RESUMO
Mastitis in cows is caused by the inflammation of the mammary glands due to an infection by external pathogenic bacteria. Mammary gland epithelial cells, which are in direct contact with the external environment, are responsible for the first line of defense of the mammary gland against pathogenic bacteria, playing an essential role in immune defense. To investigate the mechanism of bovine mammary epithelial cells in the inflammatory process, we treated the cells with LPS for 12 hours and analyzed the changes in mRNA by transcriptome sequencing. The results showed that compared to the control group, the LPS treatment group had 121 up-regulated genes and 18 down-regulated genes. GO and KEGG enrichment analysis revealed that these differential genes were mainly enriched in the IL-17 signaling pathway, Legionellosis, Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, and other signaling pathways. Furthermore, the expression of GRO1 and CXCL3 mRNAs increased significantly after LPS treatment. These findings provide new insights for the treatment of mastitis in cows in the future.
Assuntos
Doenças dos Bovinos , Mastite Bovina , Feminino , Bovinos , Animais , Lipopolissacarídeos/farmacologia , Transcriptoma , Glândulas Mamárias Animais/metabolismo , Células Epiteliais/metabolismo , Mastite Bovina/genéticaRESUMO
Tetraconazole is a type of fungicide that eliminates pathogens in plants and fruit. To date, studies have focused on the direct exposure of plants and fruits to residual tetraconazole, but no studies have been conducted on the indirect effects of tetraconzaole. Given the importance of cows as milk-producing animals and their potential exposure to pesticides via plant consumption, we analyzed the mechanism by which tetraconazole influences milk production. Here, we confirmed that tetraconazole-induced apoptosis and inhibited cell viability and proliferation by regulating the cell cycle in bovine mammary epithelial cells (MAC-T). In addition, Ca2+ homeostasis in mitochondria was disrupted by tetraconazole, leading to the depolarization of mitochondrial membrane potential. Consistent with the proliferation-related findings, tetraconazole downregulated AKT, ERK1/2, P38, and JNK signaling pathways and proliferation-related proteins such as CCND1 and PCNA in MAC-T cells. Meanwhile, it upregulated cleaved caspase 3, BAX, and Cytochrome c under the same conditions in MAC-T cells. Furthermore, MAC-T exposed to tetraconazole causes a failure of proper autophagy functioning. In summary, the results of this study indicated that tetraconazole exposure may lead to a failure of milk production from bovine mammary epithelial cells by disrupting calcium homeostasis and mitochondrial function.
Assuntos
Cálcio , Glândulas Mamárias Animais , Feminino , Bovinos , Animais , Cálcio/metabolismo , Apoptose , Células Epiteliais , MitocôndriasRESUMO
Dairy farming is the most important economic activity in animal husbandry. Mastitis is the most common disease in dairy cattle and has a significant impact on milk quality and yield. The natural extract allicin, which is the main active ingredient of the sulfur-containing organic compounds in garlic, has anti-inflammatory, anticancer, antioxidant, and antibacterial properties; however, the specific mechanism underlying its effect on mastitis in dairy cows needs to be determined. Therefore, in this study, whether allicin can reduce lipopolysaccharide (LPS)-induced inflammation in the mammary epithelium of dairy cows was investigated. A cellular model of mammary inflammation was established by pretreating bovine mammary epithelial cells (MAC-T) with 10 µg/mL LPS, and the cultures were then treated with varying concentrations of allicin (0, 1, 2.5, 5, and 7.5 µM) added to the culture medium. MAC-T cells were examined using RT-qPCR and Western blotting to determine the effect of allicin. Subsequently, the level of phosphorylated nuclear factor kappa-B (NF-κB) was measured to further explore the mechanism underlying the effect of allicin on bovine mammary epithelial cell inflammation. Treatment with 2.5 µM allicin considerably decreased the LPS-induced increase in the levels of the inflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) and inhibited activation of the NOD-like receptor protein 3 (NLRP3) inflammasome in cow mammary epithelial cells. Further research revealed that allicin also inhibited the phosphorylation of inhibitors of nuclear factor kappa-B-α (IκB-α) and NF-κB p65. In mice, LPS-induced mastitis was also ameliorated by allicin. Therefore, we hypothesize that allicin alleviated LPS-induced inflammation in the mammary epithelial cells of cows probably by affecting the TLR4/NF-κB signaling pathway. Allicin will likely become an alternative to antibiotics for the treatment of mastitis in cows.
