N-乙酰半胱氨酸对犬细小病毒诱导的MDCK细胞凋亡及活性氧的影响

为探讨N-乙酰半胱氨酸(25μg/mL)对犬细小病毒侵染MDCK细胞的保护作用,采用噻唑兰比色法来检测NAC对染毒MDCK细胞增殖率的影响,流式细胞术检测NAC对染毒MDCK细胞凋亡率及活性氧ROS水平。结果表明,与对照组相比,染毒组细胞增殖显著下降(P0.05),而NAC组细胞增殖显著高于攻毒组(P0.05);染毒组细胞凋亡率显著上升,胞内ROS水平显著升高(P0.05),呈时间-效应关系,但NAC组明显减轻上述情况。NAC可通过降低ROS的产生从而改善染毒的MDCK细胞凋亡,对细胞起到保护作用。     

动物中活性氧的产生及清除机制

各种氧化应激使动物细胞中积累大量的活性氧，从而导致畜禽生产性能的下降。动物体内有效清除活性氧的保护机制分为酶促和非酶促两类。酶促系统包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH—Px)和过氧化物酶(POD)。非酶促系统包括抗坏血酸(Vc)、谷胱甘肽、维生素E、类胡萝卜紊等。在动物日粮中添加抗氧化剂来维持抗氧化平衡，是集约化管理条件下维持畜禽良好生产性能和繁殖性能的一个重要方法。     

动物中活性氧的产生及清除机制

各种氧化应激使动物细胞中积累大量的活性氧,从而导致畜禽生产性能的下降.动物体内有效清除活性氧的保护机制分为酶促和非酶促两类.酶促系统包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化物酶(POD).非酶促系统包括抗坏血酸(Vc)、谷胱甘肽、维生素E、类胡萝卜素等.在动物日粮中添加抗氧化剂来维持抗氧化平衡,是集约化管理条件下维持畜禽良好生产性能和繁殖性能的一个重要方法.     

谷胱甘肽和活性氧对哺乳动物配子作用的研究进展

文章综述了近年来关于谷胱甘肽对哺乳动物精子、卵母细胞和胚胎早期发育的作用及活性氧对精子功能的影响。在雄性和雌性配子中,谷胱甘肽起着保护精子和卵母细胞免受氧化损害的作用。在卵母细胞减数分裂中谷胱甘肽具有维持纺锤体形态的作用,受精后,巯基在精核的形成中起着积极的作用,并促进早期胚胎发育到囊胚阶段。在卵母细胞成熟过程中,谷胱甘肽的浓度发生着变化。它的合成受促性腺激素的调节,在精子的发生过程中随着精子的成熟,谷胱甘肽的浓度逐渐降低,但活性氧对精子功能也有多方面的作用。文章对卵丘细胞中小分子巯基化合物在谷胱甘肽合成中的重要作用也进行了综述。     

马冷冻精液氧自由基的产生和清除系统

马精液中存在相对稳定的氧自由基(ROS)产生和清除系统,当精液经离心去除精清后,稳定的系统遭到破坏。马精液冷冻和解冻过程不能有效的清除ROS,将导致解冻后精子各项生理功能的下降,直接影响冻精的质量,而添加外源性的抗氧化剂则可以重新使精液的ROS产生和清除达到平衡。作者综述了马精液中精子细胞、白细胞ROS的产生系统和精液GSH-PX、CAT、SOD、VA、VE等对ROS的清除系统,旨在对人们研究马精液冷冻保存时精子中ROS的损伤机理研究提供一定的帮助。     

氧及活性氧代谢相关基因在大鼠肝脏再生中的作用

为了在基因转录水平了解氧及活性氧代谢相关基因在大鼠肝脏再生(LR)中的作用,用Rat Genome 230 2.0芯片检测了上述在大鼠再生肝中表达变化,用真、假手术比较方法确定肝再生相关基因。初步证实上述基因中81个与肝再生相关。其中,肝再生启动、G0/G1过渡、细胞增殖、细胞分化和组织结构功能重建等四个阶段起始表达的基因数为42、9、35和2;基因的总表达次数为42、31、77和50。表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共上调317次,下调161次,分为6类表达方式,表明肝再生中这些基因的表达变化多样和复杂。芯片结果分析表明,肝再生早期和前期过氧化氢和超氧化物代谢增强;几乎在整个肝再生中氧化反应和谷胱甘肽结合反应增强。     

