冬凌草甲素缓解脂多糖诱导的Caco-2肠上皮细胞屏障损伤和SIRT1/eIF2α信号通路活性的抑制

目的 探索冬凌草甲素对脂多糖（lipopolysaccharide, LPS）诱导的肠上皮屏障障碍的影响及其生物学机制。方法将Caco-2细胞进行单层培养21 d使其分化后,分为对照组、LPS组（2μg/mL LPS处理24 h）、LPS+低剂量冬凌草甲素组（10μg/mL冬凌草甲素预处理30 min,2μg/mL LPS处理24 h）和LPS+高剂量冬凌草甲素组（40μg/mL冬凌草甲素预处理30 min,2μg/mL LPS处理24 h）。采用跨上皮电阻法和FITC-dextran 4通量法评估细胞单层屏障功能;CCK-8法检测细胞活力;乳酸脱氢酶（LDH）法检测LDH释放活性;DCFH-DA探针法检测细胞内活性氧水平;ELISA法检测炎症因子IL-1β和TNF-α分泌水平;免疫荧光法检测细胞的屏障功能相关蛋白闭合蛋白（occludin）和闭锁小带蛋白1(zonula occludens protein 1, ZO-1)的表达分布情况;Western blot法检测SIRT1和p-eIF2α的相对表达水平。结果 与LPS组比较,冬凌草甲素能改善LPS诱导的肠上皮细胞单层屏障障碍,增...     

蒙脱石对大肠杆菌K88感染Caco-2细胞的屏障功能和紧密连接蛋白表达的影响

采用Caco-2细胞培养模型,分析大肠杆菌K88感染Caco-2后的单层细胞跨膜电阻值(TEER)、甘露醇透过率、紧密连接蛋白occludin分布的变化,并在培养液中加入蒙脱石,探讨蒙脱石对大肠杆菌K88感染Caco-2后的屏障功能和紧密连接蛋白表达的影响.结果表明:大肠杆菌K88感染Caco-12细胞后,细胞单层TEER值随时间的延长而降低,感染3 h后TEER值显著低于正常组(P<0.05),而添加蒙脱石组TEER值与正常组无显著差异(P>0.05).蒙脱石剂量在0～1 g/L的范围内,感染Caco-2细胞单层TEER值随着蒙脱石剂量的增加而急剧增加;蒙脱石剂量在1～1.67 g/L的范围内,TEER值变化趋平.感染Caco-2细胞的3H甘露醇表观渗透系数随着时间的延长而增加,各个时间点均显著高于正常组(P<0.05),而蒙脱石组各时间点3H甘露醇表观渗透系数均显著低于大肠杆菌K88感染组(P<0.05).大肠杆菌K88感染后,相邻Caco-2细胞间紧密连接结构遭到破坏,occludin的表达减少,而蒙脱石处理后可使大肠杆菌K88引起的紧密连接结构受损减轻、occludin表达增多.结果提示蒙脱石可有效抑制大肠杆菌K88黏附Caco-2细胞引起的通透性增加、屏障功能损坏,改善紧密连接的结构和OCtludin的表达分布.     

氧化应激、血管内皮功能障碍与高血压

高血压是心血管疾病的主要危险因素之一,降低血压对于减少心血管事件的发生有的重要的意义。高血压的病理生理机制复杂,其共同特点是活性氧的利用度增加,即氧化应激。在心血管系统中,活性氧在控制血管内皮功能、血管张力和心功能方面有着重要作用,并在炎症、肥大、增殖、凋亡、迁移、血管生成等病理生理过程中也发挥一定的作用,均参与了高血压所致的内皮功能障碍。虽然,动物实验研究的结果表明氧化应激是高血压的发病机制,但目前尚无明确的证据支持氧化应激在人群中导致高血压的发生。内皮功能障碍是血管内皮受到刺激后血管舒张反应下降,众多的研究证据表明其是介导高血压血管负面影响的重要机制之一,且有研究表明高血压相关的内皮功能障碍是由氧化应激所致。本文主要就高血压中的氧化应激、血管内皮功能障碍的实验研究、机制以及在高血压发生发展中两者的关系进行了综述。     

