[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tis...[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.展开更多
Background: Tea tree oil(TTO) plays an important role in antibacterial activity and alleviating the inflammatory responses. Bovine mammary epithelium and polymorphonuclear leukocytes(PMNL) can actively respond to bovi...Background: Tea tree oil(TTO) plays an important role in antibacterial activity and alleviating the inflammatory responses. Bovine mammary epithelium and polymorphonuclear leukocytes(PMNL) can actively respond to bovine mastitis infection. However, regulatory effects of TTO extracts on the innate immune response of bovine mammary epithelial cells(BMECs) and PMNL remain not reported. Therefore, aim of the study was to evaluate the effects of TTO extracts on the m RNA levels of the genes involved in the innate immune response of BMECs and PMNL.Results: Our results demonstrated that addition of 0.025% and 0.05% TTO increased the proliferation of BMECs, and significantly enhanced(P < 0.05) the viability of BMECs exposed to Staphylococcus aureus(S. aureus). An inhibitory effect was observed against the growth of S. aureus by TTO incubation. The 0.05% TTO reduced S. aureus biofilm formation, association and invasion of S. aureus to BMECs, and changed the morphological and structural features of S. aureus. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were decreased(P < 0.001) by the incubation of TTO. Interestingly, the expression of IL-8 known for PMNL chemotactic function was elevated(P < 0.05) by 0.05%TTO treatment. Consistently, 0.05% TTO increased the migration of PMNL in S. aureus-exposed BMECs when compared with S. aureus treatment alone(P < 0.05). In addition, PMNL incubated with 0.05% TTO decreased the levels of NFKB inhibitor alpha(NFKBIA) and TNF-α.Conclusions: Our results indicate that use of TTO can relieve the BMECs pro-inflammatory response caused by S.aureus and promote the migration of PMNL to mount the innate immune responses, and it may be novel strategy for the treatment of bovine mastitis caused by S. aureus.展开更多
Background:Bovine mammary epithelial cells after calving undergo serious metabolic challenges and oxidative stress both of which could compromise autophagy.Transcription factor EB(TFEB)-mediated autophagy is an import...Background:Bovine mammary epithelial cells after calving undergo serious metabolic challenges and oxidative stress both of which could compromise autophagy.Transcription factor EB(TFEB)-mediated autophagy is an important cytoprotective mechanism against oxidative stress.However,effects of TFEB-mediated autophagy on the oxidative stress of bovine mammary epithelial cells remain unknown.Therefore,the main aim of the study was to investigate the role of TFEB-mediated autophagy in bovine mammary epithelial cells experiencing oxidative stress.Results:H_(2)O_(2) challenge of the bovine mammary epithelial cell MAC-T increased protein abundance of LC3-II,increased number of autophagosomes and autolysosomes while decreased protein abundance of p62.Inhibition of autophagy via bafilomycin A1 aggravated H_(2)O_(2)-induced reactive oxygen species(ROS)accumulation and apoptosis in MAC-T cells.Furthermore,H_(2)O_(2) treatment triggered the translocation of TFEB into the nucleus.Knockdown of TFEB by siRNA reversed the effect of H_(2)O_(2) on protein abundance of LC3-II and p62 as well as the number of autophagosomes and autolysosomes.Overexpression of TFEB activated autophagy and attenuated H_(2)O_(2)-induced ROS accumulation.Furthermore,TFEB overexpression attenuated H_(2)O_(2)-induced apoptosis by downregulating the caspase apoptotic pathway.Conclusions:Our results indicate that activation of TFEB mediated autophagy alleviates H_(2)O_(2)-induced oxidative damage by reducing ROS accumulation and inhibiting caspase-dependent apoptosis.展开更多
