脂多糖对肠黏膜微血管内皮细胞表达TLR2和TLR4的影响

为研究TLR2、TLR4在脂多糖(lipopolysaccharide,LPS)致肠黏膜微血管内皮细胞(RIMECs)损伤中的作用,以体外培养的肠黏膜微血管内皮细胞为模型,用浓度为1、5、10μg/mL的LPS刺激RIMECs3、6、9、12、24h后,采用半定量RT-PCR法检测细胞表面TLR2和TLR4的表达情况;再以浓度为1、5、10μg/mL的LPS刺激RIMECs9h后,半定量RT-PCR法检测细胞TLR2和TLR4的表达情况。结果发现：与对照组比较,不同浓度LPS刺激均能引起RIMECs表面TLR2和TLR4mRNA表达增加。不同浓度LPS刺激不同时间后,RIMECs表面TLR2和TLR4mRNA表达基本均在9h达到峰值,且无时间依赖性。用LPS刺激RIMECs9h,5μg/mL的LPS对细胞表达TLR2mRNA作用最强且无剂量依赖性,10μg/mLLPS对细胞表达TLR4mRNA作用最强且呈剂量依赖性。TLR2和TLR4在LPS损伤RIMECs的过程中起到重要作用。     

连翘酯苷A对内毒素致鸡脾淋巴细胞白介素-17的影响

[目的]研究内毒素和连翘酯苷A对鸡脾淋巴细胞中白介素-17(IL-17)水平的影响。[方法]将1日龄雏鸡正常饲养至50日龄时,心脏采血处死,表面消毒后,剖检用脾淋巴细胞分离液分离脾淋巴细胞,将脾淋巴细胞分为20个组,具体为4个剂量组×5个时间点。4个剂量组分别为:对照组、LPS组(5μg/m L)、连翘酯苷A预防低剂量组(200μg/m L连翘酯苷A+5μg/m L LPS)、连翘酯苷A预防高剂量组(400μg/m L连翘酯苷A+5μg/m L LPS)。5个时间点分别为0、6、12、24、48 h。通过采用ELISA和Real time-PCR方法检测脾脏淋巴细胞中IL-17水平。[结果]ELISA结果显示,与正常组相比,LPS组脾脏淋巴细胞中炎症因子IL-17含量升高;与LPS组比较,连翘酯苷A组脾脏淋巴细胞中上述炎症因子含量下降,表明脾脏中炎症因子的变化呈正相关性,通过抑制LPS诱导的鸡脾脏中上述炎症因子的升高,减轻炎症反应。Real time-PCR结果显示,与正常组相比,LPS组脾脏淋巴细胞中炎症因子IL-17 mRNA表达量升高;与LPS组比较,连翘酯苷A组预防组脾脏淋巴细胞中上述炎症因子表达量下降,变化呈相关性,试验表明连翘酯苷A可以通过转录水平抑制LPS诱导的鸡脾脏淋巴细胞中上述炎症因子表达量的升高,减轻炎症反应。[结论]连翘酯苷A抑制鸡脾脏淋巴细胞中IL-17水平,可起到抗炎功能。     

内毒素的生物活性及其对猪的危害

文章就内毒素的生物活性及其对猪的危害的最新研究进展进行了综述。     

4-Phenylbutyric acid accelerates rehabilitation of barrier function in IPEC-J2 cell monolayer model

As the first line of defence against pathogens and endotoxins crossing the intestine-blood barrier, the intestinal epithelial barrier plays a determinant role in pigs' health and growth. 4-Phenylbutyric acid (4-PBA), an aromatic fatty acid, was reported to benefit homeostasis of endoplasmic reticulum and protein synthesis. However, whether 4-PBA affects intestinal epithelial barrier function in pigs is unknown. This study aimed to explore the effects of 4-PBA on the intestinal barrier function, using in vitro models of well-differentiated intestinal porcine epithelial cell (IPEC-J2) monolayers in the transwell plates. Cell monolayers with or without 4-PBA (1.0 mmol/L) treatment were challenged with physical scratch, deoxynivalenol (DON, 2.0 μg/mL, 48 h), and lipopolysaccharide (LPS, 5.0 μg/mL, 48 h), respectively. Transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FD-4) permeability were measured to indicate barrier integrity and permeability. Real-time PCR and Western blot were conducted to determine relative gene and protein expressions of tight junction proteins. As expected, physical scratch, DON, and LPS challenges decreased TEER and increased FD-4 permeability. 4-PBA treatment accelerated cell mitigation and rehabilitation of the physical scratch-damaged intestinal epithelial barrier but did not alleviate DON or LPS induced barrier damage. However, once 48-h DON and LPS challenges were removed, rehabilitation of the epithelial barrier function of IPEC-J2 monolayer was accelerated by the 4-PBA treatment. Also, the relative gene and protein expressions of zonula occludens-1 (ZO-1), occludin, and claudin-1 were further upregulated by the 4-PBA treatment during the barrier rehabilitation. Taken together, 4-PBA accelerated the IPEC-J2 cell monolayer barrier recovering from physical scratch, DON-, and LPS-induced damage, via enhancing cell mitigation and expressions of tight junction proteins.     

