MAC-T Cells Research Articles

NOTCH and IGF1 signaling systems are involved in the effects exerted by anthelminthic treatment of heifers on the bovine mammary gland.

Dairy heifers with gastrointestinal nematodes have reduced growth rates, and delayed age at puberty and milk production onset related to late mammary gland development. IGF1 and Notch signaling systems are important in this process, and an altered profile of serum IGF1 has been associated with the detrimental effect of the nematodes on parenchymal development. In this context, we aimed to study the molecular mechanisms involved in bovine mammary gland development around pre and postpuberty, focusing on proliferative and angiogenic processes that involve the Notch and IGF1 pathways. We used mammary tissue samples from pre and pubertal heifers, treated or untreated with anthelmintics, and MAC-T bovine mammary epithelial cells in vitro. Anthelminthic treatment effectively lowered EPG in feces. Mammary glands from treated heifers had increased proliferation rate (measured by PCNA) and angiogenic marker expression (VEGF and CD34), as well as increased αSMA area compared to age-matched control parasitized heifers. These changes were preceded by increased expression of Notch targets at 20 wk of age (HES1, HEY2, and HEY1), indicating a possible interaction. Similarly, IGF1R expression was increased at 30 weeks of age. To study the crosstalk between systems, bovine MAC-T cells were treated with DAPT (50 μM) to inhibit Notch signaling. DAPT decreased the proliferation of cells as evidenced by a decrease in PCNA, pERK, CYCYLIN D1; and the wound healing capacity of HMEC cells was impaired in the presence of the supernatants of DAPT-treated cells. Furthermore, DAPT decreased IGF1 and increased IGF1R mRNA levels in MAC-T cells. On the other hand, cells treated with 10 ng/mL IGF1 Increased their proliferation (MTS assay), and induced a strong tendency to increase Notch target genes (HEY1, and HES1). Furthermore, IGF1 treatment tampered the decrease in the proliferation rate induced by DAPT. Finally, a positive correlation between the IGF1R and Notch target genes (HEY1, and HES1) further suggested a relation between these two signaling systems in the bovine mammary gland. In conclusion, pubertal delay related to parasitosis is counteracted by anthelminthic treatments, which increase serum IGF1, mammary cell proliferation, and angiogenesis. We postulate the Notch pathway, mainly through the HEY1 target gene, which is modulated by the IGF1 system, may regulate both proliferative and angiogenic processes favoring normal development of the bovine mammary gland during puberty. In addition, we demonstrate that the interaction between the Notch and the IGF1 pathways may affect cell proliferation.

Pterostilbene attenuates oxidative stress induced by hydrogen peroxide in MAC-T cells through activating PINK1/Parkin-mediated mitophagy

High‐producing dairy cows are susceptible to altered redox balance owing to their high secretion and strong metabolism during transition to lactation. Previously reports demonstrated that pterostilbene (PTE) alleviate hydrogen peroxide (H2O2)-induced oxidative damage. However, molecular mechanism underlying protective role of PTE in H2O2-elicited oxidative stress in bovine mammary epithelial cells (MAC-T cells) remains unclear. The aims of our research is to clarify the potential molecular mechanisms of PTE in H2O2-triggered oxidative damage. MAC-T cells were pre-treated with PTE (0, 10, 25 and 50 μM) for 12 h and then cultured with H2O2 (0, 100, 200, 400, 600, 800 and 1000 μM) for another 24 h. The results were interpreted by CCK8, western blot, and immunofluorescence assay. Results showed that PTE significantly attenuated H2O2-mediated reduction in T-AOC, SOD and GSH-Px activity. Mechanistic studies found that PTE restricted the H2O2-induced protein expression of HO-1, GPX4 and SOD1, and PTE reversed intracellular MDA and ROS generation and decreased the MMP in MAC-T cells. Moreover, H2O2 exposure markedly inhibited mitophagy, whereas PTE pre-treatment activated PINK1/Parkin-mediated mitophagy pathway, which further limited ROS production. Overall, PTE can be used to improve oxidative stress and as a novel antioxidant agent in dairy cows. Highlights PTE restored the levels of H2O2-induced oxidative stress markers (T-AOC, SOD, GSH-Px and MDA) in MAC-T cells. PTE effectively blocked the increase of protein expression (HO-1, SOD1 and GPX4) in H2O2-induced MAC-T cells. PTE remarkably decreased the accumulation of ROS and improved MMP in H2O2-induced MAC-T cells. PTE exerted a significant protective effect against oxidative stress injury by reducing the inhibition of H2O2 on mitophagy.

A Disintegrin and Metalloproteinase 17 Disrupts Bovine Macrophage MER Proto-Oncogene Tyrosine Kinase Integrity to Impede Apoptotic Cell Clearance and Promote Inflammation in Clinical Mastitis.