Background:The biosynthesis of milk fat affects both the technological properties and organoleptic quality of milk and dairy products.MicroRNAs(miRNAs)are endogenous small non-coding RNAs that inhibit the expression o...Background:The biosynthesis of milk fat affects both the technological properties and organoleptic quality of milk and dairy products.MicroRNAs(miRNAs)are endogenous small non-coding RNAs that inhibit the expression of their mRNA targets and are involved in downstream signaling pathways that control several biological processes,including milk fat synthesis.miR-34b is a member of the miR-34 miRNA cluster,which is differentially expressed in the mammary gland tissue of dairy cows during lactation and dry periods.Previous studies have indicated miR-34b is a potential candidate gene that plays a decisive role in regulating milk fat synthesis;therefore,it is important to focus on miR-34b and investigate its regulatory effect on the biosynthesis of milk fat in bovine mammary epithelial cells(BMECs).Results:In this study,elevated miR-34b levels reduced milk fat synthesis,upregulated 1,999 genes,and downregulated 2,009 genes in BMECs.Moreover,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis of differentially expressed genes suggested that miR-34b may play an inhibitory role in milk fat synthesis via the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway by reducing phosphorylation levels.Notably,the mTOR activator MHY1485 rescued the inhibitory effect of miR-34b.Furthermore,we demonstrated that retinoic acid-induced protein 14(RAI14)is a target of miR-34b via TargetScan and immunofluorescence assays.RAI14 mRNA and protein levels were significantly decreased by the miR-34b mimic and increased by the miR-34b inhibitor.Moreover,the reduction in RAI14 levels led to the inhibition of the Akt/mTOR signaling pathway.Conclusions:Overall,our results identified a miR-34b-RAI14-Akt/mTOR regulatory network,while also providing a theoretical basis for the molecular breeding of dairy cows.展开更多
Fat is an indispensable nutrient and basic metabolite for sustaining life,and milk is particularly rich in fatty acids,including a variety of saturated and unsaturated fatty acids.MicroRNA(miRNA)and mRNA play an impor...Fat is an indispensable nutrient and basic metabolite for sustaining life,and milk is particularly rich in fatty acids,including a variety of saturated and unsaturated fatty acids.MicroRNA(miRNA)and mRNA play an important role in the regulation of milk fat metabolism in mammary gland tissue.It has been shown that lipid metabolism has a complex transcriptional regulation,but the mechanism by which milk fat synthesis is regulated through miRNA–mRNA interactions is poorly understood.In this study,we performed transcriptome sequencing with bovine mammary gland tissue in the late lactation(270 and 315 days after parturition)to identify the key gene that regulating milk fat metabolism.A total of 1207 differentially coexpressed genes were selected,828 upregulated genes and 379 downregulated genes were identified.The transforming growth factor alpha(TGFA)gene was selected as the target gene,and luciferase reporter assay,Western blotting and q RT-PCR were used for further study.The results demonstrated that miR-140 was an upstream regulator of TGFA,and miR-140 could inhibit(P<0.01)unsaturated fatty acid and triglyceride(TAGs)production in bovine mammary epithelial cells(BMECs).In contrast,TGFA promoted(P<0.01)unsaturated fatty acid and TAG production.Rescue experiments further indicated the mi R-140/TGFA regulatory mechanism.Taken together,these results suggest that the mi R-140/TGFA pathway can inhibit(P<0.01)milk fat metabolism and improve milk quality by genetic means.展开更多
Excess ammonia(NH_(3))in the circulation of dairy animals can reduce animal health and the quality of products for human consumption.To develop effective prevention and treatment methods,it is essential to examine the...Excess ammonia(NH_(3))in the circulation of dairy animals can reduce animal health and the quality of products for human consumption.To develop effective prevention and treatment methods,it is essential to examine the molecular mechanisms through which excess NH_(3) may affect the mammary gland.The present study used bovine mammary epithelial cells(BMECs)to evaluate the effects of exogenous NH_(4)Cl on the abundance of circular RNAs(circRNAs)using high-throughput sequencing.Among the identified circRNAs,circ02771 was the most significantly upregulated by exogenous NH_(4)Cl(P<0.05),with a fold change of 4.12.The results of the apoptosis and proliferation assays,transmission electron microscopy,H&E staining,and immunohistochemistry revealed that circ02771 increased apoptosis and inflammation.A double luciferase reporter assay revealed that circ02771 targeted miR-194b,and the overexpression of circ02771(pcDNA-circ02771)reduced(P<0.05)the expression of miR-194b and led to apoptosis and inflammation.Circ02771 also enhanced the expression of transforming growth factor beta-induced factor homeobox 1(TGIF1),which is a target gene of miR-194b.Overall,this study suggests that the circ02771/miR-194b/TGIF1 axis plays a role in mediating the effects of NH_(4)Cl on BMECs.Therefore,this axis provides a novel target to help control hazards within the mammary gland from high circulating NH_(4)Cl levels.展开更多
