褪黑素通过改善线粒体动力学缓解棕榈酸诱导的牛子宫内膜上皮细胞损伤

doi:10.11843/j.issn.0366-6964.2024.09.022

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (9): 3978-3987.doi: 10.11843/j.issn.0366-6964.2024.09.022

褪黑素通过改善线粒体动力学缓解棕榈酸诱导的牛子宫内膜上皮细胞损伤

王忆(), 巩建飞, 衡诺, 胡樱凡, 王蕊, 王欢, 朱妮, 何维, 胡智辉, 郝海生, 朱化彬, 赵善江*()

中国农业科学院北京畜牧兽医研究所, 农业农村部畜禽生物育种重点实验室, 北京 100193

收稿日期:2024-03-05 出版日期:2024-09-23 发布日期:2024-09-27
通讯作者: 赵善江 E-mail:yiwang86@hotmail.com;zhaoshanjiang@caas.cn
作者简介:王忆(1998-), 女, 江西萍乡人, 硕士, 主要从事动物遗传育种与繁殖研究, E-mail: yiwang86@hotmail.com
基金资助:
中央级公益性科研院所基本科研业务费(2023-YWF-ZX-07);国家奶牛产业技术体系项目(CARS-36);中国农业科学院科技创新工程(ASTIP-IAS06);河北省现代农业产业技术体系建设专项资金(HBCT2024230203);宁夏回族自治区重点研发计划(2021BEF02023);内蒙古揭榜挂帅项目(2022JBGS0021)

Melatonin Alleviates Palmitic Acid-induced Damage in Bovine Endometrial Epithelial Cells by Improving Mitochondrial Dynamics

Yi WANG(), Jianfei GONG, Nuo HENG, Yingfan HU, Rui WANG, Huan WANG, Ni ZHU, Wei HE, Zhihui HU, Haisheng HAO, Huabin ZHU, Shanjiang ZHAO*()

Key Laboratory of Livestock and Poultry Biological Breeding of the Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2024-03-05 Online:2024-09-23 Published:2024-09-27
Contact: Shanjiang ZHAO E-mail:yiwang86@hotmail.com;zhaoshanjiang@caas.cn

摘要/Abstract

摘要：

旨在探究褪黑素对棕榈酸(palmitic acid, PA)诱导的牛子宫内膜上皮细胞(bovine endometrial epithelial cells, BEECs)线粒体动力学的影响及机制。本试验以BEECs为研究对象, 分为3组: 对照组、PA组和褪黑素组, 其中, 对照组添加0.2 mmol·L^-1 BSA溶剂, PA组添加0.2 mmol·L^-1 PA, 褪黑素组同时添加0.2 mmol·L^-1 PA和100 nmol·L^-1褪黑素。本研究通过CCK8法(n=4)和Annexin V-FITC/PI双染法(n=3)检测细胞活力和凋亡情况; 利用细胞流式术检测细胞线粒体内的ROS水平(n=3);进一步使用Seahorse XFe96细胞代谢呼吸动态分析仪检测耗氧率、基础呼吸值、ATP合成能力等线粒体呼吸功能的相关指标(n=4);qRT-PCR法检测线粒体动力学相关基因MFN1、DRP1和PGC1-α的mRNA表达变化量(n=3);最后通过Western-Blot法检测线粒体动力学相关蛋白MFN1、p-DRP1、DRP1和PGC1-α的表达变化量(n=3)。结果显示: 与PA组相比, 1)添加褪黑素能显著降低BEECs线粒体ROS水平(P＜0.05), 并显著提高BEECs活力和显著降低BEECs凋亡率(P＜0.05);2) Seahorse结果提示, 添加褪黑素能显著提高线粒体基础呼吸值、ATP产量和最大耗氧量(P＜0.05), 此结果表明褪黑素能改善线粒体功能, 促进线粒体有氧呼吸, 增加ATP产量; 3) qRT-PCR结果表明, 添加褪黑素能显著降低MFN1、DRP1的mRNA表达量, 显著提高PGC1-α的mRNA表达量(P＜0.05);4) Western-Blot检测发现褪黑素组中MFN1蛋白表达量显著下降(P＜0.05), p-DRP1/DRP1和PGC1-α蛋白表达量显著上升(P＜0.05)。综上, 本研究表明褪黑素可能通过改善BEECs线粒体动力学功能, 从而缓解PA诱导下的线粒体氧化应激和提高线粒体呼吸功能, 最终增强BEECs活力和降低BEECs凋亡; 本研究初步探究了褪黑素对PA诱导BEECs损伤的作用机制, 为提高严重NEB奶牛低繁殖力提供了理论依据。

