College of Animal Science and Technology, JLAU-Borui Dairy Science and Technology R&D Center, Key Laboratory of Animal Nutrition and Feed Science of Jilin Province, Key Laboratory of Animal Production Product Quality and Security Ministry of Education, Jilin Agricultural University, Changchun 130118, China.
Institute of Animal Science, Jilin Academy of Agricultural Sciences, Changchun 130033, China.
Oxid Med Cell Longev. 2022 Mar 22;2022:4013575. doi: 10.1155/2022/4013575. eCollection 2022.
Leucine and isoleucine possess antioxidative and anti-inflammatory properties. However, their underlying protective mechanisms against oxidative damage remain unknown. Therefore, in this study, the protective mechanism of leucine and isoleucine against HO-induced oxidative damage in a bovine mammary epithelial cell lines (MAC-T cells) were investigated. Briefly, MAC-T cells exposed or free to HO were incubated with different combinations of leucine and isoleucine. The cellular relative proliferation rate and viability, oxidative stress indicators, and inflammatory factors were determined by specific commercial kits. The genes related to barrier functions was measured by real-time quantitative PCR. The protein expression differences were explored by 4D label-free quantitative proteomic analyses and validated by parallel reaction monitoring. The results revealed that leucine and isoleucine increased cell proliferation, total antioxidant status (TAS), and the relative mRNA expression of occludin, as well as decreased malondialdehyde (MDA), total oxidant status (TOS)/TAS, IL-6, IL-1, and TOS. When leucine and isoleucine were combined, MDA, TOS/TAS, and the relative mRNA expression levels of claudin-1, occludin, and zonula occludens-1 increased when compared to leucine or isoleucine alone. Proteomics analyses revealed that leucine significantly upregulated the propanoate metabolism; valine, leucine, and isoleucine degradation; and thermogenesis pathways, whereas isoleucine significantly upregulated the peroxisome and propanoate metabolism pathways. In conclusion, leucine protected MAC-T cells from HO-induced oxidative stress by generating more ATP to supplement energy demands, and isoleucine improved the deficit in peroxisome transport and promoted acetyl-CoA production. The findings of this study enhance our understanding of the protective mechanisms of leucine and isoleucine against oxidative damage.
亮氨酸和异亮氨酸具有抗氧化和抗炎特性。然而，它们对氧化损伤的潜在保护机制尚不清楚。因此，本研究旨在探讨亮氨酸和异亮氨酸对 HO 诱导的奶牛乳腺上皮细胞系（MAC-T 细胞）氧化损伤的保护机制。简要地说，用不同浓度的亮氨酸和异亮氨酸孵育暴露或未暴露于 HO 的 MAC-T 细胞。通过特定的商业试剂盒测定细胞相对增殖率和活力、氧化应激指标和炎症因子。通过实时定量 PCR 测定与屏障功能相关的基因。通过 4D 无标记定量蛋白质组学分析探索蛋白质表达差异，并通过平行反应监测进行验证。结果表明，亮氨酸和异亮氨酸增加了细胞增殖、总抗氧化状态（TAS）和闭合蛋白的相对 mRNA 表达，降低了丙二醛（MDA）、总氧化状态（TOS）/TAS、白细胞介素-6（IL-6）、白细胞介素-1（IL-1）和 TOS。当亮氨酸和异亮氨酸联合使用时，与单独使用亮氨酸或异亮氨酸相比，MDA、TOS/TAS 和闭合蛋白-1、occludin 和 zonula occludens-1 的相对 mRNA 表达水平增加。蛋白质组学分析表明，亮氨酸显著上调了丙酸代谢、缬氨酸、亮氨酸和异亮氨酸降解以及产热途径，而异亮氨酸则显著上调了过氧化物酶体和丙酸代谢途径。总之，亮氨酸通过产生更多的 ATP 来补充能量需求，从而保护 MAC-T 细胞免受 HO 诱导的氧化应激，而异亮氨酸则改善了过氧化物酶体转运的不足，促进了乙酰辅酶 A 的产生。本研究结果增强了我们对亮氨酸和异亮氨酸抵抗氧化损伤的保护机制的理解。
