Leukocyte and Cytokine Accumulation in the Ovine Teat and Udder during Endotoxin-Induced Inflammation

Persson Waller, K., Colditz, I.G., Flapper, P. and Seow, H.-F., 1997. Leukocyte and cytokine accumulation in the ovine teat and udder during endotoxin-induced inflammation. Veterinary Research Communications, 21 (2), 101-115The accumulation of leukocytes, ovine serum albumin and the cytokines interleukin-1 (IL-1), tumour necrosis factor- (TNF-), interleukin-8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon- (IFN-) was studied during endotoxin-induced inflammation in lactating and dry ovine udders, and in the teat cisterns of dry ewes after surgical closure of the passage between the teat and udder cisterns. Samples were taken before infusion and hourly up to 10 h after infusion of 0.1, 1 or 10 µg of endotoxin, or infusion of pyrogen-free saline (PFS) as a control. Rectal temperatures were measured.A significant dose- and time-dependent accumulation of leukocytes, mainly neutrophils, was observed in the lactating udders and in the teat cisterns. In the dry udders, the leukocyte accumulation was significant for time but not for dose. Peak numbers of cells were reached at 3-4 h in the dry udders and in the teat cisterns, but not until 10 h after infusion in the lactating udders. The changes in the ovine serum albumin concentrations mostly paralleled changes in leukocyte numbers.A role was indicated for TNF-, IL-8 and GM-CSF, but not for IL-1 and IFN-, during endotoxin-induced inflammation in the ovine udder. Release of TNF-, IL-8 and GM-CSF was most prominent in lactating udders, peaking at 2 or 3 h after infusion, but was also detected in dry udders and teat cisterns. Detectable levels of IL-1 and IFN- were occasionally found in all three groups.     

复方甘草酸单胺可溶性粉对恩诺沙星联合LPS 致鸡肝损伤的免疫调节

【目的】探索恩诺沙星联合脂多糖粗提液（lipopolysaccharide crude extract,LPS）诱导鸡肝损伤中机体免疫功能变化,并研究复方甘草酸单胺粉（compound ammonium glycyrrhizin soluble powder,CAG）护肝及免疫调节作用和机理。【方法】104 羽海蓝蛋鸡随机分为空白组（I）,模型组（II）,CAG预防组（III）、CAG治疗高、低剂量（IV、V）组,除I组外,各组连续3 d灌服恩诺沙星（100 mg•kg-1,1次/天）,于第3次灌药时腹注LPS（4 mL•kg-1）诱发肝损伤,III组建模前3 d以40 mg•L-1混饮CAG,持续至建模结束;IV、V组腹注LPS后分以22.5、7.5 mg•kg-1灌服CAG,2 次/天,重复3 d。建模后6、24、48 h采血并剖检。【结果】与I组相比,II组鸡血清ALT、AST、TNF-α、IL-1、IL-6水平 6、24和48h显著升高,外周血中CD3+、CD4+和CD8+ T细胞百分含量 6和24 h显著降低,48 h显著升高,肝组织病变明显。与II组相比：III组ALT、TNF-α 和IL-6 6 h,AST 6和24 h,CD3+、CD4+ 和CD8+ T细胞48 h降低显著;IV组ALT、AST、TNF-α、IL-6 24和48 h,IL-1、CD3+、 CD4+ 和CD8+ T细胞 48 h 显著降低,CD3+ T细胞6和24 h,CD4+ T细胞24 h显著升高;V组中CD8+ T细胞在48 h显著降低;其余差异不显著。IV组较其它组肝组织病变有最大改善。【结论】恩诺沙星联合LPS诱发肝损伤同时致炎性细胞因子TNF-α、IL-1、IL-6释放和CD3+细胞及其亚群CD4+ 和CD8+发生先低于正常水平,后又显著高于正常水平现象,复方甘草酸单胺可能通过抑制以上3种炎性因子释放,双向调节外周血T细胞亚群恢复正常水平而发挥护肝作用,但作用与给药剂量和时机相关。     

