玉米赤霉烯酮致彭波半细毛羊睾丸支持细胞增殖凋亡、氧化应激及NAC保护机制

doi:10.11843/j.issn.0366-6964.2025.06.020

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (6): 2752-2764.doi: 10.11843/j.issn.0366-6964.2025.06.020

玉米赤霉烯酮致彭波半细毛羊睾丸支持细胞增殖凋亡、氧化应激及NAC保护机制

朱爱文¹(), 王健¹, 朱戈辉³, 刘海霞¹^,*(), 平措班旦²^,*(), 王军¹, 德庆卓嘎², 闫伟¹, 韩大勇¹

1. 江苏农牧科技职业学院，泰州 225300
2. 西藏自治区农牧科学院畜牧兽医研究所，拉萨 850009
3. 甘肃农业大学动物医学院，兰州 730070

收稿日期:2024-08-02 出版日期:2025-06-23 发布日期:2025-06-25
通讯作者: 刘海霞,平措班旦 E-mail:631443163@qq.com;153558193@qq.com;sazz0123@126.com
作者简介:朱爱文(1972-)，男，江苏姜堰人，副教授，硕士，主要从事牛羊生产研究，E-mail: 631443163@qq.com
基金资助:
国家绒毛用羊产业技术体系拉萨半细毛羊试验站(CARS-39-33)；西藏自治区羊品种选育与健康养殖专项(XZ202101ZD0001N)；江苏现代农业(肉羊)产业技术体系建设专项资金(JATS[2023]353)；江苏农牧科技职业学院科研项目(NSF201905)

Zearalenone Induced Proliferation, Apoptosis, Oxidative Stress and NAC Protective Mechanism of Sertoli Cells in Pengbo Semi-fine Wool Sheep

ZHU Aiwen¹(), WANG Jian¹, ZHU Gehui³, LIU Haixia¹^,*(), PINGCUO Bandan²^,*(), WANG Jun¹, DEQING Zhuoga², YAN Wei¹, HAN Dayong¹

1. Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China
2. Institute of Animal Husbandry and Veterinary, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850009, China
3. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Received:2024-08-02 Online:2025-06-23 Published:2025-06-25
Contact: LIU Haixia, PINGCUO Bandan E-mail:631443163@qq.com;153558193@qq.com;sazz0123@126.com

摘要/Abstract

摘要：

旨在研究玉米赤霉烯酮(zearalenone，ZEA)对彭波半细毛羊睾丸支持细胞的生殖毒性及N-乙酰半胱氨酸(NAC)对睾丸支持细胞抗氧化应激的保护机制。本试验选取体质健康3月龄雄性彭波半细毛羊((13.87±0.46) kg)的睾丸组织用以分离支持细胞进行体外染毒试验，试验组ZEA染毒剂量分别为25、50、100、200 μmol·L^-1，对照组(NC组)仅添加0.1% DMSO，每组3个重复，筛选ZEA试验浓度。采用组合酶消化法、差速贴壁法和免疫荧光染色(IF)法进行彭波半细毛羊睾丸组织支持细胞分离、纯化培养及特异性抗体GATA4和Vimentin鉴定。CCK-8法、EdU法检测睾丸组织支持细胞活率及增殖能力。qRT-PCR和Western blot技术从转录和翻译水平检测细胞增殖(pcna)、凋亡(bax、caspase3、caspase9)和氧化应激(cat、gsh-px、sod1)等相关基因和蛋白的表达变化规律以及NAC的保护作用。结果发现，组合酶消化法和差速贴壁法能够获得满足ZEA染毒试验的SCs，随ZEA染毒浓度升高，SCs细胞活性和细胞增殖数量逐渐下降，200 μmol·L^-1 ZEA处理组SCs活性和细胞增殖数量极显著低于NC组(P < 0.01)；细胞增殖基因pcna的mRNA相对表达量和蛋白表达量均极显著低于NC组(P < 0.01)；促凋亡相关基因bax、caspase3、caspase9的mRNA相对表达量极显著高于NC组(P < 0.01)，CASPASE3蛋白表达量极显著高于NC组(P < 0.01)，BAX、CASPASE9蛋白表达量显著高于NC组(P < 0.05)；抗凋亡基因bcl-2的mRNA相对表达量和蛋白表达量显著低于NC组(P < 0.05)；氧化应激相关基因gsh-px和sod1的mRNA相对表达量极显著高于NC组(P < 0.01)，cat的mRNA相对表达量显著高于NC组(P < 0.05)，CAT、SOD1蛋白表达量极显著于NC组(P < 0.01)。N-乙酰半胱氨酸(NAC)预处理后，与NC组对比，试验组cleaved-CASPASE-9、cleaved-CASPASE-3蛋白表达量极显著下降(P < 0.01)，BAX蛋白表达量显著下降(P < 0.05)，BCL-2蛋白表达量显著上升(P < 0.05)。综上表明，ZEA暴露能够对彭波半细毛羊产生生殖毒性，抑制睾丸SCs增殖，促进SCs凋亡，导致SCs发生氧化损伤，而NAC对ZEA诱导的彭波半细毛羊睾丸SCs氧化损伤具有保护作用。

