电针预处理对脑缺血再灌注损伤大鼠神经功能和缺血半暗区Tolls样受体4、核转录因子κB蛋白的影响

目的探讨电针预处理对大鼠脑缺血再灌注损伤后神经功能的影响及其机制。方法雄性Sprague-Dawley大鼠36只随机分为假手术组(n=12)、模型组(n=12)和电针预处理组(n=12)。后两组线栓法制备大鼠脑缺血2 h再灌注模型。电针预处理组在缺血前给予电针百会干预2周。再灌注24 h后,采用改良神经功能缺损评分进行评定,HE染色观察缺血侧脑组织脑损伤程度,Western blotting检测脑缺血半暗区Tolls样受体4(TLR4)、核转录因子κB(NF-κB)蛋白表达。结果与模型组比较,电针预处理组神经功能缺损评分降低(P0.05),脑组织损伤程度减轻,缺血半暗区TLR4、NF-κB蛋白表达下调(P0.05)。结论电针预处理通过下调缺血半暗区TLR4、NF-κB蛋白表达,诱导脑保护作用,降低炎性损伤程度,改善脑缺血再灌注损伤后大鼠的神经功能。     

关于子宫内膜异位症患者内膜中TLR4、MyD88、NF-κB表达的临床研究

目的通过观察子宫内膜异位症(EMs)患者内膜中TLR4、MyD88、NF-κB的表达,探究发生该疾病的可能机制。方法选取120例EMs患者,其中Ⅰ、Ⅱ期33例,Ⅲ、Ⅳ期87例;取异位内膜120例,在位内膜89例,取同期行宫腔镜手术患者正常内膜组织100例为正常对照组。采用蛋白免疫印迹法(Western blot)测定TLR4、MyD88、NF-κB的表达情况。结果异位内膜组、在位内膜组TLR4、MyD88、NF-κB蛋白表达水平均高于对照组,差异有统计学意义(P0.05),异位内膜组高于在位内膜组(P0.05)。不同分期EMs患者内膜组织中TLR4、MyD88、NF-κB表达水平比较,差异无统计学意义(P0.05)。结论TLR4、MyD88、NF-κB通路可能是EMs发病的重要机制。     

强直性脊柱炎髋关节病变中Toll样受体4表达的初步研究

目的 研究强直性脊柱炎（AS）髋关节病变中滑膜Toll样受体2和4（TLR2和TLR4）等受体的表达,以探讨髋关节病变的发病机制.方法 免疫组化染色法检测AS患者及对照组患者滑膜组织中TLR2、TLR4、髓样分化因子88（MyD88）、NFκB-p65表达情况.结果 AS组滑膜组织中TLR4、NFκB-p65表达阳性,TLR2、MyD88表达阴性,而对照组只NFκB-p65表达阳性,TLR2、TLR4、MyD88表达阴性.结论 AS患者髋关节病变存在TLR4高表达,并可能经MyD88非依赖性信号通路引起免疫炎症反应,导致髋关节破坏.     

阻断toll样受体对脂多糖致急性肺损伤的保护作用

目的：本文旨在探讨阻断toll样受体（TRL4）对脂多糖（LPS）致急性肺损伤（ALI）的保护作用。方法将60只健康雄性清洁级SD大鼠分为正常组、损伤组和阻断组,每组20只。损伤组大鼠采用尾静脉注射LPS（6mg/kg）复制ALI模型;阻断组同时注射TLR4抗体（10mg/mL）;正常组给予等容积生理盐水。3h后处死大鼠,采用凝胶滞留法检测核因子-κB（NF-κB）活性;Westernblot印记分析法检测抑制蛋白（IκB-α）水平;检测肺组织匀浆肿瘤坏死因子α细胞因子（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-10（IL-10）水平。结果与正常组相比,损伤组和阻断组肺组织湿质量/干质量比值增高,NF-κB活性升高,IκB-α、TNF-α、IL-1β水平升高（P＜0．05）,而IL-10水平降低（P＜0．05）。而比较损伤组与阻断组发现,阻断组肺组织湿质量/干质量比值降低,NF-κB活性降低,IκB-α、TNF-α、IL-1β水平明显低于损伤组（P＜0．05）,IL-10水平高于损伤组（P＜0．05）。结论采用抗TLR4单克隆抗体阻断TLR4介导的信号传导可明显降低NF-κB活性和IκB-α、TNF-α、IL-1β水平,同时提高IL-10水平阻断TLR4,对LPS致ALI有一定的保护作用。     