关键词: 褪黑素, 棕榈酸, 牛子宫内膜上皮细胞, 线粒体动力学, 凋亡

Abstract:

The aim was to investigate the effect and mechanisms of melatonin on the mitochondrial dynamics of bovine endometrial epithelial cells (BEECs) induced by palmitic acid (PA). BEECs were used as the research subjects and divided into 3 groups: control group, PA group, and melatonin group. The control group was supplemented with 0.2 mmol·L^-1 BSA as the solvent, the PA group was supplemented with 0.2 mmol·L^-1 PA, and the melatonin group was supplemented with both 0.2 mmol·L^-1 PA and 100 nmol·L^-1 melatonin. The experiments included: assessing cell viability and apoptosis using the CCK8 method (n=4) and Annexin V-FITC/PI double staining method (n=3); detecting the ROS levels in mitochondria using flow cytometry(n=3); further analyzing mitochondrial respiration-related indicators such as oxygen consumption rate, basal respiration, ATP production capacity using Seahorse XFe96 cellular metabolism analysis system (n=4); measuring the mRNA expression changes of mitochondrial dynamics-related genes MFN1, DRP1 and PGC1-α were detected by qRT-PCR (n=3); finally, the protein expression changes of mitochondrial dynamics-related proteins MFN1, p-DRP1, DRP1 and PGC1-α were detected by Western-Blot (n=3). The results showed that: compared with the PA group, 1) the melatonin significantly reduced the mitochondrial ROS level of BEECs, increased cell viability, and decreased apoptosis rate significantly (P<0.05); 2) Seahorse results indicated that melatonin significantly increased mitochondrial basal respiration, ATP production, and maximum oxygen consumption (P<0.05), suggesting that melatonin could improve mitochondrial function, promote aerobic respiration, and increase ATP production; 3) qRT-PCR results showed that the melatonin significantly reduced the mRNA expression of MFN1 and DRP1, while significantly increasing the mRNA expression of PGC1-α(P<0.05); 4) Western-Blot analysis revealed a significant decrease in MFN1 protein expression and a significant increase in p-DRP1/DRP1 and PGC1-α protein expression in the melatonin group (P<0.05). In conclusion, the study suggests that melatonin may alleviate PA-induced mitochondrial oxidative stress and improve mitochondrial respiration by improving mitochondrial dynamics of BEECs, which ultimately enhances the viability of BEECs and reduces apoptosis. This study preliminarily explored the mechanism by which melatonin mitigates PA-induced injury in BEECs, offering a theoretical foundation for improving the low reproductive capacity of severe NEB cows.

Key words: melatonin, palmitic acid, bovine endometrial epithelial cells, mitochondrial dynamics, apoptosis

中图分类号:

S823.3

王忆, 巩建飞, 衡诺, 胡樱凡, 王蕊, 王欢, 朱妮, 何维, 胡智辉, 郝海生, 朱化彬, 赵善江. 褪黑素通过改善线粒体动力学缓解棕榈酸诱导的牛子宫内膜上皮细胞损伤[J]. 畜牧兽医学报, 2024, 55(9): 3978-3987.

Yi WANG, Jianfei GONG, Nuo HENG, Yingfan HU, Rui WANG, Huan WANG, Ni ZHU, Wei HE, Zhihui HU, Haisheng HAO, Huabin ZHU, Shanjiang ZHAO. Melatonin Alleviates Palmitic Acid-induced Damage in Bovine Endometrial Epithelial Cells by Improving Mitochondrial Dynamics[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3978-3987.

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参考文献 36

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引物名称 Primer name	GenBank accession No.	引物序列(5′→3′) Sequence	产物长度/bp PCR size
β-Actin-Forward	NM_173979.3	GCCCTGAGGCTCTCTTCCA	101
β-Actin-Reverse		GCGGATGTCGACGTCACA
MFN1-Forward	NM_001206508.1	GAAAGCTGGGTGTCTTGTGC	93
MFN1-Reverse		TCTGTGCCCGGACTGTCTA
DRP1-Forward	NM_001046494.2	GGCTGGGGTCTTGTGTGTTA	141
DRP1-Reverse		TCTTTCCACTGCTCTGCGTT
PGC1-α-Forward	NM_177945.3	GACCACAAATGATGACCCTC	90
PGC1-α-Reverse		TGGTTTGGCTTGTAGATGTT

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褪黑素通过改善线粒体动力学缓解棕榈酸诱导的牛子宫内膜上皮细胞损伤

Melatonin Alleviates Palmitic Acid-induced Damage in Bovine Endometrial Epithelial Cells by Improving Mitochondrial Dynamics

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