关键词: 彭波半细毛羊, 玉米赤霉烯酮, 睾丸, 支持细胞, 增殖凋亡, 氧化应激, N-乙酰半胱氨酸

Abstract:

The aim of this experiment was to analyze the reproductive toxicity of zearalenone (ZEA) on Pengbo semi-fine wool sheep testicular sertoli cells and the antioxidant stress protection mechanism of N-acetylcysteine (NAC) on testicular sertoli cells. The testicular tissue of 3-month-old male Pengbo semi-fine wool sheep ((13.87±0.46) kg) were isolated from sertoli cells for in vitro infection test, and the doses of ZEA in the experimental group were 25, 50, 100 and 200 μmol· L^-1, control group (NC group) added only 0.1% DMSO, with 3 replicates in each group, screening ZEA test concentration. Combined enzymatic digestion, differential attachment and immunofluorescence staining (IF) were used to isolate and purify sertoli cells, and to identify specific antibodies GATA4 and Vimentin. CCK-8 and EdU were used to detect the viability and proliferation of sertoli cells. qRT-PCR and Western blot were used to detect the expression changes of genes and proteins related to cell proliferation (pcna), apoptosis (bax, caspase3, caspase9) and oxidative stress (cat, gsh-px, sod1) and the protective effect of NAC. The results showed that combined enzyme digestion and differential adhesion could obtain the SCs for the ZEA toxicity test, cell activity and proliferation number of SCs gradually decreased with the increase of ZEA toxicity concentration, those of 200 μmol·L^-1 ZEA treatment group were extremely significantly lower than that in NC group (P < 0.01); The mRNA and protein expression levels of cell proliferation gene pcna were extremely significantly lower in 200 μmol ·L^-1 ZEA treatment group than that in NC group (P < 0.01); The mRNA expression levels of pro-apoptosis genes bax, caspase3 and caspase9 were extremely significantly higher than NC group (P < 0.01), the protein expression levels of CASPASE3 were extremely significantly higher than NC group (P < 0.01), BAX and CASPASE9 were significantly higher than NC group (P < 0.05); The mRNA and protein expression of anti-apoptosis gene bcl-2 were significantly lower than NC group (P < 0.05). The mRNA expression levels of oxidative stress-related genes gsh-px and sod1 were extremely significantly higher than NC group (P < 0.01), and gene cat were significantly higher than NC group (P < 0.05), the protein expressions of CAT and SOD1 were extremely significantly higher than NC group (P < 0.01). After pretreatment with NAC, compared with NC group, the expression levels of cleaved-CASPASE-9 and cleaved-CASPASE-3 in experimental groups were extremely significantly decreased (P < 0.01), BAX protein was significantly decreased (P < 0.05), and BCL-2 protein was significantly increased (P < 0.05). To sum up, ZEA could cause reproductive toxicity to Pengbo semi-fine wool sheep, inhibit testicular SCs proliferation, promote SCs apoptosis, and lead to oxidative damage of SCs. NAC has a protective effect on ZEA induced testicular SCs oxidative damage in Pengbo semi-fine wool sheep.