In clinical mastitis of dairy cows, the abnormal accumulation of apoptotic cells (ACs) and subsequent secondary necrosis and inflammation pose significant concerns, with macrophage-mediated efferocytosis, crucial for ACs clearance, remaining unexplored in this context. In nonruminants, MER proto-oncogene tyrosine kinase (MERTK) receptors are essential for efferocytosis and A Disintegrin and Metalloproteinase 17 (ADAM17) is thought to play a role in regulating MERTK integrity. This study aimed to delineate the in situ role of efferocytosis in clinical mastitis, with a particular focus on the interaction between MERTK and ADAM17 in bovine macrophages. In mastitic mammary tissue, a significant accumulation of ACs was observed, along with active macrophage efferocytosis. Western blotting analysis revealed elevated expressions of MERTK and ADAM17, and immunofluorescence confirmed that MERTK is predominantly localized within CD163+ macrophages. Additionally, elevated levels of soluble MERTK (sol-MER) in serum indicated impaired integrity and functionality of MERTK. In vitro experiments with the bovine macrophage cell line Bo-mac cells selectively phagocytosed apoptotic MAC-T cells, a process associated with increased MERTK phosphorylation and an anti-inflammatory phenotype. The activation of ADAM17 by Phorbol 12-myristate 13-acetate (PMA) induced sol-Mer release and impaired efferocytosis, with these effects reversed by the ADAM17 inhibitor TAPI-1. Bo-mac efferocytosis was influenced by the presence and activation of MERTK. Silencing MERTK interrupted efferocytosis, a phenomenon also observed with the inhibition of MERTK phosphorylation by UNC2025, which concurrently suppressed anti-inflammatory cytokine production. These findings suggest that targeting the MERTK-ADAM17 axis could enhance inflammatory resolution, providing a novel therapeutic strategy for dairy cattle health management.

Regulation of the expression of casein alpha S1 and S2 genes in the bovine mammary epithelial cells by STAT5A

Cow milk is rich in protein. Major cow milk proteins include casein α S1 (CSN1S1), casein α S2 (CSN1S2), casein β (CSN2), casein kappa (CSN3), lactalbumin α (LALBA), and β-lactoglobulin (LGB). These milk proteins are produced through gene expression in the mammary epithelial cells. Little is known about the molecular mechanism that mediates the expression of milk protein genes in cows. In this study, we tested the hypothesis that the expression of milk protein genes in cows is mediated by STAT5A, a transcription factor that is induced to bind and activate the transcription of target genes by extracellular signals such as prolactin. To circumvent the need of prolactin-responsive bovine mammary epithelial cells, we generated a plasmid that expresses a constitutively active bovine STAT5A variant, bSTAT5ACA. Transfection of the bovine mammary epithelial cell line MAC-T cells with the bSTAT5ACA expression plasmid caused a more than 100,000-fold and 600-fold increase in the expression of CSN1S1 and CSN1S2 mRNAs, respectively, compared with transfection of the wild-type bovine STAT5A (bSTAT5A) expression plasmid. Transfection of bSTAT5ACA, however, had no significant effect on the expression of CSN2, CSN3, LALBA, or LGB mRNA in MAC-T cells. Transfection of bSTAT5ACA caused a more than 260-fold and 120-fold increase in the expression of a luciferase reporter gene linked to the bovine CSN1S1 and CSN1S2 promoters in MAC-T cells, respectively, compared with that of bSTAT5A. The bovine CSN1S1 and CSN1S2 promoters each contain a putative STAT5 binding site, and gel-shift and super-shift assays confirmed bSTAT5ACA binding to both sites. These results together suggest that STAT5A plays a major role in regulating the expression of CSN1S1 and CSN1S2 genes in the bovine mammary epithelial cells and that STAT5A regulates the expression of these genes at least in part by binding to the STAT5 binding sites in their promoter regions. These results also suggest that STAT5A does not play a major role in regulating the expression of other major milk protein genes.

SIRT5-mediated GLS and GDH desuccinylation attenuates the autophagy of bovine mammary epithelial cells induced by ammonia.

Sirtuin 5 (SIRT5) in mitochondria possesses a strong capacity for lysine desuccinylation, involving in various biological processes. Our previous research demonstrated that NH3 regulated autophagy dependent on SIRT5 in bovine mammary epithelial cells (bMECs). Interestingly, we discovered that SIRT5 reduced the content of NH3 and glutamate by inhibiting GLS activity in bMECs, the ratio of ADP/ATP also declined. In this study, we identified that SIRT5 interacted with endogenous GLS and GDH through Co-IP assay, but had no effect on endogenous GLS and GDH expression. SIRT5 made the succinylation levels of GLS and GDH significantly declined and resulted in the reduction of GLS and GDH activity. Next, the content of ammonia and glutamate, as well as the related autophagy markers were measured, we found that SIRT5 affected the glutamine metabolism, which attenuated ammonia release in MAC-T cells, accompanying with cellular autophagy decline, reducing the formation of autophagosome. Deletion of SIRT5 gene in MAC-T cells by means of CRISPR-cas9, we found the content of NH3 and glutamate increased, as well as autophagy promoted, which could be alleviated by SIRT5 overexpression. SIRT5 KO also resulted in increase of succinylation of GLS and GDH, as well as autophagy response in bMECs. Furthermore, SIRT5 promoted the maintenance of mitochondria homeostasis. Mechanistically, SIRT5 reduced ammonia release by modulating the succinylation levels and enzymatic activities of GLS and GDH in mitochondria and promoted the maintenance of mitochondria homeostasis, as well as further attenuated ammonia-stimulated autophagy in bovine mammary epithelial cells.