Assuntos
Dissulfetos , Mastite Bovina , NF-kappa B , Ácidos Sulfínicos , Animais , Bovinos , Feminino , Camundongos , Dissulfetos/uso terapêutico , Células Epiteliais/metabolismo , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Mastite Bovina/tratamento farmacológico , NF-kappa B/metabolismo , Transdução de Sinais , Ácidos Sulfínicos/uso terapêutico , Receptor 4 Toll-Like/metabolismoRESUMO
Serotonin (5-HT) has been reported to play an important role in mammary gland involution that is defined as the process through which the gland returns to a nonlactating state. However, the overall picture of the regulatory mechanisms of 5-HT and the effects of serotonylation on mammary gland involution still need to be further investigated. The current study aimed to investigate the effects of 5-HT on global gene expression profiles of bovine mammary epithelial cells (MAC-T) and to preliminarily examine whether the serotonylation involved in the mammary gland involution by using Monodansylcadaverine (MDC), a competitive inhibitor of transglutaminase 2. Results showed that a high concentration of 5-HT decreased viability and transepithelial electrical resistance (TEER) in MAC-T cells. Transcriptome analysis indicated that 2477 genes were differentially expressed in MAC-T cells treated with 200 µg/mL of 5-HT compared with the control group, and the Notch, p53, and PI3K-Akt signaling pathways were enriched. MDC influenced 5-HT-induced MAC-T cell death, fatty acid synthesis, and the formation and disruption of tight junctions. Overall, a high concentration of 5-HT is able to accelerate mammary gland involution, which may be regulated through the Notch, p53, and PI3K-Akt signaling pathways. Serotonylation is involved in bovine mammary gland involution.
Assuntos
Lactação , Serotonina , Feminino , Animais , Bovinos , Serotonina/farmacologia , Serotonina/metabolismo , Sobrevivência Celular , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Glândulas Mamárias Animais/metabolismo , Perfilação da Expressão Gênica , Células Epiteliais/metabolismo , PermeabilidadeRESUMO
BACKGROUND: Heat stress has a substantial negative economic impact on the dairy industry. N6-methyladenosine (m6A) is the most common internal RNA modification in eukaryotes and plays a key role in regulating heat stress response in animals. In dairy cows, however, this modification remains largely unexplored. Therefore, we examined the effects of heat stress on the m6A modification and gene expression in bovine mammary epithelial cells to elucidate the mechanism of heat stress response. In this study, Mammary alveolar cells-large T antigen (MAC-T) cells were incubated at 37 °C (non-heat stress group, NH) and 40 °C (heat stress group, H) for 2 hours, respectively. HSP70, HSF1, BAX and CASP3 were up regulated in H group compared with those in the NH group. RESULTS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were conducted to identify m6A peaks and to produce gene expression data of MAC-T cells in the H and NH groups. In total, we identified 17,927 m6A peaks within 9355 genes in the H group, and 18,974 peaks within 9660 genes in the NH groups using MeRIP-seq. Compared with the NH group, 3005 significantly differentially enriched m6A peaks were identified, among which 1131 were up-regulated and 1874 were down-regulated. In addition, 1502 significantly differentially expressed genes were identified using RNA-seq, among which 796 were up-regulated and 706 were down-regulated in the H group compared to the NH group. Furthermore, 199 differentially expressed and synchronously differentially methylated genes were identified by conjoint analysis of the MeRIP-seq and RNA-seq data, which were subsequently divided into four groups: 47 hyper-up, 53 hyper-down, 59 hypo-up and 40 hypo-down genes. In addition, GO enrichment and KEGG analyses were used to analyzed the potential functions of the genes in each section. CONCLUSION: The comparisons of m6A modification patterns and conjoint analyses of m6A modification and gene expression profiles suggest that m6A modification plays a critical role in the heat stress response by regulating gene expression.
Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Feminino , Bovinos , Animais , Adenosina/metabolismo , Resposta ao Choque Térmico/genética , Células Epiteliais/metabolismo , RNA/metabolismoRESUMO
Members of the genus Enterococcus are among the most relevant etiologic agents of bovine clinical and subclinical mastitis, a major problem for the dairy industry. In Brazil, clonal diversity, and multidrug resistance profiles related to bovine infections need further investigation. In this study, 11 bacterial strains recovered from mastitis subclinical cases detected in different farms of São Paulo, Brazil, were identified as Enterococcus faecalis (n = 8) and Enterococcus mundtii (n = 3) by biochemical testing and MALDI-TOF mass spectrometry. Pulsed-field gel electrophoresis categorized the enterococcal isolates into two main clusters (A and B) with similarity ranging from 85 to 100%. The isolates were shown to be resistant tetracycline (73%), erythromycin (73%), quinupristin-dalphopristin (64%), norfloxacin (9%), fosfomycin (9%) and linezolid (9%). Moreover, seven strains (64%) were considered multidrug-resistant. All the isolates were able to produce biofilms when grown in milk for 24 h: 54·54% were classified as moderate producers and 45·45% were weak producers. Interestingly, only two strains (Ef17 and Em42) remained as moderate biofilm producers after 48 h incubation. Moreover, all isolates showed no ability to form biofilm in tryptic soy broth (TSB) after 24 and 48 h incubation. In addition, cytoskeleton components were partially involved in E. faecalis and E. mundtii entry to epithelial cells as demonstrated by induction of actin stress fibre. In conclusion, enterococci isolates recovered from bovine subclinical mastitis were resistant to several classes of antibiotics, showing the ability to form biofilms in milk and invade mammary epithelial cells, suggesting an advantageous feature in mammary gland colonization during mastitis development. In addition, they can spread along the food chain by different routes and eventually constitute a possible threat for public health, including E. mundtii specie.
Assuntos
Mastite Bovina , Animais , Antibacterianos/farmacologia , Biofilmes , Brasil/epidemiologia , Bovinos , Farmacorresistência Bacteriana , Enterococcus , Enterococcus faecalis , Células Epiteliais , Feminino , Humanos , Mastite Bovina/microbiologia , Testes de Sensibilidade MicrobianaRESUMO
Folic acid (FA) is known to be an important micronutrient in humans; however, information regarding the effect of FA supplementation on bovine mammary epithelial (BME) cells is insufficient. FA supplementation is reported to increase milk production in dairy cows, but the underlying molecular mechanisms are unknown. This study examined the effects of FA supplementation on the proliferation and apoptosis of a BME cell line (MAC-T). MAC-T cells were treated with various concentrations (deficient in FA (DF) < 0.01 ng/mL; low-level FA (LF) 3.1 ng/mL; normal FA (NF) 15.4 ng/mL; and high-level FA (HF) 30.8 ng/mL) based on serum folate (10-20 ng/mL) in milking cows. HF treatment significantly increased the proliferation of MAC-T cells. Cellular apoptosis was observed mainly in the DF group. The number of apoptotic cells in DF media was significantly higher than that in NF media. The bcl-2/bax mRNA expression ratio was significantly increased in the HF group compared to that in the DF group. FA supplementation significantly increased the ratio of Bcl-2/Bax protein levels in MAC-T cells. FA supplementation increases proliferation and decreases apoptosis in these cells. This study might provide information regarding the molecular mechanism through which FA supplementation is associated with increased milk yield.
