阻塞性黄疸肠屏障功能变化研究进展

阻塞性黄疸最重要的病理基础是肠黏膜屏障损伤导致的内毒素血症,肠黏膜屏障损伤机制十分复杂,近年来对于胆汁及其成分的作用、氧化应激及炎性介质的影响、肠道微生态的变化方面取得了一定的进展,本文将对此作一综述.     

乌司他丁在梗阻性黄疸时保护肠黏膜屏障功能的实验研究

梗阻性黄疸引起肝细胞的损伤，释放多种细胞因子及炎性介质致肠黏膜屏障损伤，肠道细菌移位产生肠源性内毒素血症，可导致肝肾等多脏器功能衰竭。乌司他丁（ulinastatin，UTI）有抑制多种水解酶活性，抑制细胞因子和炎性介质的释放，本研究探讨UTI在梗阻性黄疸大鼠模型中保护肠黏膜屏障功能的作用机制。     

脂多糖介导的感染后免疫抑制

感染及ＭＯＤＳ是严重烧伤患者主要并发症和死亡原因。烧伤感染与广泛皮肤屏障破坏、大量体液丢失、组织缺血缺氧及肠道粘膜屏障受损、肠道菌群移位和内毒素血症、机体免疫功能低下等都有着密切关系。有学者认为 ,创伤、休克和内毒素血症早期的内源性介质[1] 可能导致了免疫细胞的低反应性[2 ,3] ,间接造成了机体免疫抑制。严重感染后 ,全身性的促炎性因子ＴＮＦα、Ｌ 1、ＩＬ 6等释放增加 ,免疫抑制因子ＴＧＦ β、ＩＬ 4、ＩＬ 10、ＰＧＥ2以及糖皮质激素也持续增加 ,因此推测这些因子的释放 ,作为严重感染后全身炎症反应的一部分 ,可能…     

罗格列酮对肽聚糖作用下大鼠腹膜间皮细胞Toll样受体2表达的影响

在腹膜透析相关性腹膜炎中,革兰阳性菌感染最为多见,其胞壁成分肽聚糖(PGN)是其特征性成分,具有致炎作用.Toll样受体家族是重要的病原成分识别受体,其成员之-Toll样受体2(TLR2)是PGN的主要受体,TLR2的活化最终通过激活核因子(NF)kB导致炎性介质的释放,由TLR2信号转导途径的激活所导致的过量炎性介质的释放对腹膜透析是非常不利的.作为过氧化物酶增殖物激活受体(PPAR)3种表型之一的PPARγ可通过多种途径来抑制炎性反应.本研究通过观察PPARγ激动剂罗格列酮对肽聚糖作用下大鼠腹膜问皮细胞TLR2表达的影响,从而探讨罗格列酮能否通过抑制TLR2表达起到抑制炎性反应扩大、保护腹膜的作用.     

Toll样受体4介导心肺复苏后肠粘膜损伤的研究

目的探讨Toll样受体4（TLR4）信号在心脏骤停及复苏后肠粘膜损伤中的作用。方法采用15只雄性大鼠建立心脏骤停及心肺复苏模型（窒息法）,自主循环恢复后,留取回肠标本,观察6、24和48小时肠粘膜的形态学改变及损伤情况：Westernblot检测回肠粘膜TLR4表达,以及ELISA法测定粘膜肿瘤坏死因子-α（TNF-α）的释放。结果复苏后动物肠粘膜显著受损,损伤程度均明显高于假手术组（sham）（P〈0．01）,其中6小时组为甚;TLR4表达在3个时间段均明显强于sham（P〈0．001）,并伴随着TNF-α表达升高。TLR4表达强弱与TNF-α释放及粘膜损伤程度变化呈正相关（P〈0．05）。结论本研究与其他疾病研究结果基本相符合,心肺复苏后肠粘膜损伤伴随着TLR4的高炎症信号,TLR4信号失控通过诱导炎性介质产生导致复苏后肠粘膜损伤。     

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。     

谷氨酰胺对肝缺血再灌注损伤的影响

研究显示,门静脉淤血所致的再灌注损伤在肝缺血再灌注损伤中具有重要作用~([1]),因而维护肠道屏障以减轻肝缺血再灌注损伤,具有重要意义.为此,笔者采用肝缺血再灌注动物模型,探讨谷氨酰胺(Gln)对肝脏高迁移率族蛋白B1(HMGB1)基因表达、内毒素血症和炎性介质的影响及其与肝损伤的关系.     