PTGS2/GRP78 Activation Triggers Endoplasmic Reticulum Stress Leading to Lipid Metabolism Disruption and Cell Apoptosis, Exacerbating Damage in Bovine Mastitis.

Lipoteichoic acid (LTA), an organic acid of Gram-positive bacteria, is closely related to mastitis in dairy cows. This study evaluates the effect of LTA-induced endoplasmic reticulum stress (ER stress) in vitro using MAC-T (mammary epithelial cells) and in dairy cows with mastitis. LTA stimulation significantly increases ER stress and apoptosis-related factors in MAC-T. Further analysis suggests that the increase in ER stress may be associated with interactions involving PTGS2 and GRP78. Protein structural studies indicate a strong interaction between PTGS2 and GRP78. Lipidomics results further demonstrate that LTA disrupts lipid balance in MAC-T cells, affecting lipid metabolism in the endoplasmic reticulum, including PC, PE, TAG, and DAG, thereby exacerbating inflammation and ER stress. In dairy cows with mastitis caused by Gram-positive bacterial infection, damaged epithelial cells, inflammatory cell infiltration, and apoptotic vesicles are observed in affected tissues. In contrast, tissues from healthy cows exhibit regular epithelial cells without inflammatory cells or apoptotic vesicles. Furthermore, a significant ER stress and apoptosis increase is observed in mastitis tissues. This study demonstrates the close association between LTA-induced cell damage and ER stress, contributing to understanding the mechanisms underlying LTA-induced damage and supporting strategies for mastitis prevention and control in dairy cows.

S100A9 Affects Milk Protein Content by Regulating Amino Acid Transporters and the PI3K-Akt, WNT, and mTOR Signaling Pathways.

Calgranulin B (S100A9) was found to be strongly associated with milk protein percentage in dairy cattle in our previous genome-wide association study. SNPs in S100A9 were identified via pooled sequencing, and genotyping of 1054 cows was performed individually using MassArray with MALDI-TOFMS technology. Association analyses between the S100A9 SNPs and five milk production traits were conducted using SAS 9.2 software. Functional studies of S100A9 were conducted using quantitative PCR, Western blot, CCK-8, and immunofluorescence assays. In the present study, we further verified that two SNPs in S100A9, g.17115387 C>A and g.17115176 C>A, were significantly associated with milk protein percentage. We found that S100A9 could affect the expressions of caseins CSN1S1, CSN2, and CSN3 in MAC-T cells by regulating the expressions of amino acid transporter genes. We investigated the effects of S100A9 on the PI3K-Akt, WNT, and mTOR pathways, which are well known to play important roles in mammary gland development and milk protein synthesis. Our results suggest that S100A9 regulates the expressions of the relevant genes in these pathways, and thus potentially influences the protein synthesis in the mammary gland. This study demonstrates the important role of the S100A9 gene in the milk protein trait of dairy cattle and provides new insights into the molecular mechanism of milk protein content.

The Effect of Rumen-Protected Conjugated Linoleic Acid on Milk Composition and Milk Energy Output of Holstein Cows and Its Potential Mechanism.

Conjugated linoleic acid (CLA) can regulate fatty acid metabolism and increase the content of unsaturated fatty acids in milk during the nutritional regulation process in dairy cows. However, its effects on milk composition and milk energy output in dairy cows remain unclear. We aimed to investigate whether RP-CLA reduces milk energy output during early lactation while lowering milk fat, as well as whether it affects milk protein synthesis and the potential mechanisms. We examined the milk composition of Holstein fresh cows at different stages by administering RP-CLA for 7 days and analyzing related gene expression in the MAC-T cell line. RP-CLA did not significantly alter milk yield, lactose, or blood ketone levels in early lactation. Starting RP-CLA from day 21 postpartum reduced milk fat by about 26% (3.74% vs 2.78%) on day 28 postpartum, slightly increased milk protein, and improved blood glucose on day 24. No such effects were seen with RP-CLA beginning on day 7 postpartum. RP-CLA supplementation saved 6.45 and 11.43% milk energy output during days 7-14 and 21-28, respectively. We hypothesize that the limited RP-CLA regulation effect during days 7-21 may relate to intense lipid mobilization under negative energy balance. In MAC-T cells under low glucose, t-10, c-12 CLA decreased the expression of SREBP1 and AMPK, while increased CDK1, indicating its role in reducing fat synthesis, easing energy deficiency, promoting cell proliferation, and enhancing protein synthesis. Similarly, under normal glucose, t-10, c-12 CLA suppressed SREBP1 and elevated CDK1 expression, with AMPK reduction only at low doses. Inhibiting milk fat synthesis with an SREBP1 inhibitor slightly upregulated CDK 1 without significantly affecting AMPK. In conclusion, RP-CLA has the potential to alleviate negative energy balance in dairy cows during days 21-35 postpartum. t10, c12 CLA can inhibit milk fat synthesis by reducing the expression of SREBP1 and AMPK in mammary epithelial cells, while increasing the expression of CDK1, thereby improving the cellular energy status and promoting milk protein synthesis. The energy required for the increase in milk protein is not derived from the energy saved by the inhibition of milk fat synthesis by CLA.