四联活菌制剂对危重症患者肠屏障功能障碍的影响及疗效观察

目的探讨双歧杆菌、嗜热链球菌、保加利亚乳杆菌和嗜酸乳杆菌四联活菌制剂对危重症患者肠道屏障功能的影响及疗效观察。方法选择新疆维吾尔自治区人民医院重症监护室2014年5月至2015年2月收治的危重症患者中64例肠道屏障功能障碍的患者,按照随机数字表法随机分为观察组和对照组。两组患者均给予基础治疗加营养治疗。在营养治疗方面,对照组给予常规营养治疗,观察组在常规营养治疗的基础上加用四联活菌制剂,800mL/d。观察两组患者在治疗前及治疗后7、14d外周血二胺氧化酶(DAO),血浆D-乳酸,血清内毒素的变化;观察两组患者腹泻治疗情况。结果两组患者治疗后7、14d肠黏膜屏障功能3项指标有明显降低,观察组治疗14d后肠黏膜屏障功能3项指标较对照组降低幅度更大,差异有统计学意义(P0.01)。患者治疗腹泻总有效率观察组为96.7%,对照组为70.0%,两组间比较差异有统计学意义(P0.05)。结论四联活菌制剂可以明显改善危重症患者肠道通透性,同时改善肠道微生态平衡,修复肠黏膜屏障功能,对危重症患者腹泻有显著的治疗作用,进而改善患者的预后,提高临床疗效,应在临床上推广应用。     

副干酪乳酪杆菌BD5115代谢产物对肠上皮屏障完整性的增强作用及其效应物质的初步研究

目的 筛选具有改善肠上皮屏障功能潜力的益生菌,并进一步探究其作用机制及其生物效应物质。方法 以Caco-2细胞单层跨上皮电阻（transepithelial electrical resistance,TEER）和异硫氰酸荧光素―葡聚糖（FITC-Dextran）的透过率为评价指标,比较不同乳酪杆菌发酵脱脂乳上清对细胞单层完整性的影响及发酵时间对副干酪乳酪杆菌BD5115发酵乳上清活性的影响;采用实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测乳酪杆菌发酵乳上清及其代谢产物对Caco-2细胞间紧密连接相关基因表达的影响;采用超高效液相色谱和四极杆―静电场轨道阱高分辨质谱仪联用对发酵24 h和6 h的BD5115发酵乳上清中的差异代谢物进行筛选;采用细胞免疫荧光对Caco-2细胞中紧密连接蛋白Occludin进行定位。结果 干酪乳酪杆菌ATCC334和副干酪乳酪杆菌BD5115发酵乳上清能显著增加细胞单层TEER及降低FITC-Dextran的透过率,上调Occludin基因表达,且BD5115发酵乳上清对细胞单层完整性的增强作用优于ATCC...     

槲皮苷减轻TNF-α诱导的肠上皮屏障功能障碍

目的探究槲皮苷对TNF-α诱导的结肠腺癌细胞(colorectal adenocarcinoma cell, Caco-2)单层膜肠上皮屏障功能损伤的保护作用及其机制。方法 Caco-2细胞接种于镶嵌0.4μm孔径Transwell小室聚碳酸酯膜上,并培养21d以获得Caco-2细胞单层膜。接着,将Caco-2细胞单层膜随机分空白对照组(未用任何药物处理)、单纯TNF-α组(仅用100ng/mL TNF-α处理72h)、TNF-α+槲皮苷低、中、高剂量组(分别用25、50和100μmol/L槲皮苷预处理30min后,再用100ng/mL TNF-α处理72h)。采用MTT法检测细胞活力,采用ELISA和RT-qPCR分别检测各组中白介素1β(interleukin 1β,IL-1β)、白介素6(interleukin 6,IL-6)和环氧化酶2(cyclooxygenase 2,COX-2)分泌水平和mRNA表达水平,采用跨上皮电阻(transepithelial electrical resistance,TEER)和FITC标记葡聚糖40 kD(FITC-dextran 40kD,...     

霉菌毒索引起肠毒性的机制

霉菌毒素是主要由曲霉菌、青霉菌或镰刀菌类等真菌产生的次级代谢产物,它们是人类和动物食物中最常见的天然食品污染物.摄入受霉菌毒素污染的食物会引起以急性死亡、生殖紊乱、生长障碍和对传染病的抵抗力降低等为特点的毒理学改变.此外,霉菌毒素还具有致畸、致癌和致突变的风险.肠道是霉菌毒素摄入机体的首要屏障,与其他器官相比,霉菌毒素...     