Assuntos
Glândulas Mamárias Animais , Linfócitos T , Animais , Apoptose , Bovinos , Proliferação de Células , Suplementos Nutricionais , Células Epiteliais , Feminino , Ácido Fólico/farmacologia , Lactação , LeiteRESUMO
Deoxynivalenol (DON) is one of the most common feed contaminants, and it poses a serious threat to the health of dairy cows. The existing studies of biological toxicity of DON mainly focus on the proliferation, oxidative stress, and inflammation in bovine mammary epithelial cells, while its toxicity on the biosynthesis of milk components has not been well documented. Hence, we investigated the toxic effects and the underlying mechanism of DON on the bovine mammary alveolar cells (MAC-T). Our results showed that exposure to various concentrations of DON significantly inhibited cell proliferation, induced apoptosis, and altered the cell morphology which was manifested by cell distortion and shrinkage. Moreover, the transepithelial electrical resistance (TEER) values of MAC-T cells exposed to DON were gradually decreased in a time- and concentration- dependent manner, but lactate dehydrogenase (LDH) leakage was significantly increased with the maximum increase of 2.4-fold, indicating the cell membrane and tight junctions were damaged by DON. Importantly, DON significantly reduced the synthesis of ß-casein and lipid droplets, along with the significantly decreases of phospho-mTOR, phospho-4EBP1, phospho-JAK2, and phospho-STAT5. Gene expression profiles showed that the expressions of several genes related to lipid synthesis and metabolism were changed, including acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid binding protein 3 (FABP3), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), and insulin-induced gene 1 (INSIG1). GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in ribosome, glutathione metabolism, and lipid biosynthetic process, which play important roles in the toxicological process induced by DON. Taken together, DON affects the proliferation and functional differentiation of MAC-T cells, which might be related to the cell junction disruption and morphological alteration. Our data provide new insights into functional differentiation and transcriptomic alterations of MAC-T cells after DON exposure, which contributes to a comprehensive understanding of DON-induced toxicity mechanism.
Assuntos
Leite , Junções Íntimas , Animais , Bovinos , Células Epiteliais , Feminino , Lipídeos , Junções Íntimas/metabolismo , TricotecenosRESUMO
Fluroxypyr-1-methylheptyl ester (FPMH) is an auxin herbicide that is widely applied to crops and pastures to block growth of post-emergence weeds. Several studies have reported the toxicity of FPMH in aquatic vertebrates. However, the adverse impacts of FPMH on mammals, including domestic animals, have not been reported. The purpose of our current study is to assess the impact of FPMH on the bovine mammary system and milk production. To evaluate the toxicity of FPMH on the mammary glands of lactating cows, the bovine mammary gland epithelial cell line, MAC-T, was exposed to various concentrations (0, 5, 7.5, 10, 15, and 20 µM) of FPMH for 24 h, and then various assessments were performed. The results showed that FPMH dose-dependently reduced MAC-T cell viability following exposure to FPMH and induced mitochondrial depolarization and apoptosis. FPMH also modulated signaling through the PI3K and MAPK pathways. In addition, the expression levels of proteins related to endoplasmic reticulum (ER) stress were upregulated, indicating induction of ER stress, and calcium homeostasis was disrupted following FPMH treatment. In conclusion, our investigation suggests that FPMH may be toxic to the bovine mammary system and may decrease dairy production.
Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Células Epiteliais/efeitos dos fármacos , Transdução de Sinais , Acetatos , Animais , Bovinos , Células Cultivadas , Ésteres , Lactação , Sistema de Sinalização das MAP Quinases , Glândulas Mamárias Animais/citologia , Fosfatidilinositol 3-Quinases , PiridinasRESUMO
Herbicides are chemicals that have been established to have adverse impacts. However, they are still widely used in agriculture. Pendimethalin (PDM) is an herbicide that is widely used in many countries to control annual grasses. The possibility of livestock being exposed to PDM is relatively high, considering the half-life of PDM and its residues in water, soil and crops. However, the toxicity of PDM in cattle, especially in the mammary glands, has not been reported. Therefore, we investigated whether PDM has toxic effects in the mammary epithelial cells (MAC-T) of cattle. MAC-T cells were treated with various doses (0, 2.5, 5 and 10 µM) of PDM. We found that PDM affected cell viability and cell proliferation and causes cell cycle arrest. Furthermore, PDM triggered cell apoptosis, induced excessive ROS production and mitochondrial membrane potential (MMP) loss, and disrupted calcium homeostasis. In addition, PDM altered the activation of proteins associated with the endoplasmic reticulum (ER) stress response and modified PI3K and MAPK signaling cascades. In conclusion, our current study unveiled the mechanism of PDM in MAC-T cells and we suggest that PDM might be harmful to the mammary gland system of cattle, possibly affecting milk production.