茶多酚对活性氧引起的鸡胚睾丸细胞氧化损伤的保护作用

利用精原细胞-体细胞共培养模型研究茶多酚对鸡胚睾丸细胞氧化损伤的缓解作用。结果表明:由次黄嘌呤-黄嘌呤氧化酶体系产生的活性氧可引起脂质过氧化产物丙二醛(MDA)的生成量增加、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性降低,而添加0.1μg/mL的茶多酚能降低MDA的生成量、使SOD和GSH-Px的活性恢复到对照组水平。这些结果说明,茶多酚通过抗氧化作用对活性氧引起的鸡胚睾丸细胞的氧化损伤具有保护作用。     

辣蓼黄酮对脂多糖诱导下RAW264.7细胞活性氧及炎性因子分泌的影响

采用酶解-超声偶联法提取辣蓼全草中的总黄酮,并经过萃取分离结合大孔树脂纯化富集获得辣蓼乙酸乙酯部分黄酮(FEA)与辣蓼正丁醇部分黄酮(FNB),并通过MTT法测定药物安全浓度。采用不同剂量的FEA与FNB作用于脂多糖(LPS)刺激所诱导的RAW264.7细胞炎症体外模型,测定细胞内活性氧(ROS)、一氧化氮(NO)及炎症因子TNF-α、IL-1β、IL-6、IL-8、IL-10水平。结果表明,FEA与FNB能明显减少LPS诱导的ROS释放量;不同浓度的FEA与FNB可以抑制LPS诱导的RAW264.7细胞分泌NO水平的升高。FEA与FNB均能减少LPS刺激所诱导的促炎因子TNF-α、IL-1β、IL-6、IL-8释放,FEA能促进抗炎因子IL-10的生成。说明一定浓度的FEA和FNB具有显著的抗炎效果,且其抗炎作用的产生可能与抗氧化途径有关。     

NaCl胁迫对碱蒿活性氧产生及抗氧化酶活性的影响

为了解NaCl胁迫对碱蒿(Artemisia anethifolia)耐盐性的影响,在温室盆栽条件下,通过研究碱蒿幼苗在不同浓度NaCl(0,25,50,100,200,400 mmol·L^-1)胁迫下的相对盐害率、超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性、丙二醛(MDA)含量、脯氨酸(Pro)含量、H₂O₂含量和超氧阴离子(O₂^-)产生速率的变化,结果表明:随着NaCl浓度增加,碱蒿幼苗相对盐害率和H₂O₂含量增加,而SOD活性、POD活性、Pro含量、O₂^-产生速率和MDA含量均呈先升高后降低的变化趋势。这表明碱蒿具有较强的耐盐性,SOD活性、POD活性、MDA和Pro含量均可作为碱蒿的耐盐生理指标。     

山豆根多糖对鸡免疫器官发育及体内活性氧水平的影响

为探讨山豆根多糖(SSP)对鸡免疫功能及体内抗氧化能力的影响,将山豆根多糖分别按50、100、200 mg/kg体重对14日龄公鸡胸部肌肉注射,在21、28、35、42日龄分别测定了鸡血浆中超氧化物歧化酶(SOD)活性、总抗氧化力(TAOC)、丙二醛(MDA)水平、谷胱甘肽(GSH)含量和一氧化氮(NO)的水平及鸡胸腺指数、脾脏指数和法氏囊指数。结果表明,鸡28日龄时,50～200 mg/kg山豆根多糖能显著提高鸡血浆中还原型谷胱甘肽水平(P0.05,P0.01);鸡35日龄时,50 mg/kg山豆根多糖能显著提高鸡的胸腺指数(P0.05),100 mg/kg山豆根多糖组鸡血浆中SOD活性极显著升高(P0.01),200 mg/kg山豆根多糖组鸡血浆NO水平显著升高(P0.01);鸡42日龄时,50 mg/kg山豆根多糖能显著提高鸡脾脏指数和法氏囊指数(P0.05),100 mg/kg山豆根多糖组鸡血浆中MDA水平极显著升高(P0.01)。提示山豆根多糖能够促进鸡免疫器官发育,并对鸡体内活性氧水平具有调节作用。     

Non-enzymatic Antioxidative Defence Mechanisms Against Reactive Oxygen Species in Bovine-retained and not-retained Placenta: Vitamin C and Glutathione

Vitamin C and glutathione (GSH) are water-soluble antioxidants which take part in defence mechanisms against reactive oxygen species (ROS). They may also be involved in processes of releasing/retaining bovine fetal membranes. Hence vitamin C, reduced (GSH) and oxidised (GSSG) glutathione levels were determined in retained and not-retained bovine fetal membranes in order to describe the non-enzymatic antioxidative status. Placental samples were collected immediately after spontaneous delivery or during caesarean section before term and at term, and 6 groups were formed as follows: (A) pre-term caesarean section without retained placenta; (B) pre-term caesarean section with retained placenta; (C) term caesarean section without retained placenta; (D) term caesarean section with retained placenta; (E) spontaneous delivery without retained placenta and (F) spontaneous delivery with retained placenta. Homogenates of maternal and fetal placental tissues were prepared, and vitamin C, GSH and GSSG were measured spectrophotometrically. Vitamin C levels were significantly higher in the maternal part than in the fetal part of the placenta in all groups examined. In retained placenta cases the levels were significantly lower than in control cows, except in pre-term groups. GSH concentrations were significantly higher in placentas without retention than with retention. GSSG levels showed the opposite relationship and were significantly higher in samples with retention of fetal membranes than in controls. Further experiments on antioxidative as well as oxidative status in bovine placenta are necessary.     

