Echigoya Yusuke, Morita Shohei, Itou Takuya, Sakai Takeo
Nihon University Veterinary Research Center, 1866 Kameino, Fujisawa, Kanagawa, Japan.
Am J Vet Res. 2012 Aug;73(8):1290-8. doi: 10.2460/ajvr.73.8.1290.
To evaluate effects of extracellular lactate on viability, shape change, lactate metabolism, and reactive oxygen species (ROS) production in equine polymorphonuclear leukocytes (PMNs).
PMNs isolated from equine venous blood samples.
PMNs were incubated with 0 to 300mM lactate for 30 minutes before each experiment. Viability was assessed via trypan blue exclusion. Shape change was assessed via flow cytometry and light microscopy. Relative quantification of monocarboxylic acid transporter and lactate dehydrogenase lactate dehydrogenase (LDH) isotype mRNAs was performed with a real-time PCR assay. Effects of lactate at a pH of 7.4 to 6.0 on ROS production in response to phorbol 12-myristate 13-acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-phenylalanine was assessed by luminol-dependent chemiluminescence.
Lactate had no effect on viability of PMNs but did alter their size and density. Monocarboxylic acid transporter 1 and lactate dehydrogenase B mRNA values were not altered. Monocarboxylic acid transporter 4 and lactate dehydrogenase A mRNA values were significantly decreased. Lactate incubation of cells significantly decreased PMN-derived luminol-dependent chemiluminescence and induced different sensitivities to stimulants (phorbol 12-myristate 13-acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine). The response ratio to N-formyl-methionyl-leucyl-phenylalanine revealed that PMNs were primed by incubation with up to 50mM lactate, significantly increasing the production of ROS. Incubation with lactate and acidic pH caused a synergistic effect on ROS production.
Extracellular lactate potentially has a direct effect on the capacity to produce ROS by equine PMNs, which may be associated with alterations in innate immune functions within a short period after high-intensity exercise.
评估细胞外乳酸对马多形核白细胞（PMN）活力、形态变化、乳酸代谢及活性氧（ROS）生成的影响。
从马静脉血样本中分离出的PMN。
每次实验前，将PMN与0至300mM乳酸孵育30分钟。通过台盼蓝排斥法评估活力。通过流式细胞术和光学显微镜评估形态变化。采用实时PCR检测法对单羧酸转运体和乳酸脱氢酶（LDH）同工型mRNA进行相对定量。通过鲁米诺依赖性化学发光法评估pH值为7.4至6.0的乳酸对佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯、调理酵母聚糖或N - 甲酰 - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸刺激下ROS生成的影响。
乳酸对PMN活力无影响，但会改变其大小和密度。单羧酸转运体1和乳酸脱氢酶B的mRNA值未改变。单羧酸转运体4和乳酸脱氢酶A的mRNA值显著降低。细胞经乳酸孵育后，显著降低了PMN来源的鲁米诺依赖性化学发光，并诱导了对刺激物（佛波醇12 - 肉豆蔻酸酯乙酸酯、调理酵母聚糖和N - 甲酰 - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸）的不同敏感性。对N - 甲酰 - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸的反应率显示，PMN经高达50mM乳酸孵育后被致敏，显著增加了ROS的生成。乳酸与酸性pH孵育对ROS生成产生协同作用。
细胞外乳酸可能直接影响马PMN产生ROS的能力，这可能与高强度运动后短时间内固有免疫功能的改变有关。