Assuntos
Herbicidas , Fosfatidilinositol 3-Quinases , Bovinos , Animais , Espécies Reativas de Oxigênio , Células Epiteliais , Herbicidas/toxicidade , Transdução de SinaisRESUMO
Sestrin2 (SESN2) is a highly conservative oxidative stress protein that can regulate energy metabolism, cell proliferation, apoptosis, and mitochondria autophagy processes. It plays a role as an antioxidant in various diseases. The aims of the present study were to explore the underlying role of SESN2 after hydrogen peroxide (H2 O2 ) treatment in bovine mammary epithelial cells (MAC-T cells) by the methods of knockout or overexpression of SESN2. The results show that knockout of Sestrin2 exacerbate apoptosis, upregulate the expressions of Bax/Bcl2 in H2 O2 -treated MAC-T cells. Moreover, knockout of SESN2 also promoted reactive oxygen species (ROS) generation and exacerbated oxidative damage in H2 O2 -treated MAC-T cells. On the contrary, overexpression of SESN2 decreased apoptosis by downregulation of Bax/Bcl2 level decreased ROS generation and blocked oxidative damage in H2 O2 -treated MAC-T cells. In addition, results indicate that the Kelch-like ECH-associated protein-1 (Keap1)-nuclear factor (erythroid-derived 2) like2 (Nrf2)/antioxidant response element (ARE) signaling pathway was activated by H2 O2 ; upregulation of SESN2 could relieve oxidative stress by inducing the expression of Keap1, Nrf2, HO-1, and NDPH: quinone oxidoreductase-1 protein. In conclusion, this study demonstrates that expression of SESN2 was significantly increased after H2 O2 treatment and that SESN2 can alleviate oxidative stress and cell apoptosis in H2 O2 -treated MAC-T cells through activation of the Keap1-Nrf2/ARE pathway.
Assuntos
Células Epiteliais/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas Nucleares/genética , Estresse Oxidativo/genética , Transdução de Sinais/genética , Regulação para Cima/genética , Animais , Antioxidantes/metabolismo , Apoptose/genética , Hidrolases de Éster Carboxílico/genética , Bovinos , Proliferação de Células/genética , Células Cultivadas , Regulação para Baixo/genética , Células Epiteliais/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Glândulas Mamárias Animais/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Pesticides, which are used in agriculture and forestry to eliminate insects, are a major cause of environmental pollution. Among them, diflubenzuron (DFB), 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl) urea, is a common benzoylurea insecticide that hinders larval development, primarily in Aedes aegypti larvae. Many experts have announced the biological toxicity of DFB in various species. However, the toxicity of benzoylurea pesticides, including DFB, to bovine mammary epithelial cells (MAC-T) is unclear. Therefore, in this study, we confirmed the cytotoxic effects of DFB on the viability and proliferation of MAC-T cells. Additionally, we observed that DFB induced lipid peroxidation through reactive oxygen species (ROS) production, resulting in an increase in transcriptional gene expression related to inflammatory response. Moreover, we demonstrated mitochondrial dysfunction including depolarization of the mitochondrial membrane, perturbation of calcium homeostasis, and, eventually, apoptosis. Furthermore, we identified DFB-triggered signaling pathways related to ROS generation and cell proliferation, as well as their interactions, by treating the cells with pharmacological inhibitors in combination with DFB. DFB attenuated the phosphorylation of AKT, P70S6K, S6, and ERK1/2 and facilitated the phosphorylation of JNK and c-Jun. These results show that DFB can induce apoptotic cell death via ROS generation and mitochondrial dysfunction in MAC-T cells.
