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

Abstract

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)

CLC Number:

S826.3

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.

Figures/Tables 9

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

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