未足月胎膜早破孕妇胎盘中Toll样受体4/髓样分化因子/核因子-κB通路表达及临床意义

目的 探究并分析未足月胎膜早破（PPROM）孕妇胎盘中Toll样受体4(TLR4)/髓样分化因子（MyD88）/核因子-κB(NF-κB)通路表达及临床意义。方法 选取2017年6月—2020年6月湖北省妇幼保健院收治的PPROM患者90例，按照是否合并宫内感染将患者分为感染组（32例）和非感染组（58例），另选同期正常分娩孕妇50名作为对照组。对感染组患者阴道分泌物进行病原菌鉴定，收集PPROM患者临近破膜处胎盘组织和对照组正常胎盘组织，实时荧光定量聚合酶链反应（qRT-PCR）和Western blot法分别检测胎盘组织中TLR4/MyD88/NF-κB通路的mRNA表达和蛋白表达水平，受试者工作特征（ROC）曲线评估TLR4/MyD88/NF-κB通路对于PPROM并发宫内感染的诊断效能，并对影响PPROM并发宫内感染的危险因素进行分析。结果 32例感染患者阴道分泌物中检出78株病原菌，其中革兰阳性菌36株，占46.2%，革兰阴性菌33株，占42.3%，真菌9株，占11.5%。与对照组相比，非感染组和感染组患者胎盘组织中TLR4、MyD88和NF-κB的mRNA和蛋白水平均显著升...     

跑台训练对大鼠脑缺血再灌注损伤后胶质纤维酸性蛋白和脑源性神经营养因子表达的影响

目的观察跑台训练对大鼠脑缺血再灌注损伤后胶质纤维酸性蛋白(GFAP)和脑源性神经营养因子(BDNF)表达的影响。方法成年雄性Wistar大鼠30只随机分为假手术组、模型组和跑台训练组,每组10只。采用线栓法制备大脑中动脉阻塞2 h再灌注模型。假手术组插线10 mm后即刻退出。跑台训练组在造模成功后第3天进行跑台训练12 d,在造模后第4、8、15天采用改良神经功能缺损评分(m NSS)对各组大鼠评分,造模后第15天HE染色观察脑组织病理学变化,Western blotting检测BDNF和GFAP表达。结果造模后15 d,与模型组比较,跑台训练组m NSS评分明显降低(F=9.931,P0.01),缺血侧皮质脑组织病理损伤减轻,GFAP(t=6.73)和BDNF(t=3.78)表达明显升高(P0.01)。结论跑台训练可促进大鼠脑缺血再灌注损伤后GFAP和BDNF的表达,促进神经功能的恢复。     

β受体阻滞剂阻断TLR4炎症通路减轻脓毒症心肌损伤的研究

目的探讨β受体阻滞剂(艾司洛尔)对脓毒症大鼠的心肌保护作用及对Toll样受体4(Toll-like receptor4, TLR4)炎症通路的影响。方法选取雄性Wistar大鼠60只, 采用随机数字法随机分为假手术组(Shame组)、脓毒症组(CLP组)、艾司洛尔组(CLP+ES组)、TLR4抑制剂组(CLP+TAK-242组)每组15只。Shame组实施盲肠探查术, CLP组、CLP+ES组与CLP+TAK-242组通过盲肠结扎穿孔法(CLP法)建立脓毒症模型;CLP+ES组于CLP术后12 h给予腹腔注射艾司洛尔稀释液20 mg/kg;CLP+TAK-242组于上述相同时间点给予腹腔注射TAK-242 3 mg/kg;Shame组与CLP组给予等量生理盐水。各组均于术后24 h处死大鼠, 采集并处理标本。取心肌组织行苏木精-伊红染色观察大鼠心肌病理学变化;采用免疫组织化学法观察心肌组织中TLR4、髓样分化蛋白88(myeloid differentiation factor88, MyD88)及核转录因子-κB(nuclear factor-κB,NF-κB)的表达;采用马松染色(...     

