[Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/ß-catenin Signaling Pathway]. / 催乳颗粒通过分泌Frizzledç›¸å ³è›‹ç™½2-Wnt/ß-cateninä¿¡å·é€šè·¯æ”¹å–„å¤§é¼ äº§åŽç¼ºä¹³.

Objective: Based on the secreted frizzled-related protein 2 (SFRP2)-Wnt/ß-catenin signaling pathway, this study explored the effect and mechanism of Cuiru Keli (CRKL) in the treatment of postpartum hypogalactia. Methods: A rat model of postpartum hypogalactia was established by gavaging 2 mL of 1.6 mg/mL bromocriptine mesylate to female rats on the third day after delivery. Female rats with a delivery time difference of less than 48 hours were selected and randomly assigned to 7 groups, including a normal group (without any modeling or medication), a model group, a CRKL low-dose group of model group model rats receiving CRKL at the dose of 3 g/kg, a CRKL medium-dose group of model rats receiving CRKL at the dose of 6 g/kg, a CRKL high-dose group of model rats receiving CRKL at the dose of 9 g/kg, a positive drug group of model rats receiving domperidone at the dose of 3 mg/kg, and a negative control (NC) group of model rats receiving normal saline. Each group contained 6 rats. Except for the normal and model groups, the remaining 5 groups were continuously administered with the respective intervention drugs at the specified doses by gavage once a day for 10 days. Changes in the total litter mass of the offspring in the 7 groups within 10 days were measured, and HE staining was performed to identify pathological changes in the mammary tissue (MT). Six groups of rats (excluding the positive control group) were used to observe the pathological changes of eosinophils in pituitary tissue. ELISA was performed to determine the content of prolactin (PRL) in serum, immunohistochemical staining was used to determine the expression of prolactin receptor (PRLR) in MT, and RT-qPCR was used to determine the mRNA expression of genes related to lactation in MT. Network pharmacology and molecular docking were used to study the therapeutic effect and mechanism of CRKL on postpartum hypogalactia, particularly whether it acted through the SFRP2-Wnt/ß-catenin signaling pathway. The mechanism of CRKL treatment was further validated by detecting mRNA (RT-qPCR) and protein expression (Western blot) of related pathway genes. Cell experiments were conducted using primary culture rat mammary epithelial cells (RMEC) from rat MT. RMEC were divided into four groups, including a normal group (primary culture RMEC, untreated), SFRP2 overexpression group (primary cultured RMEC treated with SFRP2 overexpression vector), SFRP2 overexpression+CRKL group (receiving treatment for SFRP2 overexpression group plus 10% drug-containing serum), and negative control group (primary culture RMEC treated with empty vector). The effect of CRKL on the expression of lactation-related genes FASN, CSN2, and GLUT1 mRNA after SFRP2 overexpression was detected by RT-qPCR. Results: In this study, CRKL was administered at a dose of 3 g/kg in the CRKL low-dose group, 6 g/kg in the medium-dose group, and 9 g/kg in the high-dose group (P<0.05 or P<0.01). Compared with the model group, CRKL at all doses significantly increased the total litter weight gain of the offsprings within 10 days (P<0.05 or P<0.01), and effectively increased lactation (P<0.01), the area of mammary lobules, and the size and filling of acinar cavities. CRKL at all doses also increased the number of eosinophils that secreted PRL in the pituitary gland of the postpartum hypogalactia rat model, and increased the content of PRL in the serum (P<0.05 or P<0.01). CRKL promoted the secretion and expression of PRL in postpartum hypogalactic model rats. In addition, it significantly promoted the expression of genes related to milk fat, milk protein, and lactose synthesis in MT (P<0.05 or P<0.01). Network pharmacology predicted that the Wnt signaling pathway might be a key pathway for CRKL in treating postpartum hypogalactia. The molecular docking results showed that related chemical components in CRKL had good binding ability with CCND1 and SFRP2. Compared with the model group, CRKL at all doses inhibited the expression of SFRP2 gene in vivo (P<0.01) and activated the mRNA and protein expression of CCND1 and c-Myc in the Wnt/ß-catenin signaling pathway in MT (P<0.05 or P<0.01). Cell experiments showed that, compared to the normal group, SFRP2 overexpression reduced the mRNA expression of milk synthesis-related genes FASN, CSN2, and GLUT1 in RMEC (P<0.01). The CCK8 results indicated that 10% of the drug-containing serum was the effective concentration administered to cells (P<0.01). After administering drug-containing serum, the expression of the lactation-related genes FASN, CSN2, and GLUT1 were up-regulated (compared with the SFRP2 overexpression group, P<0.01). Conclusion: CRKL alleviates postpartum hypogalactia through the SFRP2-Wnt/ß-catenin signaling pathway. SFRP2 might be a potential new target for the diagnosis and treatment of postpartum hypogalactia. This reveals a new mechanism of CRKL in treating postpartum hypogalactia and promotes its clinical application.