姜黄素通过调节肠道菌群可改善脂多糖诱导糖尿病

姜黄素是从姜科植物姜黄的根茎中提取的多酚类物质,具有重要的药用价值。本研究将30只健康雄性Wistar大鼠,随机分为3组：正常对照组（n=10）、糖尿病模型组（n=10）、糖尿病模型＋姜黄素治疗组（n=10简称姜黄素治疗组）。以LPS（300ug·kg·-1day·-1）皮下注射8周建立2型糖尿病模型。测定空腹血糖逸11.1 mmol/L诊断为糖尿病。造模成功后,予姜黄素200 mg/（kg·-1day·-1）灌胃。连续治疗8周后,观察各组动物的一般情况,进行口服糖耐量试验（OGTT）及采用16S rRNA基因扩增子测序比较各组大鼠的肠道菌群。研究姜黄素对脂多糖（lipopolysaccharide,LPS）诱导糖尿病大鼠肠道菌群的影响。结果表明：姜黄素可改善LPS所致糖尿病大鼠的多饮、多食等症状并对糖耐量有明显的改善（p〈0.05）。LPS诱导的糖尿病大鼠肠道中提升的Melainabacteria含量可被姜黄素灌服降低（p〈0.05）。本研究揭示姜黄素具有降低LPS诱导的糖尿病大鼠血糖的作用,其机制可能与调节肠道微生态有关。     

Xylooligosaccharide attenuates lipopolysaccharide-induced intestinal injury in piglets via suppressing inflammation and modulating cecal microbial communities

Xylooligosaccharide (XOS) has been considered to be an effective prebiotic, but its exact mechanisms remain unknown. This research was conducted to evaluate the effects of XOS on pig intestinal bacterial community and mucosal barrier using a lipopolysaccharide (LPS)-caused gut damage model. Twenty-four weaned pigs were assigned to 4 treatments in a 2 × 2 factorial design involving diet (with or without XOS) and immunological challenge (saline or LPS). After 21 d of feeding 0% or 0.02% commercial XOS product, piglets were treated with saline or LPS. After that, blood, small intestinal mucosa and cecal digesta were obtained. Dietary XOS enhanced intestinal mucosal integrity demonstrated by higher villus height, villus height-to-crypt depth ratio, disaccharidase activities and claudin-1 protein expression and lower crypt depth. XOS also caused down-regulation of the gene expression of toll-like receptor 4 and nucleotide-binding oligomerization domain protein signaling, accompanied with decreased pro-inflammatory cytokines and cyclooxygenase 2 contents or mRNA expression and increased heat shock protein 70 mRNA and protein expression. Additionally, increased Bacteroidetes and decreased Firmicutes relative abundance were observed in the piglets fed with XOS. At the genus level, XOS enriched the relative abundance of beneficial bacteria, e.g., Faecalibacterium, Lactobacillus, and Prevotella. Moreover, XOS enhanced short chain fatty acids contents and inhibited histone deacetylases. The correlation analysis of the combined datasets implied some potential connections between the intestinal microbiota and pro-inflammatory cytokines or cecal metabolites. These results suggest that XOS inhibits inflammatory response and beneficially modifies microbes and metabolites of the hindgut to protect the intestine from inflammation-related injury.     

Lipopolysaccharide binding protein in the acute phase response of experimental murine Trypanosoma brucei brucei infection

Cellular responses to lipopolysaccharide (LPS) are enhanced by LPS-binding protein (LBP). The present study investigated the acute phase response of LBP during Trypanosoma brucei brucei infection in mice. Mean plasma concentrations of LBP increased two-fold by the seventh day following infection, but decreased to intermediate levels by the 14th day. There were no significant differences in LBP concentrations of infected/antibiotic-treated and infected/untreated mice. At 35 days post-infection, the infected mice were treated with the anti-trypanosomal diminazine aceturate (Berenil^®). LBP levels of the mice then decreased to pre-infection levels within one-week. This demonstrated that LBP is an acute phase protein during murine trypanosomosis. Furthermore, opportunistic secondary bacterial infection during trypanosomosis did not seem to play an important role in the changes in plasma LBP levels. We speculate that the marked concomitant increases in plasma LBP and endotoxin-like activity following murine trypanosome infection might play an important role in the pathogenesis of trypanosomosis.     

