人参皂苷Rg_1通过作用于炎性因子和糖皮质激素受体减少金葡菌对肺上皮细胞的损伤(英文)

本文旨在探讨人参皂苷Rg_1体外对金葡菌感染肺上皮细胞所致病变的作用及其可能的机制。实验采用体外大鼠原代肺上皮细胞感染模型。RT-PCR和Western blot方法观察肺细胞中integrinβ1,NF-κB和糖皮质激素受体(GR)等相关因子的表达。结果表明人参皂苷Rg_1可以明显抑制金葡菌感染后肺上皮细胞整合素integrinβ1的表达,下调炎性因子NF-κB,ICAM-1,TNF-α,IL-2和IL-6,上调GR的表达。人参皂苷Rg_1可以明显抑制金葡菌感染肺上皮细胞后的炎性因子,从而对肺脏起到一定的保护作用。     

α-硫辛酸抑制炎症信号TLR4和NLRP3的活化在糖尿病大鼠肾组织纤维化中的保护作用

目的观察α-硫辛酸(ALA)对糖尿病(DM)大鼠肾组织Notch2、TLR4、NLRP3及炎性因子表达的影响,探讨ALA在糖尿病肾病(DN)中抗炎、抗纤维化的可能保护机制。方法 STZ复制DM模型,分为DM组和ALA组,同时设对照组。8周后处死大鼠,测定生化指标和氧化应激水平,免疫组化观察肾皮质Notch2、TLR4、NLRP3的表达部位,Western blot检测肾组织中Notch2、TLR4、NLRP3和Col-Ⅳ蛋白的表达,ELISA检测肾组织炎性因子IL-6和TNF-α的含量。结果与对照组相比,DM组大鼠血糖、24 h尿蛋白、总胆固醇和甘油三酯升高,T-AOC水平降低,MDA增多;ALA组除了血糖外,其余生化指标均低于DM组,而T-AOC活性增高,MDA含量降低。DM组肾组织Notch2、TLR4、NLRP3、Col-Ⅳ蛋白表达增多,炎性因子增多,ALA组各指标均降低。结论 ALA可下调DM大鼠肾组织TLR4和NLRP3炎症信号,减少炎性因子的表达和细胞外基质的沉积,从而减轻DN炎症反应和纤维化病变的发生发展,其机制可能与抑制Notch2蛋白的表达有关。     

基于UPLC-Q-Exactive-Orbitrap-MS整合网络药理学探讨金银花抗RSV肺炎的作用机制

目的： 基于超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（UPLC-Q-Exactive-Orbitrap-MS）技术、网络药理学和整体动物实验药理学的方法,初步探讨金银花抗RSV肺炎的作用机制。方法： 采用滴鼻法建立呼吸道合胞病毒（respiratory syncytial virus,RSV）肺炎小鼠模型,设立空白组、模型组、阳性对照组、金银花低、中、高剂量给药组,以小鼠体质量、肛温、肺指数以及IFN-γ、TNF-α、MCP-1、IL-10、IL-6、IFN-β的变化为指标,探讨金银花抗RSV肺炎的作用机制。采用UPLC-Q-Exactive-Orbitrap-MS对金银花中成分进行表征。采用网络药理学,将已定性成分-靶点网络与RSV肺炎-靶点网络进行映射,构建成分-RSV肺炎靶点网络。同时利用STRING数据库筛选网络中核心靶点,导入Cytoscape 3.6.1软件,构建核心靶点PPI网络,计算拓扑参数,推测核心靶点。结果： 与模型组比较,各金银花给药组小鼠体质量均增加,肛温及肺指数均下降,炎性因子（IFN-γ、TNF-α、MCP-1、IL-10、IL-6）含量增加或减少,且有显著性差异;对金银花中39个成分进行表征,结合网络药理学获得成分对应靶点277个,疾病靶点390个,去重后生成PPI网络,PPI网络中根据度值确定核心靶点为TNF、AKTI、TLR4、IL-10、IL-2。结论： 通过网络药理技术和整体动物实验推测,金银花可以通过下调TLR4/NK-κB通路,减少TNF-α、IL-6、IL-10等炎性因子的表达;作用于T细胞、自然杀伤细胞进而上调免疫因子IFN-γ的表达;降低MCP-1含量,减少炎性损伤、巨噬细胞的产生来协同发挥抗RSV感染肺炎的作用。     