提高消化系统在危重症救治中的认识

<正>危重病常致胃肠屏障功能障碍,肠道微生态平衡失调,细菌和内毒素进入血液循环导致炎症介质失控性释放而引起全身炎症反应综合征(SIRS),从而导致多脏器功能不全综合征。而大量临床和基础研究资料表     

大鼠肠道缺血再灌注损伤对远隔组织Toll样受体4内源性配体表达的影响

目的 研究大鼠缺血再灌注损伤和淋巴液引流后,Toll样受体4(TLR4)的内源性配体高迁移率族蛋白1(HMGB1)的表达以及远隔组织的损伤.方法 24只健康雄性SPF级SD大鼠被随机分为假手术组(Sham)、肠道缺血再灌注组(I/R)和肠道缺血再灌注+淋巴液引流组(I/R+引流),每组8只.在肠道缺血再灌注损伤后检测远隔器官肺、肝和肾脏的损伤程度,肠道以及远隔器官肺、肝脏中TLR4内源性配体HMGBI的表达.结果 HE染色以及HMGB1免疫组织化学染色结果显示I/R组和I/R+引流组损伤较Sham组重,L/R组细胞出现大量黄染,表明I/R损伤时HMGB1的表达增加,而I/R+引流组的大鼠损伤显著轻于I/R组.Western blot检测显示I/R组的空肠、回肠、肝脏和肺组织中的HMGB1表达显著增加,其HMGB1/β-actin灰度值分别为0.3145±0.0549、1.7352±0.3280、1.4443±0.0926、3.1382±0.4202;引流淋巴液可以减轻损伤,其HMGB1表达均显著降低(P＜0.05),HMGB1/β-actin灰度值分别为0.1745±0.0327、1.1083±0.2098、1.1862±0.1221、2.1095±0.1993.结论 大鼠肠道缺血再灌注损伤时,肠道和远隔组织的损伤与TLR4的内源性配体HMGB1表达增加呈正相关.淋巴引流能阻断"肠-淋巴"途径,减少肠道来源的HMGB1对肠道以及远隔组织的损伤.     

体外循环炎性反应防治措施的研究进展

体外循环（CPB）可通过多种因素诱发全身性炎性反应（SIRS），包括（1）血液与管道等异物接触激活补体系统；（2）CPB过程中心肺等多脏器的缺血再灌注损伤；（3）肠道菌群移位引起的内毒素血症；（4）手术创伤。这些致炎性反应因素均可激活全身性的细胞与体液免疫系统而产生大量的炎性介质，如过敏毒素、缓激肽、细胞因子等，炎性介质进一步激活中性粒细胞、血小板及血管内皮细胞，促进其表面各种黏附分子如选择素、内皮细胞黏附分子等的表达，中性粒细胞在黏附分子的作用下与内皮细胞结合，通过释放细胞毒性蛋白酶及氧自由基引起内皮细胞的损伤，最终导致终末器官的功能障碍。     

Toll-Like Receptor Signaling in Liver Ischemia and Reperfusion

ABSTRACT

Liver ischemia–reperfusion (I/R) injuries are significant clinical challenges implicated in various hepatic surgical procedures and transplantations. Associated with varying degrees of insult, the hallmark of I/R is the excessive inflammatory response potentiated by the host immune system. Toll-like receptors (TLRs), known to play an important role in pathogen-derived inflammation, are now thought to participate in I/R injury-derived inflammation signaling pathways. Endogenous particles (proteins, cytokines, nucleic acids) that are released from damaged host cells bind to TLR2, TLR4, and TLR9, resulting in even further injury by subsequent inflammatory reactions and activation of the innate immune system. This review aims to systematically examine the current literature about TLR signaling mechanisms, allowing for a greater understanding of the precise role of TLRs in hepatic I/R injuries.     

Toll-like receptor 4 is protective against neonatal murine ischemia-reperfusion intestinal injury

Purpose

Premature infants receiving probiotics have a decreased incidence of necrotizing enterocolitis. This may be mediated by intestinal bacterial signaling via toll-like receptors (TLRs) 2 and 4 maintaining intestinal homeostasis. We hypothesized that TLRs 2 and 4 are protective against ischemia-reperfusion (I/R) intestinal injury.

Methods

Two-week-old C57BL/6 wild-type (WT), B6.TLR2^−/−, B6.TLR4^−/−, B6.TLR2^−/−4^−/−, and microbially reduced (antibiotic-treated) mice (MR) underwent 60 minutes of superior mesenteric artery occlusion (I) followed by 90 minutes of reperfusion (R). Small intestine was harvested for analysis of microscopic injury, apoptosis, and inflammatory gene expression using quantitative polymerase chain reaction.

Results

After I/R, the median histologic injury scores of the B6.TLR4^−/−, B6.TLR2^−/−4^−/−, and MR pups were higher than the WT or B6.TLR2^−/− pups that corresponded with greater apoptosis based on terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling and activated caspase-3 immunostaining. B6.TLR4^−/−, B6.TLR2^−/−4^−/−, and MR also had elevated tissue innate immunity-associated chemokine and cytokine expression.