In Situ Expression of Yak IL-22 in Mammary Glands as a Treatment for Bovine Staphylococcus aureus-Induced Mastitis in Mice.

Since the development of dairy farming, bovine mastitis has been a problem plaguing the whole industry, which has led to a decrease in milk production, a reduction in dairy product quality, and an increase in costs. The use of antibiotics to treat mastitis can cause a series of problems, which can bring a series of harm to the animal itself, such as the development of bacterial resistance and dramatic changes in the gut flora. However, the in vivo and in vitro antibacterial activity of yak Interleukin-22 (IL-22) and its application in mastitis caused by Staphylococcus aureus have not been reported. In this study, the mammary gland-specific expression plasmid pLF-IL22 of the yak IL-22 gene was constructed and expressed in MAC-T cells and mammary tissue of postpartum female mice. The coding region of the IL-22 gene in yaks is 573 bp, which can encode 190 amino acids, and the homology difference in the IL-22 gene in yaks is less than 30%, which indicates certain conservation. IL-22 is a hydrophilic protein with a total positive charge of four, the presence of a signal peptide, and the absence of a transmembrane domain. Sufficient expression of IL-22 effectively inhibited the high expression of inflammatory factors caused by Staphylococcus aureus, reduced the symptoms of mammary gland histopathology, and alleviated mastitis. Under the action of IL-22, the intestinal flora of mastitis mice also changed, the abundance of intestinal Bacilli, Prevotellaceae, and Alloprevotella in mice increased after treatment, and the pathogenic bacteria decreased. These findings provide new insights into the potential application of the yak IL-22 gene in the treatment of bovine mastitis in the future.

Difenoconazole Induced Damage of Bovine Mammary Epithelial Cells via ER Stress and Inflammatory Response.

Difenoconazole (DIF) is a fungicide used to control various fungi. It is absorbed on the surface of different plants and contributes significantly to increased crop production. However, DIF is reported to exhibit toxicity to fungi and to aquatic plants, fish, and mammals, including humans, causing adverse effects. However, research on the impact of DIF on the mammary epithelial cells of herbivorous bovines is limited. DIF-induced damage and accumulation in the mammary glands can have direct and indirect effects on humans. Therefore, we investigated the effects and mechanisms of DIF toxicity in MAC-T cells. The current study revealed that DIF reduces cell viability and proliferation while triggering apoptotic cell death through the upregulation of pro-apoptotic proteins, including cleaved caspase 3 and Bcl-2-associated X protein (BAX), and the downregulation of leukemia type 2 (BCL-2). DIF also induced endoplasmic reticulum (ER) stress by increasing the expression of genes or proteins of Bip/GRP78, protein disulfide isomerase (PDI), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and endoplasmic reticulum oxidoreductase 1 Alpha (ERO1-Lα). We demonstrated that DIF induces mitochondria-mediated apoptosis in MAC-T cells by activating ER stress pathways. This cellular damage resulted in a significant increase in the expression of inflammatory response genes and proteins, including cyclooxygenase 2 (COX2), transforming growth factor beta 3 (TGFB3), CCAAT enhancer binding protein delta (CEBPD), and iNOS, in DIF-treated groups. In addition, spheroid formation by MAC-T cells was suppressed by DIF treatment. Our findings suggest that DIF exposure in dairy cows may harm mammary gland function and health and may indirectly affect human consumption of milk.

Caveolin 1 ameliorates nonesterified fatty acid-induced oxidative stress via the autophagy regulator Beclin 1 in bovine mammary gland epithelial cells

High blood concentrations of nonesterified fatty acids (NEFA) during ketosis enhance uptake by the mammary gland and impair autophagy while causing oxidative stress. Caveolin 1 (CAV1) is closely related to autophagy and plays a role in regulating oxidative stress. The aim of this study was to explore the potential role of CAV1 on oxidative stress and autophagy during a high NEFA challenge in the immortalized bovine mammary epithelial cell line MAC-T. Mammary gland tissue biopsies and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) Holstein cows at 3 to 10 (average = 6) days in milk. Compared with healthy cows, ketotic cows had lower dry matter intake (DMI), daily milk yield, serum glucose and greater serum NEFA and BHBA, accompanied by greater milk fat and lower milk protein. Malondialdehyde (MDA) was greater but activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) were lower in cows with clinical ketosis. A lower protein abundance of CAV1, Beclin 1, autophagy relative gene 5 (ATG5), and microtubule-associated protein 1 light chain 3 (LC3) as well as greater protein abundance of sequestosome-1 (SQSTM1, also called p62) were detected in mammary tissue of cows with clinical ketosis. In vitro, the MAC-T cells were treated with 0, 0.6 and 1.2 mM NEFA for 12 h or treated with 1.2 mM NEFA for various time points (0, 0.5, 1, 2, 4, 8, 12 and 24 h). Compared with 0 mM NEFA, protein abundance of CAV1, Beclin 1, ATG5 and LC3 was greater in the MAC-T challenged with 0.6 mM NEFA, but lower in the 1.2 mM NEFA group. Protein abundance of p62 was lower with 0.6 mM NEFA, but higher with 1.2 mM NEFA. In response to increasing doses of NEFA, mRNA abundance of CAV1, total antioxidant capacity (T-AOC) and SOD activity decreased while the level of reactive oxygen species (ROS) and content of MDA increased. The protein abundance of CAV1, Beclin 1, ATG5 and LC3 peaked at 0.5 h and 1 h, resulting in both linear and quadratic effects. The protein abundance of p62 decreased, reaching a nadir at 4 h in both a linear and quadratic manner. The silencing of CAV1 in MAC-T cells aggravated the 1.2 mM NEFA-induced decrease in Beclin 1 expression, impaired autophagy, and increase in oxidative stress, whereas the overexpression of CAV1 alleviated these effects. Pretreatment of MAC-T cells with Beclin 1 siRNA (si-Beclin 1) and overexpressing CAV1 followed by challenged with 1.2 mM NEFA reversed the CAV1 induced autophagy, thereby enhancing oxidative stress. Overall, these data suggest that CAV1 protects bovine mammary epithelial cells from NEFA-induced oxidative stress through enhancing the expression of Beclin 1 and activating autophagy.

