Se通过miR-200a/NRF2途径调节绵羊亮德细胞增殖和凋亡的机制

IF 2.4 2区农林科学 Q3 REPRODUCTIVE BIOLOGY

Theriogenology Pub Date : 2025-01-06 DOI:10.1016/j.theriogenology.2025.01.003

Kexin Li , Xiaolei Wang , Liang Ma , Youshe Ren , Lei Shi

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引用次数: 0

摘要

本研究旨在探讨硒通过miR-200a/NRF通路调控绵羊间质细胞增殖和凋亡的机制。通过Percoll密度梯度从8月龄羊的睾丸中分离纯化细胞。不同浓度Se（0、2.0、4.0、6.0、8.0 μmol/L Se）处理细胞18 h后，检测miR-200a水平。将MiR-200a模拟物和抑制物转染到细胞中，分为五组（对照组、NC模拟物、MiR-200a模拟物、NC抑制剂和MiR-200a抑制剂）。通过CCK8和抗氧化试验分别测定细胞活力和抗氧化状态。采用real-time PCR和Western blot检测促凋亡基因（BAX、CASPASE 3和CASPASE 8）、细胞周期基因（P21、P27和CDK1）和NRF2相关基因（NRF2、HO-1、NQO1和KEAP1）的丰度。结果显示，miR-200a模拟组与对照组相比，差异有统计学意义（P < 6.0）。与Se （6.0 μmol/L）组相比，Se + miR-200a抑制剂组细胞活力降低（P < 0.05）

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The mechanism of Se in regulating the proliferation and apoptosis of sheep Leydig cells through the miR-200a/NRF2 pathway

This study aimed to investigate the mechanism by which Se in regulates the proliferation and apoptosis of sheep Leydig cells via the miR-200a/NRF pathway. The cells were isolated and purified from the testes of 8-month-old sheep via a Percoll density gradient. After the cells were treated with different concentrations of Se (0, 2.0, 4.0, 6.0, and 8.0 μmol/L of Se) for 18 h, the miR-200a levels was detected. MiR-200a mimics and inhibitors were transfected into the cells, resulting in five groups (control, NC mimics, miR-200a mimics, NC inhibitor and miR-200a inhibitor). Cell viability and antioxidant status were measured via CCK8 and antioxidant assays, respectively. The abundances of pro-apoptotic (BAX, CASPASE 3 and CASPASE 8), cell cycle (P21, P27 and CDK1), and NRF2-related (NRF2, HO-1, NQO1 and KEAP1) genes were detected by real-time PCR and Western blot analysis.

The results revealed that miR-200a mimics group presented greater (P < 0.05) abundances of NRF2, HO-1 and NQO1 mRNA transcripts and proteins. Compared with those both in the NC mimics and the miR-200a inhibitor groups, the activities of GSH-Px and SOD, as well as cell viability in the miR-200a mimics group were significantly greater (P < 0.05). In contrast, the ROS levels, MDA content and abundances of KEAP1, P21, P27 and apoptosis-related genes mRNA transcripts and proteins were decreased (P < 0.05). The highest (P < 0.05) miR-200a expression level was detected in the Se_6.0 group. Compared with that in the Se (6.0 μmol/L) group, cell viability in the Se + miR-200a inhibitor group was lower (P < 0.05). The abundances of NRF2, HO-1 and NQO1 in the Se + miR-200a inhibitor group were lower (P < 0.05) than those in the Se (6.0 μmol/L) group but greater (P < 0.05) than those in the inhibitor group, while KEAP1 displayed the opposite trend (P < 0.05).

These results indicate that Se can activate the NRF2 antioxidant signaling pathway to regulate the proliferation and apoptosis of sheep Leydig cells and that miR-200a plays a vital role in this process. The regulatory effect of Se on male reproduction and spermatogenesis may be related to the number of Leydig cells. This study aimed to provide experimental data for Se regulation of spermatogenesis.

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来源期刊

Theriogenology 农林科学-生殖生物学

CiteScore

5.50

自引率

14.30%

发文量

387

审稿时长

72 days

期刊介绍： Theriogenology provides an international forum for researchers, clinicians, and industry professionals in animal reproductive biology. This acclaimed journal publishes articles on a wide range of topics in reproductive and developmental biology, of domestic mammal, avian, and aquatic species as well as wild species which are the object of veterinary care in research or conservation programs.