Conclusions

Neonatal mice deficient in TLR4, either alone or also deficient in TLR2, as well as those lacking a normal commensal intestinal microbiome are more susceptible to an I/R model of intestinal injury. These results may provide a mechanism for commensal bacterial-mediated protection, which may help to direct further studies to elucidate the mechanism of probiotic protection.     

Toll-like receptors 2 and 4 in renal ischemia/reperfusion injury

Toll-like receptors (TLRs) are an evolutionarily conserved family of cell membrane receptors that are part of the innate immunity system playing an important role as a first response to tissue injury. TLR2 and TLR4 are constitutively expressed on renal epithelium, and their expression is enhanced following renal ischemia/reperfusion (I/R) injury. Genetic deletion of either TLR2 or TLR4 protects from renal I/R injury. However, it is not known whether deletion of both combined protects the kidney more than a deletion of either one alone. Therefore, we performed renal I/R injury in mice lacking TLR2, TLR4, and TLR2/4, respectively. Our results demonstrate that there are no significant differences regarding protection from renal I/R injury in TLR2/4^(?/?) compared with either TLR2^(?/?) or TLR4^(?/?) gene-targeted mice as determined by histological evaluation and renal functional parameters. Furthermore, there was no difference in the number of apoptotic tubular cells and in nuclear translocation of nuclear factor kappa-B (NF-κB) between the TLR-gene-targeted groups. In parallel, in vitro experiments did not demonstrate an additional effect of the double genetic deletion compared with the single gene deletion with respect to tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 production in hypoxic isolated proximal tubular epithelial cells of the respective animals. In conclusion, a double genetic deletion of TLR2 and TLR4 confers a similar protection following renal I/R injury compared with single deletions of TLR2 and TLR4.     

The role of toll-like receptor-4 in the development of multi-organ failure following traumatic haemorrhagic shock and resuscitation

Haemorrhagic shock and resuscitation (HS/R) following major trauma results in a global ischaemia and reperfusion injury that may lead to multiple organ dysfunction syndrome (MODS). Systemic activation of the immune system is fundamental to the development of MODS in this context, and shares many features in common with the systemic inflammatory response syndrome (SIRS) that complicates sepsis. An important advancement in the understanding of the innate response to infection involved the identification of mammalian toll-like receptors (TLRs) expressed on cells of the immune system. Ten TLR homologues have been identified in humans and toll-like receptor-4 (TLR4) has been studied most intensively. Initially found to recognise bacterial lipopolysaccharide (LPS), it has also recently been discovered that TLR4 is capable of activation by endogenous 'danger signal' molecules released following cellular injury; this has since implicated TLR4 in several non-infectious pathophysiologic processes, including HS/R. The exact events leading to multi-organ dysfunction following HS/R have not yet been clearly defined, although TLR4 is believed to play a central role as has been shown to be expressed at sites including the liver, lungs and myocardium following HS/R. Multi-organ dysfunction syndrome remains an important cause of morbidity and mortality in trauma patients, and current therapy is based on supportive care. Understanding the pathophysiology of HS/R will allow for the development of targeted therapeutic strategies aimed at minimising organ dysfunction and improving patient outcomes following traumatic haemorrhage. A review of the pathogenesis of haemorrhagic shock is presented, and the complex, yet critical role of TLR4 as both a key mediator and therapeutic target is discussed.     

TLR2/4蛋白在小鼠全肝缺血再灌注损伤肝脏的表达及意义

目的观察Toll样受体2／4蛋白在小鼠全肝缺血再灌注损伤中肝脏的表达，并分析其与肝功能损伤的关系。方法通过夹闭BALB／c小鼠肝门，复制小鼠全肝缺血再灌注损伤模型。采用Western blot方法定量检测缺血肝叶中TLR2／4蛋白的表达变化，并检测门静脉血浆丙氨酸氨基转移酶（pALT）、肿瘤坏死因-α（TNF-α）及门静脉血清内毒素（endotoxin，EN）水平。结果与假手术组（sham-operated group，SH组）相比：（1）全肝缺血20min并行再灌注后，缺血再灌注组（ischemic／reperfusion group，I／R组）血清ALT在再灌注1h即明显升高，且在再灌注3h时较1h时明显升高；（2）I／R组缺血肝脏TLR2／4蛋白的表达（OD值）明显升高，TLR2蛋白的表达在再灌注3h较1h明显高；而TLR4蛋白的表达以再灌注1h时水平最高。（3）I／R组中门静脉血清TNF-α在再灌注1h即开始高，在再灌注3h达高峰。（4）门静脉血清内毒素水平明显升高（与SH相比，P〈0．01），但I／R组在不同灌注时间点之间无显著性差异（P〉0．05）。结论TLR2／4蛋白表达的上调参与了小鼠全肝脏缺血再灌注中肝脏的损伤。     

The effect of lower limb ischaemia-reperfusion on intestinal permeability and the systemic inflammatory response.