In vitro and in silico assessment of cytotoxicity of dinitramine via calcium dysregulation and mitochondrial dysfunction in bovine mammary glands

The herbicide market is expanding rapidly due to the global increase in herbicide usage. Dairy cows are susceptible to herbicide exposure through the ingestion of contaminated plants, which can adversely affect the mammary gland health and reduce milk production. Dinitramine, a synthetic herbicide in the dinitroaniline family, is typically used to control weeds by inhibiting their sprouting and root development. While previous studies have demonstrated the cytotoxicity of dinitramine in aquatic organisms, research on its toxicity in cattle is limited. In this study, bovine mammary epithelial cells (MAC-T) were used to verify the detrimental effects on dairy cows, especially on the mammary glands. First, we evaluated the cytotoxic effects of dinitramine on MAC-T cells and examined various cellular responses to dinitramine treatment, including alterations in apoptotic cells, mitochondrial dysfunction, and calcium dysregulation. Moreover, the expression levels of AKT and MAPK signaling proteins were confirmed in response to dinitramine treatment. Alterations in the mRNA levels of genes related to milk production and inflammatory response following dinitramine exposure were evaluated using quantitative PCR. Finally, we assessed the binding affinity between dinitramine and the target proteins using in silico molecular docking analysis. Overall, the cumulative evidence of the various toxic effects of dinitramine on MAC-T cells suggests its potential to reduce both milk yield and quality.

Minthostachys verticillata essential oil modulates cytokine synthesis and Staphylococcus aureus internalization in MAC-T cells at least through TLR4/MyD88/NFkB pathway.

The aim of this study was to evaluate the activation pathway(s) triggered by Minthostachys verticillata essential oil (EO) in bovine mammary epithelial cells (MAC-T) challenged with a strain of bovine Staphylococcus aureus. MAC-T cells were stimulated with EO, S. aureus or pre-treated with EO and then challenged with S. aureus. Cytokine's release was measured by ELISA. The mRNA for TLR2, TLR4, NOD2, MyD88 and NFκB was quantified by RT-qPCR. S. aureus adherence and internalization was also evaluated. MAC-T cells stimulated with S. aureus synthesized high levels of IL-1ß and IL-6 were kept up to 48 h, while IL-4 levels were not altered.Cells pre-treated with EO for 2 and 6h and then challenged with S. aureus showed a significant increase of IL-1β and IL-6. However, in these cells, a decrease in IL-1ß and IL-6 levels and an increase of IL-4 values was observed from 24 h. No significant increase in the expression levels of TLR2 or NOD2 was detected in all stimulated cells. However, the expression of TLR4, MyD88 and NFκB was increased in cells stimulated with S. aureus at 2 and 6 h as well as in cells pre-treated with EO between 2 and 6h and then challenged with S. aureus. The NFκB expression levels was similar to control at 24h in all stimulated cells, although pro-inflammatory cytokine levels and TLR4 and MyD88 expression levels remained high in cells stimulated with S. aureus. This results suggested the activation of other pathways independent of MyD88 by the pathogen that involucrated the activation of others transcription factors. Pre-treatment with EO during 2, 6 and 24h did not affect S. aureus adherence but decreased its internalization. In conclusion, pre-treatment with EO increased the IL-1β and IL-6 synthesis during the first hours post-challenged with S. aureus up-regulating TLR4/MyD88/NFκB pathway. Furthermore, EO increased the IL-4 levels from 6 to 24 h down-regulating the NFκB and possibly other transcription factors activated by the pathogen, which decreased its internalization into MAC-T cells.

Palmitic acid-induced cell death: impact of endoplasmic reticulum and oxidative stress, mitigated by L-citrulline.