Key words: Pengbo semi-fine wool sheep, zearalenone, testicle, sertoli cells, proliferation and apoptosis, oxidative stress, N-acetylcysteine(NAC)

中图分类号:

S826.3

朱爱文, 王健, 朱戈辉, 刘海霞, 平措班旦, 王军, 德庆卓嘎, 闫伟, 韩大勇. 玉米赤霉烯酮致彭波半细毛羊睾丸支持细胞增殖凋亡、氧化应激及NAC保护机制[J]. 畜牧兽医学报, 2025, 56(6): 2752-2764.

ZHU Aiwen, WANG Jian, ZHU Gehui, LIU Haixia, PINGCUO Bandan, WANG Jun, DEQING Zhuoga, YAN Wei, HAN Dayong. Zearalenone Induced Proliferation, Apoptosis, Oxidative Stress and NAC Protective Mechanism of Sertoli Cells in Pengbo Semi-fine Wool Sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2752-2764.

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基因 Gene	引物序列(5′→3′) Primer sequence	产物长度/bp Product length	登录号 Accession number
pcna	F：CTGCAGATGTACCCCTTGTTGT	173	XM_004014340.5
	R：GACTTAAGTGTGTGCTGGCATC
bax	F：ATGCATCCACCAAGAAGCTG	143	XM_027978594.3
	R：AAACATTTCAGCCGCCACTC
bcl-2	F：CATCGTGGCCTTCTTTGAGTTC	108	XM_027960877.2
	R：TTCAGGTACTCGGTCATCCAC
Caspase3	F：TCAGGGAAACCTTCACGAGC	274	XM_015104559.3
	R：CCTCGGCAGGCCTGAATAAT
Caspase9	F：GCCAAGCCAAGGAAAACTCG	236	XM_042257438.1
	R：CACGGCAGAAGTTCACGTTG
β-actin	F：CTTCCAGCCTTCCTTCCTGG	180	NM_001009784.3
	R：GCCAGGGCAGTGATCTCTTT
cat	F：TTTCAATGCCATCGCCACAG	129	XM_004016396.5
	R：TAGTCACTGTGAGGCCAAACC
Gsh-px	F：ACATTGAAACCCTGCTGTCC	216	XM_004018462.5
	R：TCATGAGGAGCTGTGGTCTG
Sod1	F：TTCCACGTCCATCAGTTTGG	140	NM_001145185.2
	R：TTGTCAGCCTTCACATTGCC

抗体 Antibody	蛋白印记稀释比例 Dilution of WB	分子质量/ku Predicted band size
Anti-PCNA rabbit polyclonal antibody	1∶2 000	29
Anti-BAX rabbit polyclonal antibody	1∶5 000	21
Anti-BCL2 rabbit polyclonal antibody	1∶1 000	26
Anti-CASP3 rabbit polyclonal antibody	1∶500	32
Anti-CASP9 rabbit monoclonal antibody	1∶500	46
Anti-ACTB rabbit polyclonal antibody	1∶1 000	42
Anti-CAT rabbit polyclonal antibody	1∶1 000	60
Anti-SOD1 rabbit polyclonal antibody	1∶1 000	16
Cleaved Caspase-3 (Asp175) Antibody(rabbit)	1∶1 000	17/19
Cleaved-Caspase-9 (Asp315) Antibody(rabbit)	1∶1 000	35/37
HRP-conjugated Goat Anti-Rabbit IgG	1∶5 000

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玉米赤霉烯酮致彭波半细毛羊睾丸支持细胞增殖凋亡、氧化应激及NAC保护机制

Zearalenone Induced Proliferation, Apoptosis, Oxidative Stress and NAC Protective Mechanism of Sertoli Cells in Pengbo Semi-fine Wool Sheep

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