严重脓毒症患者外周血单个核细胞Toll样受体2、4和髓样分化因子88在胸腺肽α1治疗过程中的基因表达变化

目的 观察严重脓毒症患者外周血单个核细胞Toll样受体（TLR）2、4基因及其信号传导通路上的髓样分化因子88（MyD88）基因表达水平在胸腺肽α1治疗过程中的变化。方法 54例严重脓毒症患者被随机分为对照组（常规治疗,28例）和治疗组（胸腺肽α1治疗,26例）,采用实时荧光定量PCR监测两组患者入院时及治疗后第3、7天外周血单个核细胞TLR2、TLR4和MyD88基因表达水平变化,并观察两组的28 d病死率。结果 治疗组的28 d病死率虽低于对照组,但差异无统计学意义（23.1%vs 35.7%,P=0.310）。治疗组的患者单核细胞TLR2、TLR4和MyD88基因表达水平逐渐上升,而对照组未见该规律,与对照组相比,治疗后第3天时治疗组的TLR2（2.31±0.79 vs 1.83±0.51）、TLR4基因表达增高（7.31±0.79 vs 6.55±0.92）;第7天时治疗组TLR2（2.75±1.17 vs 1.63±0.36）、TLR4（7.75±1.03 vs 6.39±0.72）和MyD88（4.26±0.77 vs 3.77±0.68）水平明显增高,均P〈0.05。结论 胸腺肽α1在治疗严重脓毒症患者过程中可以上调患者单核细胞的TLR2、4和MyD88基因的表达水平。     

TLR4/MyD88依赖性通路和Caveolin-1的表达与2型糖尿病神经病变患者炎症状态的相关性

目的探讨单核细胞Toll样受体4(TLR4)/下游髓样分化蛋白(MyD88)依赖性通路和陷窝蛋白-1(Caveolin-1)的表达与2型糖尿病神经病变患者炎症状态的相关性。方法 120例受试者均分为三组:健康志愿者组(NC组)、2型糖尿病不伴有神经病变组(TDM组)和2型糖尿病伴有神经病变组(DPN组);单次抽取各研究对象清晨空腹血8ml;应用密度梯度离心法分离和提取外周血单核细胞;分别通过RT-PCR检测Caveolin-1mRNA和TLR4表达,Western blot检测Caveolin-1和TLR4及其下游信号(MyD88、IκB、p-IκB)蛋白表达,ELISA检测血浆肿瘤坏死因子α(TNF-α)和白细胞介素-6(IL-6)的水平。结果与TDM和NC组比较,DPN组患者血浆TNF-α、IL-6和TLR4及其下游信号蛋白表达明显增加(P0.05),而Caveolin-1表达明显降低(P0.05);Spearman相关性分析显示TLR4mRNA与TNF-α(r=0.723,P0.01)和IL-6(r=0.486,P0.01)呈正相关,且与TNF-α密切程度高于IL-6,与Caveolin-1mRNA呈负相关(r=-0.782,P0.01)。结论 DPN患者炎症状态与单核细胞TLR4/MyD88依赖性通路介导的炎症级联反应相关;低表达的Caveolin-1与DPN患者TLR4/MyD88依赖性通路异常活化有关。     

