中国农业科学 ›› 2022, Vol. 55 ›› Issue (14): 2850-2861.doi: 10.3864/j.issn.0578-1752.2022.14.014

• 畜牧·兽医 • 上一篇    下一篇

咖啡酸苯乙酯通过AMPK/FOXO3a信号通路缓解猪精液常温保存氧化应激的作用机制

蓝群(),谢颖瑜,曹嘉程,薛丽娥,陈德军,饶勇勇,林瑞意,方绍明(),肖天放()   

  1. 福建农林大学动物科学学院,福州 350002
  • 收稿日期:2021-05-16 接受日期:2021-06-21 出版日期:2022-07-16 发布日期:2022-07-26
  • 通讯作者: 方绍明,肖天放
  • 作者简介:蓝群,E-mail: lanqunstrive@163.com
  • 基金资助:
    福建省现代农业生猪产业技术体系项目(MNZ2019-144);福建省自然科学基金(2020J01537)

Effect and Mechanism of Caffeic Acid Phenethyl Ester Alleviates Oxidative Stress in Liquid Preservation of Boar Semen Via the AMPK/FOXO3a Signaling Pathway

LAN Qun(),XIE YingYu,CAO JiaCheng,XUE LiE,CHEN DeJun,RAO YongYong,LIN RuiYi,FANG ShaoMing(),XIAO TianFang()   

  1. College of Animal Sciences Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2021-05-16 Accepted:2021-06-21 Online:2022-07-16 Published:2022-07-26
  • Contact: ShaoMing FANG,TianFang XIAO

摘要:

【目的】探究咖啡酸苯乙酯(caffeic acid phenethyl ester,CAPE)通过AMPK/FOXO3a信号通路缓解氧化应激对猪精液的影响及其分子机制。【方法】选取8头成年(1—2岁)、健康状况良好长白种公猪进行鲜精采集,将质检合格的样品混池待用。试验设计如下:首先,在常温基础稀释液中分别添加0、0.02、0.04、0.06、0.08和0.10 g·L-1浓度的CAPE,将精液样品与各个浓度组依次混合后放置室温平衡,随即转入17 ℃电子恒温冰箱进行常温保存。全自动精子分析仪(CASA)测定1—5 d精子运动学参数(总活率与渐进性活力),筛选出最佳适宜浓度为0.06 g·L-1。其次,将0.06 g·L-1 CAPE与400 μmol·L-1的H2O2建立氧化胁迫模型,在第1、3、5天分别采用CASA测定精子总活率与渐进性活力,荧光探针技术评估质膜完整性与顶体完整性,抗氧化试剂盒检测过氧化氢酶(catalase,CAT)与超氧化物歧化酶(superoxide dismutase,SOD)活性,在第5 天利用实时荧光定量 PCR技术测定AMPK/FOXO3a信号通路下游靶向基因(AMPK、FOXO3a、SOD1、SOD2、CAT)及凋亡基因BAX的mRNA表达水平,蛋白免疫印记技术测定AMPK、p-AMPK蛋白表达。【结果】(1)浓度筛选试验中,0.06 g·L-1的CAPE在保存第3 天显著提高精子的总活率并维持至第5天(P<0.05),不仅如此,精子的渐进性活力在第1 天显著高于其他处理组(P<0.05)。(2)氧化胁迫试验中,H2O2处理组的精子质膜完整性、顶体完整性、总活率、渐进性活力、SOD与CAT活性在保存的第5天均极限显著低于空白组与CAPE+H2O2混合组(P<0.001),而空白组与CAPE+H2O2混合组的总活率、顶体完整性、CAT与SOD活性不存在显著差异(P>0.05)。与CAPE+H2O2混合组相比,H2O2处理组AMPK、FOXO3a、SOD1、SOD2、CAT的mRNA表达水平显著降低(P<0.05),BAX的表达水平显著升高(P<0.05),而空白组与CAPE+H2O2混合组的AMPK、SOD1、CAT与BAX不存在显著差异(P>0.05)。在蛋白表达水平中,CAPE+H2O2混合组与H2O2组相比,AMPK、p-AMPK 与p-AMPK/AMPK比率显著提升(P <0.05)。【结论】在常温基础稀释液中添加0.06 g·L-1CAPE可通过促进磷酸化AMPK的表达,诱导AMPK/FOXO3a信号下游抗氧化分子的转录,继而缓解H2O2介导的氧化危害对精液品质产生的影响,延长精液的保存时间,但CAPE保护精子氧化损伤更为深入的分子机制仍需作进一步探究。