Palmitic acid (PA), the most abundant saturated free fatty acids, induces apoptosis in bovine mammary epithelial cells. It is suggested that oxidative stress and endoplasmic reticulum (ER) stress are key mechanisms underlying PA-induced cell death. This study aimed to investigate the interaction between ER stress and oxidative stress during PA-induced cell death in MAC-T cells. Additionally, we examined whether L-citrulline can protect against PA-induced damage of MAC-T cells. MAC-T cells were treated with 4-phenyl butyric acid (4-PBA) or N-acetyl-L-cysteine (NAC) to inhibit PA-induced ER stress and oxidative stress, respectively. MAC-T cells were pretreated with or without L-citrulline for 48 h followed by PA treatment. Cell viability was measured with MTT assays. Intracellular reactive oxygen species (ROS) levels in MAC-T cells were assessed using 5-(and-6)-chloromethyl- 2`,7`-dichlorodihydrofluorescein diacetate acetyl ester dye. Real-time qPCR was used to explore the regulation of genes associated with oxidative stress, and ER stress genes. Western blotting analysis was also carried out. 4-PBA significantly reduced PA-induced mRNA expressions of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and intracellular ROS levels. Furthermore, NAC dramatically reduced PA-induced ROS levels and the mRNA expressions of NRF2, ATF4, and CHOP. L-citrulline pretreatment effectively rescued cell viability decreased by PA. Moreover, L-citrulline pretreatment significantly downregulated the PA-induced upregulation of GRP78, ATF4, and CHOP mRNA expression, and protein expression of p-PERK and cleaved caspase-3. PA increased intracellular ROS levels and NRF2 mRNA expression, whereas L-citrulline pretreatment remarkably reduced these levels. Both ER and oxidative stresses interact during PA-induced cell death in MAC-T cells, and L-citrulline could attenuate this cell death by inhibiting ER and oxidative stresses. Therefore, L-citrulline may be a promising supplement for protecting against PA-induced cell death in bovine MECs during the lactation period of dairy cows.

DNA methylation patterns in the peripheral blood of Xinjiang brown cattle with variable somatic cell counts.

The use of wide-ranging dairy herd improvement (DHI) measurements has resulted in the investigation of somatic cell count (SCC) and the identification of many genes associated with mastitis resistance. In this study, blood samples of Xinjiang brown cattle with different SCCs were collected, and genome-wide DNA methylation was analyzed by MeDIP-seq. The results showed that peaks were mostly in intergenic regions, followed by introns, exons, and promoters. A total of 1,934 differentially expressed genes (DEGs) associated with mastitis resistance in Xinjiang brown cattle were identified. The enrichment of differentially methylated CpG islands of the TRAPPC9 and CD4 genes was analyzed by bisulfate genome sequencing. The methylation rate of differentially methylated CpGs was higher in the TRAPPC9 gene of cattle with clinical mastitis (mastitis group) compared with healthy cattle (control group), while methylation of differentially methylated CpGs was significantly lower in CD4 of the mastitis group compared with the control group. RT-qRCR analysis showed that the mastitis group had significantly reduced expression of CD4 and TRAPPC9 genes compared to the control group (p < 0.05). Furthermore, Mac-T cells treated with lipopolysaccharide and lipoteichoic acid showed significant downregulation of the TRAPPC9 gene in the mastitis group compared with the control group. The identified epigenetic biomarkers provide theoretical reference for treating cow mastitis, breeding management, and the genetic improvement of mastitis resistance in Xinjiang brown cattle.

In vivo and in vitro anti-inflammation of Rhapontici Radix extract on mastitis via TMEM59 and GPR161

Ethnopharmacological relevanceRhapontici Radix ethanol extract (RRE) is derived from the dried root of Rhaponticum uniflorum (L.) DC belonging to the Asteraceae family. RRE exhibits significant anti-inflammatory and antioxidant properties; however, the potential of RRE in mastitis treatment requires further investigation. Aim of this studyThis research was performed to examine the protective properties of RRE against mastitis and the mechanisms underlying the effects of RRE. Material and methodsRRE components were analyzed by HPLC-MS/MS and DPPH methods. Isochlorogenic acid B (ICAB) was obtained commercially. MTT assay was utilized to assess RRE or ICAB cytotoxicity in bovine mammary alveolar (MAC-T) cells. Immunohistochemistry were used to investigate the pathological alterations in mammary tissue. The protein levels of inflammatory cytokines and mediators were analyzed using ELISA, and the expression of MAPK and NF-κB signaling pathways, as well as p65 nuclear translocation, were analyzed through Western blotting and immunofluorescence techniques, respectively. Target proteins of RRE were screened by RNA-seq and tandem mass tag analyses. Protein interaction was revealed and confirmed using co-immunoprecipitation and CRISPR/Cas9-based knockdown and overexpression of target genes. ResultsICAB was revealed as one of the main components in RRE, and it was responsible for 84.33% of RRE radical scavenging activity. Both RRE and ICAB mitigated the infiltration of T lymphocytes in the mammary glands of mice, leading to decreased levels of inflammatory mediators (COX-2 and iNOS) and cytokines (TNF-α, IL-6, and IL-1β) in lipopolysaccharide (LPS)-induced MAC-T cells. Furthermore, RRE and ICAB suppressed the LPS-induced phosphorylation of NF-κB inhibitor and p65, thereby impeding p65 nuclear translocation in mouse mammary glands and MAC-T cells. In addition, RRE and ICAB attenuated the LPS-triggered activation of c-Jun N-terminal kinase 1/2, p38, and extracellular regulated protein kinase 1/2. Importantly, co-treated with LPS and ICAB in MAC-T cells, an upregulation of G-protein coupled receptor 161 (GPR161) and transmembrane protein 59 (TMEM59) was observed; the interact between TMEM59 and was found, leading to inhibition of NF-κB activity and inflammatory cytokine production. ConclusionICAB is a prominent antioxidant in RRE. RRE and ICAB reduce mammary inflammation via MAPK and NF-κB pathways and the interaction between TMEM59 and GPR161 mediates the control of ICAB in NF-κB signaling.