鹰嘴豆多肽的免疫活性研究

【目的】探究鹰嘴豆多肽对机体免疫功能的调节作用。【方法】用复合酶解方法制备鹰嘴豆多肽,对其分子质量分布及氨基酸组成进行分析。给小鼠腹腔注射环磷酰胺建立免疫功能低下的动物模型,建模成功后将小鼠随机均分为模型组（灌胃生理盐水）、阳性对照组（灌胃匹多莫德25 mg/kg）和鹰嘴豆多肽低、中、高剂量组(分别灌胃鹰嘴豆多肽50,100和200 mg/kg),另取10只健康小鼠作为空白组（灌胃生理盐水）。各组小鼠每日灌胃1次相应物质,连续灌胃21 d后采样,研究鹰嘴豆多肽对小鼠体质量、脾脏和胸腺指数及形态变化、脾淋巴细胞增殖率、血清细胞因子（IFN-γ、IL-6、IL-2）及免疫球蛋白（IgG、IgA和IgM）质量浓度的影响。研究不同质量浓度（50,100,200和400 μg/mL）鹰嘴豆多肽对RAW264.7细胞功能的免疫调节作用,以MTT法、中性红法和酶联免疫吸附检测试剂盒来检测细胞增殖率、吞噬活性以及分泌的细胞因子（NO、TNF-α、IL-6、IL-1β）水平。【结果】制得分子质量在1 000 u左右、纯度为97.12 %的鹰嘴豆多肽,其中富含谷氨酸、天冬氨酸、精氨酸、赖氨酸、亮氨酸、丝氨酸等氨基酸。与空白组相比,模型组小鼠脾脏和胸腺受损严重,多项检测指标显著降低;而鹰嘴豆多肽各剂量组小鼠的各项检测指标均较模型组小鼠显著增加,且呈剂量效应关系。鹰嘴豆多肽可促进RAW264.7细胞的增殖率和吞噬能力,并提升其产生的NO、IL-6、IL-1β和TNF-α的水平。【结论】鹰嘴豆多肽可以显著改善环磷酰胺导致的小鼠多项免疫相关指标的下降情况,同时可刺激巨噬细胞,增强其吞噬能力,促进其分泌白介素等细胞因子,表明鹰嘴豆多肽具有良好的免疫增强作用。     

姜黄素对大黄鱼组织中磷酸酶活力及血清中细胞因子含量的影响

为促进大黄鱼免疫增强剂的开发利用,以大黄鱼为研究对象,在饵料中添加不同剂量的姜黄素,研究其对大黄鱼组织中磷酸酶活力及血清中细胞因子含量的影响。试验设置3个试验组(D1-100 mg/kg、D2-150 mg/kg、D3-300 mg/kg)和1个对照组,并分别在第30天、45天取样并测定组织中磷酸酶活力和血清中细胞因子的含量。结果显示:(1)与对照组相比,当姜黄素的添加量为100 mg/kg时,肝脏、前肠中的ACP活力及鳃、胃、肌肉、肝脏中的AKP活力显著提高(P0.05)。添加量为150 mg/kg时,肌肉、肝脏、前肠中的ACP活力显著提高,鳃、胃、肌肉、肝脏及前肠中的AKP活力显著提高。添加量为300 mg/kg时,除后肠中的ACP、AKP活力及中肠中的AKP活力无显著影响外(P0.05),其余各组织中的磷酸酶活力均显著提高。(2)与对照组相比,姜黄素添加量为100 mg/kg时,仅TNF-α含量显著升高。添加量为150 mg/kg或300 mg/kg时,GM-CSF、IL-2及TNF-α含量均显著提高。3个试验组IFN-γ含量虽与对照组间无显著差异,但2段时间内均呈下降的趋势。综合考虑,以300 mg/kg姜黄素的添加量对大黄鱼各组织中磷酸酶活力及血清中细胞因子的含量的影响效果最佳。     

阿魏酸抗LPS诱导的奶牛子宫内膜上皮细胞炎性作用

[目的]为了探明阿魏酸的抗炎机制.[方法]采用组织块培养法获得奶牛子宫内膜上皮细胞.用噻唑蓝(MTT法)检测细胞活力.采用荧光定量PCR方法,分别对对照组、模型组、药物作用高、中、低剂量组在LPS作用3h、6h炎症基因IL-1β、IL-6、IL-8和TNF-α的mRNA变化进行检测.[结果]阿魏酸在一定浓度范围内对细胞增殖无显著影响.药物预处理细胞能够显著降低LPS诱导的IL-1β、IL-6、IL-8和TNF-α的mRNA表达.[结论]阿魏酸能够显著降低LPS刺激细胞后炎症细胞因子的表达,说明阿魏酸是通过抑制炎症因子的表达发挥其抗炎作用.     

The effects of the Recombinant Mycobacterium Tuberculosis CFP10 Protein on the TLR Signal-mediated Inflammatory Responses in A549 Cells

The purpose of this study was to investigate the effects of recombinant 10 kDa culture filtrate protein (CFP10) of Mycobacterium tuberculosis on the inflammatory responses mediated by Toll-like receptor (TLR) signaling in A549 cells. The recombinant plasmid pCzn1-CFP10 was obtained by amplifying the cfp10 gene fragment using PCR and cloning it into the prokaryotic expression vector pCzn1. The obtained recombinant plasmid pCzn1-CFP10 was transformed into Escherichia coli BL21(DE3) and induced by IPTG to express the recombinant protein CFP10 (rCFP10). The purified rCFP10 protein was preserved after endotoxin was removed and desalted before use. The effect of rCFP10 treatment on the survival rate of A549 cells was detected by MTT assay. A549 cells were treated with rCFP10 to detect changes of key molecules of TLR signaling pathway and downstream inflammatory factors in A549 cells by qRT-PCR,Western blot and ELISA. The results showed that the recombinant expression vector pCzn1-CFP10 was successfully constructed and the high-purity rCFP10 protein was expressed and purified in this study. MTT assay showed that rCFP10 could inhibit the survival rate of A549 cells in a time- and concentration-dependent manner. In addition, rCFP10 could significantly (P<0.05) up-regulate the key molecules of TLR pathways TLR2, TLR4, MyD88, TRAF6, NF-κBp65 and downstream inflammatory cytokines IL-6 and TNF-α in A549 cells as compared to the control group. The recombinant Mycobacterium tuberculosis protein CFP10 could promote the secretion of cytokines by activating TLR receptor signaling pathway in A549 cells, which will provide a theoretical basis for further understanding of the pathogenesis of Mycobacterium tuberculosis.     