Expression and Function of Mammary Epithelial Cell-Derived Immunoglobulins.

Over the past 20 years, increasing evidence has demonstrated that immunoglobulins (Igs) can be widely generated from non B cells, including normal and malignant mammary epithelial cells. In normal breast tissue, the expression of IgG and IgA has been identified in epithelial cells of mammary glands during pregnancy and lactation, which can be secreted into milk, and might participate in neonatal immunity. On the other hand, non B-IgG is highly expressed in breast cancer cells, correlating with the poor prognosis of patients with breast cancer. Importantly, a specific group of IgG, bearing a unique N-linked glycan on the Asn162 site and aberrant sialylation modification at the end of the novel glycan (referred to as sialylated IgG (SIA-IgG)), has been found in breast cancer stem/progenitor-like cells. SIA-IgG can significantly promote the capacity of migration, invasiveness, and metastasis, as well as enhance self-renewal and tumorigenicity in vitro and in vivo. These findings suggest that breast epithelial cells can produce Igs with different biological activities under physiological and pathological conditions. During lactation, these Igs could be the main source of milk Igs to protect newborns from pathogenic infections, while under pathological conditions, they display oncogenic activity and promote the occurrence and progression of breast cancer.

Sirtuin 5 alleviates apoptosis and autophagy stimulated by ammonium chloride in bovine mammary epithelial cells.

Ammonia (NH3) is an irritating and harmful gas that affects cell apoptosis and autophagy. Sirtuin 5 (SIRT5) has multiple enzymatic activities and regulates NH3-induced autophagy in tumor cells. In order to determine whether SIRT5 regulates NH3-induced bovine mammary epithelial cell apoptosis and autophagy, cells with SIRT5 overexpression or knockdown were generated and in addition, bovine mammary epithelial cells were treated with SIRT5 inhibitors. The results showed that SIRT5 overexpression reduced the content of NH3 and glutamate in cells by inhibiting glutaminase activity in glutamine metabolism, and reduced the ratio of ADP/ATP. The results in the SIRT5 knockdown and inhibitor groups were comparable, including increased content of NH3 and glutamate in cells by activating glutaminase activity, and an elevated ratio of ADP/ATP. It was further confirmed that SIRT5 inhibited the apoptosis and autophagy of bovine mammary epithelial cells through reverse transcription-quantitative PCR, western blot, flow cytometry with Annexin V FITC/PI staining and transmission electron microscopy. In addition, it was also found that the addition of LY294002 or Rapamycin inhibited the PI3K/Akt or mTOR kinase signal, decreasing the apoptosis and autophagy activities of bovine mammary epithelial cells induced by SIRT5-inhibited NH3. In summary, the PI3K/Akt/mTOR signal involved in NH3-induced cell autophagy and apoptosis relies on the regulation of SIRT5. This study provides a new theory for the use of NH3 to regulate bovine mammary epithelial cell apoptosis and autophagy, and provides guidance for improving the health and production performance of dairy cows.

RNA-seq screening and gene function analysis uncover GPR183 as a key mediator for methionine to stimulate milk synthesis in mouse mammary epithelial cells.

