Expression of let-7i is associated with Toll-like receptor 4 signal in coronary artery disease: effect of statins on let-7i and Toll-like receptor 4 signal

Toll-like receptor (TLR) 4 signal plays an important role in immunity in coronary artery disease (CAD). A recent report has demonstrated that one of the let-7 family microRNAs, let-7i, directly regulates Toll-like receptor 4 (TLR4) expression and contributes to immune response. The aim of this study was to determine whether let-7i is expressed with TLR4 in patients with CAD, and whether statins (atorvastatin or rosuvastatin) might affect these levels. To determine the effects of let-7i on TLR4 expression, human THP-1 cells transfected with let-7i were analyzed for TLR4 levels. This study included 98 patients with CAD and 48 subjects without CAD (non-CAD). Patients with CAD were randomized to 12 months of treatment with atorvastatin or rosuvastatin. Monocytes were obtained from peripheral blood at baseline and after 12 months of each type of therapy. Levels of let-7i and TLR4 were measured by real-time RT-PCR and FACS. Functional approaches to let-7i showed that transfection of let-7i into human THP-1 cells resulted in regulation of TLR4 expression. Levels of let-7i were lower in the CAD group than in the non-CAD group (0.98±0.42 vs. 4.65±1.21, P<0.01). There was a negative correlation between let-7i and TLR4 levels in patients with CAD (let-7i vs. TLR4 mRNA: r=-0.60, P<0.01; let-7i vs. TLR4 MFI: r=-0.32, P<0.01). The atorvastatin group had markedly increased let-7i levels and diminished TLR4 levels (all P<0.01), whereas the rosuvastatin group showed no change in these levels. This study suggests that atorvastatin down-regulates TLR4 signal via let-7i expression in CAD patients, possibly contributing to the beneficial effects of atorvastatin on let-7i-mediated TLR4 signal in this disorder.     

TLR4与TNF-α在脑缺血再灌注小鼠海马CA1区神经元中的表达

目的:探讨Toll样受体4(TLR4)在脑缺血再灌注损伤炎症反应中的作用。方法:采用抗TLR4抗体封闭阻断TLR4,应用HE染色观察小鼠海马CA1区组织病理学改变、Western Blot和RT-PCR检测海马TLR4蛋白和mRNA表达量,免疫组化方法检查TNF-α表达。小鼠随机分为3组,即假手术组(S组)、缺血再灌注组(I组)和TLR4阻断组(T组),各组又分12,24,48和72h4个时间点组。结果:缺血再灌注组TLR4蛋白、TLR4 mRNA和TNF-α表达水平明显高于假手术组表达水平(P0.05),而TLR4阻断组TLR4蛋白、TLR4 mRNA和TNF-α表达水平明显少于缺血再灌注组(P0.05)。相关分析表明,TLR4 mRNA表达水平与TNF-α含量呈显著正相关(P0.01)。结论:本结果提示缺血再灌注可激活TLR4,TLR4在脑缺血再灌注损伤中起重要作用;炎性因子TNF-α的产生、分泌可能与TLR4 mRNA表达存在密切联系。     

骨髓间充质干细胞下调类风湿性关节炎小鼠脾脏单个核细胞TLR8及TLR9的表达

背景：间充质干细胞能够缓解类风湿性关节炎小鼠的症状,但是其机制还不清楚。 目的：观察骨髓间充质干细胞对类风湿性关节炎小鼠脾脏单个核细胞表达TLR8及TLR9等的影响。 方法：DBA/1J小鼠随机分3组,非造模组不造模,阳性对照组和实验组制备Ⅱ型胶原诱导的小鼠类风湿性关节炎模型。实验组尾静脉注射移植大鼠骨髓间充质干细胞。 结果与结论：与阳性对照组比较,实验组小鼠关节直径明显减小,关节炎症细胞浸润程度明显降低,但比非造模组略高;小鼠脾脏单个核细胞表达TLR8、TLR9和白细胞介素1β的水平较阳性对照组明显降低(P < 0.01或P < 0.05);阳性对照组与实验组中TLR8与TLR9的表达均无明显相关性(P > 0.05)。说明骨髓间充质干细胞下调了类风湿关节炎小鼠脾脏单个核细胞表达TLR8及TLR9的水平,但TLR8与TLR9的表达无相关性。     