Assuntos
Diflubenzuron , Animais , Apoptose , Bovinos , Diflubenzuron/metabolismo , Células Epiteliais , Mitocôndrias , Espécies Reativas de Oxigênio/metabolismoRESUMO
The objective of this study was to investigate the effects of supplementing with L-tryptophan (L-Trp) on milk protein synthesis using an immortalized bovine mammary epithelial (MAC-T) cell line. Cells were treated with 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mM of supplemental L-Trp, and the most efficient time for protein synthesis was determined by measuring cell, medium, and total protein at 0, 24, 48, 72, and 96 h. Time and dose tests showed that the 48 h incubation time and a 0.9 mM dose of L-Trp were the optimal values. The mechanism of milk protein synthesis was elucidated through proteomic analysis to identify the metabolic pathway involved. When L-Trp was supplemented, extracellular protein (medium protein) reached its peak at 48 h, whereas intracellular cell protein reached its peak at 96 h with all L-Trp doses. ß-casein mRNA gene expression and genes related to milk protein synthesis, such as mammalian target of rapamycin (mTOR) and ribosomal protein 6 (RPS6) genes, were also stimulated (p < 0.05). Overall, there were 51 upregulated and 59 downregulated proteins, many of which are involved in protein synthesis. The results of protein pathway analysis showed that L-Trp stimulated glycolysis, the pentose phosphate pathway, and ATP synthesis, which are pathways involved in energy metabolism. Together, these results demonstrate that L-Trp supplementation, particularly at 0.9 mM, is an effective stimulus in ß-casein synthesis by stimulating genes, proteins, and pathways related to protein and energy metabolism.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Triptofano/farmacologia , Animais , Bovinos , Linhagem Celular Transformada , Meios de Cultura , Feminino , Glândulas Mamárias Animais/citologiaRESUMO
Staphylococcus aureus is a vital bovine mastitis pathogen causing huge economic losses to the dairy industry worldwide. In our previous studies, leukotoxin ED (LukED) was detected in most S. aureus strains isolated from bovine mastitis. Here, four single-chain fragment variables (scFvs) (ZL8 and ZL42 targeting LukE, ZL22 and ZL23 targeting LukD) were obtained using purified LukE and LukD proteins as the antigens after five rounds of bio-panning. The complementarity-determining region 3 (CDR3) of the VH domain of these scFvs exhibited significant diversities. In vitro, the scFvs significantly decreased LukED-induced cell killing by inhibiting the binding of LukED to chemokine receptors (CCR5 and CXCR2) and reduced the death rates of bovine neutrophils and MAC-T cells caused by LukED and S. aureus (p < 0.05). In an S. aureus-induced mouse mastitis model, histopathology and MPO results revealed that scFvs ameliorated the histopathological damages and reduced the infiltration of inflammatory cells (p < 0.05). The ELISA and qPCR assays showed that scFvs reduced the transcription and expression levels of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-8 and IL-18 (p < 0.05). The overall results demonstrated the protective anti-inflammatory effect of scFvs in vitro and in vivo, enlightening the potential role of scFvs in the prevention and treatment of S. aureus-induced mastitis.
Assuntos
Inflamação/patologia , Leucocidinas/metabolismo , Mastite/microbiologia , Anticorpos de Cadeia Única/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Animais , Bovinos , Morte Celular , Citocinas/metabolismo , Feminino , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Leucocidinas/isolamento & purificação , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/metabolismo , Biblioteca de Peptídeos , Peroxidase/metabolismo , Receptores de Quimiocinas/metabolismo , Anticorpos de Cadeia Única/isolamento & purificação , Infecções Estafilocócicas/patologia , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
The interactions between Mycobacterium avium subsp. paratuberculosis (MAP) and the causative agents of bovine mastitis are still relatively unknown. Still, it is suspected that they may contribute to the worsening and persistence of mastitis within the mammary epithelial cells. Considering the growing economic implications of paratuberculosis and subclinical mastitis in dairy herds, this study aimed to determine the coinfection interaction between MAP and S. aureus or S. agalactiae in bovine mammary epithelial cells (MAC-T) in an ex-vivo model. For this purpose, internalisation tests of MAP + S. aureus or MAP + S. agalactiae were performed in MAC-T cells for 10, 30 and 120 min. The qPCR was performed to quantify internalised MAP at the time of exposure. Colony-forming units were counted on BHI agar medium for internalised subclinical mastitis bacteria at each time of infection. Viability tests of MAC-T cells, using the lactate dehydrogenase assay, were performed. The results showed that in the MAC-T cells previously infected by MAP and subsequently by S. aureus, there was a rapid internalisation in the first 10 min, maintaining a higher number of internalised bacteria during all exposure times. Regarding MAP + S. agalactiae, there were no changes in the internalisation patterns. The amount of MAP remained constant at all times evaluated, and there was no compromise in the viability of MAC-T cells during the tests. Thus, the results demonstrate the existence of an interaction between MAP + S. aureus, favouring internalisation and being able to contribute to the persistence of subclinical mastitis in dairy herds.