Methionine (Met) can activate mTOR to promote milk synthesis in mammary epithelial cells (MECs). However, it is largely unknown which G protein-coupled receptor (GPCR) can mediate the stimulation of Met on mTOR activation. In this study, we employed transcriptome sequencing to analyze which GPCRs were associated with the role of Met, and further used gene function study approaches to explore the role of GPR183 in Met stimulation on mTOR activation in HC11 cells. We identified 9 GPCRs including GPR183 which expression levels were upregulated by Met treatment through RNA-seq and subsequent RT-qPCR analysis. Using GPR183 knockdown and overexpression technology, we demonstrate that GPR183 is a positive regulator of milk protein and fat synthesis and proliferation of HC11 cells. Met affected GPR183 expression in a dose-dependent manner, and GPR183 mediated the stimulation of Met (0.6 mM) on milk protein and fat synthesis, cell proliferation, and mTOR phosphorylation and mRNA expression. The inhibition of PI3K blocked the phosphorylation of mTOR and AKT stimulated by GPR183 activation. In summary, through RNA-seq and gene function study, we uncover that GPR183 is a key mediator for Met to activate the PI3K-mTOR signaling and milk synthesis in mouse MECs.

Soluble CD14 produced by bovine mammary epithelial cells modulates their response to full length LPS.

Bovine mastitis remains a major disease in cattle world-wide. In the mammary gland, mammary epithelial cells (MEC) are sentinels equipped with receptors allowing them to detect and respond to the invasion by bacterial pathogens, in particular Escherichia coli. Lipopolysaccharide (LPS) is the major E. coli motif recognized by MEC through its interaction with the TLR4 receptor and the CD14 co-receptor. Previous studies have highlighted the role of soluble CD14 (sCD14) in the efficient recognition of LPS molecules possessing a full-length O-antigen (LPSS). We demonstrate here that MEC are able to secrete CD14 and are likely to contribute to the presence of sCD14 in milk. We then investigated how sCD14 modulates and is required for the response of MEC to LPSS. This study highlights the key role of sCD14 for the full activation of the Myd88-independent pathway by LPSS. We also identified several lncRNA that are activated in MEC in response to LPS, including one lncRNA showing homologies with the mir-99a-let-7c gene (MIR99AHG). Altogether, our results show that a full response to LPS by mammary epithelial cells requires sCD14 and provide detailed information on how milk sCD14 can contribute to an efficient recognition of LPS from coliform pathogens.

Regulatory roles of dopamine D2 receptor in milk protein production and apoptosis in mammary epithelial cells.

Dopamine D2 receptors (D2Rs) play crucial roles in regulating diverse physiological functions of the central nervous system and peripheral organs. D2Rs are also expressed in mammary glands. However, which cell types express D2Rs and whether they are involved in milk production remains unclear. The present findings revealed that D2Rs are expressed in the apical regions of the lateral membranes of mammary epithelial cells (MECs) in lactating mice. We also investigated the effects of the D2R agonist bromocriptine and/or antagonist domperidone on intracellular cAMP levels, milk protein production, and apoptosis in a lactation culture model of MECs that produce major milk components like lactating MECs in vivo. We found that bromocriptine decreased intracellular cAMP levels, whereas domperidone dose-dependently neutralized this effect. Bromocriptine also inhibited casein and lactoferrin production and suppressed activities of STAT5 and glucocorticoid receptors (GRs). Domperidone neutralized the inhibition of casein production as well as STAT5 and GR inactivation induced by bromocriptine. Furthermore, D2R activation by bromocriptine induced apoptosis and inactivated ERK, a signaling molecule responsible for promoting cell proliferation and survival. Domperidone attenuated ERK inactivation and apoptosis induced by bromocriptine. These findings suggest that D2Rs play regulatory roles in milk protein production and apoptosis in MECs.

Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland's defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1ß concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.

Regulatory impacts of PPARGC1A gene expression on milk production and cellular metabolism in buffalo mammary epithelial cells.