舍得老酒对大鼠肠屏障功能及肠道菌群的影响

探究舍得老酒对大鼠肠屏障功能及其肠道菌群构成的影响。

将28只SPF级180～220 g SD雄性大鼠随机分为4组（n = 7）：对照组、乙醇组、舍得老酒1组及舍得老酒2组。分别灌胃生理盐水、酒精（7.6 mL/kg）及舍得老酒（3.8 mL/kg和7.6 mL/kg 2个剂量）,连续灌胃4周。检测结肠紧密连接蛋白mRNA表达水平,血清炎症相关分子水平;采用流式细胞仪检测脾脏免疫细胞水平;采用测序检测粪便肠道菌群构成。

乙醇组大鼠紧密连接蛋白mRNA表达水平较低,血清中的肿瘤坏死因子-α浓度较高,白介素10浓度较低,而舍得老酒2个剂量组均不同程度地改善了乙醇对大鼠肠屏障功能的不良反应。舍得老酒7.6 mL/kg剂量组可能通过降低CD4⁺ T细胞比例而减少炎症的发生。舍得老酒3.8 mL/kg组大鼠肠道菌群alpha多样性高于其他3组。4组大鼠肠道菌群组成具有一定差异,门水平上,乙醇组拟杆菌门相对丰度较其他组高,厚壁菌门与拟杆菌门比值（F/B）低于其他3组;属水平上,乙醇组Ruminiclostridium_6、Prevotella_9及Lachnospiraceae_NK4A136_group丰度较对照组增加,舍得老酒3.8 mL/kg组的接近对照组,乙醇组Lactobacillus丰度降低,舍得老酒3.8 mL/kg组Lactobacillus丰度增加。

低剂量组舍得老酒可改善大鼠肠屏障功能,并可通过增加肠道菌群丰度及多样性、抑制部分肠道致病菌的生长及增加有益菌,从而起到一定的调节肠道菌群的作用。

     

活血化瘀法对肠道菌群结构及肠屏障功能影响的研究

随着人们对于肠道菌群种类以及作用认识的逐渐深入,我们发现作为人体庞大而又复杂的微生态系统,肠道菌群的结构及和菌群分布有紧密联系的肠屏障功能的改变与人体的健康息息相关。中药作为传统医学的一种治疗方法,其对人体的治疗作用是十分显著的,而活血化瘀法是使用具有消散作用及攻逐体内淤血作用的药物来治疗人类各种疾病的一种方法,这种方法对于肠道菌群以及肠屏障功能也产生了深远的影响。本文围绕肠道菌群结构改变及肠黏膜屏障功能的变化,对近十年关于肠道菌群与活血化瘀方药的相关文献进行综述,探究活血化瘀法（中药及中药复方）对于肠道菌群的影响,为临床治疗提供新思路。     

布拉酵母对小儿重症肺炎患儿肠黏膜屏障功能的影响

目的 探讨布拉酵母对小儿重症肺炎患儿肠黏膜屏障功能的影响。方法 将90例重症肺炎患儿按随机数字表分为对照组和观察组各45例。对照组患儿采用常规治疗,观察组患儿在常规治疗的同时加用布拉酵母治疗1周。比较两组患儿治疗前后血浆Ｄ-乳酸及肠型脂肪酸结合蛋白变化情况、胃肠功能障碍及腹泻发生率以及患儿不良反应。结果 治疗1周后,两组患儿血浆D-乳酸和肠型脂肪酸结合蛋白水平均低于治疗前（均P<0.01）,且不同时间点观察组指标水平下降幅度大于对照组（均P<0.01）。观察组患儿胃肠功能障碍发生率为6.7％、对照组为22.2％;观察组患儿腹泻发生率为15.6%,对照组为33.3%,二者比较差异均有统计学意义（均P<0.05）。观察组患儿无药物不良反应发生。结论 布拉酵母对重症肺炎患儿肠黏膜屏障具有保护作用,能减少胃肠功能障碍及腹泻发生率。     

Role of oxidative stress in the pathogenesis of alcohol-induced liver disease

Abstract

Chronic alcohol consumption is a well-known risk factor for liver disease, which represents a major cause of morbidity and mortality worldwide. The pathological process of alcohol-induced liver disease is characterized by a broad spectrum of morphological changes ranging from steatosis with minimal injury to more advanced liver damage, including steato-hepatitis and fibrosis/cirrhosis. Experimental and clinical studies increasingly show that the oxidative damage induced by ethanol contribute in many ways to the pathogenesis of alcohol hepatotoxicity. This article describes the contribution of oxidative mechanisms to liver damage by alcohol.     