miR-146a在大鼠缺血再灌注肝损伤中的表达及作用

目的:评价肝组织微小RNA-146a(miR-146a)表达变化与大鼠缺血再灌注(I/R)炎性肝损伤的关系。方法:72只SD大鼠随机分为非手术组(N组)、假手术组(S组)及I/R组,按分组处理后分别检测各组大鼠0、2、12和24 h(每组各时点n=6)血浆丙氨酸转氨酶(ALT)活性及白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)含量,real-time PCR检测肝组织Toll样受体4(TLR4)、IL-6、TNF-α和miR-146a表达,Western blot分析肝组织中TLR4、IL-6和TNF-α表达。结果:N组大鼠各时点血浆ALT活性及IL-6和TNF-α含量与肝组织中TLR4、IL-6和TNF-α表达量无显著差异。S组大鼠血浆IL-6和TNF-α含量升高(P0.01),但血浆ALT活性与肝组织TLR4、IL-6和TNF-αm RNA及蛋白的表达量却无明显变化。I/R后,大鼠血浆ALT活性及IL-6和TNF-α含量明显升高(P0.01),肝组织TLR4、IL-6和TNF-αm RNA及蛋白的表达量亦显著升高(P0.01),且随着时间延长,各指标仍持续升高;肝组织miR-146a表达量显著下降,其中在缺血12 h时下降最为明显,到24 h表达量再次升高,但始终低于I/R前(P0.01)。N组及S组大鼠各时点肝组织miR-146a表达量显著高于I/R组I/R后相应时点的表达(P0.01)。结论:I/R大鼠肝组织miR-146a表达下降的同时存在TLR4信号通路的激活及炎性肝损伤的发生。     

Toll样受体在重症肌无力患者胸腺组织中的表达及其临床意义

目的 研究Toll样受体(Toll-like receptor,TLRs)在重症肌无力(myasthenia gravis,MG)患者胸腺组织中的表达,分析其与MG发生、发展的关系.方法 收集我科2007年7月至2008年2月手术治疗的36例MG患者胸腺标本,分为胸腺瘤组、瘤旁胸腺组、非瘤MG胸腺组,以无MG的正常胸腺21例为对照(正常胸腺组),采用逆转录-PCR技术,检测TLR2、TLR3、TLR4在胸腺组织中的mRNA转录表达水平,进一步采用实时定量逆转录-PCR检测TLR4 mRNA转录表达水平以及与MG临床特点间的关系.结果 逆转录-PCR结果显示,TLR2 mRNA和TLR3 mRNA在4组之间的差别没有统计学意义,TLR4 mRNA的表达在4组之间的差别有统计学意义.进一步的实时定量逆转录.PCR检测TLR4 mRNA转录表达的结果显示,瘤旁胸腺组的TLR4 mRNA的表达比胸腺瘤组有明显的增加(0.8214±0.1019 vs 0.7101±0.0916,P=0.005),非瘤MG胸腺组TLR4 mRNA的表达量比正常胸腺组明显增加(0.8544±0.1200 vs 0.6851±0.1524,P=0.018),瘤旁胸腺组和非瘤MG胸腺组TLR4 mRNA的表达量之间的差别没有统计学意义,且都比正常胸腺组明显增加;TLR4 mRNA的表达与患者的Osserman分型呈正相关(R=0.609;P=0.004).结论 TLR4异常表达可能在MG发病中有重要作用,MG合并胸腺瘤患者瘤旁胸腺组织中TLR4的表达状态对胸腺瘤患者是否并发MG可能有重要影响.     