Varying the ratio of Lys: Met through enhancing methionine supplementation improved milk secretion ability through regulating the mRNA expression in bovine mammary epithelial cells under heat stress.

The ratio of lysine (Lys) to methionine (Met) with 3.0: 1 is confirmed as the "ideal" profile for milk protein synthesis, but whether this ratio is suitable for milk protein synthesis under HS needs to be further studied. To evaluate the molecular mechanism by which HS and Lys to Met ratios affect mammary cell functional capacity, an immortalized bovine mammary epithelial cell line (MAC-T) is incubated with 5 doses of Met while maintaining a constant concentration of Lys. The MAC-T cells was treated for 6 h as follow: Lys: Met 3.0: 1 (control 37°C and IPAA 42°C) or treatments under HS (42°C) with different ratios of Lys: Met at 2.0: 1 (LM20), 2.5: 1 (LM25), 3.5: 1 (LM35) and 4.0: 1 (LM40). RNA sequencing was used to assess transcriptome-wide alterations in mRNA abundance. The significant difference between control and other groups was observed base on PCA analysis. A total of 2048 differentially expressed genes (DEGs) were identified in the IPAA group relative to the control group. Similarly, 226, 306, 148, 157 DEGs were detected in the LM20, LM25, LM35 and LM40 groups, respectively, relative to the IPAA group. The relative mRNA abundance of HSPA1A was upregulated and anti-apoptotic genes (BCL2L1 and BCL2) was down-regulated in the IPAA group, compared to the control group (p < 0.05). Compared with the IPAA group, the relative mRNA abundance of anti-apoptotic genes and casein genes (CSN1S2 and CSN2) was up-regulated in the LM25 group (p < 0.05). The DEGs between LM25 and IPAA groups were associated with the negative regulation of transcription RNA polymerase II promoter in response to stress (GO: 0051085, DEGs of BAG3, DNAJB1, HSPA1A) as well as the mTOR signaling pathway (ko04150, DEGs of ATP6V1C2, WNT11, WNT3A, and WNT9A). Several DEGs involved in amino acids metabolism (AFMID, HYKK, NOS3, RIMKLB) and glycolysis/gluconeogenesis (AFMID and MGAT5B) were up-regulated while DEGs involved in lipolysis and beta-oxidation catabolic processes (ALOX12 and ALOX12B) were down-regulated. These results suggested that increasing Met supply (Lys: Met at 2.5: 1) may help mammary gland cells resist HS-induced cell damage, while possibly maintaining lactation capacity through regulation of gene expression.

Unveiling the regulatory role of SIRT1 in the oxidative stress response of bovine mammary cells

High-yield dairy cows typically undergo intense cellular metabolism, leading to oxidative stress in their mammary tissues. Our study found that compared with ordinary cows, these high-yield cows had significantly elevated levels of H2O2, lipoperoxidase, and total antioxidant capacity in their blood. This increased oxidative stress is associated with heightened expression of genes such as GCLC, GCLM, and SIRT1 and proteins such as SIRT1 in the mammary tissue of high-yield cows. We stimulated MAC-T cells with H2O2 at a concentration equal to the average H2O2 level in the serum of ethically high-yielding cows, as detected by an assay kit. Our observations revealed that short-term exposure (12 h) to H2O2 upregulated the expression of the SIRT1 gene and SIRT1 protein. It also increased gene expression for SOD2, CAT, GCLC, GCLM, PGC-1α, and NQO1, elevated the phosphorylation of AMPK, and enhanced protein expression of PGC-1α, NQO1, Nrf2, and HO-1, as well as reduced the phosphorylation of NF-κB. Additionally, short-term H2O2 stimulation resulted in increased total antioxidant capacity and levels of superoxide dismutase, glutathione, and catalase in the mammary epithelial cells of dairy cows. In contrast, prolonged exposure to H2O2 (24 h) yielded opposite results, indicating reduced antioxidant capacity. Further investigation showed that the SIRT1 inhibitor EX 527 could reverse the enhanced cellular antioxidant capacity triggered by short-term oxidative stress. However, it is crucial to note that although 12 h of H2O2 stimulation improved antioxidant capacity, reactive oxygen species (ROS) and malondialdehyde (MDA) levels inside the cell gradually increased over time, suggesting greater damage under long-term stimulation. Conversely, the SIRT1 activator SRT 2104 could reverse the reduced cellular antioxidant capacity caused by long-term oxidative stress and significantly inhibit the accumulation of ROS and MDA. Notably, SRT 2104 demonstrated similar effects in MAC-T cells during lactation. In summary, SIRT1 plays a crucial role in regulating the antioxidant capacity of mammary epithelial cells in dairy cows. This discovery provides valuable insights into the antioxidant mechanisms of mammary cells, which can serve as a theoretical foundation for future mammary health strategies.

