Methionine alleviates heat stress-induced ferroptosis in bovine mammary epithelial cells through the Nrf2 pathway.

Heat stress (HS) triggers mammary gland degradation, accompanied by apoptosis and autophagy in bovine mammary epithelial cells, negatively affecting milk performance and mammary gland health. Ferroptosis is iron-mediated regulated cell death caused by over production of lipid peroxides, however, the relationship between ferroptosis and HS in bovine mammary epithelial cells has not been clarified. Methionine (Met) plays a notable role in alleviating HS affecting the mammary glands in dairy cows, but the underlying mechanisms require further exploration. Therefore, we evaluated the regulatory effect and mechanism of Met in alleviating HS-induced ferroptosis by using bovine mammary epithelial cell line (MAC-T) as an in vitro model. The results showed that Met improved cell vitality, restored mitochondrial function; reduced the content of various reactive oxygen species (ROS), especially hydrogen peroxide (H2O2) and superoxide anion (O2·-); had positive effects on antioxidant enzyme activity, namely glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). More importantly, Met reduced labile iron protein (LIP) levels; increased iron storage and simultaneously decreased the levels of lipid reactive oxygen species (lipid ROS) and malondialdehyde (MDA), which all caused by HS in MAC-T. Mechanistically, Met increased the protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7, member 11 (SLC7A11) and ferritin heavy chain 1 (FTH1) by activating nuclear factor E2-related factor 2 (Nrf2) expression. Additionally, the protection effect of Met was cut off in MAC-T cells after interference with Nrf2, manifesting in decresing the protein expression levels of GPX4, SLC7A11 and FTH1,and increasing the levels of LIP and lipid ROS. Our findings indicate that Met eases HS-induced ferroptosis in MAC-T through the Nrf2 pathway, revealing that Met produces a marked effect on easing HS-induced bovine mammary gland injury in dairy cows.

Nicotinamide mononucleotide alleviates heat stress-induced oxidative stress and apoptosis in BMECs through reducing mitochondrial damage and endoplasmic reticulum stress.

Heat stress is directly correlated to mammary gland dysfunction in dairy cows, especially in summer. Abnormally high environmental temperature induces oxidative stress and apoptosis in bovine mammary epithelial cells (BMECs). Nicotinamide mononucleotide (NMN) has beneficial effects in maintaining the cellular physiological functions. In this study, we evaluate the protective effect of NMN on heat stress-induced apoptosis of BMECs and explore the potential underlying mechanisms. Our results showed that heat stress considerably decreased cell viability in BMECs, whereas pretreatment of BMECs with NMN (150 µM) for 24 h significantly alleviated the negative effects of heat stress on cells. NMN protected BMECs from heat stress-induced oxidative stress by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited apoptosis by reducing the ratio of Bax/Bcl2 and blocking proteolytic the cleavage of Caspase-3 in heat stressed-BMECs. Importantly, NMN treatment could reduce mitochondrial damage through mediating the expression of mitochondrial fission and fusion-related genes, including Dynamin related protein 1 (Drp1), Mitochondrial fission 1 protein (Fis1), and Mitofusin1, 2 (MFN1, 2); and suppress endoplasmic reticulum stress through unfolded protein response regulator Glucose regulated protein 78 (GRP78), and downstream elements Recombinant activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Above all, our results demonstrate that NMN supplemention attenuates heat stress-induced oxidative stress and apoptosis in BMECs by maintaining mitochondrial fission and fusion, and regulating endoplasmic reticulum stress, which provides the convincing evidence that NMN has valuable potential in alleviating mammary gland injury of dairy cows caused by environmental heat stress.

Identification and bioinformatics analysis of differentially expressed milk exosomal microRNAs in milk exosomes of heat-stressed Holstein cows.

In summer, heat stress is one of the primary reasons for the compromised health and low milk productivity of dairy cows. Hyperthermia affects milk synthesis and secretion in the mammary glands of dairy cows. As molecules for intercellular communication, milk-derived exosomes carry genetic material, proteins, and lipids, playing a crucial role in mammary tissue growth and milk synthesis in dairy cows. The aim of this study was to explore the milk exosomal miRNA profile of heat-stressed and normal Holstein cows. We isolated and identified milk exosomes to screening for differentially expressed miRNAs using small RNA sequencing. Then, TargetScan and miRanda algorithms were used to predict the putative targets of the differentially expressed miRNAs, whereas GO and KEGG pathway enrichment analyses were performed for the differentially expressed miRNA-target genes. Our results showed that 215 miRNAs were significantly differentially expressed in heat-stressed milk exosomes, of which one was upregulated and 214 were significantly downregulated. GO and KEGG enrichment analyses indicated that differentially expressed miRNAs might play a role in apoptosis, autophagy, and the p38 MAPK pathway. qRT-PCR assay verified that the expression of miRNAs was consistent with the sequencing results, warranting further verification of their specific targets of action. In conclusion, changes in the miRNA expression profile of milk exosomes indicated the role of exosomal miRNAs in regulating heat stress resistance and apoptosis in dairy cows. Our results suggested that milk-derived exosomal miRNAs could increase mammary gland resistance to heat stress, thereby enhancing milk synthesis in dairy cows.

SIRT3 protects bovine mammary epithelial cells from heat stress damage by activating the AMPK signaling pathway.

With global warming, heat stress has become an important challenge for the global dairy industry. Sirtuin 3 (SIRT3), an important mitochondrial NAD+dependent decarboxylase and a major regulator of cellular energy metabolism and antioxidant defense, is integral to maintaining normal mitochondrial function. The aim of this study was to assess the protective effect of SIRT3 on damage to bovine mammary epithelial cells (BMECs) induced by heat stress and to explore its potential mechanism. Our results indicate that SIRT3 is significantly downregulated in heat-stressed mammary tissue and high-temperature-treated BMECs. SIRT3 knockdown significantly increased the expression of HSP70, Bax, and cleaved-caspase 3 and inhibited the production of antioxidases, thus promoting ROS production and cell apoptosis in BMECs. In addition, SIRT3 knockdown can aggravate mitochondrial damage by mediating the expression of genes related to mitochondrial fission and fusion, including dynamin-related protein 1, mitochondrial fission 1 protein, and mitochondrial fusion proteins 1and 2. In addition, SIRT3 knockdown substantially decreased AMPK phosphorylation in BMECs. In contrast, SIRT3 overexpression in high-temperature treatment had the opposite effect to SIRT3 knockdown in BMECs. SIRT3 overexpression reduced mitochondrial damage and weakened the oxidative stress response of BMECs induced by heat stress and promoted the phosphorylation of AMPK. Taken together, our results indicate that SIRT3 can protect BMECs from heat stress damage through the AMPK signaling pathway. Therefore, the reduction of oxidative stress by SIRT3 may be the primary molecular mechanism underlying resistance to heat stress in summer cows.