Double‐blinded cross‐over study on the efficacy of pentoxifylline for canine atopy

The pathogenesis of canine atopy has not been established completely. Recent studies have shown that tumour necrosis factor alpha and Interleukin‐6 play a role in allergic reactions in humans and mice. Pentoxifylline (PTX) suppresses synthesis of these cytokines and may be a useful therapy for modulating the symptoms of canine atopy. The objectives of this study were to investigate the effects of PTX (10mg kg^?1 twice daily for 4 weeks) on clinical signs (erythema and pruritus) and intradermal skin test reactivity in atopic dogs (n = 10). The study was a double‐blinded, placebo controlled, crossover clinical trial with a washout period of 2 weeks between treatments. Clinical signs were evaluated and scored by the investigator and owners. During PTX treatment, scores of pruritus and erythema decreased significantly. PTX did not affect intradermal skin test reactivity to house dust mite at 15 min (allergen of reference for this study).     

紫草素抑制炎症促进驴皮伤口愈合的效果

试验旨在探究紫草素凝胶对驴伤口愈合的效果，为皮肤无瘢痕愈合提供参考。用不同浓度的紫草素对体外培养的驴巨噬细胞进行处理，分别检测细胞中TGF-β1、IL-10、TNF-α和IL-6的mRNA与细胞上清液中蛋白的表达水平，以确定紫草素的最佳添加浓度；建立驴皮伤口模型，在伤口表面涂抹紫草素凝胶，拍照、HE染色观察伤口愈合情况，并检测驴皮肤中伤口愈合相关基因和血清的表达水平。结果表明，与对照组相比，添加不同浓度的紫草素均能降低相关炎症因子的表达，其中10 μmol/L紫草素效果最佳。在驴皮伤口上涂抹紫草素凝胶第11天时，紫草素组的伤口收缩率极显著高于对照组（P<0.01）；HE染色发现，第11天时紫草素中的组织逐渐排列整齐；第60天时，紫草素组中表皮层的厚度相对减少；实时荧光定量PCR结果表明，第11天时，紫草素组中TGF-β1、IL-10 mRNA表达极显著升高（P<0.01），TNF-α、IL-6 mRNA的表达极显著降低（P<0.01）；第3天时，紫草素组血清中TGF-β1、IL-10的浓度升高，TNF-α、IL-6浓度极显著降低（P<0.01）；第7天时，紫草素组TGF-β1浓度显著降低（P<0.05），TNF-α浓度极显著降低（P<0.01），且IL-10的浓度显著升高（P<0.05）。综上，与对照组相比，添加不同浓度的紫草素均能降低相关炎症因子的表达，其中10 μmol/L紫草素效果最佳，能促进驴皮伤口的愈合。     

寡果糖诱导瘤胃急性酸中毒对山羊瘤胃发酵、蹄组织结构及炎症因子、金属蛋白酶表达的影响

本试验旨在研究寡果糖诱导瘤胃酸中毒对山羊瘤胃发酵、蹄组织结构、蹄部炎症因子及金属蛋白酶表达的影响。采用随机区组设计,将8头健康装有永久性瘤胃瘘管的波杂山羊(波尔山羊×长江三角洲白山羊)随机分为对照组和诱导酸中毒的试验组,每组4头。其中试验组山羊瘤胃寡果糖灌注量为21 g/kg BW。分别于灌注前(0 h)和灌注后4、8、12、24和48 h采集瘤胃液,同时分别在0、4、8、24和48 h通过颈静脉采集血液,灌注后48 h屠宰2组山羊,采集蹄组织。结果表明:与对照组相比,试验组山羊瘤胃液p H、挥发性脂肪酸浓度平均值显著降低(P0.05),血液和瘤胃液中乳酸和脂多糖浓度平均值显著提高(P0.05);组织形态学结果显示,与对照组相比,试验组山羊蹄组织中次级表皮蹄小叶和次级真皮蹄小叶的长度变短,蹄小叶形状不规则;实时定量PCR检测结果显示,较对照组比较,试验组山羊蹄组织中基质金属蛋白酶组织抑制物-1的mRNA的相对表达量显著降低(P0.05),白细胞介素-6、膜型基质金属蛋白酶-1和基质金属蛋白酶-2的mRNA的相对表达量显著升高(P0.05)。结果提示,寡果糖诱导山羊急性瘤胃酸中毒可导致山羊瘤胃发酵紊乱,提高瘤胃液和血液中脂多糖与乳酸浓度,导致山羊蹄组织相关炎症因子与金属蛋白酶表达改变,最终引发山羊急性蹄叶炎。