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

doi:10.3864/j.issn.0578-1752.2022.14.014

Abstract

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+H₂O₂group, 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 H₂O₂mediated 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

LAN Qun,XIE YingYu,CAO JiaCheng,XUE LiE,CHEN DeJun,RAO YongYong,LIN RuiYi,FANG ShaoMing,XIAO TianFang. Effect and Mechanism of Caffeic Acid Phenethyl Ester Alleviates Oxidative Stress in Liquid Preservation of Boar Semen Via the AMPK/FOXO3a Signaling Pathway[J].Scientia Agricultura Sinica, 2022, 55(14): 2850-2861.

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References 38

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基因 Gene	引物序列 Primer sequence (5´-3´)	退火温度 Tm (℃)	产物长度 Length (bp)	基因编号 GenBank ID
AMPK	F:AACATGGACGGGTTGAAGAG	60	193	NM_001167633.1
AMPK	R:CGCAGAAACTCACCATCTGA	60	193	NM_001167633.1
FOXO3a	F:CACTACGGCAACCAGACACTC	60	153	NM_001135959.1
FOXO3a	R:GTCACTGCGAAGCATCACG	60	153	NM_001135959.1
SOD1	F:ACACAAGGCTGTACCAGTGC	60	135	NM_001190422.1
SOD1	R:GTACACAGTGGCCACACCAT	60	135	NM_001190422.1
SOD2	F:GGGAACCCAAAGGGGAATTG	60	171	NM_214127.2
SOD2	R:CCTGGTTAGAACAAGCGGCA	60	171	NM_214127.2
CAT	F:CCTTGCACAAACAAAACCTGA	60	182	NM_214301.2
CAT	R:CTCCTTTGGCATGCACAACTC	60	182	NM_214301.2
BAX	F:ACCGACCCAGAGATGGTCAC	60	209	XM_013998624.2
BAX	R:CAGTTGATGCCACTCTCGAA	60	209	XM_013998624.2
GAPDH	F:GTCGGAGTGAACGGATTTGG	60	96	NM_001206359.1
GAPDH	R:GAAGGGGTCATTGATGGCGA	60	96	NM_001206359.1

保存时间 Preservation time (d)	咖啡酸苯乙酯 CAPE (g·L^-1)
保存时间 Preservation time (d)	0	0.02	0.04	0.06	0.08	0.10
1	85.2±0.86	83.9±0.75	82.9±1.67	82.3±0.95	82.6±1.03	82.7±1.62
2	79.3±1.05	80.5±2.36	79.3±1.89	83.2±0.74	79.9±3.28	79.6±1.78
3	71.4±0.59c	74.0±1.41b	75.4±1.17b	80.0±1.40a	75.3±0.69b	74.0±0.40b
4	68.2±0.30d	70.3±0.71cd	71.1±1.07c	79.0±0.47a	75.4±1.44b	73.7±1.83b
5	62.1±1.20d	66.0±2.12c	68.6±1.24bc	79.3±0.68a	70.6±1.57b	67.5±1.51bc

保存时间 Preservation time (d)	咖啡酸苯乙酯CAPE (g·L^-1)
保存时间 Preservation time (d)	0	0.02	0.04	0.06	0.08	0.10
1	61.3±1.88bc	61.9±0.91bc	60.4±1.82bc	62.9±0.16a	60.65±1.49bc	59.4±1.48c
2	50.8±0.44c	57.2±0.11b	58.6±1.44b	61.5±0.56a	56.7±1.22b	57.3±1.57b
3	49.4±2.10c	51.4±1.65bc	52.5±2.38bc	59.0±0.57a	54.9±1.50b	52.3±0.92bc
4	47.2±0.49c	49.8±0.95bc	52.4±1.02bc	58.5±0.82a	53.6±3.20ab	49.8±3.97bc
5	42.2±1.35c	42.7±1.39c	50.1±0.76b	56.0±0.47a	48.6±0.69b	41.7±1.69c

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

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