“芩部丹”中三种单体对结核分枝杆菌作用下TLR2表达的影响

目的观察"芩部丹"中3种单体对巨噬细胞TLR2的调节作用,以探寻其抗结核的可能作用机制。方法取黄芩苷、对叶百部碱、丹参多酚酸盐单体作用于感染结核杆菌的U937单核巨噬细胞,分别采用RT-QPCR、FACS方法检测用药前后巨噬细胞表面TLR2的表达差异。结果与模型组相比,黄芩苷、对叶百部碱、丹参多酚酸盐均具有上调TLR2表达的作用。结论 "芩部丹"中3种单体抗结核作用可能通过纠正TLR2的表达而发挥作用。     

紫花苜蓿总黄酮对LPS诱导乳腺上皮细胞炎性因子的抑制作用

目的:研究紫花苜蓿总黄酮对LPS诱导小鼠乳腺上皮细胞炎症反应的抑制作用及机理。方法:1μg/m L LPS刺激细胞之前,分别用50μg/m L、100μg/m L和200μg/m L紫花苜蓿总黄酮预先处理细胞1.5h;采用MTT法测定细胞的活性;分别利用qRT-PCR法和Western boltting法检测炎性因子TNF-α和IL-6 mRNA表达和NF-κBp56蛋白表达。结果:紫花苜蓿总黄酮可以抑制NF-κB核转位,降低TNF-α和IL-6 mRNA的表达量,提高细胞的活力。结论:紫花苜蓿总黄酮可以通过下调NF-κB信号通路抑制炎性因子的产生。     

阿托伐他汀治疗对2型糖尿病患者单核细胞toll样受体4表达和炎性因子的影响

目的：观察2型糖尿病患者单核细胞toll样受体4表达和炎性因子的变化情况,以及阿托伐他汀对其表达的影响。方法：选取2013年1月至2015年1月期间收治的60例2型糖尿病患者作为观察组,并选取同期在我院进行健康体检的60例正常人作为对照组。采用流式细胞仪检测单核细胞toll样受体4表达;采用ELISA(酶联免疫吸附)分别对血清相关因子水平进行测定。将观察组中的患者分为A组(n=30)和B组(n=30),其中A组患者予以常规的降糖治疗,B组患者在常规治疗基础上予以阿托伐他汀药物进行治疗。分别对两组患者的单核细胞toll样受体4表达和炎性因子水平变化情况进行客观的评定和比较,同时对两组患者治疗后临床疗效进行对比。结果：较对照组而言,观察组的单个核细胞TRL4 mRNA和蛋白表达以及血浆TNF-α等水平明显升高(P<0.05);与治疗后相比,A组患者的单个核细胞TRL4 mRNA和蛋白表达以及血浆TNF-α等水平无显著差异(P>0.05),而B组患者治疗后的单个核细胞TRL4 mRNA和蛋白表达以及血浆TNF-α等水平较治疗前明显降低,且更低于A组患者(P<0.05)。结论：阿托伐他汀可以降低血清炎性因子的水平,且可以下调2型糖尿病患者的单核细胞toll样受体4表达,其的抗炎和抑制TLR4效果显著,可以发挥治疗作用。     