Assuntos
Doenças dos Bovinos , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Bovinos , Células Epiteliais , Feminino , Staphylococcus aureus , Streptococcus agalactiaeRESUMO
OBJECTIVES: Hydrogen sulfide (H2S) is involved in regulating cell apoptosis and proliferation. However, The effects and mechanism of H2S on the apoptosis of mammary epithelial cells that suffer from an inflammatory response remain unknown. RESULTS: An inflammatory cell model was used to explore whether exogenous H2S regulates lipopolysaccharides (LPS)-induced cell proliferation and apoptosis. We found that H2S affected cell viability, the inflammatory response and apoptosis in LPS-treated cells in a concentration-dependent manner. Moreover, exogenous H2S rescued LPS-induced cystathionine γ-lyase (CSE) inhibition and cystathionine ß-synthase (CBS) synthesis. Interestingly, in cells undergoing inflammation-induced apoptosis, H2S activated the PI3K/Akt and NFκB signal pathways both tested concentrations. Akt appeared to be a key crosstalk molecule that played a "bridge" role. CONCLUSIONS: H2S regulates LPS-induced inflammation and apoptosis by activating the PI3K/Akt/NFκB signaling pathway. Hence, NaHS may be clinically useful for preventing or treating mastitis.
Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/metabolismo , Sulfeto de Hidrogênio/farmacologia , Glândulas Mamárias Animais/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Bovinos , Linhagem Celular , Células Epiteliais/patologia , Feminino , Inflamação/metabolismo , Inflamação/patologia , Glândulas Mamárias Animais/patologiaRESUMO
A previous study in the bovine mammary epithelial cell line BME-UV1 demonstrated that suppression of the phosphatidylinositol-4,5-biphosphate 3 kinase (PI3K)/AKT (somatotropic) signaling pathway was required for transforming growth factor ß1 (TGFß1)-induced programmed cell death (PCD). To investigate whether this is a universal mechanism for TGFß1 to induce PCD in bovine mammary epithelium, we compared TGFß1 modulation of PI3K/AKT and its role in PCD in 2 bovine mammary epithelial cell lines: MAC-T and BME-UV1. In MAC-T cells, TGFß1 promoted cell survival, and this paralleled a reduction in PI3K/AKT activity, rather than an increase. In BME-UV1 cells, TGFß1 induced PCD, and this was accompanied by a time-dependent effect on PI3K/AKT activity, including an initial significant increase in the phosphorylation of AKT at 3 h, followed by a reduction between 12 and 24 h, and then an increase at 48 h. Inhibition of AKT activity enhanced TGFß1-induced PCD in BME-UV1 cells but had no effect on MAC-T cells, suggesting that TGFß1 mediates PCD in BME-UV1 cells through suppression of AKT activity. Inhibition of TGFß receptor type I (TßRI) kinase activity completely abrogated TGFß1-induced PCD in BME-UV1 cells but had no effect on TGFß1-induced suppression of PCD in MAC-T cells, demonstrating that TGFß1-induced PCD in BME-UV1 cells is dependent on TßRI/SMAD signaling. These and previous observations suggest that the different effects of TGFß1 on PCD in these cell lines might involve noncanonical signaling pathways other than PI3K/AKT, and may reflect their different lineages. Future studies should address this finding, taking into consideration the effect that different culture conditions might have on cell phenotype.
Assuntos
Apoptose , Bovinos/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Células Epiteliais/metabolismo , Feminino , Glândulas Mamárias Animais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , FosforilaçãoRESUMO
OBJECTIVE: Caseins and fatty acids of milk are synthesized and secreted by the epithelial cells of the mammary gland. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been shown to promote mammary development. This study was conducted to determine the effect of ATRA on casein synthesis and fatty acid composition in MAC-T cells. METHODS: MAC-T cells were allowed to differentiate for 4 d, treated with ATRA (0, 1.0, 1.5, and 2.0 µM), and incubated for 3 d. We analyzed the fatty acid composition, the mRNA expression of casein and fatty acid synthesis-related genes, and the phosphorylation of casein synthesis-related proteins of MAC-T cells by gas chromatography, quantitative polymerase chain reaction, and western blotting, respectively. RESULTS: In MAC-T cells, ATRA increased the mRNA levels of αS1-casein and ß-casein, janus kinase 2 (JAK2) and E74-like factor 5 of the signal transducer and activator of transcription 5 ß (STAT5-ß) pathway, ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 of the mammalian target of rapamycin (mTOR) pathway, inhibited the mRNA expression of phosphoinositide 3-kinase and eukaryotic initiation factor 4E of the mTOR pathway, and promoted the phosphorylation of STAT5-ß and S6K1 proteins. Additionally, ATRA increased the de novo synthesis of fatty acids, reduced the content of long-chain fatty acids, the ratio of monounsaturated fatty acids to saturated fatty acids (SFA), the ratio of polyunsaturated fatty acids (PUFA) to SFA, and the ratio of ω-6 to ω-3 PUFA. The mRNA levels of acetyl-CoA carboxylase 1, fatty acid synthase, lipoprotein lipase, stearoyl-CoA desaturase, peroxisome proliferator-activated receptor gamma, and sterol regulatory element-binding protein 1 (SREBP1) were enhanced by ATRA. CONCLUSION: ATRA promotes the synthesis of casein by regulating JAK2/STAT5 pathway and downstream mTOR signaling pathway, and it improves the fatty acid composition of MAC-T cells by regulating SREBP1-related genes.