Background:In early lactation,bovine mammary epithelial cells undergo serious metabolic challenges and oxidative stress both of which could be alleviated by activation of autophagy.Nuclear factor erythroid 2 related f...Background:In early lactation,bovine mammary epithelial cells undergo serious metabolic challenges and oxidative stress both of which could be alleviated by activation of autophagy.Nuclear factor erythroid 2 related factor 2(NFE2L2),a master regulator of cellular redox homeostasis,plays an important role in the regulation of autophagy and oxidative stress.Thus,the objective of this study was to investigate the role of NFE2L2-mediated autophagy on oxidative stress of bovine mammary epithelial cells in response to exogenous free fatty acids(FFA).Results:Exogenous FFA induced linear and quadratic decreases in activities of glutathione peroxidase(GSH-Px),catalase(CAT),and superoxide dismutase(SOD),and increases in the contents of reactive oxygen species(ROS)and malondialdehyde(MDA).Protein abundance of LC3-phosphatidylethanolamine conjugate(LC3-Ⅱ)and the number of autophagosomes and autolysosomes decreased in a dose-dependent manner,while protein abundance of p62 increased in cells challenged with FFA.Activation of autophagy via pre-treatment with Rap attenuated the FFAinduced ROS accumulation.Importantly,FFA inhibited protein abundance of NFE2L2 and the translocation of NFE2L2 into the nucleus.Knockdown of NFE2L2 by siRNA decreased protein abundance of LC3-Ⅱ,while it increased protein abundance of p62.Furthermore,sulforaphane(SFN)pre-treatment attenuated the FFA-induced oxidative stress by activating NFE2L2-mediated autophagy.Conclusions:The data suggested that NFE2L2-mediated autophagy is an important antioxidant mechanism in bovine mammary epithelial cells experiencing increased FFA loads.展开更多
Cow mastitis is the most common disease that affects the dairy farming industry and causes serious harm to dairy cows and humans,and Staphylococcus(S.)aureus is one of the main pathogens that cause mastitis in dairy c...Cow mastitis is the most common disease that affects the dairy farming industry and causes serious harm to dairy cows and humans,and Staphylococcus(S.)aureus is one of the main pathogens that cause mastitis in dairy cows.In this study,a mastitis model was established through the infection of bovine mammary epithelial cells(BMECs)with S.aureus(bacterial concentration of 1×10^(9)/mL),and these cells and a blank group(untreated)were analyzed by flow cytometry(10000 cells,200 cells collected per second),hematoxylin and eosin(H&E)staining and immunohistochemistry.In addition,the lncRNAs(long non-coding RNAs)in the normal and S.aureus-infected BMEC group were screened by second-generation sequencing.Flow cytometry,H&E staining,and immunohistochemistry assays were performed to verify the successful construction of an S.aureus infection model in BMECs.A close relationship was found between the differential expression of lncRNAs and S.aureus mastitis.The total original sequencing reads were 627.13 M,and the average reads from each sample were approximately 104.52 M.After removing the unwanted reads,the total clean reads were 606.43 M,and the average reads from each sample were approximately 101.07 M.After S.aureus infection,30 lncRNAs were differentially expressed,and these included 21 upregulated and nine down-regulated lncRNAs.This research will not only expand our understanding of the lncRNA map in dairy cows but also help us hypothesize the function of lncRNAs in the genome and identify novel molecular markers of mastitis.展开更多