黄芩苷下调沙眼衣原体感染小鼠TLR2和TLR4基因表达的研究

目的:研究黄芩苷对沙眼衣原体(Chlamydia trachomatis,Ct)感染小鼠宫颈组织Toll样受体2和Toll样受体4(TLR2,TLR4)基因表达的影响。方法:6~8周龄雌性C57BL/6小鼠,分为5组:阿奇霉素组,模型组,黄芩苷高、中、低剂量组。通过阴道接种Ct感染小鼠,建立Ct感染小鼠生殖道的动物模型。接种前7 d皮下注射黄体酮以增加小鼠对Ct感染的敏感性。通过比较各组小鼠阴道分泌物中的Ct数量,评价黄芩苷抗沙眼衣原体感染的能力。感染27 d之后,用RT-PCR法和Western Blot法检测宫颈组织TLR2和TLR4基因表达情况。结果:在阴道接种Ct后第3天和第6天,模型组小鼠阴道分泌物中Ct数量显著升高。通过黄芩苷治疗后,各药物组小鼠阴道分泌物中,Ct数量均明显少于模型组,黄芩苷高剂量组和阿奇霉素组显著性降低。同时,黄芩苷还可明显抑制各组宫颈组织内TLR2和TLR4基因的表达。结论:黄芩苷可有效地抑制Ct感染的小鼠宫颈组织TLR2和TLR4的高表达,这可能是黄芩苷抗Ct感染的作用机制之一。     

巨噬细胞移动抑制因子siRNA对肺泡上皮细胞Toll样受体及其下游信号转导通路的影响

目的:研究巨噬细胞移动抑制因子siRNA对脂多糖刺激的肺泡上皮细胞Toll样受体(TLR)及其下游信号转导通路的影响和作用机制.方法:用LPS刺激A549细胞,脂质体法分别将巨噬细胞移动抑制因子(MIF) siRNA和非特异性siRNA加入A549细胞进行干预.RTPCR法检测MIF和TLR2、TLR4 mRNA的表达,Western Blot法分析TLR下游信号转导通路元件髓样分化因子88 (MyD88)和干扰素调节因子3(IRF3)蛋白表达,细胞免疫荧光法观察NF-κB核迁移,ELISA法测定细胞培养上清炎症介质TNF-α、IL-1β、IL-6,免疫介质IFN-β分泌水平.结果:MIF siRNA对LPS刺激诱导的MIF和TLR2、TLR4 mRNA高表达有显著的下调作用和部分阻断NF-κB(p65)核迁移.LPS刺激诱导后,MIF siRNA显著降低MyD88蛋白表达和炎症介质TNF-α、IL-1β和IL-6的分泌水平,但MIF siRNA对IRF3蛋白表达和免疫介质IFN-β分泌水平无影响.结论:MIF siRNA能抑制A549细胞的炎症介质TNF-α、IL-1β、IL-6过度分泌,其机制可能是MIF siRNA降低了LPS刺激A549细胞诱导的MIF和TLR2、TLR4高表达,及阻断TLR下游信号转导通路元件MyD88和NF-κB核迁移.     

健脾清化中药复方对大鼠慢性萎缩性胃炎TLR4-MyD88依赖途径蛋白表达及TNF-α的影响

目的观察健脾清化中药复方对慢性萎缩性胃炎(CAG)大鼠TLR4及下游My D88依赖途径相关蛋白表达及炎性因子TNF-α的影响,探讨健脾清化中药复方治疗CAG的分子机制。方法将53只Wistar大鼠随机分为空白组8只和CAG造模组45只,以"氨水+去氧胆酸钠+乙醇"法复制CAG大鼠模型。确认造模成功后,将造模组余下40只CAG大鼠随机分为模型组、维酶素组、中药低、中、高剂量组,各8只。各组给予相应药物灌胃,连续30 d。HE染色观察病理组织学改变,Western blot法检测TLR4、My D88、NF-κB、COX-2的蛋白表达量,ELISA法检测血清中TNF-α含量。结果模型组大鼠TLR4、My D88、NF-κB、COX-2蛋白表达水平明显增高(P<0.01),血清TNF-α含量明显增高(P<0.01)。与模型组比较,健脾清化中药复方低、中、高剂量组胃黏膜病变明显改善,TLR4、My D88、NF-κB、COX-2蛋白表达水平均明显下降(P<0.05或P<0.01),血清TNF-α含量降低(P<0.05或P<0.01)。结论健脾清化中药复方可有效改善CAG大鼠胃黏膜组织病理改变,其治疗机制可能与降低组织中TLR4-My D88依赖途径中相关蛋白表达,以及抑制炎症因子的表达有关。     