Application of Ionized Reactive Oxygen Species for Disinfection of Carcasses,Table Eggs,and Fertile Eggs

We evaluated the effect of ionized reactive oxygen species created using Binary Ionization Technology (BIT) for disinfection of broiler carcasses, table eggs, and treatment of fertile eggs. Previous research has indicated that BIT creates a high concentration of reactive oxygen species (ROS) that lyse bacterial cells on contact. Application of BIT to broiler carcasses that had been intentionally inoculated with 1.58 × 10⁶ Salmonella enterica Enteritidis (SE) caused a 1 to 3 log reduction in recoverable SE, depending on the duration of the treatment. Additionally, after inoculation of table eggs with 6.8 × 10⁸ cfu of SE, we recovered SE from 95% fewer eggs following enrichment and found significantly fewer (7.77 and 7.41 log reduction) colony-forming units recovered from eggs treated with BIT compared with nontreated control eggs. We also evaluated whether application of the BIT treatment had any effect on hatchability of broiler breeder eggs to determine whether use of this technology could be feasible in a hatchery environment for disinfection of eggs. There were no significant effects of BIT on the hatchability (of total set) of treated eggs as compared with nontreated control eggs; however, there was a slight numerical increase in hatchability, between 5 and 10% in 2 trials. These data suggest that application of BIT technology to carcasses and table eggs could reduce contamination with pathogens and that the application to fertile eggs may not have effects on hatchability of eggs set.     

稀释液中添加海藻糖对猪冷冻精液质量参数和活性氧的影响

本试验旨在探讨海藻糖对猪精子冷冻效果以及精子内活性氧(ROS)的含量影响。试验分对照组和4个海藻糖处理组(0.025,0.05,0.1和0.2 mol/L)。精子冷却后,添加0.1 mol/L海藻糖处理组精子活力显著(P<0.05)高于对照组,而精子内和稀释液中各组间ROS发生量都没有显著的差异。精子冷冻解冻后,添加0.05 mol/L海藻糖处理组的精子活力显著(P<0.05)高于对照组;添加0.05 mol/L和0.1 mol/L海藻糖处理组的精子生存率显著(P<0.05)高于对照组;添加0.1 mol/L海藻糖处理组顶体完整的精子百分比显著(P<0.05)高于对照组;4个海藻糖处理组膨胀精子百分比都显著(P<0.05)高于对照组;添加0.025 mol/L海藻糖处理组ROS水平显著(P<0.05)低于对照组。结果表明:海藻糖对精子冷冻保存是有益的,且能抑制精子内ROS的发生,但它的作用机制有待于进一步的研究。     

Light and Electron Microscopic Investigation of Equine Synovial Membrane: A Comparison Between Healthy Joints and joints with Intraarticular Fractures and Osteochondrosis Dissecans

Light and electron microscopic examination was made on equine synovial membrane from 23 healthy joints, nine joints with synovitis caused by intraarticular fracture and 10 joints with synovitis caused by osteochondrosis dissecans. Histologically as well as ultrastructurally the equine synovial membrane from healthy joints was of principally the same character as described in other species. Three types of synovial membrane — areolar, fibrous and adipose — and two types of lining cell were distinguished histologically. Ultrastructurally three types of lining cells were distinguished: A and Β type and an intermediate cell type. In healthy joints they were loosely arranged, parallel to the joint surface in an intercellular matrix, which was in direct continuity with the joint space. In joints with intraarticular fracture there was mild inflammation of the synovial membrane. There was elongation and hyperplasia of the lining cells with a relative increase in type A cells. The cell surface of lining cells was increased through filopodia. There was also an increase in cytoplasmic organelles i.e. hyperplasia of rough endoplasmic reticulum and Golgi complexes in Β type cells and an increase in lysosomes, and increased numbers of vesicles of varying types in A cells. In joints with osteochondrosis dissecans the lining cell hyperplasia and the inflammation in the synovial membrane were more prominent. Ultrastructurally the same alterations as in the previous group were seen including a relative increase in the number of A cells but degenerative changes were common in the lining cells. These changes were dilatation and vesiculation of rough endoplasmic reticulum, mitochondrial condensation, dilatation of the nuclear envelope and loss of plasma membranes, leading to disintegration of cells.     

