法：用LPS诱导RAW264.7细胞建立炎症模型。CCK-8法检测细胞活力;ELISA检测细胞因子(TNF-α、IL-1β、IL-6和IL-10)的水平;Griess法检测细胞培养上清液中一氧化氮(NO)的分泌情况;荧光染色双标法观察巨噬细胞的极化状态;免疫荧光染色检测NF-κB定位及表达;Transwell检测TAAE对RAW264.7细胞迁移和趋化性的影响;RT-qPCR检测TNF-α、IL-1β、IL-6、IL-10、Arg-1、NF-κB和TLR4的mRNA水平;Western blot检测iNOS、COX-2、TLR4、NF-κB、p-NF-κB、IκBα和p-IκBα蛋白水平。使用TLR4通路抑制剂TAK-242进一步验证TAAE对TLR4/NF-κB信号通路的影响。结果：与模型组相比较,TAAE降低LPS诱导的炎症模型RAW264.7细胞中M1型促炎因子(TNF-α、IL-1β和IL-6)及NO水平,促使M2型抑炎因子(IL-10和Arg-1)分泌...     

RGS1对RAW264.7细胞共刺激分子和细胞因子表达的调节

目的:研究RGS1基因对细胞表面分子表达以及细胞因子分泌的影响.方法:通过克隆RGS1基因、转染RAW264.7细胞、建立稳定转染的细胞系, 利用流式细胞术检测, 基因芯片分析初步探讨RGS1基因的功能.结果:RGS1基因能够抑制NF-κB转录因子活性; RGS1基因明显增加RAW264.7细胞表面CD86分子的表达, 降低CD80表达; 在不同Toll样受体配体刺激后, CD40、 CD80和PD1水平低于对照组.同时, RGS1基因使IL-6、IL-15的表达明显升高, IL-10、 IL-1的表达明显降低.结论:RGS1基因可调节免疫相关细胞表面分子和细胞因子的分泌, 影响Toll 样受体信号通路,表明RGS1可能在免疫调节方面起重要作用.     

TGF—β信号通路中负性调节的核内转录因子

TGF β超家族成员参与多种细胞生物学功能。TGF β超家族的信号转导由两型跨膜受体介导 ,活化的受体调节型Smad与Smad4结合形成寡聚物 ,并转移到核内 ,从而调节基因的表达。Smad寡聚物进入核内后 ,可受到多种蛋白的调节 ,本文就其中 8种核内抑制分子作一介绍     

CLP-19预处理RAW264.7细胞对炎性因子的调节作用初探

目的探讨CLP-19对RAW264.7细胞的免疫调节及其在炎症中的作用。方法以50μg/ml的CLP-19作用于RAW264.7细胞后不同时间(0.5、1、2.5、5、10和20 h),及不同质量浓度(1、10、50、100和200μg/ml)的CLP-19作用于RAW264.7细胞20 h,观察细胞TNF-α、IL-10、MCP-1、ICAM-1的mRNA水平的变化情况。另用50μg/ml的CLP-19预处理细胞5、10、和20 h后,再以LPS刺激,4 h后提取细胞RNA,观察上述细胞因子的变化,以CLP-19与LPS预混组为对照。结果 CLP-19单独作用于RAW264.7细胞后,CLP-19对MCP-1的mRNA调节呈显著的时间依赖性和剂量依赖性,高剂量的CLP-19可上调ICAM-1、TNF-α和IL-10的mRNA水平。以CLP-19预处理RAW264.7细胞不同时间(5、10和20 h)后,均可显著抑制LPS所刺激的炎症因子上调,各预处理组间无统计学差异。结论 CLP-19能改变细胞的免疫状态,并抑制LPS诱导的免疫反应。     

TGF-β1下调炎性巨噬细胞RAW264.7 TLR4受体的表达

目的探讨TGF-β1对小鼠巨噬细胞RAW264.7 TLR4受体表达的调节作用。方法 Teal-Time PCR法检测RAW264.7细胞TGF-β1和TLR4受体mRNA表达,ELISA法检测RAW264.7细胞TGF-β1的分泌,流式细胞术检测RAW264.7细胞TLR4受体的表达。结果一定剂量的LPS促进RAW264.7分泌TGF-β1,且呈剂量依赖关系,一定剂量的TGF-β1单独刺激下调RAW264.7的TLR4受体的表达,TGF-β1能够下调LPS活化的RAW264.7细胞TLR4受体表达。结论 TGF-β1可以通过下调巨噬细胞TLR4受体表达来控制机体炎性反应,这可能是TGF-β1作为免疫抑制剂抑制炎症反应的重要途径之一。     

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.     

Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells

Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.     

TLR2 and TLR4 agonists synergistically up-regulate SR-A in RAW264.7 through p38

It is known that macrophage scavenger receptor A (SR-A) can protect mice from endotoxemia. In addition, Escherichia coli O111:B4 LPS from Sigma (sLPS), which contains both TLR4 and TLR2 agonists, was previously reported to be able to induce SR-A expression on murine macrophage cell line RAW264.7. However, the relative role of both TLR4 and TLR2 agonists from Sigma (sLPS) in the up-regulation of SR-A on RAW264.7 is still undefined. Here, we found that sLPS could only slightly up-regulate SR-A on RAW264.7 following removing its TLR4 and TLR2 agonists, respectively. In contrast, the combination of TLR4 agonist uLPS (re-extracted sLPS) and TLR2 agonist Pam3CSK4 dramatically induced SR-A expression, and synergistically promoted RAW264.7 to bind and internalize FITC-LPS specifically through SR-A. The combination had no such effect either on TLR2 or TLR4 expression, and incubation with IL-6, IL-10, IL-12 or TNF-alpha alone could not induce SR-A expression on RAW264.7. In addition, treatment with a NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) could only weakly suppress the up-regulation of SR-A by the combination. However, the combination synergistically promoted MAPK p38 phosphorylation, and p38 specific inhibitor SB203580 completely suppressed its inducible effect on SR-A expression. Hence, we demonstrated that up-regulation of SR-A by sLPS was resulted from the cooperation of its TLR4 and TLR2 agonists through p38, and we also presented a novel synergy effect of TLR2 and TLR4 agonists.     