KAE神经保护及抗神经炎症作用机制

目的神经炎症与神经退行性疾病的发生密切相关,KAE是一种具有抗炎活性的黄酮类化合物,本研究评价KAE的抗神经炎症作用,并探讨其作用机制。方法 LPS刺激BV2细胞建立细胞水平神经炎症模型;LPS腹腔注射小鼠、侧脑室注射建立动物水平神经炎症模型;ELISA法检测炎症相关因子水平;Griess试剂检测NO水平;RT-PCR及Western蛋白印迹法检测炎症相关蛋白的表达;RNA-seq、非标记定量蛋白质组学分析方法探讨KAE调控靶点和信号通路。结果在LPS刺激BV2细胞模型上,KAE显著降低LPS诱导的BV2细胞上清中炎症因子水平,下调LPS诱导的BV2细胞中炎症蛋白TLR4和MYD88的表达。在LPS刺激小鼠模型上,KAE显著改善纹状体神经元损伤,提高纹状体TH和PSD95水平;抑制脑纹状体小胶质细胞的活化,降低IL-1β,IL-6,TNF-α,MCP-1和ICAM-1水平,抑制COX-2,TLR4和My D88等炎症蛋白的表达,保护血脑屏障的完整。在LPS侧脑室注射大鼠模型上,KAE显著降低纹状体组织中炎症因子水平,RNA-seq、非标记定量蛋白质组学分析方法检测发现KAE调控靶点和信号通路。结论 KAE具有神经保护及抗神经炎症作用,机制与下调TLR4/My D88炎症通路、保护血脑屏障相关。     

Artesunate in combination with oxacillin protect sepsis model mice challenged with lethal live methicillin-resistant Staphylococcus aureus (MRSA) via its inhibition on proinflammatory cytokines release and enhancement on antibacterial activity of oxacillin

Sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA) has worse outcome because of multiresistance to a large group of antibiotics, which may lead to death from septic shock. In the present study, we firstly found that artesunate in combination with oxacillin was capable of protecting mice challenged with live MRSA WHO-2 (WHO-2) and the protection was related to the reduced TNF-α and IL-6 levels and decreased bacterial load. Based on above results, artesunate was further investigated from two aspects in vitro, anti-inflammation effect and antibacterial enhancement effect on antibiotics. Artesunate not only inhibited TNF-α and IL-6 release but also inhibited mRNA and protein expressions of TLR2 and Nod2, two important receptors, in murine peritoneal macrophages stimulated with heat-killed WHO-2, further demonstrating anti-inflammatory effect of artesunate was related to the inhibition of TLR2- and Nod2-mediated proinflammatory cytokines. Significantly, artesunate enhanced antibacterial activity of oxacillin and ampicillin not levofloxacin against WHO-2 and a clinical MRSA strain; the fractional inhibitory concentration indexes were lower than 0.5. Further, artesunate possessed moderate affinity for penicillin-binding protein 2a (PBP2a) and reduced the mecA mRNA expression up-regulated by oxacillin, suggesting that artesunate's enhancement on antibacterial activity of β-lactams was related to the inhibition of PBP2a and down-regulation of mecA mRNA expression. In conclusion, our results demonstrated that artesunate in combination with oxacillin protected mice challenged with lethal live MRSA via its inhibition on proinflammatory cytokines release and enhancement on antibacterial activity of oxacillin. Artesunate could be further investigated as a candidate drug for MRSA sepsis.     