Long-Time Exposure of Mouse Embryos to the Sperm Produces High Levels of Reactive Oxygen Species in Culture Medium and Relates to Poor Embryo Development

Small amounts of reactive oxygen species (ROS), metabolites of oxygen, are necessary for sperm-fertilizing capability. However, in excessive levels, their role in infertility has been extensively studied. The conventional in vitro fertilization (IVF) method employs a prolonged co-incubation of gametes for 16–18 h to reach fertilization. However, it has been shown that this long period might create high levels of ROS. We aimed at finding out whether ROS increases in vitro during prolonged incubation with fertilized oocytes and whether high level of ROS relates to poor embryo development. To confirm if levels of ROS relate to length of time, we measured the ROS levels in fertilization medium (FM), which contained mouse embryos exposed to spermatozoa. To evaluate the contribution of sperm in production of ROS, we measured the ROS in the medium with only sperm. The measurements were performed by chemiluminescence assay using luminol as a probe after 4 and 18 h of incubation separately. The ROS levels were significantly increased after 18 h as compared with 4 h (p < 0.0001). Moreover, ROS in the medium with only sperm was also increased after 18 h (p < 0.0001), demonstrating that they were generated either by spermatozoa or as a result of possible reaction of sperm with medium during prolonged incubation. In addition, we compared embryo development after 2, 4, 6, 8, 10, 12 and 18 h of incubation. The number of degenerated embryos exposed to sperm for 12 and 18 h was significantly higher than those exposed for 4 or 6 h (p < 0.01). These results demonstrate that ROS concentrations appear to be related to the length of incubation time, and their excessive levels have a negative effect on embryo development. We suggest reducing incubation time to at least 4 h.     

Prostaglandin F2α Stimulates Endothelial Nitric Oxide Synthase Depending on the Existence of Bovine Granulosa Cells: Analysis by Co‐culture System of Endothelial Cells,Smooth Muscle Cells and Granulosa Cells

Prostaglandin F_2α (PGF_2α) induces luteolysis in the mid but not in the early luteal phase; despite this, both the early and the mid corpus luteum (CL) have PGF_2α receptor (FPr). We previously indicated that the luteal blood flow surrounding the CL drastically increases prior to a decrease of progesterone (P) in the cows, suggesting that an acute increase of luteal blood flow may be an early sign of luteolysis in response to PGF_2α and that this may be induced by a vasorelaxant nitric oxide (NO). The aim of this study was to investigate the luteal stage‐dependent and the site‐restricted effect of PGF_2α and NO on the mRNA expressions and P secretion. To mimic the local luteal region both of peripheral and central areas of the CL, we utilized co‐cultures using bovine aorta endothelial cells (EC), smooth muscle cells (SMC) and luteinizing granulosa cells (GC) or fully‐luteinized GC. PGF_2α stimulated the expression of endothelial NO synthase (eNOS) mRNA at 0.5 h in mix‐cultures of EC and SMC with fully‐luteinized GC but not with luteinizing GC. The expression of eNOS mRNA in EC was increased by PGF_2α at 1 h only when EC was cultured together with fully‐luteinized GC but not with luteinizing GC. In all co‐cultures, PGF_2α did not affect the mRNA expression of FPr. Treatment of NO donor inhibited P secretion at 0.5 h. In conclusion, the present study suggests that the coexistence of the mature luteal cells (fully‐luteinized GC) with EC/SMC may be crucial for acquiring functional NO synthesis induced by PGF_2α.     

The Relationship of Mitochondrial Membrane Potential,Reactive Oxygen Species,Adenosine Triphosphate Content,Sperm Plasma Membrane Integrity,and Kinematic Properties in Warmblood Stallions

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.     

Equine Infectious Anemia: Preliminary Investigation of the Complement-Fixation Test for the Demonstration of Antibodies and Antigen

Clinical field cases of equine infectious anemia were studied and the disease was reproduced experimentally in horses. Attempts were made to adapt the complement-fixation test to the detection of antibodies in the serum of infected animals and to the demonstration of antigens in tissue extracts.

A moderate complement-fixing antibody response was demonstrated in the serum of horses shortly after primary exposure to the infectious agent. However, this reactivity was of short duration and occurred with normal as well as with infected saline tissue extracts. It was therefore concluded that this reaction was not specific for equine infectious anemia. Possibly it is due to the appearance of auto-tissue antibodies. The value of this reaction in the diagnosis of the infection was limited because of its short duration and absence in chronic infection and following re-exposure to the infectious agent.