OBJECTIVES: a relationship has been demonstrated between increased intestinal permeability, endotoxaemia and the development of the systemic inflammatory response syndrome (SIRS) after aortic surgery. The aim of this study was to evaluate whether isolated lower limb ischaemia-reperfusion (I/R) injury affects intestinal mucosal barrier function and cytokine release. PATIENTS AND METHODS: four groups of patients were investigated, group I, patients with critical limb ischaemia (CLI) undergoing infra-inguinal bypass surgery (n=18); group II, patients with intermittent claudication (IC) undergoing infra-inguinal bypass surgery (n=14); group III, patients with CLI unsuitable for arterial reconstruction, undergoing major amputation (n=12); and group IV, patients undergoing carotid endarterectomy for symptomatic carotid stenosis (n=13). Intestinal permeability, endotoxaemia and urinary soluble tumour necrosis factor receptors were assessed (p55TNF-R). RESULTS: an increase in intestinal permeability was observed on the 3rd postoperative day only in CLI group. This was found to correlate with arterial clamp time. Patients who had a femoro-distal bypass had significantly higher intestinal permeability compared to those who had femoro-popliteal bypass. Endotoxaemia was not detected in any of the groups. Postoperative urinary p55TNF-R concentrations were significantly higher in CLI group compared to the other groups. These did not correlate with the increased intestinal permeability. CONCLUSIONS:our results support the hypothesis that revascularisation of critically ischaemia limbs leads to intestinal mucosal barrier dysfunction and cytokine release. They also suggest that the magnitude of the inflammatory response following I/R injury is related to the degree of initial ischaemia.     

Oleuropein ameliorated lung ischemia-reperfusion injury by inhibiting TLR4 signaling cascade in alveolar macrophages

Lung ischemia–reperfusion (I/R) injury is a common postoperative complication in patients with lung transplantation, pulmonary embolism, and cardiopulmonary bypass. Lung I/R injury is a sterile inflammatory process that leads to lung dysfunction, and is an important cause of patient death. Effectively alleviating lung I/R injury can thus improve the prognosis of patients. In this study, we created a mouse model of lung I/R injury by transient unilateral left pulmonary artery occlusion. 6–8 weeks male C57BL/6 mice were randomly assigned to four groups: Sham, I/R, I/R + oleuropein (OLE) and OLE. OLE (50 mg/kg) was orally 24 h and 30 min before anesthesia. Measurement of lung pathohistological, isolated alveolar macrophages (AMs), inflammatory mediators, TLR4 and its downstream factors (MyD88, NF-κB) were performed. We then evaluated the ability of oleuropein (OLE) to ameliorate I/R-induced lung injury and explored the possible molecular mechanisms. OLE ameliorated I/R-induced lung injury and edema and decreased inflammatory factors in lung tissue and bronchoalveolar lavage fluid. This protection required toll-like receptor 4 (TLR4). OLE significantly inhibited I/R-induced expression of TLR4 and its downstream factors in lung tissue and alveolar macrophages. In addition, hypoxia-inducible factor 1α protein accumulated in TLR4-mediated lung I/R injury, and further induced the production of inflammatory factors. Collectively, these data suggest that OLE ameliorates I/R-induced lung injury. The mechanism responsible for its protective effect may involve inhibition of the I/R-induced inflammatory response by downregulating the TLR4 signaling cascade in AMs.     

Evidence for the pivotal role of endogenous toll-like receptor 4 ligands in liver ischemia and reperfusion injury

BACKGROUND: Although toll-like receptor 4 (TLR4) activation has been demonstrated to play a key role in the induction of intrahepatic inflammation, leading to hepatocellular damage in liver ischemia/reperfusion injury (IRI), the nature of TLR4 ligands generated during tissue injury remains to be elucidated. We hypothesized that endogenous TLR4 ligands, rather than endotoxin (lipopolysaccharide [LPS]), are instrumental in the activation of liver TLR4 leading to local inflammation response that culminates in ultimate organ IRI. METHODS AND RESULTS: By using the LPS-neutralizing agent, recombinant bactericidal/permeability-increasing protein, we showed that the endotoxin blockade failed to protect mouse livers from warm IRI, as assessed by serum alanine aminotransferase levels, intrahepatic inflammatory gene induction profile, and liver pathology. The recombinant bactericidal/permeability-increasing protein did not cause any hepatocytoxicity by itself if injected into normal naive mice. Furthermore, we demonstrated that liver perfusates, generated by isolated liver perfusion system, contained LPS-independent, heat-sensitive protein molecules that activated macrophages to produce tumor necrosis factor (TNF)-alpha through TLR4 but not TLR2 pathway. CONCLUSION: This study provides a definitive evidence that endogenous TLR4 ligands are critical in the pathogenesis of liver IRI.