金茵清热口服液对人PBMC中TLR2信号通路的调节机制研究

目的：研究金茵清热口服液（JYQR）对人外周血单个核细胞（PBMC）中Toll样2受体（TLR2）信号通路免疫调节作用机制。方法：体外分离、培养人PBMC,设空白对照组和JYQR组,JYQR组加入JYQR刺激PBMC,与空白组经37℃,5% CO₂培养24 h后,分别提取各组PBMC中总RNA、总蛋白、细胞上清液,RT-PCR和Western Blot法检测空白对照组和JYQR组PBMC中相关信号分子TLR2、MyD88、IRAK4、TRAF6、IRF3 mRNA和蛋白的表达情况,ELISA法检测细胞因子IL-1α、IL-6、TNF-α、IFN-α的分泌水平;分别设空白对照组：不作处理;JYQR组：JYQR 2.015 mg·mL^-1;TLR2激动剂组：Pam3CSK4 300 ng·mL^-1;TLR2抑制剂组：OxPAPC 30 μg·mL^-1+JYQR 2.015 mg·mL^-14组,提取核蛋白和浆蛋白,Western Blot法检测IRF3和NF-κB p65的核转位情况。结果：与空白对照组相比,JYQR组中TLR2、IRAK4、TRAF6、IRF3 mRNA表达水平分别上调5.93倍、1.77倍、2.95倍、1.43倍（^*P<0.05,^**P<0.01）;MyD88、TRAF6蛋白的表达分别上调1.26倍、1.24倍（^*P<0.05）,IRAK4和IRF3的相对表达水平差异无统计学意义;细胞因子IL-1α、IL-6的表达下调55%、61%,TNF-α、IFN-α的分泌水平上调4.3倍、2.9倍（^*P<0.05,^**P<0.01）。JYQR预处理PBMC后,IRF3及NF-κB核转位增加,但是经过TLR2抑制剂预处理后,IRF3和NF-κB的核转位减弱;相反,TLR2激动剂预处理可以进一步上调IRF3和NF-κB的核转位。结论：金茵清热口服液对人PBMC中TLR2信号通路的作用机制可能是通过MyD88依赖型信号通路激活中、下游的IRAK4、TRAF6、IRF3以及NF-κB调控终端效应因子发挥免疫调节作用。     

中药热毒清对人巨细胞病毒抑制作用的实验与临床研究(英文)

目的:探讨中药热毒清对人巨细胞病毒(HCMV)体内外的抑制作用及机制。方法:采用定量逆转录聚合酶链反应(RT-PCR)方法检测中药热毒清干预前后感染细胞HCMV晚期mRNA的表达水平,观察感染细胞的病变进展,并在体外实验的基础上将热毒清应用于HCMV活动性感染孕妇的临床治疗,测定治疗前后血清肿瘤坏死因子α(TNF-α)和白细胞介素2(IL-2)的水平。结果:热毒清对     

肿瘤坏死因子-α对原代大鼠肾近端小管上皮细胞Toll样受体2表达的调控及机制

目的探讨肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)对原代大鼠肾近端小管上皮细胞Toll样受体2(tolllike receptor2,TLR2)表达的调控以及核因子-κB(nuclearfactor-κB,NF-κB)在其中的作用。方法体外分离与培养原代大鼠肾近端小管上皮细胞,以TNF-α按不同时间给予刺激,Western blot检测TLR2,I-κBα,磷酸化I-κBα(pI-κBα),GAPDH蛋白水平。分别以TNF-α、NF-κB特异性抑制剂Bay11-7082处理25h、Bay11-7082预处理1h后加入TNF-α刺激24h,检测TLR2表达的变化。结果TNF-α刺激6~24h,TLR2高于基线水平;Bay11-7082处理组和Bay11-7082+TNF-α处理组的TLR2蛋白表达水平高于对照组。Bay11-7082+TNF-α处理组与单独TNF-α处理组TLR2蛋白表达水平无差异。TNF-α刺激后5~120min,pI-κBα蛋白水平升高。结论TNF-α促进原代大鼠肾近端小管上皮细胞TLR2蛋白表达,NF-κB对TLR2的表达可能起负调节作用。     

Microglia and cyclooxygenase-2: possible therapeutic targets of progesterone for stroke