The PPARGC1A gene plays a fundamental role in regulating cellular energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, adipogenesis, gluconeogenesis, and glucose/fatty acid metabolism. In a previous study, our group investigated seven SNPs in Mediterranean buffalo associated with milk production traits, and the current study builds on this research by exploring the regulatory influences of the PPARGC1A gene in buffalo mammary epithelial cells (BuMECs). Our findings revealed that knockdown of PPARGC1A gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis. Additionally, we observed downregulated triglyceride secretion after PPARGC1A knockdown. Furthermore, the critical genes related to milk production, including the STATS, BAD, P53, SREBF1, and XDH genes were upregulated after RNAi, while the FABP3 gene, was downregulated. Moreover, Silencing the PPARGC1A gene led to a significant downregulation of ß-casein synthesis in BuMECs. Our study provides evidence of the importance of the PPARGC1A gene in regulating cell growth, lipid, and protein metabolism in the buffalo mammary gland. In light of our previous research, the current study underscores the potential of this gene for improving milk production efficiency and overall dairy productivity in buffalo populations.

Hesperidin ameliorates H2O2-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway.

BACKGROUND: Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells (bMECs) exposed to oxidative stress have not been elucidated. RESULTS: In this study, we investigated the effects of hesperidin on H2O2-induced oxidative stress in bMECs and the underlying molecular mechanism. We found that hesperidin attenuated H2O2-induced cell damage by reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels, increasing catalase (CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent. CONCLUSIONS: Our results suggest that hesperidin could protect bMECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

Cross-Oxygen Gradients Transcriptomic Comparison Revealed the Central Role of MAPK and Hippo in Hypoxia-Mediated Mammary Proliferation Inhibition.

The role of hypoxia in terms of affecting mammary epithelial cells (MECs) proliferation is closely associated with the milk synthesis of lactating mammals. Primary bovine MECs were cultured at 1, 6, 11, 16, and 21% O2 for 24 h. The results showed that cell proliferation decreased linearly, and hypoxic inducible factor (HIF)-1α expression increased linearly along with the declining O2. The linear increase in oxidative stress resulted in the accumulation of malondialdehyde and reactive oxygen species and decreased antioxidant enzyme activities following the reduced O2. Concerning mitochondria, the dynamin-related protein 1 showed improved expression, and optin atrophy protein 1 decreased along with the decreasing O2 gradient, which led to decreased mitochondrial mass and mitophagy emerging under 1% O2. Oxygen concentration-trend RNA-seq analysis was conducted. Specifically, HIF-1-MAPK (1% O2), PI3K-Akt-MAPK (6% O2), and p53-Hippo (11 and 16% O2) were found to primarily regulate cell proliferation in response to hypoxia compared with normoxia (21%), respectively. In conclusion, our study suggests that bMEC proliferation is suppressed in low-oxygen conditions, and is exacerbated following the reduced oxygen supply. The cross-oxygen gradient comparisons suggest that MAPK and Hippo, which are core pathways of mammary cell proliferation, are repressed by hypoxia via oxidative-stress-dependent signals.

Isolation and characterization of mammary epithelial cells derived from Göttingen Minipigs: A comparative study versus hybrid pig cells from the IMI-ConcePTION Project.

The value of pig as "large animal model" is a well-known tool for translational medicine, but it can also be beneficial in studying animal health in a one-health vision. The ConcePTION Project aims to provide new information about the risks associated with medication use during breastfeeding, as this information is not available for most commonly used drugs. In the IMI-Conception context, Göttingen Minipigs have been preferred to hybrid pigs for their genetic stability and microbiological control. For the first time, in the present research, three primary cell cultures of mammary epithelial cells were isolated and characterized from Göttingen Minipigs (mpMECs), including their ability to create the epithelial barrier. In addition, a comparative analysis between Göttingen Minipigs and commercial hybrid pig mammary epithelial cells (pMECs) was conducted. Epithelial markers: CKs, CK18, E-CAD, ZO-1 and OCL, were expressed in both mpMECs and pMECs. RT2 Profiler PCR Array Pig Drug Transporters showed a similar profile in mRNA drug transporters. No difference in energy production under basal metabolic condition was evidenced, while under stressed state, a different metabolic behaviour was shown between mpMECs vs pMECs. TEER measurement and sodium fluorescein transport, indicated that mpMECs were able to create an epithelial barrier, although, this turned out to be less compact than pMECs. By comparing mpMECs with mammary epithelial cells isolated from Hybrid pigs (pMECs), although both cell lines have morphological and phenotypic characteristics that make them both useful in barrier studies, some specific differences exist and must be considered in a translational perspective.

