高硒通过PI3K/AKT/FoxO1信号通路对绵羊睾丸间质细胞自噬的影响

doi:10.11843/j.issn.0366-6964.2025.08.024

Abstract

Abstract:

This study aimed to investigate the effects of selenium (Se, from sodium selenite) on the autophagy of ram leydig cells (LCs) and its underlying mechanism. In this study, the LCs collected from 3 healthy Dorper Hu crossbred ram aged 8 months were isolated and treated with different concentrations of Se (0, 2, 4 and 8 μmol·L^-1) for 18 h. The experiment was repeated 3 times and each treatment group included 6 replicates. The proliferation of LCs was detected by CCK-8 assay, autophagy level was detected by MDC, the expression of autophagy-related genes (ATG5, P62 and LC3) and key molecules of PI3K/AKT/FoxO1 signaling pathway were detected by qRT-PCR and Western blot. The AKT activator SC79 or FoxO1 siRNA were used to examine the mechanism of PI3K/AKT/FoxO1 pathway in excessive Se (8 μmol·L^-1) regulating the autophagy of LCs. The results showed that the highest and lowest (P < 0.05) cell viability was obtained in the 2 μmol·L^-1 and 8 μmol·L^-1 group, respectively. The ROS level in the 2 μmol·L^-1 group was significantly (P < 0.05) lower than those in other groups. The 8 μmol·L^-1 group had the highest (P < 0.05) autophagy level of LCs. Excessive Se (8 μmol·L^-1) induced ROS accumulation and significantly (P < 0.05) increased the expressions of ATG5 and LC3-Ⅱ/Ⅰ, and significantly (P < 0.05) decreased the expression of P62. Compared with the control group, the 8 μmol·L^-1 group significantly inhibited (P < 0.05) the activities of PI3K and p-Akt, significantly increased (P < 0.05) the expression of FoxO1 in nucleus and total, significantly reduced the abundance of p-FoxO1 (P < 0.05). In order to further investigate the role of PI3K/AKT/FoxO1 pathway in excessive Se-induced autophagy of LCs, the abundance of LC3-Ⅱ/Ⅰ ratio and ATG5 proteins significantly decreased (P < 0.05) after the cells were pre-treated with SC79 (15 mg·L^-1) for 1 h or the FoxO1 was downregulated by siRNA. P62 significantly (P < 0.05) upregulated the autophagy induced by excessive Se. The SC79 significantly (P < 0.05) inhibited the dephosphorylation of FoxO1 induced by excessive Se and reduce nuclear translocations. In summary, excessive Se (8 μmol·L^-1) regulates the phosphorylation of FoxO1 and its nuclear translocation by inhibiting PI3K/AKT pathway, inducing oxidative stress and autophagy of sheep leydig cells. The results provide some experimental evidences for elucidating the molecular mechanism of the negative effects caused by excessive Se in LCs.

Key words: Se, leydig cells, PI3K/AKT/FoxO1 pathway, autophagy

CLC Number:

S826.3

DONG Jianing, HU Yingfan, DOU Yufei, LI Jun, SHI Lei, REN Youshe. Effects of Excessive Selenium on Autophagy of Leydig Cells in Sheep through PI3K/AKT/FoxO1 Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3837-3848.

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小干扰RNA siRNA	正义序列(5′→3′)Sense	反义序列(5′→3′)Antisense
si-NC	UUCUCCGAACGUGUCACGUTT	ACGUGACACGUUCGGAGAATT
si-FoxO1-1	GCUGUCAAUGCCGACUUCATT	UGAAGUCGGCAUUGACAGCTT
si-FoxO1-2	GCAAUGACGACUUUGACAATT	UUGUCAAAGUCGUCAUUGCTT
si-FoxO1-3	CGGUGAAGACAGCUUUACATT	UGUAAAGCUGUCUUCACCGTT

基因Gene	序列号GenBank No.	引物序列(5′→3′)Primer sequence	产物长度/bp Product length
ATG5	XM_042253359.1	F: GCCTTTCATCCAGAAGCTGT R: TCATCACCTGGCTCCTCTTC	174
LC3-Ⅱ	XM_004014953.4	F: ATGCCGTCCGAGAAAACCTT R: GCTGCTCTCGGATAAGTCGG	79
P62	XM_015104932.3	F: CGCAGAACAGAGTTACGAAGG R: TCCCATTCCAGTCATCTTGTC	183
PI3K	XM_042233720.2	F: GGCGGCTGAGTATCGTGAGATTG R: CGGACACCTTTCTGAGTCAACCAC	114
AKT	XM_027960258.2	F: CAGGAGGAGGAGACGATGGACTTC R: CCCAGCAGCTTCAGGTACTCAAAC	134
FoxO1	XM_027973596.2	F: TGTCCTACGCCGACCTCATCAC R: GCACGCTCTTGACCATCCACTC	96
β-actin	NM_001009784.3	F: AAGACCTCTACGCCAACACG R: GCCAGGGCAGTGATCTCTTT	91

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Effects of Excessive Selenium on Autophagy of Leydig Cells in Sheep through PI3K/AKT/FoxO1 Pathway

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