Previous studies have demonstrated that progesterone (PROG) may be a pleiotropic neuroprotective agent. Although there have been reports about the neurotoxicity of activated microglia and cyclooxygenase-2 (COX-2) in animal models of ischemic stroke, the influence of PROG on the activation of microglia and the expression of COX-2 after stroke has not been examined in detail. In this investigation, we carried out research about the influence of PROG on cultured microglia by detection of the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in their supernatant fluid before and after induced with lipopolysaccharide (LPS) or influenced by PROG with Enzyme-Linked Immunosorbent Assay technique in vitro. Moreover, the expression of COX-2 and ionized calcium-binding adapter molecule 1 (Iba1) was also detected in the cortex of rats that underwent permanent middle cerebral artery occlusion and received PROG or vehicle treatment by immunohistochemistry and western blot technique. The results revealed that PROG significantly reduced the expression of TNF-α and IL-1β in cultured microglia after activated with LPS in vitro. In addition, PROG also valuably inhibited the expression of Iba1 and COX-2 after stroke in vivo. These observations raised the possibility that PROG can exert its neuroprotective effects by inhibiting the activation of microglia and the over expression of COX-2 after stroke.     

小檗碱对Toll样受体2信号通路和炎症因子的影响

目的:研究小檗碱(Ber)对Toll样受体2(TLR2)信号通路和炎症因子的影响。方法:100mol·L-1Ber作用于细菌脂蛋白(BLP)刺激的RAW264.7细胞,RT-PCR法检测TLR2表达;Western-blot法检测抗核因子κB抑制性蛋白(IκB)磷酸化水平;ELISA法检测炎症因子分泌情况。结果:作用6、12h时,Ber可促进TLR2的表达、IκB的激活和促炎因子IFNγ、TNFγ的分泌;作用24、48h时,Ber可抑制TLR2的表达、IκB的激活和促炎因子IFNγ、TNFα的分泌;且Ber可能具有促进抗炎因子IL-10和IL-13分泌的作用。结论:Ber能够双向调控TLR2信号通路的激活和促进抗炎因子的分泌,从而起到抗菌消炎的作用。     

葡萄糖及细胞因子对胰岛素瘤相关蛋白-2表达水平的影响

目的探讨葡萄糖、肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ对胰岛细胞自身抗原胰岛素瘤相关蛋白(IA)-2表达水平及胰岛素分泌水平的影响。方法以不同浓度的葡萄糖(3、7、11、30mmol/L)、10ng/mlIFN-γ及100ng/mlTNF-α分别刺激胰岛细胞系RIN5F24h和48h,用反转录聚合酶链反应(RT-PCR)方法检测各组RIN5F细胞中IA-2的mRNA表达水平,并用放射免疫法检测上清中胰岛素的浓度。结果RT-PCR结果显示以不同浓度葡萄糖(3、7、11、30mmol/L)分别刺激RIN5F细胞系后IA-2mRNA水平呈浓度依赖性和时间依赖性增加;TNF-α可抑制IA-2mRNA的水平,并且作用48h后IA-2表达水平显著降低(P<0.05);IFN-γ作用24h及48h均可显著抑制IA-2的表达(P<0.05)。TNF-α或IFN-γ刺激RIN5F细胞系后,上清中胰岛素水平均较基础值降低,并且作用48h后差异有统计学意义(P<0.05)。结论葡萄糖可使IA-2的mRNA水平呈剂量依赖性和时间依赖性增加,TNF-α、IFN-γ可抑制IA-2的mRNA表达和胰岛素分泌浓度。     