革兰氏阴性、阳性菌感染后外周血单个核细胞TLR4、TLR2的表达

目的:研究革兰氏阴性菌(G- 菌)、革兰氏阳性菌(G+ 菌)感染患者与正常人外周血单个核细胞Toll样受体4(TLR4 )mRNA、TLR2mRNA的表达情况。方法:提取经检验科体液镜检或培养阳性,证实为G- 菌、G+ 菌感染患者35例,正常对照2 4例。运用Taqman实时定量PCR方法测定G- 菌、G+ 菌感染患者外周血单个核细胞的TLR4mRNA、TLR2mRNA并测定表达水平,正常人作为对照。结果:G- 菌感染患者较正常对照和G+ 菌感染外周血单个核细胞TLR4mRNA表达显著升高(P <0 .0 1) ;G- 菌感染伴发热较不伴发热患者TLR4表达显著升高(P <0 . 0 1)。G+ 菌感染患者TLR4表达与正常对照无显著差异(P>0 .0 5 )。G+ 菌、G- 菌感染患者和正常对照之间TLR2mRNA表达无显著差异(P >0 . 0 5 )。结论:TLR4在G- 菌感染患者外周血单个核细胞中表达升高,在G- 菌感染伴发热的患者中升高尤为显著,表明TLR4是G- 菌的识别受体。在临床上当G- 菌感染尚未或不能证实时,可通过TLR4mRNA表达水平的检测,为患者是否存在G- 菌感染提供依据。     

脓毒症婴幼儿Toll样信号传导途径相关因子改变的临床意义

目的 探讨脓毒症婴幼儿Toll样信号传导途径相关因子改变的临床意义。方法 选取广东省肇庆市端州区妇幼保健院和广州市儿童医院2009年6月至2011年3月收治脓毒症患儿20例（脓毒症组）,重症脓毒症感染性休克及多器官功能障碍综合征患儿20例（严重脓毒症组）,以同时期20例健康婴幼儿为健康对照组。用流式细胞仪检测外周血单个核细胞(PBMC)表面Toll样受体2(TLR2)、TLR4的表达情况;酶联免疫吸附法检测血浆中前炎性细胞因子肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)的浓度变化。结果脓毒症组和严重脓毒症组婴幼儿外周血PBMC表面TLR2、TLR4的表达阳性率比健康对照组均明显增高[(52.48±27.37)％和（80.56.±42.95）％比(15.63±9.28)％;(26.58±14.26)％和(49.32±23.74)％比(2.84±2.0r7)％;均P＜0.01];严重脓毒症组婴幼儿PBMC表面TLR2、TLR4的表达明显高于脓毒症组(P＜0.01)。与健康对照组比较,脓毒症组血浆TNF-α和IL-6水平均明显增高,且严重脓毒症组较脓毒症组升高更加明显(P＜0.01)。结论 TLR2和TLR4可能共同参与了婴幼儿脓毒症时机体对病原微生物的识别,激活TLR信号通路。婴幼儿脓毒症时TLR2和TLR4可能介导了血清前炎性因子TNF-α,IL-6的产生和释放。     

Toll样受体4与肿瘤坏死因子α在脑缺血再灌注小鼠顶叶皮质神经元中的表达及意义

目的:探讨Toll样受体4(TLR4)在脑缺血再灌注损伤炎症反应中的作用.方法:采用TLR4抗体封闭TLR4受体,H-E染色观察小鼠顶叶皮质组织病理学改变、免疫印迹法检测顶叶皮质TLR4蛋白表达、RT-PCR检测顶叶皮质TLR4 mRNA表达量、免疫组织化学显色法检测顶叶皮质TNF-α表达情况,TLR4 mRNA表达水平与TNF-α含量相关分析.小鼠随机分为假手术组、缺血再灌注组和TLR4阻断组,各组又分12、 24、 48、 72h 4个时间点组.结果:缺血再灌注组TLR4蛋白、TLR4 mRNA、 TNF-α表达水平明显高于假手术组表达水平,而TLR4阻断组TLR4蛋白、TLR4 mRNA、 TNF-α表达水平明显低于缺血再灌注组,相关分析表明,TLR4 mRNA表达水平与TNF-α含量呈显著正相关.结论:缺血再灌注可激活TLR4,TLR4在脑缺血再灌注损伤中起重要作用;炎性因子TNF-α的产生、分泌可能与TLR4 mRNA表达存在着密切联系.     

Possible involvement of toll-like receptor 4 in endothelial cell activation of larger vessels in response to lipopolysaccharide.