跑台训练对大鼠脑缺血再灌注后脑组织基质金属蛋白酶-2和血管内皮生长因子表达的影响

摘要 目的：探讨跑台训练对大鼠脑缺血再灌注神经功能恢复和缺血脑组织中MMP-2和VEGF表达的影响。 方法：用线栓法制作Wistar大鼠大脑中动脉梗死再灌注模型,35只大鼠随机分为假手术组、跑台训练组和手术对照组。跑台训练和手术对照组又分为跑3天、跑7天、跑14天3个亚组,各亚组及假手术组每组5只大鼠。跑台组于术后第3天开始给予跑台训练,假手术组及手术对照组不予跑台训练。于跑3天、跑7天、跑14天3个时间点进行神经功能评估后处死大鼠。采用RT-PCR技术测定缺血区脑组织中MMP-2及VEGF的水平。 结果：跑台训练组在跑7天、跑14天神经功能评分明显低于对照组(P＜0.05)。跑台训练组缺血区脑组织在跑7天、跑14天MMP-2水平高于对照组（P＜0.05）,各时间点VEGF水平均高于对照组(P＜0.05)。 结论：跑台训练能通过上调MMP-2及VEGF的表达,促进血管形成和神经再生等,有利于脑损伤后神经功能的恢复。     

抑制核因子-κB对Toll样受体4在急性坏死性胰腺炎大鼠胰腺组织中表达的影响

目的：观察急性坏死性胰腺炎（ANP）大鼠胰腺组织中Toll样受体4（TLR4）的表达情况。并且应用核因子（NF）-κB活化抑制剂四氢化吡咯二硫代氨基甲酸酯（PDTC）进行干预，观察阻断NF-κB以后，TLR4在ANP大鼠胰腺组织中表达的变化，探讨ANP中TLR4与NF-κB的关系及NF-κB的活性对TLR4表达的影响。方法：72只SD大鼠随机分为假手术组、ANP组和PDTC预处理组。采用胆胰管内逆行注射5%牛磺胆酸钠溶液诱导大鼠ANP模型。PDTC预处理组在建立模型前1h按100mg·kg^-1腹腔注射给药。分别于建模后3h、6h、12h将大鼠分批处死。观察胰腺病理，用免疫组化及WesternBlot检测胰腺组织TLR4与NF-κB表达变化，RT-PCR检测胰腺组织TNFα、IL-1β和IL-6mRNA表达。结果：同假手术组相比，ANP组胰腺组织TLR4、NF-κB表达及TNFα、IL-1β和IL-6mRNA表达明显升高（P〈0.05）；同ANP组相比，PDTC预处理组胰腺组织炎症明显减轻，胰腺组织TLR4、NF-κB表达及TNFα、IL-1β和IL-6mRNA表达明显降低（P〈0.05）。结论：TLR4和NF-κB在ANP大鼠胰腺组织中的表达水平明显增高，PDTC抑制ANP大鼠胰腺组织中NF-κB表达的同时，下调TLR4蛋白水平表达和TNFα、IL-1β和IL-6mRNA表达。TLR4和NF-κB是介导炎症反应的枢纽，因而它们在ANP发生发展中起重要作用。TLR4和NF-κB可能不是简单的上下游关系，其关系还需进一步研究。     

MAPK通过调控TLR4/Myd88/NF-κB通路对妊娠期肠梗阻胃肠功能的影响

目的 分析MAPK通过调控TLR4/MyD88/NF-κB通路对妊娠期肠梗阻胃肠功能的影响。方法 选取105只SPF级Wistar大鼠,雌性、雄性分别为70、35只,将所有大鼠同笼放置后,共有33只雌性大鼠妊娠成功,将妊娠成功的8只作为空白组,剩余25只雌性大鼠建立肠梗阻模型,最终建模成功24只,将其分为模型组、上调组、下调组各8只。结果 与模型组相比,上调组MAPK表达量、VIP、GAS、TNF-α、IL-6水平、TLR4、Myd88、NF-κB mRNA表达量及相对表达量上升,MOT、IL-4、IL-10水平下降,下调组MAPK表达量、VIP、GAS、TNF-α、IL-6水平、TLR4、Myd88、NF-κB mRNA表达量及相对表达量下降,MOT、IL-4、IL-10水平上升（P <0.05）。结论 妊娠期肠梗阻大鼠经下调MAPK干预后胃肠功能改善,炎性反应减轻,其机制可能与TLR4/Myd88/NF-κB通路得到抑制有关。     

The future of toll-like receptor therapeutics

Since the discovery of human TLRs, manipulating the activity of these receptors to modulate immune responses for therapeutic purposes has initiated intense activity in the pharmaceutical industry. The focus of these activities has been largely in the areas of infectious diseases, cancer, allergic diseases and vaccine adjuvants. Although initial clinical trials for infectious diseases and cancer showed early promise, subsequent longer-term trials have been disappointing and more research is required to find dosing regimes that balance efficacy with acceptable side-effect profiles. As only a small number of doses are given, TLR agonists as vaccine adjuvants appear to hold greater potential and have less safety concerns than for other applications. Several innovative strategies for using TLR agonists in vaccine development are currently being pursued, and these are discussed in more detail in this review.     