肺炎克雷伯菌引起大鼠小气道上皮细胞损伤及与肺内α肿瘤坏死因子关系的探讨

目的探讨肺炎克雷伯菌引起大鼠小气道上皮细胞损伤及机制。方法将Wistar大鼠随机分为两组:对照组和感染组采用光镜动态观察小气道上皮细胞损伤改变。以免疫组化及原位分子杂交分析方法,检测小气道上皮细胞α肿瘤坏死因子(TNF-α)蛋白和TNF-αmRNA表达的变化。并用放射免疫分析法测定肺组织中TNF-α含量。结果显示感染组大鼠小气道上皮细胞病理改变明显。感染第2周起至第4周小气道壁各种炎症细胞增加,可见上皮细胞部分脱落坏死,损伤明显加重。感染组在2～4周TNF-αmRNA表达与对照组比较显著增强(P <0 .0 1)。在第4周TNF α蛋白明显增高(P <0 .0 1)。从第1周起肺组织中TNF-α含量增加并保持到第8周。结论肺炎克雷伯菌在体内可引起小气道上皮细胞损伤,肺内TNF-α水平升高与气道上皮细胞损伤程度平行。并且TNF-α蛋白和TNF-αmRNA表达上调在加重上皮细胞损伤中又具有重要作用     

Differential effects of Gram-positive versus Gram-negative bacteria on NOSII and TNFalpha in macrophages: role of TLRs in synergy between the two

1. Gram-negative and Gram-positive bacteria are sensed by Toll-like receptor (TLR)4 and TLR2, respectively. TLR4 recruits MyD88 and TRIF, whereas TLR2 recruits MyD88 without TRIF. NOSII and TNFalpha are central genes in innate immunity and are thought to be differentially regulated by the MyD88 versus TRIF signalling pathways. Here, we have used Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and highly selective TLR ligands to establish the precise relationship between TLR2, TLR1, TLR6 and TLR4 for NOSII versus TNFalpha induction. 2. In murine macrophages at 24 h, E. coli or LPS (TLR4) induced NO and TNFalpha release. In contrast, S. aureus (TLR2/TLR1/TLR6) or Pam(3)CSK4 (TLR2/TLR1), or FSL-1 and LTA (TLR2/TLR6) induced TNFalpha without an effect on NO. 3. At later time points (48-72 h), S. aureus induced NO release. The ability of S. aureus, but not E. coli or LPS, to induce NO release was inhibited by anti-TNFalpha-binding antibodies. 4. At 24 h, LPS synergised with TLR2 ligands to induce NO release and NOSII protein expression. LPS also induced the expression of TLR2 gene expression without affecting levels of TLR4. 5. Using cells from TLR2(-/-) or TLR4(-/-) mice, the ability of LPS to synergise with S. aureus or Pam(3)CSK4 was found to be dependent on both TLR2 and TLR4. 6. These observations are the first to clearly delineate the role of separately activating TLR2 and TLR4 in the induction of NOSII and TNFalpha genes compared with their coinduction when both receptor pathways are activated.     

Poly(I:C) reduces expression of JAM-A and induces secretion of IL-8 and TNF-α via distinct NF-κB pathways in human nasal epithelial cells

Human nasal epithelium is an important physical barrier and innate immune defense protecting against inhaled substances and pathogens. Toll-like receptor (TLR) signaling, which plays a key role in the innate immune response, has not been well characterized in human nasal epithelial cells (HNECs), including the epithelial tight junctional barrier. In the present study, mRNAs of TLR1-10 were detected in hTERT-transfected HNECs, which can be used as an indispensable and stable model of normal HNECs, similar to primary cultured HNECs. To investigate the changes of tight junction proteins and the signal transduction pathways via TLRs in HNECs in vitro, hTERT-transfected HNECs were treated with TLR2 ligand P₃CSK₄, TLR3 ligand poly(I:C), TLR4 ligand LPS, TLR7/8 ligand CL097, TLR8 ligand ssRNA40/LyoVec, and TLR9 ligand ODN2006. In hTERT-transfected HNECs, treatment with poly(I:C) significantly reduced expression of the tight junction protein JAM-A and induced secretion of proinflammatory cytokines IL-8 and TNF-α. Both the reduction of JAM-A expression and the induction of secretion of IL-8 and TNF-α after treatment with poly(I:C) were modulated by distinct signal transduction pathways via EGFR, PI3K, and p38 MAPK and finally regulated by a TLR3-mediated NF-κB pathway. The control of TLR3-mediated signaling pathways in HNECs may be important not only in infection by viral dsRNA but also in autoimmune diseases caused by endogenous dsRNA released from necrotic cells.