Expression of macrophage CD44 receptor in the course of experimental inflammatory response of bovine mammary gland induced by lipopolysaccharide and muramyl dipeptide

The aim of this study was to investigate development over time of the surface expression of CD44 on macrophages during an inflammatory response of bovine mammary gland. Intramammary instillation of muramyl dipeptide (MDP) and lipopolysaccharide (LPS) resulted in a significant increase in the total count of CD44+ non-vacuolised macrophages (_NMAC) after 24 h. During resolution of the inflammatory response, there was observed a gradual decrease in the total count CD44+ _NMAC. The lower total count and proportion of CD44 + vacuolised macrophages (_VMAC) was observed as the effect of MDP and LPS at 24 h after induction (P < 0.01). During resolution, the total count and proportion of CD44 + _VMAC increased. We have demonstrated CD44 receptor is expressed during the inflammatory response caused by LPS and MDP and the effect of these components on CD44 expression was particularly evident during initiation of the inflammatory response. High expression of CD44 in resolution of inflammatory response may relate to macrophages´ involvement in the processes leading to restitution of injured tissues.     

Temporal pattern and effect of sex on lipopolysaccharide-induced stress hormone and cytokine response in pigs

The temporal pattern and sex effect of immune and stress hormone responses to a lipopolysaccharide (LPS) challenge were assessed using a pig model. Secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 increased in a time-dependent manner following LPS infusion. There was also a time-dependent increase in secretion of the stress-related hormones cortisol, epinephrine (E), and norepinephrine (NE) following LPS, with peak concentrations attained within 30 min. The magnitude of the TNF-α and IL-1β responses were both positively associated (P < 0.05) with the magnitude of cortisol response following LPS, whereas serum IL-1β and IL-6 were positively correlated with the magnitude of E and NE responses following LPS. Acute-phase protein production was also time-dependently increased following LPS. The concentration of immune cells in circulation was decreased (P < 0.05) at 5.5 h post-LPS and negatively correlated with pro-inflammatory cytokine production. By 24 h post-LPS, immune cell counts increased (P < 0.05) and were positively associated with both pro-inflammatory cytokine and stress hormone production. The amplitude of pro-inflammatory cytokine response following LPS was affected (P < 0.05) by sex classification; however, the magnitude of elevated cytokine concentrations was not. The magnitude of the NE response, but not of the E and cortisol responses, to LPS was influenced by sex (P < 0.05). Similar to the pro-inflammatory cytokines, the magnitude of exposure to the stress hormones following LPS was not influenced by sex. The production of serum amyloid A (SAA) was influenced by sex, with barrows producing more SAA than gilts at 24 h post-LPS (P < 0.05). Collectively, these results demonstrate sex-specific, concomitant temporal changes in innate immune- and stress-related hormones.     

Effects of chitooligosaccharide supplementation on growth performance, nutrient digestibility, blood characteristics and immune responses after lipopolysaccharide challenge in weanling pigs

The objective of this study was to determine the effects of dietary supplementation with chitooligosaccharide (COS) on growth performance, nutrient digestibility, blood characteristics and immune response in lipopolysaccharide-challenged weanling pigs. A total of 90 crossbred weanling pigs (5.44 ± 0.50 kg BW) were employed in Exp. 1. The three dietary treatments were basal diets supplemented with 0, 2.5, and 5 g COS/kg, and fed for 28 d. Each treatment had 6 replications with 5 pigs per pen. Increasing the level of supplemental COS tended to linearly (P < 0.10) improve ADG and ADFI during phase 2 and overall period, while there were no differences in G:F. The linear improvement in the apparent DM (P < 0.05) and N (P < 0.10) digestibility in pigs fed COS supplemented diets was noticed. The tested blood characteristics were not influenced under non-challenge conditions. In Exp. 2, a total of 20 pigs (5.22 ± 0.31 kg BW) were initially assigned to two dietary treatments and fed basal diets supplemented with 0 or 0.5 g COS/kg for 28 d. At the end of d 28, half of the pigs in each treatment (n = 5) were injected i.p. with Escherichia coli lipopolysaccharide at a concentration of 100 μg/kg of BW. The other half of the pigs in each treatment were injected with sterile saline solution at a concentration of 100 μg/kg of BW. This arrangement resulted in a 2 × 2 factorial design with diet and LPS challenge as the main effects. Blood sample and rectal temperature data were collected at 0, 2, 4 and 12 h post-challenge. Rectal temperatures increased as the result of LPS injection at 4 and 12 h post-challenge (P < 0.05). Serum cortisol, IGF-1, and TNF-α concentration were also increased as the result of LPS challenge (P < 0.05). The COS treatments resulted in lower cortisol concentrations at 2 h and higher IGF-1 concentrations at 4 h post-challenge (P < 0.05). COS and LPS interactions were also observed on cortisol and IGF-1 when the COS effects were presented (P < 0.05). Haptoglobin concentrations remained unaffected throughout the challenge period. White blood cell counts were increased in the LPS-treated pigs at 2 and 4 h post-challenge (P < 0.01). Lymphocyte count was elevated at 2 h and reduced at 12 h post-challenge as the result of LPS challenge (P < 0.05). However, there were no COS main effects observed on lymphocyte count throughout the challenge period. The comparison between two LPS challenged treatments also indicated that COS treatment has beneficial effects on rectal temperature, cortisol and IGF-1 concentrations. In conclusion, dietary supplementation with COS had little effect on nutrient digestibility and inflammatory stress markers in weanling pigs.