Fumonisin B1 damages the barrier functions of porcine intestinal epithelial cells in vitro

Fumonisins (Fums) are mycotoxins widely distributed in crops and feed, and ingestion of Fums‐contaminated crops is harmful to animal health. The purpose of this study is to explore the effect of Fum B₁ (FB₁) on barrier functions of porcine intestinal epithelial cells, IPEC‐J2, to clarify the intestinal toxicity of Fums in pigs. The results showed that the persistent treatment of FB₁ significantly decreased the viability of IPEC‐J2. Moreover, the expressions of Claudin 1, Occludin, Zonula Occluden‐1 (ZO‐1) on the messenger RNA (mRNA), and protein levels and MUC1 on the mRNA level were significantly inhibited after FB₁ treatment, while the mRNA relative expression level of MUC2 was clearly increased. FB₁ also enhanced the monolayer cell permeability of IPEC‐J2. Importantly, FB₁ promoted the expression of phosphorylated extracellular regulated protein kinase (p‐ERK_1/2). These data suggest that long‐term treatment of FB₁ can suppress IPEC‐J2 proliferation, damage tight junctions of IPEC‐J2, and regulate expression of mucins to induce the damage of barrier functions of porcine intestinal epithelial cells, which may be associated with the ERK_1/2 phosphorylation pathway.     

Enzymatically synthesized megalo-type isomaltosaccharides enhance the barrier function of the tight junction in the intestinal epithelium

Abstract

Megalo-type isomaltosaccharides are an enzymatically synthesized foodstuff produced by transglucosylation from maltodextrin, and they contain a mid-chain length polymer of D-glucose with α-1,6-glycoside linkages. The injection of a solution of megalo-type isomaltosaccharides (1–4%(w/v), average DP = 12.6), but not oligo-type isomaltosaccharides (average DP = 3.3), into the intestinal lumen dose-dependently reduced the transport rates of tight junction permeable markers in a ligated loop of the anesthetized rat jejunum. Application of the megalosaccharide also suppressed the transport of tight junction markers and enhanced transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Cholesterol sequestration by methyl-β-cyclodextrin in the Caco-2 monolayers abolished the effect of megalosaccharide. Treatment with anti-caveolin-1 and a caveolae inhibitor, but not clathrin-dependent endocytosis and macropinocytosis inhibitors, suppressed the increase in TEER. These results indicate that isomaltosaccharides promote the barrier function of tight junctions in the intestinal epithelium in a chain-length dependent manner and that caveolae play a role in the effect.     

Changes in the antigenic properties of proteins of laboratory mice during oxidative stress

Changes in the level of oxidative damage to proteins in CD1 outbred mice γ irradiated with a dose of 3 Gy have been studied. The changes were estimated from the amount of carbonyl groups (CG) in the proteins. It was found that two hours after exposure to γ radiation, the amount of CG in the cytoplasmic and nuclear fractions of the liver, heart, brain, and spleen sharply increased. Two months after irradiation, the level of CG in the cytoplasmic and nuclear subcellular fractions of the liver and brain decreased to the level of CG in the control animals, which were not exposed to radiation. In the subcellular fractions of the heart and spleen, the increase in the degree of damage was more significant and a high level of damage was observed even two months after irradiation. An enhancement of the antigenic properties of proteins from the liver, heart, and spleen in the postirradiation period was found. Spleen proteins were most immunogenic. A comparison of the antigenic properties of proteins isolated from the tissues 60 days after irradiation revealed a correlation between the level of oxidative damage and the immunogenicity of the total protein fraction.     

Up-regulation of P-glycoprotein expression by glutathione depletion-induced oxidative stress in rat brain microvessel endothelial cells

Glutathione (GSH) depletion has been implicated in the pathogenesis of neurological diseases. During GSH depletion, cells of the blood-brain barrier (BBB) are subjected to chronic oxidative stress. In this study, we investigated the effect of such stress, produced with the GSH synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO), on expression of P-glycoprotein (Pgp) in primary cultured rat brain microvessel endothelial cells that comprise the blood-brain barrier (BBB). Application of BSO to cell monolayers at concentrations up to 800 microm caused increases in expression of Pgp. Concentrations >or= 400 microm BSO decreased cell viability. Application of 200 microm BSO caused a significant increase in Pgp function activity, as assessed by rhodamine 123 (Rh123) accumulation experiments. At this concentration, BSO produced time-dependent decreases in levels of intracellular GSH and increases in levels of intracellular reactive oxygen species (iROS). The increases were also observed within 48 h following BSO treatment in mdr1a and mdr1b mRNA. Exposure of cells to BSO for 24 h produced maximal effects in the accumulation of iROS, and in expression and function of Pgp. The ROS scavenger N-acetylcysteine prevented ROS generation and attenuated the changes of both expression and activity of Pgp induced by BSO. Therefore, the transport of Pgp substrates may be affected by changing Pgp expression under conditions of chronic oxidative stress induced by GSH depletion.     