血管紧张素Ⅱ通过TLR4-MyD88途径诱导大鼠肾小管上皮细胞炎性因子释放

目的 观察血管紧张素Ⅱ（AngⅡ）刺激肾小管上皮细胞（NRK-52E）后肿瘤坏死因子α（TNF-α）和热休克蛋白47（HSP47）的表达,分析Toll样受体4（TLR4）信号通路的变化与上述因子的关联,探讨AngⅡ促进NRK-52E细胞炎性反应、纤维化的天然免疫机制。 方法 细胞同步化后,将其分为4组：对照组、AngⅡ（10-7 mmol/L）组、坎地沙坦（10-5 mmol/L）+AngⅡ组、TLR4阻断剂（20 mg/L）+AngⅡ组,培养6 h后RT-PCR法检测TLR4及转接信号髓分化因子88（MyD88）mRNA表达水平;12 h后免疫荧光法检测细胞表面TLR4蛋白表达;24 h后以ELISA法检测细胞上清液TNF-α及HSP47的浓度。 结果 与对照组相比,AngⅡ显著上调NRK-52E细胞 TLR4、MyD88 mRNA和TLR4蛋白表达（均P < 0.01）,并诱导细胞TNF-α和HSP47的释放（均P < 0.01）。与AngⅡ组相比,TLR4阻断剂和坎地沙坦干预均显著抑制 AngⅡ对细胞TLR4、MyD88的刺激效应（均P < 0.01）;坎地沙坦抑制AngⅡ诱导的细胞 TNF-α、HSP47的释放（均P < 0.01）,TLR4阻断剂对细胞 TNF-α、HSP47的下调呈剂量依赖性。 结论 AngⅡ对NRK-52E细胞天然免疫信号TLR4、MyD88具有激活效应,该信号激活可能是AngⅡ促进肾小管细胞炎性相关因子释放的重要机制之一。坎地沙坦抑制肾小管细胞炎性因子的体外效应也与其调节该信号通路有关。     

Toll样受体4与肾缺血再灌注损伤关系研究进展

Toll样受体4（Toll-like receptor4,TLR4）是一种介导免疫反应的病原分子识别受体,通过MyD88（myeloid differentiation factor 88）依赖途径和MyD88非依赖途径两条主要信号转导通路介导的炎症级联反应引起肾脏缺血再灌注（renal ischemia reperfusion injury,RIRI）.通过阻断信号转导通路上的相关因子,可以抑制炎症级联反应的发生发展,减少炎症因子的产生,从而为临床治疗肾缺血再灌注损伤提供新思路.     

TLR4/MyD88信号通路在膝骨关节炎滑膜炎中的作用

Toll样受体4是重要的固有免疫模式识别受体,通过识别损伤相关分子模式及病原相关分子模式,启动髓样分化因子 88依赖途径后激活NF-κB信号通路,引发炎性介质及细胞因子的分泌,触发固有免疫应答,在膝骨关节炎滑膜炎的发生中发挥作用。研究TLR4/MyD88信号通路在膝骨关节炎滑膜炎中的作用,以期更深入的阐明膝骨关节炎滑膜炎发病机制,为膝骨关节炎滑膜炎的诊疗提供理论依据和参考。     

Toll样受体与炎症性肠病关系研究进展

炎症性肠病（IBD）包括溃疡性结肠炎（UC）和克罗恩病（CD）,其病因和发病机制至今尚未完全明确。近几年来研究显示,炎症性肠病与易感个体在肠腔内正常菌群诱导下过度激活黏膜免疫系统有关。Toll样受体家族（TLRs）在肠黏膜免疫中发挥重要的作用,通过识别病原相关分子结构（PAMPs）激活固有免疫。不同的TLRs识别不同的配体,通过MyD88依赖型信号传导通路或MyD88非依赖型信号传导通路引起不同的效应。近年来研究涉及TLRs的多态性与IBD联系。重点介绍不同TLRs的分布、表达及TLRs多态性与IBD的联系。     