Hexokinase 1 and 2 mediates glucose utilization to regulate the synthesis of kappa casein via ribosome protein subunit 6 kinase 1 in bovine mammary epithelial cells.

Glucose plays a vital part in milk protein synthesis through the mTOR signaling pathway in bovine mammary epithelial cells (BMEC). The objectives of this study were to determine how glucose affects hexokinase (HK) activity in BMEC and investigate the regulatory effect of HK in kappa casein (CSN3) synthesis via the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in BMEC. For this, HK1 and HK2 were knocked out in BMEC using the CRISPR/Cas9 system. The gene and protein expression, glucose uptake, and cell proliferation were measured. We found that glucose uptake, cell proliferation, CSN3 gene expression levels, and expression of HK1 and HK2 increased with increasing glucose concentrations. Notably, glucose uptake was significantly reduced in HK2 knockout (HK2KO) BMEC treated with 17.5 mM glucose. Moreover, under the same glucose treatment conditions, the proliferative ability and abundance of CSN3 were significantly diminished in both HK1 knockout (HK1KO) and HK2KO BMEC compared with that in wild-type BEMC. We further observed that the phosphorylation levels of ribosome protein subunit 6 kinase 1 (S6K1) were reduced in HK1KO and HK2KO BMEC following treatment with 17.5 mM glucose. As expected, the levels of glucose-6-phosphate and the mRNA expression levels of glycolysis-related genes were decreased in both HK1KO and HK2KO BMEC following glucose treatment. These results indicated that the knockout of HK1 and HK2 inhibited cell proliferation and CSN3 expression in BMEC under glucose treatment, which may be associated with the inactivation of the S6K1 and inhibition of glycolysis.

Chromatograms and Mass Spectra of High-Mannose and Paucimannose N-Glycans for Rapid Isomeric Identifications.

N-Linked glycosylation is one of the most essential post-translational modifications of proteins. However, N-glycan structural determination remains challenging because of the small differences in structures between isomers. In this study, we constructed a database containing collision-induced dissociation MSn mass spectra and chromatograms of high-performance liquid chromatography for the rapid identification of high-mannose and paucimannose N-glycan isomers. These N-glycans include isomers by breaking of arbitrary numbers of glycosidic bonds at arbitrary positions of canonical Man9GlcNAc2 N-glycans. In addition, some GlcMannGlcNAc2 N-glycan isomers were included in the database. This database is particularly useful for the identification of the N-glycans not in conventional N-glycan standards. This study demonstrated the application of the database to structural assignment for high-mannose N-glycans extracted from bovine whey proteins, soybean proteins, human mammary epithelial cells, and human breast carcinoma cells. We found many N-glycans that are not expected to be generated by conventional biosynthetic pathways of multicellular eukaryotes.

Linc-smad7 is involved in the regulation of lipid synthesis in mouse mammary epithelial cells.

Long intergenic non-coding RNA(lincRNA) is transcribed from the intermediate regions of coding genes and plays a pivotal role in the regulation of lipid synthesis. N6-methyladenosine (m6A) modification is widely prevalent in eukaryotic mRNAs and serves as a regulatory factor in diverse biological processes. This study aims to delineate the mechanism by which Linc-smad7 mediates m6A methylation to regulate milk fat synthesis. Tissue expression analysis in this study revealed a high expression of Linc-smad7 in breast tissue during pregnancy. Cell proliferation assays, including CCK8 and EdU assays, demonstrated that Linc-smad7 had no significant impact on the proliferation of mammary epithelial cells. However, during mammary epithelial cell differentiation, the overexpression of Linc-smad7 led to reduced lipid formation, whereas interference with Linc-smad7 promoted lipogenesis. Mechanistically, Linc-smad7 was found to modulate RNA m6A levels, as evidenced by dot blot assays and methylated RNA immunoprecipitation sequencing (MeRIP-Seq). Subsequent validation through RT-qPCR corroborated these findings, aligning with the m6A sequencing outcomes. Furthermore, co-transfection experiments elucidated that Linc-smad7 regulates lipid synthesis in mammary epithelial cells by influencing the expression of METTL14. In summary, these findings underscore the regulatory role of Linc-smad7 in controlling METTL14 gene expression, thereby mediating m6A modifications to regulate lipid synthesis in mammary epithelial cells.