Mitochondrial dysfunction, oxidative stress, regulation of exocytosis and their relevance to neurodegenerative diseases

A common feature in the early stages of many neurodegenerative diseases lies in mitochondrial dysfunction, oxidative stress, and reduced levels of synaptic transmission. Many genes associated with neurodegenerative diseases are now known to regulate either mitochondrial function, redox state, or the exocytosis of neurotransmitters. Mitochondria are the primary source of reactive oxygen species and ATP and control apoptosis. Mitochondria are concentrated in synapses and significant alterations to synaptic mitochondrial localization, number, morphology, or function can be detrimental to synaptic transmission. Mitochondrial by-products are capable of regulating various steps of neurotransmission and mitochondrial dysfunction and oxidative stress occur in the early stages of many neurodegenerative diseases. This mini-review will highlight the prospect that mitochondria regulates synaptic exocytosis by controlling synaptic ATP and reactive oxygen species levels and that dysfunctional exocytosis caused by mitochondrial abnormalities may be a common underlying phenomenon in the initial stages of some human neurodegenerative diseases.     

Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept

Plants suffering from abiotic stress are commonly facing an enhanced accumulation of reactive oxygen species (ROS) with damaging as well as signalling effects at organellar and cellular levels. The outcome of an environmental challenge highly depends on the delicate balance between ROS production and scavenging by both enzymatic and metabolic antioxidants. However, this traditional classification is in need of renewal and reform, as it is becoming increasingly clear that soluble sugars such as disaccharides, raffinose family oligosaccharides and fructans – next to their associated metabolic enzymes – are strongly related to stress‐induced ROS accumulation in plants. Therefore, this review aims at extending the current concept of antioxidants functioning during abiotic stress, with special focus on the emanate role of sugars as true ROS scavengers. Examples are given based on their cellular location, as different organelles seem to exploit distinct mechanisms. Moreover, the vacuole comes into the picture as important player in the ROS signalling network of plants. Elucidating the interplay between the mechanisms controlling ROS signalling during abiotic stress will facilitate the development of strategies to enhance crop tolerance to stressful environmental conditions.     

Metabolic defence against oxidative stress: the road less travelled so far

Bacteria have survived, and many have thrived, since antiquity in the presence of the highly‐reactive chalcogen—oxygen (O₂). They are known to evoke intricate strategies to defend themselves from the reactive by‐products of oxygen—reactive oxygen species (ROS). Many of these detoxifying mechanisms have been extensively characterized; superoxide dismutase, catalases, alkyl hydroperoxide reductase and the glutathione (GSH)‐cycling system are responsible for neutralizing specific ROS. Meanwhile, a pool of NADPH—the reductive engine of many ROS‐combating enzymes—is maintained by metabolic enzymes including, but not exclusively, glucose‐6 phosphate dehydrogenase (G6PDH) and NADP‐dependent isocitrate dehydrogenase (ICDH‐NADP). So, it is not surprising that evidence continues to emerge demonstrating the pivotal role metabolism plays in mitigating ROS toxicity. Stemming from its ability to concurrently decrease the production of the pro‐oxidative metabolite, NADH, while augmenting the antioxidative metabolite, NADPH, metabolism is the fulcrum of cellular redox potential. In this review, we will discuss the mounting evidence positioning metabolism and metabolic shifts observed during oxidative stress, as critical strategies microbes utilize to thrive in environments that are rife with ROS. The contribution of ketoacids—moieties capable of non‐enzymatic decarboxylation in the presence of oxidants—as ROS scavengers will be elaborated alongside the metabolic pathways responsible for their homeostases. Further, the signalling role of the carboxylic acids generated following the ketoacid‐mediated detoxification of the ROS will be commented on within the context of oxidative stress.     

Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction

Oxidative stress is one of the earliest events in Alzheimer's disease (AD). A chemical genetic screen revealed that deregulated cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by compromising the cellular anti-oxidant defense system. Using novel Cdk5 modulators, we show the mechanism by which Cdk5 can induce oxidative stress in the disease's early stage and cell death in the late stage. Cdk5 dysregulation upon neurotoxic insults results in reactive oxygen species (ROS) accumulation in neuronal cells because of the inactivation of peroxiredoxin I and II. Sole temporal activation of Cdk5 also increases ROS, suggesting its major role in this process. Cdk5 inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing Cdk5 as an upstream regulator of mitochondrial dysfunction. As mitochondrial damage results in elevated ROS and Ca(2+) levels, both of which activate Cdk5, we propose that a feedback loop occurs in late stage of AD and leads to cell death (active Cdk5 --> ROS --> excess ROS --> mitochondrial damage --> ROS --> hyperactive Cdk5 --> severe oxidative stress and cell injury --> cell death). Cdk5 inhibition upon neurotoxic insult prevents cell death significantly, supporting this hypothesis. As oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, Cdk5 could be a viable therapeutic target for AD.