七氟醚预处理对大鼠肺缺血-再灌注损伤的保护作用及对TLR4/MyD88/NF-κB信号通路的影响

目的探讨七氟醚预处理对肺缺血-再灌注损伤(IRI)的保护作用及对Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核因子(NF)-κB信号通路的影响。方法将40只健康成年SD大鼠随机分为对照组(Sham组)、模型组(LIRI组)、七氟醚预处理组(Sev组)和TLR4抑制剂TAK-242联合七氟醚预处理组(TAK+Sev组),每组各10只。采用苏木素-伊红(HE)染色观察肺组织病理学变化并进行病理损伤评分;采用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记(TUNEL)法检测肺组织细胞凋亡并计算细胞凋亡率;测定肺组织湿重/干重(W/D)比值以确定肺组织含水量;检测肺组织中氧化应激相关指标水平以及肺组织和血清中炎症因子水平;采用蛋白质印迹法检测肺组织中TLR4/MyD88/NF-κB信号通路相关蛋白的表达水平。结果与Sham组比较,LIRI组和Sev组大鼠肺组织病理损伤评分、W/D比值、细胞凋亡率、丙二醛(MDA)水平、炎症因子水平以及TLR4、MyD88、NF-κB p65蛋白相对表达量均升高,超氧化物歧化酶(SOD)水平和NF-κB抑制蛋白α(IκBα)相对表达量均降低(均为P0.05);与LIRI组比较,Sev组和TAK+Sev组大鼠肺组织病理损伤评分、W/D比值、细胞凋亡率、MDA水平、炎症因子水平以及TLR4、MyD88、NF-κB p65蛋白相对表达量均降低,SOD水平和IκBα相对表达量均升高(均为P0.05);与Sev组比较,TAK+Sev组大鼠肺组织病理损伤评分、W/D比值、细胞凋亡率、MDA水平、炎症因子水平以及TLR4、MyD88、NF-κB p65蛋白相对表达量均降低,IκBα相对表达量升高(均为P0.05)。结论七氟醚预处理能够抑制TLR4/MyD88/NF-κB信号通路的激活,抑制炎症反应和氧化应激,从而有效减轻肺IRI。     

Toll样受体4与肾脏纤维化研究进展

<正>肾脏纤维化是各种慢性肾脏病进展至终末期肾病的共同病理特征,由多种细胞、细胞因子及生长因子共同参与,最终导致细胞外基质合成增多而致纤维化,纤维化的发生发展中与炎症反应共存[1,2]。Toll样受体4(toll-like receptor4,TLR4)是人类发现的第一个TLR相关蛋白,TLR4通过识别病原相关分子模式的特有抗原成分而进行信号识别和传导,在急性炎症反应的调节、细胞信号的转导和纤维化发生发展中起重要作用。目前研究提示,TLR4是介导肾脏炎症及纤维化的重要信号分子,TLR4与其配体结合后,可通过激活My D88依赖的信号通路和非My D88依赖的信号通路(TRIF通路),诱导炎症及致纤维化等因子的激活及释放,从而导致肾脏纤维化的发生发展。     

TLR4介导大鼠肾脏缺氧复氧炎症反应依赖于MyD88

目的：探讨MyD88是否参与了TLR介导大鼠肾脏缺氧复氧（hypoxia/reoxygenation,H/R）引起的炎症反应.方法：体外分离获得Wistar大鼠原代肾小管上皮细胞（proximal tubule epithelial cells,PTECs）,建立H/R模型.采用TLR4siRNA干扰PTECs,检测白细胞介素8（interleukin-8,IL-8）和肿瘤坏死因子α（tumor necrosis factor α,TNF-α）的表达.之后,加入髓样分化因子88（myeloid differentiation factor 88,MyD88）的抑制剂ST2825后,检测IL-8和TNF-α的表达.结果：TLR4表达沉默后,IL-8、TNF-α和MyD88的表达均显著下降（P〈0.05）;加入ST2825后,IL-8、TNF-α和MyD88的表达均显著下降（P〈0.05）,且抑制MyD88活性后显著降低细胞的凋亡水平（P〈0.05）.结论：MyD88参与了TLR4介导的大鼠肾脏缺氧复氧引起的炎症反应.     

TLR4/NF-κB通路与骨质疏松症关系的研究进展

骨质疏松症(osteoporosis, OP)是一种普遍存在的与年龄相关的骨骼疾病，雌激素缺乏和衰老是主要病因，近年来越来越多的研究表明炎症反应在OP的发生发展过程中发挥重要作用。Toll样受体4(toll like receptor 4,TLR4)是Toll样受体家族的主要成员，是内毒素最重要的模式识别受体。TLR4是经典的炎症信号转导通路，当TLR4信号通路激活，与核因子κB (nuclear factorκB,NF-κB)转移到细胞核并释放炎性细胞因子(TNF-α、IL-1β、IL-6等),并抑制骨髓间充质干细胞成骨分化及钙盐沉积，促进破骨细胞成熟，最终导致OP。对TLR4信号通路与OP关系的研究不仅可以从炎症角度深入揭示OP的发病机制，还可以为OP的防治提供新的思路。     