关键词: 猪精液, CAPE, 常温保存, 精液品质, AMPK/FOXO3a信号通路

Abstract:

【Objective】The aim of this study was to explore the effect and mechanism of caffeic acid phenethyl ester (CAPE) relieves oxidative stress in boar semen through AMPK / FOXO3a signaling pathway. 【Method】 Eight adult (1-2 years) and healthy Landrace boars were selected to collect fresh semen, and the qualified samples were pooled for determination. Firstly, CAPE was added to basic diluent at the concentration of 0, 0.02, 0.04, 0.06, 0.08 and 0.10 g·L-1 at room temperature, respectively. To transferred the mixed specimens into electronic thermostat refrigerator for room temperature storage, semen samples were equilibrated at room temperature. The sperm kinematic parameters, including total motility and progressive motility, were determined by Computer-Aided Sperm Analysis (CASA) within 1-5 days, the best concentration of CAPE (0.06 g·L-1) was then selected. Secondly, the oxidative stress models were established by the ensure concentration of CAPE (0.06 g·L-1) and 400 μmol·L-1 hydrogen peroxide. At 1st, 3rd and 5th day, the total motility and progressive motility were measured by CASA, the plasma membrane integrity and acrosome integrity were evaluated by fluorescence probe technique, and the activities of catalase (CAT) and superoxide dismutase (SOD) were detected by antioxidant kit. After five days preservation, the mRNA expression of AMPK/FOXO3a signaling pathway related to genes, like AMPK, FOXO3a, SOD1, SOD2, CAT and apoptosis gene BAX, were determined by Real-time quantitative PCR (qPCR) technique, and the AMPK and p-AMPK protein expression were measured by western blot (WB). 【Result】(1) In the concentration screening experiment, 0.06 g·L-1 CAPE significantly improved the total motility on the 3rd day and maintained until the 5th day (P<0.05). Moreover, the progressive motility of sperm on the 1st day was significantly higher than other treatments (P<0.05). (2) In the oxidative stress experiment, the plasma membrane integrity, acrosome integrity, total motility, progressive motility, SOD and CAT activities of sperm in H2O2 group were significantly lower than blank group and CAPE+H2O2 group (P<0.05); however, there were no significant differences in total motility, acrosome integrity, CAT and SOD activities between the blank group and CAPE+H2O2 (P>0.05). Compared with CAPE+H2O2 group, the mRNA expression level of AMPK, FOXO3a, SOD1, SOD2, CAT in H2O2 group were significantly decreased (P<0.05), the expression level of BAX was significantly increased (P<0.05), while there was no significant difference in AMPK, SOD1, CAT and BAX between control group and CAPE+H2O2 group (P>0.05). In the expression level of proteins, AMPK, p-AMPK and p-AMPK/AMPK ratio were significantly higher in CAPE+H2O2 group in compared with H202 group (P<0.05). 【Conclusion】 The supplementation of 0.06 g·L-1 CAPE to basic diluent at room temperature could induce the transcription of downstream antioxidant molecules of AMPK/FOXO3a signaling pathway by promoting phosphorylation of AMPK expression, then alleviate the effect of H2O2 mediated oxidation which affected semen quality, and prolong its storage time. However, the in-depth molecular mechanism of CAPE protecting sperm fight with oxidative damage is still worth for further investigation.

Key words: boar semen, CAPE, room temperature preservation, semen quality, AMPK/FOXO3a signaling pathway