Hu Huijie, Liu Juxiong, Yin Junxi, Hu Guiqiu, Huang Bingxu, Tu Liqun, Liu Xuanting, Xu Bin, Cao Yu, Guo Wenjin, Fu Shoupeng
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, China.
Department of Laboratory Animals, College of Animal Sciences, Jilin University, Changchun 130062, China.
J Dairy Sci. 2025 Sep;108(9):10151-10172. doi: 10.3168/jds.2025-26746. Epub 2025 Jul 9.
Bovine mastitis is a major challenge in the dairy industry, leading to persistent oxidative stress and mammary epithelial cell senescence, which impairs mammary gland function and hinders milk yield recovery. The mitochondria-associated membrane (MAM), a critical interface between mitochondria and the endoplasmic reticulum, plays an important role in redox balance and mitochondrial homeostasis. This study aimed to investigate the role of MAM in oxidative stress-induced cellular senescence in lactating Holstein dairy cows. We first examined oxidative stress markers and key proteins related to the MAM pathway in mammary tissues using Western blotting and commercial assay kits, and found that MAM pathway alterations were negatively correlated with oxidative stress. Transcriptome analysis further confirmed this association, with differentially expressed genes enriched in the mitochondria-endoplasmic reticulum network. Subsequently, an HO-induced oxidative stress model was established in bovine mammary epithelial cells. The results showed that oxidative stress inhibited MAM formation, promoted mitochondrial fission, and induced cellular senescence. In our previous experiments, we identified mitofusin 2 (MFN2) as a critical regulator in this process. Adenoviral overexpression of MFN2 enhanced MAM formation, alleviated oxidative stress, and delayed senescence. Further investigations revealed that MFN2 undergoes proteasomal degradation under oxidative stress. When the MAM structure was disrupted, MFN2 lost its antioxidative and antisenescence functions, indicating that MAM is essential for its activity. Based on this mechanism, we identified Gracilaria lemaneiformis polysaccharide (GLP) as a potential MFN2 activator. The GLP was found to upregulate MFN2 transcription, inhibit its ubiquitination, and enhance its protein stability. When combined with antibiotic therapy, GLP effectively reduced oxidative stress in mastitic cows, restored mammary gland function, and downregulated the expression of senescence-related markers. These findings suggest that oxidative stress-induced degradation of MFN2 impairs MAM formation, resulting in excessive mitochondrial fission and cellular senescence. Mitofusin 2 overexpression restores MAM integrity and mitigates oxidative stress. Activation of MFN2 by GLP offers a promising therapeutic strategy for mastitis, with potential to reduce recurrence and improve mammary gland health in dairy cows.
奶牛乳腺炎是乳制品行业面临的一项重大挑战，会导致持续的氧化应激和乳腺上皮细胞衰老，进而损害乳腺功能并阻碍产奶量恢复。线粒体相关膜（MAM）是线粒体与内质网之间的关键界面，在氧化还原平衡和线粒体稳态中发挥着重要作用。本研究旨在探讨MAM在氧化应激诱导的泌乳期荷斯坦奶牛细胞衰老中的作用。我们首先使用蛋白质免疫印迹法和商业检测试剂盒检测了乳腺组织中氧化应激标志物和与MAM途径相关的关键蛋白，发现MAM途径改变与氧化应激呈负相关。转录组分析进一步证实了这种关联，差异表达基因富集于线粒体 - 内质网网络。随后，在牛乳腺上皮细胞中建立了过氧化氢诱导的氧化应激模型。结果表明，氧化应激抑制MAM形成，促进线粒体分裂，并诱导细胞衰老。在我们之前的实验中，我们确定线粒体融合蛋白2（MFN2）是这一过程中的关键调节因子。腺病毒介导的MFN2过表达增强了MAM形成，减轻了氧化应激，并延缓了衰老。进一步研究表明，MFN2在氧化应激下会经历蛋白酶体降解。当MAM结构被破坏时，MFN2失去其抗氧化和抗衰老功能，这表明MAM对其活性至关重要。基于这一机制，我们确定龙须菜多糖（GLP）为潜在的MFN2激活剂。发现GLP可上调MFN2转录，抑制其泛素化，并增强其蛋白质稳定性。当与抗生素治疗联合使用时，GLP有效降低了患乳腺炎奶牛的氧化应激，恢复了乳腺功能，并下调了衰老相关标志物的表达。这些发现表明，氧化应激诱导的MFN2降解损害了MAM形成，导致线粒体过度分裂和细胞衰老。MFN2过表达可恢复MAM完整性并减轻氧化应激。GLP激活MFN2为乳腺炎提供了一种有前景的治疗策略，有可能降低复发率并改善奶牛的乳腺健康。