Exogenous administration of PACAP alleviates traumatic brain injury in rats through a mechanism involving the TLR4/MyD88/NF-κB pathway

Pituitary adenylate cyclase-activating polypeptide (PACAP) is effective in reducing axonal damage associated with traumatic brain injury (TBI), and has immunomodulatory properties. Toll-like receptor 4 (TLR4) is an important mediator of the innate immune response. It significantly contributes to neuroinflammation induced by brain injury. However, it remains unknown whether exogenous PACAP can modulate TBI through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. In this study, we investigated the potential neuroprotective mechanisms of PACAP pretreatment in a weight-drop model of TBI. PACAP38 was microinjected intracerebroventricularly before TBI. Brain samples were extracted from the pericontusional area in the cortex and hippocampus. We found that TBI induced significant upregulation of TLR4, with peak expression occurring 24?h post-trauma, and that pretreatment with PACAP significantly improved motor and cognitive dysfunction, attenuated neuronal apoptosis, and decreased brain edema. Pretreatment with PACAP inhibited upregulation of TLR4 and its downstream signaling molecules MyD88, p-IκB, and NF-κB, and suppressed increases in the levels of the downstream inflammatory agents interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), in the brain tissue around the injured cortex and in the hippocampus. Administration of PACAP both in vitro and in vivo attenuated the ability of the TLR4 agonist lipopolysaccharide (LPS) to increase TLR4 protein levels. Therefore, PACAP exerts a neuroprotective effect in this rat model of TBI, by inhibiting a secondary inflammatory response mediated by the TLR4/MyD88/NF-κB signaling pathway in microglia and neurons, thereby reducing neuronal death and improving the outcome following TBI.     

TH1 Immune Responses to Fully MHC Mismatched Allografts are Diminished in the Absence of MyD88, a Toll-Like Receptor Signal Adaptor Protein

Toll‐like receptors (TLRs) are innate immune receptors that are critical for recognizing conserved microbial motifs by inducing TH1 immunity. The majority of TLRs utilize the adaptor protein MyD88 for signal transduction, although other adaptors have been recently described. As the role of innate immunity in transplantation is unclear, we examined the importance of the MyD88 pathway in acute rejection of fully MHC‐mismatched murine allografts and specifically investigated whether MyD88 signaling is important for DC (dendritic cell) function and TH1 alloimmune responses. Our results demonstrate that acute rejection of both fully allogeneic skin and cardiac allografts occurs in the absence of MyD88. However, priming of naïve recipient T cells by allogeneic DCs and TH1 immune responses were diminished in the absence of MyD88, although TH2 immunity remained intact. Thus, these results demonstrate that MyD88 signaling is important for DC function and TH1 responses during fully MHC‐mismatched solid‐organ transplantation, although graft rejection occurs independently of MyD88.     

Myeloid differentiation factor 88-silenced bone marrow-derived dendritic cells exhibit enhanced tolerogenicity in intestinal transplantation in rats

Myeloid differentiation factor 88 (MyD88) is a key adaptor of Toll-like receptor signaling, which plays a critical role in dendritic cell (DC) function. However, its role in the induction of transplant tolerance by immature DCs is unknown. In this study, we silenced MyD88 expression of bone marrow-derived immature DCs with small interference RNA demonstrating that this maneuver significantly prolonged the survival of intestinal allografts. This study provided evidence that the absence of MyD88 enhances the tolerogenicity of DCs and suggested that inhibiting innate immunity may be a potential strategy to facilitate induction of transplant tolerance by DCs.     

Rezeptoren und Signalproteine des angeborenen Immunsystems als neue Zielstrukturen der Sepsistherapie

During polymicrobial sepsis,microbial pathogens and their products activate the innate immune system through signaling receptors of the Toll-like receptor (TLR) family, resulting in hyperinflammation and organ injury.The analysis of preclinical mouse models has shown that inactivation of the common TLR signaling adaptor protein MyD88 prevents the hyperinflammatory response and improves survival.Importantly, MyD88 deficiency does not impair antibacterial defense mechanisms.Thus,TLRs and proteins involved in TLR signaling may represent interesting targets for the development of new drugs for reprogramming pathophysiological immune responses during sepsis.     