Prophylactic and therapeutic implications of toll-like receptor ligands

The evolutionary conserved Toll-like receptors (TLRs) are the first identified and best characterized pattern recognition receptors (PRRs) which discriminate self from nonself, providing an early and effective immune response against invading pathogens. The ever expanding knowledge of TLR signaling network make it one of the most promising therapeutic strategies to modulate the immune response in various human diseases. Immune modulating strategies based on TLR-specific agonists elicit a potent immune response to adjuvant vaccine immunotherapy, cancers, allergic diseases, and chronic viral infections while minimizing the risk of uncontrolled provocation of systemic inflammatory response. Moreover, the contribution of TLR signaling in the pathogenesis of chronic noninfectious inflammatory and autoimmune diseases provides the rationale for the development and clinical implementation of TLR-specific antagonists. At present, a few TLR-specific agonists have been approved for both prophylactic and therapeutic applications, while the ongoing preclinical and clinical studies show promising results on various novel therapeutic molecules as an adjunctive to conventional pharmacotherapy or stand-alone therapeutic strategy.     

Toll样受体4与结直肠癌的相关性

Toll样受体4(toll-like receptor 4,TLR4)在炎症诱发的结直肠癌(colorectal cancer,CRC)中的作用日益受到关注.活化的TLR4通过髓样分化因子88 (myeloid differentiation factor 88,MyD88)激活核转录因子-κB (nuclear factor-κB,NF-κB)产生大量炎症介质并募集炎症细胞形成肿瘤微环境,从而成为炎症与肿瘤的纽带贯穿于CRC发生、发展的整个过程中.本文着重对TLR4信号通路参与CRC的起始、进展、转移、遗传变异和表观遗传调控进行了讨论.尤其,文章中强调了TLR4在CRC发展和发病机制中的角色,并提出调节TLR4信号通路可能为CRC的治疗带来新的前景.     

Increased toll-like receptor activity in patients with metabolic syndrome

OBJECTIVE

The metabolic syndrome (MetS) is highly prevalent and confers an increased risk for diabetes and cardiovascular disease (CVD). While MetS is a proinflammatory state, there is a paucity of data on cellular inflammation in MetS. Toll-like receptors (TLRs) are classical pattern recognition receptors of the innate immune response.

RESEARCH DESIGN AND METHODS

The aim of this study was to examine monocyte TLR2 and TLR4 in MetS patients without diabetes or CVD and control subjects since both of the receptors have been implicated in atherosclerosis and insulin resistance. Fasting blood was obtained for TLR expression and activity.

RESULTS

Circulating levels of high-sensitivity C-reactive protein, interleukin (IL)-1β, IL-6, IL-8, and soluble tumor necrosis factor receptor 1 (sTNFR1) were significantly increased in MetS versus control subjects following adjustment for waist circumference. There was a significant increase in both TLR2 and TLR4 surface expression and mRNA on monocytes after adjustment for waist circumference. In addition to increased nuclear factor-κB nuclear binding, there was significantly increased release of IL-1β, IL-6, and IL-8 in MetS versus control subjects following priming of the monocytes with lipopolysaccharides. While both plasma free fatty acids and endotoxin were increased in MetS, they correlated significantly with TLR4 only.

CONCLUSIONS

In conclusion, we make the novel observation that both TLR2 and TLR4 expression and activity are increased in the monocytes of patients with MetS and could contribute to increased risk for diabetes and CVD.Metabolic syndrome (MetS) affects one in three U.S. adults and confers an increased risk for both diabetes and cardiovascular disease (CVD) (1,2). It is clearly becoming apparent that, in addition to the diagnostic criteria used for MetS by the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III), both insulin resistance and low-grade inflammation, as documented by circulating biomarkers, are also evident (3–5). However, with regards to the low-grade inflammation, there is a paucity of data with regards to the cellular basis for the proinflammatory phenotype of MetS uncomplicated by diabetes and/or CVD (6). Recently, we showed that subcutaneous adipose tissue of nascent MetS patients without diabetes or CVD produced increased levels of adipokines compared with matched control subjects, and they correlated with both the proinflammatory state and the insulin resistance of these patients (6). However, the sentinel cell of inflammation and innate immunity is the monocyte/macrophage. There is scanty data on monocyte function in patients with MetS. Natal et al. (7) showed increased monocyte CD40L/CD40 expression in MetS but did not report on downstream signaling or biomediators of this dyad. In this study, we provide further evidence for the proinflammatory state of MetS by studying the classical pathogen recognition receptors of the innate immune response, Toll-like receptors (TLRs). The TLRs that we studied included TLR2 and TLR4 since both of the receptors have been incriminated in both atherosclerosis and insulin resistance (8–13).     

Antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes

Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thought of as a causative mechanism of septic shock pathology. Infections with Gram-negative or Gram-positive bacteria provide TLR2-specific agonists and are the major cause of severe sepsis. In order to intervene in TLR2-driven toxemia, we raised mAb's against the extracellular domain of TLR2. Surface plasmon resonance analysis showed direct and specific interaction of TLR2 and immunostimulatory lipopeptide, which was blocked by T2.5 in a dose-dependent manner. Application of mAb T2.5 inhibited cell activation in experimental murine models of infection. T2.5 also antagonized TLR2-specific activation of primary human macrophages. TLR2 surface expression by murine macrophages was surprisingly weak, while both intra- and extracellular expression increased upon systemic microbial challenge. Systemic application of T2.5 upon lipopeptide challenge inhibited release of inflammatory mediators such as TNF-alpha and prevented lethal shock-like syndrome in mice. Twenty milligrams per kilogram of T2.5 was sufficient to protect mice, and administration of 40 mg/kg of T2.5 was protective even 3 hours after the start of otherwise lethal challenge with Bacillus subtilis. These results indicate that epitope-specific binding of exogenous ligands precedes specific TLR signaling and suggest therapeutic application of a neutralizing anti-TLR2 antibody in acute infection.     

Regulation of cytokine and toll-like receptor signaling by SOCS family genes

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through the Toll-like receptor (TLR) 4. Regulation of TLR signaling is a key step for inflammation, septic shock and innate/adaptive immunity. TLR signaling is shown to be regulated by cytokines, such as interferon-gamma (positive) and interleukin-10 and IL-4 (negative). However, molecular mechanisms of the regulation of LPS signaling by cytokines have not been clarified. Cytokine signaling is regulated by CIS/SOCS family proteins. Both SOCS1 and SOCS3 can inhibit JAK tyrosine kinase activity. We demonstrate that SOCS1 and SOCS3 play an important regulatory role in macrophages and dendritic cells (DCs) by modulating TLR signaling. SOCS1 negatively regulates not only the JAK/STAT pathway, but also the TLR-NF-kappaB pathway. SOCS3 protein was strongly induced by both IL-6 and IL-10 in the presence of LPS, but selectively inhibited IL-6 signaling. Therefore lack of SOCS3 gene in macrophages resulted in suppression of TLR signaling by hyperactivation of STAT3.     

Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4.

Low molecular weight fragmentation products of the polysaccharide of Hyaluronic acid (sHA) produced during inflammation have been shown to be potent activators of immunocompetent cells such as dendritic cells (DCs) and macrophages. Here we report that sHA induces maturation of DCs via the Toll-like receptor (TLR)-4, a receptor complex associated with innate immunity and host defense against bacterial infection. Bone marrow-derived DCs from C3H/HeJ and C57BL/10ScCr mice carrying mutant TLR-4 alleles were nonresponsive to sHA-induced phenotypic and functional maturation. Conversely, DCs from TLR-2-deficient mice were still susceptible to sHA. In accordance, addition of an anti-TLR-4 mAb to human monocyte-derived DCs blocked sHA-induced tumor necrosis factor alpha production. Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-kappa B, all components of the TLR-4 signaling pathway. Blockade of this pathway by specific inhibitors completely abrogated the sHA-induced DC maturation. Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4. In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs.     

Toll样受体7激动剂对K562细胞抑制作用的研究

本研究旨在探讨Toll样受体(TLR)7激动剂gardiquimod对人慢性髓系白血病细胞K562的抑制作用。以异戊烯焦磷酸法扩增人外周血γδT细胞。用不同浓度的gardiquimod处理γδT细胞和K562细胞48 h,用MTT法检测细胞增殖情况。以流式细胞术检测gardiquimod处理前后K562细胞内TLR7表达、细胞周期及凋亡的变化。用CCK-8试剂盒检测γδT细胞对K562的杀伤活性。结果表明,gardiquimod 0.69-11.0μg/ml可明显促进γδT细胞增殖,在5.5-11.0μg/ml浓度下抑制K562细胞增殖;gardiquimod处理K562细胞后未检测到凋亡,但细胞周期有明显变化,而且处理后的K562细胞更易被γδT细胞杀伤。结论:gardiquimod能够抑制K562细胞增殖,阻滞细胞周期,并增强其对γδT细胞杀伤的敏感性。