RESUMO
Bovine mammary epithelial cells (MAC-Ts) are a common cell line for the study of mammary epithelial inflammation; these cells are used to mechanistically elucidate molecular underpinnings that contribute to bovine mastitis. Bovine mastitis is the most prevalent form of disease in dairy cattle that culminates in annual losses of two billion dollars for the US dairy industry. Thus, there is an urgent need for improved therapeutic strategies. Histone deacetylase (HDAC) inhibitors are efficacious in rodent models of inflammation, yet their role in bovine mammary cells remain unclear. HDACs have traditionally been studied in the regulation of nucleosomal DNA, in which deacetylation of histones impact chromatin accessibility and gene expression. Using MAC-T cells stimulated with tumor necrosis factor α (TNF-α) as a model for mammary cell inflammation, we report that inhibition of HDACs1 and 2 (HDAC1/2) attenuated TNF-α-mediated inflammatory gene expression. Of note, we report that HDAC1/2-mediated inflammatory gene expression was partly regulated by c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation. Here, we report that HDAC1/2 inhibition attenuated JNK and ERK activation and thus inflammatory gene expression. These data suggest that HDACs1 and 2 regulate inflammatory gene expression via canonical (i.e., gene expression) and noncanonical (e.g., signaling dependent) mechanisms. Whereas, further studies using primary cell lines and animal models are needed. Our combined data suggest that HDAC1/2-specific inhibitors may prove efficacious for the treatment of bovine mastitis.
Assuntos
Células Epiteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Glândulas Mamárias Animais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Bovinos , Linhagem Celular , Células Epiteliais/enzimologia , Feminino , Regulação da Expressão Gênica , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 2/antagonistas & inibidores , Inibidores de Histona Desacetilases/uso terapêutico , Glândulas Mamárias Animais/enzimologia , Mastite Bovina/tratamento farmacológico , Mastite Bovina/enzimologia , Fosforilação , Transdução de SinaisRESUMO
MiR-24-3p, a broadly conserved, small, noncoding RNA, is abundantly expressed in mammary tissue. However, its regulatory role in this tissue remains poorly understood. It was predicted that miR-24-3p targets the 3' untranslated region (3'-UTR) of multiple endocrine neoplasia type 1 (MEN1), an important regulatory factor in mammary tissue. The objective of this study was to investigate the function of miR-24-3p in mammary cells. Using a luciferase assay in mammary epithelial cells (MAC-T), miR-24-3p was confirmed to target the 3'-UTR of MEN1. Furthermore, miR-24-3p negatively regulated the expression of the MEN1 gene and its encoded protein, menin. miR-24-3p enhanced proliferation of MAC-T by promoting G1/S phase progression. MiR-24-3p also regulated the expression of key factors involved in phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin and Janus kinase/signal transducer and activators of transcription signaling pathways, therefore controlling milk protein synthesis in epithelial cells. Thus, miR-24-3p appears to act on MAC-T by targeting MEN1. The expression of miR-24-3p was controlled by MEN1/menin, indicating a negative feedback loop between miR-24-3p and MEN1/menin. The negatively inhibited expression pattern of miR-24-3p and MEN1 was active in mammary tissues at different lactation stages. The feedback mechanism is a new concept to further understand the lactation cycle of mammary glands and can possibly to be manipulated to improve milk yield and quality.