Deferoxamine preconditioning activated hypoxia-inducible factor-1α and MyD88-dependent Toll-like receptor 4 signaling in intestinal stem cells

Background/Purpose

Toll-like receptors (TLRs) are important regulators of innate immunity, and TLR4 pathway can regulate the survival, migration, and differentiation of stem cells, including intestinal stem cells (ISCs). Deferoxamine (DFO), a hypoxia-mimic compound, can activate the proliferation of ISCs. In this study, we investigated the response of TLR4 signaling to DFO-induced hypoxia in cultured ISCs in vitro.

Angiotensin II up-regulates the release of inflammation factors via Toll-like receptor 4 signal in rat mesangial cells under high glucose

Objective To observe the regulation of Toll-like receptor 4 (TLR4) signal and the release of inflammation factors after angiotensin II (AngⅡ) stimulation in rat mesangial cells under high glucose condition, revealing the innate immune-related mechanism of injury by AngⅡ on mesangial cells under high glucose. Methods After synchronization, cells incubated with AngⅡ(10-7 mmo/L) and/or high glucose (25 mmol/L) were used as the stimulation group, cells without stimulation were as normal control (5.6 mmol/L glucose). To determine the role of TLR4 and the adaptor myeloid differentiation factor 88 (MyD88), equal number of HBZY-1 cells were added with 10-5 mmol/L irbesartan and/or TLR4 blocker (10 mg/L) for 1 h and then incubated with AngⅡ (10-7 mmo/L) and/or high glucose (25 mmol/L) for 12 h or 24 h respectively. Real-time PCR was used to analyze TLR4 mRNA and MyD88 mRNA expression after 12 h. Immunofluorescence was used to observe TLR4 protein expression after 24 h; Western blotting was used to observe TLR4, MyD88 and nuclear factor κB (NF-κB) protein; ELISA was used to detect the concentration of MCP-1, IL-6 in cell supernatant respectively. Results Compared with normal control group, TLR4 mRNA and MyD88 mRNA were highly expressed in high glucose or AngⅡ-induced HBZY-1 cells (P＜0.01), TLR4, MyD88 and NF-κB protein as well as MCP-1, IL-6 were also up-regulated significantly (P＜0.01). Compared with high glucose or AngⅡ group, MyD88 and NF-κB protein as well as MCP-1, IL-6 were further up-regulated markedly in AngⅡ and high glucose costimulated group (P＜0.01)．In HBZY-1 cells that were preincubated with irbesartan and/or TLR4 blocker, TLR4 and MyD88 protein expression were obviously inhibited, IL-6 and MCP-1 production were also decreased remarkably compared with high glucose and/or AngⅡ group (P＜0.01). Conclusions High glucose and AngⅡ stimulate the release of proinflammatory factors in rat glomerular mesangial cells via TLR4-MyD88 pathway. This process is inhibited by irbesartan or TLR4 blocker via modulation of the signal. AngⅡ has the positive-regulation potential on the release of inflammation factors via TLR4 signal in rat mesangial cells under high glucose condition.     

Human umbilical cord mesenchymal stem cells co-culture ameliorates the innate immunity of podocytes induced by high glucose