MicroRNAs in the regulation of TLR and RIG-I pathways

The innate immune system recognizes invading pathogens through germline-encoded pattern recognition receptors (PRRs), which elicit innate antimicrobial and inflammatory responses and initiate adaptive immunity to control or eliminate infection. Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I) are the key innate immune PRRs and are tightly regulated by elaborate mechanisms to ensure a beneficial outcome in response to foreign invaders. Although much of the focus in the literature has been on the study of protein regulators of inflammation, microRNAs (miRNAs) have emerged as important controllers of certain features of the inflammatory process. Several miRNAs are induced by TLR and RIG-I activation in myeloid cells and act as feedback regulators of TLR and RIG-I signaling. In this review, we comprehensively discuss the recent understanding of how miRNA networks respond to TLR and RIG-I signaling and their role in the initiation and termination of inflammatory responses. Increasing evidence also indicates that both virus-encoded miRNAs and cellular miRNAs have important functions in viral replication and host anti-viral immunity.     

Regulation of airway inflammation and remodeling in asthmatic mice by TLR3/TRIF signal pathway

This paper aims to investigate the effect of Toll-like receptors 3 (TLR3)/TIR-domain-containing adapter-inducing interferon-β (TRIF) signal pathway on the airway inflammation and remodeling in asthmatic mice. C57BL/6 and TLR3^−/− mice were randomly divided into three groups (10 mice per group), including Control group (mice inhaled phosphate buffer saline (PBS)), Asthma group (mice inhaled ovalbumin (OVA)) and polyriboinosinic-ribocytidylic acid (poly (I: C)) group (asthmatic mice were injected intraperitoneally with TLR3 agonist poly (I: C)). Hematoxylin-eosin (HE) staining, Wright-Giemsa staining, Enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry, Hydroxyproline assay, quantitative real time polymerase chain reaction (qRT-PCR) and Western blot were used to assess for the indices of airway inflammation and remodeling. In terms of WT mice, all asthma groups with or without the addition of poly (I: C) showed exaggerated inflammation and remodeling in the airways as compared to Control group, which were more seriously in poly (I: C) group than Asthma group. Furthermore, we observed the significant inhibition of airway inflammation and remodeling in the TLR3^−/− mice in both Asthma no matter with or without addition of poly (I: C) than the WT mice. TLR3 knockout could obviously relieve the airway inflammation and remodeling in asthma through inhibiting TLR3/TRIF signaling pathway.     

Atractylenolide I inhibits lipopolysaccharide-induced inflammatory responses via mitogen-activated protein kinase pathways in RAW264.7 cells

Atractylenolide I (ATL-I) is a bioactive component of Rhizoma Atractylodis macrocephalae. Although increasing evidence shows that ATL-I has an anti-inflammatory effect, the anti-inflammatory molecular mechanism of ATL-I is still unknown. In this study, we investigated the effect of ATL-I on cell viability by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and the level of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) by enzyme-linked immunosorbent assay (ELISA) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Further, we examined the effect of ATL-I on the activation of nuclear factor-kappaB (NF-κB) and phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38) by Western blot. We also investigated the effect of ATL-I on the expression of myeloid differentiation protein-2 (MD-2), CD14, complement receptor 3 (CR3), scavenger receptor class A (SR-A), toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). We found that ATL-I showed no inhibitory effect on cell viability at concentrations ranging from 1?µM to 100?µM and markedly reduced the release of IL-6 and TNF-α at a concentrate-dependent manner. In addition, ATL-I suppressed the activity of nuclear NF-κB and the phosphorylation of ERK1/2 and p38 in LPS-treated RAW264.7 cells. Further analysis showed that ATL-I inhibited the expression of MD-2, CD14, SR-A, TLR4 and MyD88, but the expression of CR3 was unaffected. These data suggest that ATL-I shows an anti-inflammatory effect by inhibiting TNF-α and IL-6 production. The anti-inflammatory effects of ATL-I may be associated with the inhibition of the NF-κB, ERK1/2 and p38 signaling pathways.     

胶球藻多糖对RAW264.7细胞凋亡的影响

目的探讨胶球藻多糖对RAW264.7细胞凋亡的影响及其可能机制。方法培养RAW264.7细胞,分为对照组、胶球藻多糖(2mg/ml)组及胶球藻多糖(4mg/ml)组;利用流式细胞仪检测各实验组中细胞的凋亡率;采用Western blot检测各实验组中凋亡相关因子Bad、Bax、Bcl-2、Bcl-xl及Caspase-3蛋白的表达。结果相较于对照组,胶球藻多糖处理组不仅可以诱导RAW264.7细胞发生凋亡,增加促凋亡蛋白(Bad,Bax)的表达及减少抗凋亡蛋白(Bcl-2, Bcl-xl)的表达,而且还能上调剪切型Caspase-3蛋白的水平,且均呈剂量依赖性。结论胶球藻多糖显著促进RAW264.7细胞发生凋亡,其机制可能与Bad、Bax表达上调,Bcl-2、Bcl-xl表达下调及Caspase-3的激活有关。