精氨酸及其代谢物抑制热应激诱导仔猪支持细胞凋亡的机制

doi:10.11843/j.issn.0366-6964.2024.02.016

摘要/Abstract

摘要： 旨在分析精氨酸及其代谢物在调节热应激诱导仔猪支持细胞凋亡中的作用。本试验选择3周龄健康雄性仔猪25只,采集睾丸并从中分离支持细胞,随机分为3组,每组3个重复。将体外培养的仔猪睾丸支持细胞在44℃条件下处理30 min作为热应激模型,质谱多重反应监测(multiple reaction monitoring,MRM)氨基酸代谢物,添加外源精氨酸和精胺,运用流式细胞术检测细胞的凋亡率,使用Western blotting检测iNOS、BAX、FAS、ARG1,ODC 和 SSAT1的表达,通过荧光定量PCR检测Arg1、Arg2和 Odc mRNA 水平,通过试剂盒检测精胺水平。结果显示,热应激显著降低了精氨酸(P<0.01)和瓜氨酸(P<0.05)水平,提高了腐胺(P<0.05)和鸟氨酸(P<0.01)水平,但精胺(P>0.05)水平无显著变化;热应激也显著增加了Inos(P<0.01)、Arg1(P<0.01)、Arg2(P<0.01)和 Odc(P<0.05)mRNA的表达以及 NO(P<0.01)的含量并导致细胞凋亡率上升(P<0.01)。添加0.05 mmol·L^-1 外源精氨酸提高了热应激条件下 SCs 的活力(P<0.05)和精氨酸(P<0.01)含量,降低了BAX(P<0.05)和 FAS(P<0.05)蛋白水平和相对凋亡率(P<0.01),同时也显著降低了 iNOS(P<0.01)蛋白水平和 NO(P<0.01)含量,提高了精胺(P<0.01)含量,缓解了热应激下SCs的凋亡(P<0.01);添加外源精胺也得到了类似的结果。以上结果表明,热处理通过增强 Arg-NO 代谢途径诱导 SCs 凋亡;添加外源性精氨酸和精胺可增强 Arg-精胺代谢,减少NO 的产生,从而抑制热应激诱导的 SCs 细胞凋亡。

关键词: 热应激, 精氨酸, 睾丸支持细胞, 细胞凋亡

Abstract: The aim of this study was to analyze the regulatory role of arginine and its metabolites in the induction of apoptosis in piglet sertoli cells under heat stress conditions. In this study, 25 3-week-old healthy male piglets were selected, testes were collected and sertoli cells were isolated, which were randomly divided into 3 groups with 3 replicates. The cultured sertoli cells were treated at 44 ℃ for 30 min as a heat stress model. Mass spectrometry multiple reaction monitoring (MRM) was used to analyze the metabolites of amino acid. The apoptosis rate of cells was detected by flow cytometry. Western blotting was used to detect the expression of iNOS, BAX, FAS, ARG1, ODC and SSAT. Quantitative real-time PCR was used to detect the mRNA levels of Arg1, Arg2 and Odc. The contents of spermine were analyzed by the kit.The results showed that heat stress significantly decreased the levels of arginine (P<0.01) and citrulline (P<0.05), increased the levels of putrine (P<0.01) and ornithine (P<0.01), but did not affect the level of spermine (P>0.05). Heat stress also significantly increased the mRNA expression of Inos (P<0.01), Arg1 (P<0.01), Arg2 (P<0.01) and Odc(P<0.05) and the content of NO (P<0.01), leading to an increase in the apoptosis rate (P<0.01). Addition of 0.05 mmol·L^-1 exogenous arginine increased the viability of SCs (P<0.05) and arginine content (P<0.01) under heat stress, and decreased BAX (P<0.05) and FAS (P<0.05) protein levels and relative apoptosis rate (P<0.01), significantly reduced the protein level of iNOS (P<0.01) and the content of NO (P<0.01), increased the content of spermine (P<0.01), and alleviated the apoptosis of SCs under heat stress (P<0.01). Similar results were obtained with the addition of exogenous spermine. These results indicate that heat treatment induces apoptosis of SCs by enhancing Arg-NO metabolic pathway. Addition of exogenous arginine and spermine enhances Arg-spermine metabolism and reduces NO production, thereby inhibiting heat stress-induced apoptosis of SCs.

Key words: heat stress, arginine, sertoli cells, apoptosis

中图分类号:

S828.3

霍元楠, 邱美佳, 张姣姣, 杨炜蓉, 王鲜忠. 精氨酸及其代谢物抑制热应激诱导仔猪支持细胞凋亡的机制[J]. 畜牧兽医学报, 2024, 55(2): 587-597.

HUO Yuannan, QIU Meijia, ZHANG Jiaojiao, YANG Weirong, WANG Xianzhong. Arginine and Its Metabolites Attenuate Heat Stress-induced Apoptosis of Immature Boar Sertoli Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 587-597.

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