Objective To explore the effects of human umbilical cord mesenchymal stem cells(HUC-MSCs) on the innate immunity of podocytes mediated by Toll-like receptor (TLR) signaling pathway under high glucose (HG) condition. Methods Podocytes were divided into four groups according to the treatment: normal glucose group (NG), mannitol control group (NG+MA), high glucose group (HG) and HUC-MSCs co-culture group (HG+MSC). After 72 hours treatment, the protein levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), heat shock protein 70 (HSP70), high-mobility group box-1 (HMGB1) in culture medium were measured by ELISA. Real-time PCR was used to detect the mRNA expressions of TLR2 and TLR4. Western blotting was used to detect the protein expressions of TLR2, TLR4, myeloid differentiation factor 88 (MyD-88) and phospho-P65 (p-P65). Immunofluorescence staining was used to study the localization of p-P65 in podocytes. Results High glucose induced the inflammation of podocytes by activating the TLR signaling, which increased the secretion of IL-6, TNF-α, HSP70, HMGB1, the mRNA level of TLR2, TLR4 and the protein level of TLR2, TLR4, MyD-88 and p-P65 (all P＜0.05). High glucose also activated NF-κB and induced its nuclear translocation. HUC-MSCs co-culture decreased the inflammation and restrained the TLR signaling. Conclusions HUC-MSCs co-culture decreases the inflammation and innate immunity of podocytes induced by HG.     

Induction of Alloimmune Tolerance in Heart Transplantation Through Gene Silencing of TLR Adaptors

Toll‐like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell (DC) maturation and initiation of adaptive immune responses that provoke allograft rejection. We aimed to prolong allograft survival by selectively inhibiting expression of the common adaptors of TLR signaling, namely MyD88 and TRIF, using siRNA. In vitro we demonstrated that blocking expression of MyD88 and TRIF led to reduced DC maturation. In vivo treatment of recipients with MyD88 and TRIF siRNA significantly prolonged allograft survival in the BALB/c > C57BL6 cardiac transplant model. Moreover, the combination of MyD88 and TRIF siRNA along with a low dose of rapamycin further extended the allograft survival (88.8 ± 7.1 days). Tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction and hemorrhage in mice treated with MyD88 and TRIF siRNA vector plus rapamycin. Furthermore, treatment was associated with an increase in the numbers of CD4⁺CD25⁺FoxP3⁺ regulatory T cells and Th2 deviation. To our knowledge, this study is the first demonstration of prolonging the survival of allogeneic heart grafts through gene silencing of TLR signaling adaptors, highlighting the therapeutic potential of siRNA in clinical transplantation.     

Impact of TLR4-siRNA transfection on the expression of TLR4/MyD88 Signaling in rat tubular epithelial cells under high glucose condition

Objective To observe the expression of toll like receptor 4(TLR4) Signaling and the release of inflammation factors in rat tubular epithelial cell(NRK-52E) under high glucose condition after TLR4-siRNA transfection. Methods Three TLR4-siRNA sequences were designed and synthesized. The transfection efficiency was observed by fluorescence microscope after transfection, and the expression of TLR4 mRNA was detected by real time PCR. The most effective siRNA was selected to be used for forward experiments. After transfection for 24 h, cells were stimulated with 25 mmol/L glucose and/or 10-7 mmol/L Angiotension Ⅱ(AngⅡ) for 12 h, 24 h; cells without stimulation were as normal control. Real-time PCR was used to analyze TLR4 and myeloid differentiation factor 88 (MyD88) mRNA expression; Western blot was used to observe TLR4/MyD88 and NF-κB protein expression. ELISA assay was used to detect the concentration of monocyte chemoattractant protein-1(MCP-1), interleukin-6(IL-6) in cell supernatant after cells were stimulated for 24 h. Results TLR4/MyD88 mRNA and TLR4/MyD88/NF-κB protein were highly expressed under high glucose or AngⅡ co-incubated NRK-52E(P＜0.01), the MCP-1 and IL-6 levels were also increased markedly compared with normal control group (P＜0.01). TLR4/MyD88 mRNA and TLR4/MyD88/NF-kB protein expressions were obviously inhibited in cells that were transfected with TLR4-siRNA compared with high glucose group(P＜0.01), MCP-1 and IL-6 production decreased remarkably compared with high glucose or AngⅡ co-stimulated group(P＜0.01). Conclusions High glucose can lead to the activation of TLR4/MyD88/NF-kB signaling and the secretion of inflammation factors in NRK-52E, AngⅡ further augments these effects. The effect can be blocked efficiently by specific siRNA gene silence. TLR4 signaling plays a pivotal role in the innate-immune inflammatory reaction in NRK-52E.