Toll-like receptors (TLRs) serve as recognition and signaling elements for bacterial substances. To examine the role of TLRs in endothelial cells of larger vessels in lipopolysaccharide (LPS)-induced signaling, the expression and function of TLRs in human umbilical vein endothelial cells (HUVEC) were analyzed. A high level of TLR4 mRNA expression was found in HUVEC, human peripheral blood mononuclear cells (PBMC) and human monocyte cell line THP-1 cells. Little or no TLR2 mRNA expression was observed in HUVEC. In contrast, strong TLR2 mRNA expression was observed in PBMC and THP-1 cells. Moderate and high levels of TLR1 mRNA expression were found in HUVEC, PBMC and THP-1 cells, respectively. TLR3 mRNA expression was moderate in PBMC but weak in HUVEC and THP-1 cells. Little or no TLR5 and RP105 mRNA expression was observed in HUVEC, whereas a moderate level was detected in PBMC and THP-1 cells. The LPS-induced E-selectin expression in HUVEC was significantly inhibited by pretreatment with an anti-TLR4 mAb. Preincubation of HUVEC with an anti- TLR4 mAb significantly reduced the LPS-induced IL-6 production. LPS induced E-selectin and IL-6 production by HUVEC only in the presence of human serum, suggesting the involvement of soluble CD14. Anti-CD14 mAb strongly inhibited the LPS-induced E-selectin and IL-6 production by HUVEC. The inhibition with the concomitant treatment with anti-TLR4 and anti-CD14 mAbs was stronger than that with anti-CD14 mAb only, although it was slight. These results show that TLR4 in the presence of soluble CD14 plays a major role in the signaling of LPS in endothelial cells of larger vessels.     

瑞香素对人PBMC的细胞因子和TLRs mRNA表达的影响

目的观察瑞香素对人外周血单个核细胞的细胞因子和Toll样受体表达的影响,研究瑞香素增强淋巴细胞功能的作用机制。方法分离人外周血T、B细胞和单核细胞,分别加入瑞香素,37℃,5%CO2培养48 h后,采用实时荧光定量PCR检测瑞香素对T细胞IL-2、IFN-γ,B细胞IL-12,单核细胞IL-1 mRNA及T、B细胞TLR1～10的mRNA表达的影响;同时检测先用抗TLR4单抗封闭,再用瑞香素处理细胞后上述细胞因子和TLRs的mRNA表达变化。结果瑞香素能明显上调T细胞IL-2及IFN-γ、B细胞IL-12、单核细胞IL-1 mRNA和T细胞TLR1、TLR4,B细胞TLR4、TLR9 mRNA的表达(P﹤0.05),其中T细胞IL-2、B细胞IL-12、单核细胞IL-1 mRNA的表达和T细胞TLR4,B细胞TLR4、TLR9 mRNA表达可被抗TLR4单抗部分阻断。结论瑞香素增强淋巴细胞的免疫功能的可能机制是通过上调T细胞TLR1、TLR4,B细胞TLR4、TLR9的表达,分别参与其细胞内信号转导,从基因转录水平促进T、B细胞分泌细胞因子。     

Toll样受体7及I型干扰素通路在系统性红斑狼疮中的作用研究

探讨Toll样受体7(TLR7)及I型干扰素(IFN-α)通路在系统性红斑狼疮(SLE)发病中的作用。采用实时荧光定量PCR方法检测42例SLE患者和34例正常人外周血TLR7mRNA以及4个干扰素调节基因mRNA的表达水平,同时观察TLR7mRNA的表达量与SLE疾病活动相关指标和干扰素积分(IFN score)的关系。结果,SLE患者外周血TLR7mRNA的表达水平显著增高;TLR7mRNA的表达水平与SLEDAI积分、肾脏损伤指数、抗双链DNA(dsDNA)抗体、抗RNA相关抗体水平及干扰素积分呈正相关;与补体C3、C4、白细胞数呈负相关。TLR7—IFN-α通路可能参与了SLE的病理过程。     

Effect of high mobility group box 1 on Toll-like receptor 9 in B cells in myeloperoxidase-ANCA-associated vasculitis