Effect of sodium butyrate treatment at the basolateral membranes on the tight junction barrier function via a monocarboxylate transporter in goat mammary epithelial cells.

In lactating mammary glands, tight junctions (TJs) prevent blood from mixing with milk and maintain epithelial cell polarity, which is important for milk production. This study aimed to investigate the effect of sodium acetate and sodium butyrate (SB) stimulation direction on the TJ barrier function, which is measured with regard to transepithelial electrical resistance and fluorescein flux, in goat mammary epithelial cells. The expression and localization of the TJ proteins claudin-3 and claudin-4 were examined using Western blotting and immunofluorescence. SB treatment in the lower chamber of cell culture inserts adversely affected the TJ barrier function, whereas sodium acetate barely had any effect, regardless of stimulation direction. In addition, SB treatment in the lower chamber significantly upregulated claudin-3 and claudin-4, whereas TJ proteins showed intermittent localization. Moreover, SB induced endoplasmic reticulum (ER) stress. ARC155858, a monocarboxylate transporter-1 inhibitor, alleviated the adverse impact of SB on TJs and the associated ER stress. Interestingly, sodium ß-hydroxybutyrate, a butyrate metabolite, did not affect the TJ barrier function. Our findings indicate that sodium acetate and SB influence the TJ barrier function differently, and excessive cellular uptake of SB can disrupt TJs and induce ER stress.

dCas9-guided demethylation of the AKT1 promoter improves milk protein synthesis in a bovine mastitis mammary gland epithelial model induced by using Staphylococcus aureus.

Mastitis is among the main factors affecting milk quality and yield. Although DNA methylation is associated with mastitis, its role in mastitis remains unclear. In this study, a bovine mastitis mammary epithelial cells (BMMECs) model was established via Staphylococcus aureus infection of bovine mammary gland epithelial cells (BMECs). Bisulfite sequencing PCR was used to determine the methylation status of the AKT1 promoter in BMMECs. We found that the degree of the AKT1 promoter methylation in BMMECs was significantly greater than that in BMECs, and the expression levels of genes related to milk protein synthesis were significantly decreased. We used the pdCas9-C-Tet1-SgRNA 2.0 system to regulate the methylation status of the AKT1 promoter. High-efficiency sgRNAs were screened and dCas9-guided AKT1 promoter demethylation vectors were constructed. Following transfection with the vectors, the degree of methylation of the AKT1 promoter was significantly reduced in BMMECs, while AKT1 protein levels increased. When the methylation level of the AKT1 promoter decreased, the synthesis of milk proteins and the expression levels of genes related to milk protein synthesis increased significantly. The viability of the BMMECs was enhanced. Taken together, these results indicate that demethylation guided by the pdCas9-C-Tet1-SgRNA 2.0 system on the AKT1 promoter can reactivate the expression of AKT1 and AKT1/mTOR signaling pathway-related proteins by reducing the AKT1 promoter methylation level and promoting the recovery milk protein expression in BMMECs, thereby alleviating the symptoms of mastitis.

Metabolites of non-aureus staphylococci affect the ability of Staphylococcus aureus to adhere to and internalize into bovine mammary epithelial cells.

This study investigated whether cell-free supernatants (SN) from four bovine non-aureus staphylococcal (NAS) isolates prevent Staphylococcus aureus adhesion to and internalization into bovine mammary epithelial cells (MAC-T cells) and if so, to determine whether such effects were potentially associated with the S. aureus accessory gene regulator (agr) system. Overall, we demonstrated that all SN obtained from the NAS isolates promoted adhesion of a S. aureus agr+ strain to, yet reduced the internalization into MAC-T cells, while similar effects were not observed for its agr- mutant strain. Our findings provide novel anti-virulence strategies for treating and controlling bovine S. aureus mastitis.

