Mechanism of mitofusin 2, mitochondria-associated membrane, and the mitochondrial pathway in alleviating oxidative stress and cell senescence in bovine mammary epithelial cells

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 H₂O₂-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.

MAM; MFN2; cell senescence; mastitis; oxidative stress.