Non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) are the metabolites of fat mobilization initiated by negative energy balance (NEB) during the perinatal period in dairy cows, which have an adverse effect on cell physiology of various bovine cell types. The aim of this study was to explore the biological roles of NEFA and BHBA on provoking oxidative stress and inflammatory responses in bovine mammary epithelial cells (BMECs). RNA sequencing analysis showed that there are 1343, 48, and 1725 significantly differentially expressed genes (DEGs) in BMECs treated with NEFA, BHBA and their combination. GO functional analysis revealed that the DEGs were significantly enriched in “response to oxidative stress” and “inflammatory response”. Further study demonstrated that NEFA and BHBA elevated the malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation and reduced the total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activity to cause oxidative stress. In addition, expression of inflammatory markers (NO, TNF-α, IL-6, and IL-1β) were increased after NEFA and BHBA stimulation. Mechanistically, our data showed that NEFA and BHBA activated the MAPK signaling pathway. Collectively, our results indicate that NEFA and BHBA induce oxidative stress and inflammatory response probably via the MAPK signaling pathway in BMECs.

Elevated concentrations of non-esterified fatty acid (NEFA) induced by negative energy balance (NEB) during the transition period of dairy cows is known to be toxic for multiple bovine cell types. However, the effect of NEFA in bovine mammary epithelial cells (BMECs) remains unclear. The present study aimed to explore the role and molecular mechanism of NEFA in endoplasmic reticulum (ER) stress and the subsequent apoptosis in BMECs. The results showed that NEFA increased ER stress and activated the three unfolded protein response (UPR) signaling sub-pathways by upregulating the expression of GRP78, HSP70, XBP1, ATF6, phosphor-PERK, and phosphor-IRE1α. We also found that NEFA dose-dependently induced apoptosis in BMECs, as indicated by flow cytometry analysis and increased apoptotic gene expression. RNA-seq analysis revealed that NEFA induced apoptosis in BMECs, probably via the ATF4-CHOP axis. Mechanistically, our data showed that NEFA increased reactive oxygen species (ROS) levels, resulting in the activation of the MAPK signaling pathway. Moreover, quercetin, a well-known antioxidant, was found to alleviate ER stress-mediated apoptosis in NEFA-treated BMECs. Collectively, our results suggest that NEFA induces ER stress-mediated apoptosis, probably via the ROS/MAPK signaling pathway, as quercetin has been shown to alleviate ER stress-mediated apoptosis in NEFA-treated BMECs.

In periparturient dairy cows, high non-esterified fatty acids (NEFAs) caused by a severe negative energy balance induce oxidative stress and metabolic dysfunction, which pose a severe challenge to the dairy industry. Resveratrol (RES) is a polyphenolic compound with antioxidant, anti-inflammatory and multiple other physiological effects. However, its effect on oxidative damage triggered by NEFAs in bovine mammary epithelial cells is rarely reported. This study aimed to investigate the antioxidant effects and underlying molecular mechanisms of RES in NEFA-challenged BMECs. The results showed that RES ameliorated NEFA-induced oxidative damage by upregulating antioxidant enzyme expression and reducing malondialdehyde (MDA) and reactive oxygen species (ROS). Furthermore, exogenous NEFAs resulted in a decrease in mitochondrial membrane potential (MMP), cellular adenosine triphosphate (ATP) production, energy metabolism (NAD⁺/NADH ratio), abnormal mitochondrial structure and an increase in apoptosis levels. RES treatment restored mitochondrial function in NEFA-stressed BMECs, as evidenced by the increase in MMP, ATP generation and NAD⁺/NADH ratio accompanying the decline in mitochondrial structural abnormalities and cell apoptosis. In addition, in vivo studies in a mouse model of oxidative damage induced by high-fat diet (HFD) demonstrated that RES alleviated oxidative damage (decreased MDA content) and mitochondrial dysfunction (decreased expression of Drp1 and Fis1 and increased levels of Mfn2, Cyt C mRNA and ATP production) in mammary gland tissue. Overall, these findings suggested that RES could alleviate NEFA-induced oxidative damage in BMECs by modulating mitochondrial function, thereby contributing to the prevention and treatment of oxidative damage in perinatal dairy cows with a negative energy balance.

Objective: Negative energy balance in transition cows elevates the circulating concentrations of non-esterified fatty acids (NEFA), potentially leading to mastitis and posing a significant threat to the dairy industry. Resveratrol is a polyphenolic compound with anti-inflammatory properties, yet its role in NEFA-induced inflammation in bovine mammary epithelial cells (BMECs) remains unclear. This study aimed how resveratrol protects against mastitis and its underlying mechanisms.Methods: BMECs were pre-treated with 100 μM resveratrol for 24 h, and then treated with 0.9 mM NEFAs for 4 h, and a PINK1 inhibitor was used to assess the mechanisms involved. Furthermore, a mouse model of mastitis was utilized to evaluate the hepatoprotective effects of resveratrol against mastitis in vivo.Results: Resveratrol significantly attenuated the NEFA-induced inflammatory response, evidenced by reduced levels of NLRP3 inflammasome components (NLRP3, caspase1, IL-1β) and pro-inflammatory cytokines (IL-6, IL-1β and TNF-α). Mechanistically, resveratrol promoted mitophagy by upregulating levels of LC3-II, PINK1, and Parkin, while downregulating P62 expression. The anti-inflammatory effect of resveratrol was reversed when PINK1 was inhibited. In vivo experiments confirmed that resveratrol alleviated mammary gland inflammation and enhanced PINK1-mediated mitophagy.Conclusion: This study demonstrates that resveratrol mitigates NLRP3-mediated inflammation by activating PINK1-mediated mitophagy, suggesting its potential as a therapeutic option for mastitis in perinatal dairy cows with negative energy balance.