The FoxO1-ATGL axis alters milk lipolysis homeostasis through PI3K/AKT signaling pathway in dairy goat mammary epithelial cells.

Goat milk is enriched in fatty acids which are beneficial to human health. Previous research has revealed that 98% of milk fat is composed of triglycerides. However, the mechanisms regulating milk fat composition remain unclear. Forkhead box protein O1 (FoxO1) is a crucial regulatory factor involved in lipid metabolism across various cell types. Chromatin immunoprecipitation sequencing (ChIP)-seq data) and RNA sequencing (RNA-seq) data revealed that have indicated a close association between FoxO1 was closely related to lipid metabolism during lactation in dairy goats. The objective of this study was to investigate the mechanisms by which FoxO1 regulates lipid metabolism in goat mammary epithelial cells (GMECs). FoxO1 knockdown significantly downregulated the expression of adipose triglyceride lipase (ATGL) and suppressed the activity of the ATGL promoter. Consistently, the number of lipid droplets decreased significantly in FoxO1-overexpressing cells and increased in ATGL-knockdown cells. To further verify the effect of FoxO1 on ATGL promoter activity, cells were transfected with four promoter fragments of different lengths. We found that the core region of the ATGL promoter was located between -882 bp and -524 bp, encompassing two FoxO1 binding sites (FKH1 and FKH2). Mutations in the FoxO1 binding sites significantly downregulated ATGL promoter activity in GMECs. Luciferase reporter assays demonstrated that FoxO1 overexpression markedly enhanced ATGL promoter activity. Furthermore, site-directed mutation confirmed that FKH1 and FKH2 sites were simultaneously mutated significantly attenuated the stimulatory effect of FoxO1 on ATGL promoter activities simultaneous mutation of FKH1 and FKH2 sites significantly attenuated the stimulatory effect of FoxO1 on ATGL promoter activity. ChIP assays showed that FoxO1 directly binds to the FKH2 element located in the ATGL promoter in vivo. Finally, immunofluorescence staining revealed that insulin promotes the translocation of FoxO1 from the nucleus to the cytoplasm, thereby attenuating the FoxO1-induced activation of the ATGL promoter. Collectively, these findings uncover a novel pathway where by FoxO1 may regulate lipid metabolism in GMECs specifically by modulating the transcriptional activity of ATGL.

Forkhead box protein O1(FoxO1) is a key cellular regulatory factor that was involved in lipid metabolism in several cell types. This study was performed to explore the regulatory mechanism of FoxO1 in adipose triglyceride lipase (ATGL) promoter-driven transcription during lactation in dairy goats. Chromatin immunoprecipitation (ChIP)-seq and RNA sequencing (RNA-seq) data revealed that FoxO1 was closely related to lipid metabolism and inflammation during lactation in dairy goats. FoxO1 overexpression significantly decreased cellular triglyceride (TAG) content lipid droplet accumulation in goat mammary epithelial cells (GMECs), while ATGL knockdown attenuated this effect of FoxO1. Furthermore, the relative content of free fatty acid (FFAs) was markedly increased in FoxO1-overexpressed cells. Additionally, site-directed mutation and ChIP assays confirmed that FoxO1 promotes ATGL transcription through FoxO1 binding sites (FKH) located in the ATGL promoter. Moreover, insulin attenuated the FoxO1-induced activation of the ATGL promoter. Our data reveal that FoxO1 regulates the activity of ATGL in GMECs by binding to FKH elements located in the ATGL promoter.

CRISPR-Cas9 based knockout of S100A8 in mammary epithelial cells enhances cell proliferation and triggers oncogenic transformation via the PI3K-Akt pathway: Insights from a deep proteomic analysis.