Abstract

High mobility group box 1 (HMGB1) played pathogenic role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Recent findings demonstrated that Toll-like receptor 9 (TLR9) was involved in B cell tolerance breaking of autoimmune disease, including AAV. Here, we investigated the effect of HMGB1 on TLR9 in B cells of AAV. In the present work, patients with myeloperoxidase (MPO)-AAV in active stage were recruited. Intracellular TLR9 expression in various B cell subpopulations of the whole blood was detected by flow cytometry and the correlation with clinical data was analysed. Our results showed that intracellular TLR9 expression in B cells, memory B cells and plasmablasts correlated with erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP). In particular, TLR9 expression in plasma cells correlated with ESR, CRP, serum creatinine, eGFR, and Birmingham Vasculitis Activity Score. To further explore the effect of HMGB1 on B cell, peripheral blood mononuclear cells (PBMCs) from AAV patients were isolated. After stimulated with HMGB1, TLR9 expression in various B cell subpopulations and proliferation ratio of live B cells were analysed by flow cytometry. We found that TLR9 expression in plasma cells and the proliferation ratio of live B cells by HMGB1 stimulation were significantly upregulated compared with the control group. Therefore, TLR9 expression in plasma cells was associated with disease activity of MPO-AAV. HMGB1 could enhance TLR9 expression in plasma cells and B cell proliferation. These indicated a role of HMGB1 on TLR9 in B cells in MPO-AAV, which would provide potential clues for intervention strategies.     

Age-related changes and distribution of T cell markers (CD3 and CD4) and toll-like receptors(TLR2, TLR3,TLR4 and TLR7) in the duck lymphoid organs

T lymphocytes and Toll-like receptors have been confirmed to have correlation with the ability to resistance to pathogenic challenges and play an important role in duck immune system. However, the information of ontogeny of T lymphocytes and Toll-like receptors is scarcely in duck. Therefore, to address these questions, we report the development and distribution of CD3 and CD4 by immunocytochemistry and the age-related mRNA level of duck T cell markers (CD3 and CD4) and Toll-like receptors (TLR2, TLR3, TLR4 and TLR7) by real time quantitative PCR in duck lymphoid organs (thymus, bursa of Fabricius and spleen). Results indicated that CD3 and CD4 positive cells can be observed in all test organs and partly change in an age-related way. CD4 positive T cell of duck spleen mainly distributed in periarterial lymphatic sheaths and red pulp, not in white pulp. Both of CD3 and CD4 were experienced significant increased wave twice in duck lymphoid organs and T cell dependent cellular immunity of duck may well established until 5 weeks old. The mRNA expression levels of duck TLRs were age and organ dependent, and duck TLR3 and TLR7 were significantly lower abundance in the spleen but higher in thymus and bursa of Fabricius, respectively. This study provide the essential knowledge of the ontogeny of T cells and Toll-like receptors in duck, which may shed lights on the T-cell mediate immunity and innate immunity in duck.     

急性脑梗死患者PBMC中TRAM、TLR4、IRF-3mRNA水平与NIHSS评分关系及预测预后的价值

目的 探究急性脑梗死(acute cerebral infarction,ACI)患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中Toll样受体相关分子(TRAM)、Toll样受体4(TLR4)、 干扰素调节因子-3(IRF-3)mRNA表达水平与斑块稳定性、 神经...     

Inflammatory unbalance of TLR3 and TLR4 in PCI patients with or without type 2 diabetes mellitus

Background

Toll-like receptors, the most characterized innate immune receptors, have recently been demonstrated to play an important role in coronary atherosclerotic disease and diabetes mellitus (DM). TLR3 and TLR4 are known to act as anti-inflammatory and pro-inflammatory factors respectively in multi-factorial inflammatory disease states. However, there is less research about TLR3 and TLR4 expression in percutaneous transluminal coronary intervention (PCI) patients, particularly those with type 2 diabetes mellitus (DM2).

Methods

We examined TLR3 and TLR4 expression and their downstream signaling pathway in PCI patients with (n = 31) or without (n = 32) DM2 compared with controls (n = 35).

Results

TLR3 and downstream anti-inflammatory factors (IRF-3, INF-β and IL-10) were significantly down-regulated in PCI patients with or without DM2 compared with controls, as determined by the quantification of both mRNA and protein. In contrast, TLR4 and downstream proinflammatory factors (MyD88 and TNF-α) were up-regulated in PCI patients with or without DM2 compared with controls.

Conclusions

Patients undergoing PCI were shown to have a TLR-dependent pro-inflammatory state, mediated by a downregulation of TLR3 pathway, and upregulation of TLR4. This occurred in both with or without type 2 diabetes mellitus compared with controls in this research. The inflammatory imbalance observed in PCI patients was exacerbated in patients with DM2, consistent with a likely contribution of DM2 to the inflammatory state of coronary atherosclerotic disease, via impact on the innate immune response. This data supports the potential of TLRs as a novel therapeutic target in diabetics with coronary atherosclerotic disease.