Targeting PHB2-mediated mitophagy alleviates nonesterified fatty acid-induced mitochondrial dysfunction in bovine mammary epithelial cells

Mitochondrial dysfunction has been reported to occur in the mammary gland of dairy cows suffering from ketosis. Prohibitin 2 (PHB2) plays a crucial role in regulating mitophagy, which clears impaired mitochondria to maintain normal mitochondrial function. Therefore, the current study aimed to investigate how PHB2 mediates mitophagy, thereby influencing mitochondrial function in the immortalized bovine mammary epithelial cell line (MAC-T cells). First, mammary gland tissue and blood samples were collected from healthy cows (n = 15, BHB <0.6 mM) and cows with clinical ketosis (n = 15, BHB >3.0 mM). Compared with healthy cows, cows with clinical ketosis exhibited lower DMI, milk production, milk protein, milk lactose, and serum glucose. In contrast, milk fat, serum nonesterified fatty acids (NEFA) and BHB were greater in cows with clinical ketosis. The protein abundance of PHB2, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), mitofusin 2 (MFN2) in whole cell lysates (WCL), as well as PHB2, sequestosome-1 (SQSTM1, also called p62), microtubule-associated protein 1 light chain 3-II (MAP1LC3-II, also called LC3-II), and ubiquitinated proteins in mitochondrial fraction were significantly lower in cows with clinical ketosis. The ATP content of mammary gland tissue in cows with clinical ketosis was lower than that of healthy cows. Second, MAC-T were cultured and treated with NEFA (0, 0.3, 0.6, 1.2 mM). The MAC-T treated with 1.2 mM NEFA displayed decreased protein abundance of PHB2, PGC-1α, and MFN2 in WCL, as well as protein abundance of PHB2, p62, LC3-II, and ubiquitinated proteins in mitochondrial fraction. The content of ATP and JC-1 aggregates in 1.2 mM NEFA group were lower than in the 0 mM NEFA group. Additionally, 1.2 mM NEFA disrupted the fusion between mitochondria and lysosomes. The MAC-T were then pretreated with 100 nM rapamycin, followed by treatment with or without NEFA. Rapamycin alleviated impaired mitophagy and mitochondria dysfunction induced by 1.2 mM NEFA. Third, MAC-T were transfected with small interfering RNA to silence PHB2 or a plasmid for overexpression of PHB2, followed by treatment with or without NEFA. The silencing of PHB2 aggravated 1.2 mM NEFA-induced impaired mitophagy and mitochondrial dysfunction, whereas the overexpression of PHB2 alleviated these effects. Overall, this study provides evidence that PHB2, in regulation of mitophagy, is a mechanism for bovine mammary epithelial cells to counteract NEFA-induced mitochondrial dysfunction.

The role of interleukin-10 receptor alpha (IL10Rα) in Mycobacterium avium subsp. paratuberculosis infection of a mammary epithelial cell line

BackgroundJohne’s disease is a chronic wasting disease caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Johne’s disease is highly contagious and MAP infection in dairy cattle can eventually lead to death. With no available treatment for Johne’s disease, genetic selection and improvements in management practices could help reduce its prevalence. In a previous study, the gene coding interleukin-10 receptor subunit alpha (IL10Rα) was associated with Johne’s disease in dairy cattle. Our objective was to determine how IL10Rα affects the pathogenesis of MAP by examining the effect of a live MAP challenge on a mammary epithelial cell line (MAC-T) that had IL10Rα knocked out using CRISPR/cas9. The wild type and the IL10Rα knockout MAC-T cell lines were exposed to live MAP bacteria for 72 h. Thereafter, mRNA was extracted from infected and uninfected cells. Differentially expressed genes were compared between the wild type and the IL10Rα knockout cell lines. Gene ontology was performed based on the differentially expressed genes to determine which biological pathways were involved.ResultsImmune system processes pathways were targeted to determine the effect of IL10Rα on the response to MAP infection. There was a difference in immune response between the wild type and IL10Rα knockout MAC-T cell lines, and less difference in immune response between infected and not infected IL10Rα knockout MAC-T cells, indicating IL10Rα plays an important role in the progression of MAP infection. Additionally, these comparisons allowed us to identify other genes involved in inflammation-mediated chemokine and cytokine signalling, interleukin signalling and toll-like receptor pathways.ConclusionsIdentifying differentially expressed genes in wild type and ILR10α knockout MAC-T cells infected with live MAP bacteria provided further evidence that IL10Rα contributes to mounting an immune response to MAP infection and allowed us to identify additional potential candidate genes involved in this process. We found there was a complex immune response during MAP infection that is controlled by many genes.