S100A8 is a calcium-binding protein with multiple functions, including being a chemoattractant for phagocytes and playing a key role in the inflammatory response. Its expression has been shown to influence epithelial-mesenchymal transition (EMT) and metastasis in colorectal cancer. However, the role of S100A8 in cell proliferation and differentiation remains unknown. In this study, we used the CRISPR-Cas9 system to knock out S100A8 in healthy mammary epithelial cells and investigated the resulting changes in proteome profiling and signaling pathways. Our results showed that S100A8 knockout led to an increase in cell proliferation and migration, reduced cell-cell adhesion, and increased apoptosis compared to wildtype cells. Proteomics data indicated that S100A8 significantly affects cell cycle progression, cell proliferation, and cell survival through the PI3K-Akt pathway. Furthermore, our findings suggest that S100A8 function is associated with Pten expression, a negative regulator of the PI3K-Akt pathway. These results indicate that S100A8 dysregulation in healthy cells can lead to altered cellular physiology and higher proliferation, similar to cancerous growth. Therefore, maintaining S100A8 expression is critical for preserving healthy cell physiology. This study provides novel insights into the role of S100A8 in cell proliferation and differentiation and its potential relevance to cancer biology. SIGNIFICANCE: The study suggests that maintaining S100A8 expression is critical for preserving healthy cell physiology, and dysregulation of S100A8 in healthy cells can lead to altered cellular physiology and higher proliferation, similar to cancerous growth. Therefore, targeting the PI3K-Akt pathway or regulating Pten expression, a negative regulator of the PI3K-Akt pathway, may be potential strategies for cancer treatment by controlling S100A8 dysregulation. Additionally, S100A8 and S100A9 have been shown to promote metastasis of breast carcinoma by forming a metastatic milieu. However, the differential expression of S100A8 in tumors and its dual effects of antitumor and protumor make the relationship between S100A8 and tumors complicated. Currently, most research focuses on the function of S100A8 as a secretory protein in the microenvironment of tumors, and its function inside healthy cells without forming dimers remains unclear. Furthermore, the study provides insight into the role of S100A8 in cell proliferation and differentiation, which may have implications for other diseases beyond cancer. The functional role of S100A8 in normal mammary epithelial cells remains completely uncertain. Therefore, the objective of this study is to investigate the function of S100A8 on proliferation in mammary epithelial cells after its deletion and to elucidate the underlying proteins involved in downstream signaling. Our findings indicate that the deletion of S100A8 leads to excessive proliferation in normal mammary epithelial cells, reduces apoptosis, and affects cell-cell adhesion molecules required for cellular communication, resulting in a cancer-like phenotype.

Optimizing hormonal and amino acid combinations for enhanced cell proliferation and cell cycle progression in bovine mammary epithelial cells.

OBJECTIVE: The number of bovine mammary epithelial cells (BMECs) is closely associated with the quantity of milk production in dairy cows; however, the optimal levels and the combined effects of hormones and essential amino acids (EAAs) on cell proliferation are not completely understood. Thus, the purpose of this study was to determine the optimal combination of individual hormones and EAAs for cell proliferation and related signaling pathways in BMECs. METHODS: Immortalized BMECs (MAC-T) were treated with six hormones (insulin, cortisol, progesterone, estrone, 17ß-estradiol, and epidermal growth factor) and ten EAAs (arginine, histidine, leucine, isoleucine, threonine, tryptophan, lysine, methionine, phenylalanine, and valine) for 24 h. RESULTS: Cells were cultured in a medium containing 10% fetal bovine serum (FBS) as FBS supplemented at a concentration of 10% to 50% showed a comparable increase in cell proliferation rate. The optimized combination of four hormones (insulin, cortisol, progesterone, and 17ß-estradiol) and 20% of a mixture of ten EAAs led to the highest cell proliferation rate, which led to a significant increase in cell cycle progression at the S and G2/M phases, in the protein levels of proliferating cell nuclear antigen and cyclin B1, cell nucleus staining, and in cell numbers. CONCLUSION: The optimal combination of hormones and EAAs increased BMEC proliferation by enhancing cell cycle progression in the S and G/2M phases. Our findings indicate that optimizing hormone and amino acid levels has the potential to enhance milk production, both in cell culture settings by promoting increased cell numbers, and in dairy cows by regulating feed intake.