Mastitis is a common inflammatory cow mammary infection;that causes significant economic loss in dairy industry.Given the interesting connection between metformin’s anti-inflammatory function and mastitis model induc...Mastitis is a common inflammatory cow mammary infection;that causes significant economic loss in dairy industry.Given the interesting connection between metformin’s anti-inflammatory function and mastitis model induced by LTA in pbMECs,our objective was to prove that metformin was beneficial in suppressing proinflammatory response induced by LTA through modulation of mitogen-activated protein kinase(MAPK)and nuclear factor kappa B(NF-κB)signaling pathways and activation of peroxisome proliferator-activated receptor-γ(PPARγ)in pbMECs.The proliferation of cells and mRNA expression were measured using EdU assay and quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR).Immunoblotting and immunofluorescence analysis were conducted to evaluate the expression of target proteins in inflammatory and anti-inflammatory responses to metformin and LTA.Finally,pbMECs were allowed to treat with the PPAR antagonist GW9662,and inflammatory markers were detected in the cells.Our results showed that LTA concentration at 100μg/mL significantly stimulated the MAPK14,IL-6 and IL-1βmRNA expressions compared to the control cells(P<0.05)in dose-dependent tests for LTA.Metformin suppressed the phosphorylation expressions of MAPK(ERK1/2,p38,and JNK)in LTA-stimulated pbMECs.Metformin also reduced the protein expression of NF-κB,interleukin-8(IL-8),interleukin-1β(IL-1β)and interleukin-6(IL-6)in pbMECs pretreated with LTA.Metformin administration activated PPARγphosphorylation by up-regulating the expression of PPARγin LTA-stimulated pbMECs.Treatment with GW9662 resulted in increased IL-6 expression,which was reversed by metformin.These findings collectively indicated that metformin act to attenuate LTA-stimulated inflammatory response in pbMECs by suppressing MAPK and NF-κB activation via a mechanism partially dependent on PPARγactivation.These results suggested that metformin could function as an anti-inflammatory drug in the treatment of mastitis.展开更多
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)activit...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 m RNA 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.展开更多
Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucid...Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-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 b MECs 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.展开更多
Short-chain fatty acids are important nutrients that regulate milk fat synthesis.They regulate milk syn-thesis via the sterol regulatory element binding protein 1(SREBP1)pathway;however,the details are still unknown.H...Short-chain fatty acids are important nutrients that regulate milk fat synthesis.They regulate milk syn-thesis via the sterol regulatory element binding protein 1(SREBP1)pathway;however,the details are still unknown.Here,the regulation and mechanism of sodium acetate(SA)in milk fat synthesis in bovine mammary epithelial cells(BMECs)were assessed.BMECs were treated with SA supplementation(SAþ)or without SA supplementation(SA-),and milk fat synthesis and activation of the SREBP1 pathway were increased(P=0.0045;P=0.0042)by SAþand decreased(P=0.0068;P=0.0031)by SA-,respectively.Overexpression or inhibition of SREBP1 demonstrated that SA promoted milk fat synthesis(P=0.0045)via the SREBP1 pathway.Overexpression or inhibition of TATA element modulatory factor 1(TMF1)demon-strated that TMF1 suppressed activation of the SREBP1 pathway(P=0.0001)and milk fat synthesis(P=0.0022)activated by SAþ.Overexpression or inhibition of TMF1 and SREBP1 showed that TMF1 suppressed milk fat synthesis(P=0.0073)through the SREBP1 pathway.Coimmunoprecipitation analysis revealed that TMF1 interacted with SREBP1 in the cytoplasm and suppressed the nuclear localization of SREBP1(P=0.0066).The absence or presence of SA demonstrated that SA inhibited the expression of TMF1(P=0.0002)and the interaction between TMF1 and SREBP1(P=0.0001).Collectively,our research sug-gested that TMF1 was a new negative regulator of milk fat synthesis.In BMECs,SA promoted the SREBP1 pathway and milk fat synthesis by suppressing TMF1.This study enhances the current understanding of the regulation of milk fat synthesis and provides new scientific data for the regulation of milk fat synthesis.展开更多
Lipopolysaccharide(LPS) was selected as a stimulus to investigate its effect on cell viability and oxidative stress in bovine mammary epithelial cells(BMEC) by detecting the cell relative growth rate(RGR),antioxidant ...Lipopolysaccharide(LPS) was selected as a stimulus to investigate its effect on cell viability and oxidative stress in bovine mammary epithelial cells(BMEC) by detecting the cell relative growth rate(RGR),antioxidant indicators and inflammatory factors. This information was used to provide the theoretical basis for the establishment of a LPS-induced oxidative damage model. The experiment was divided into two parts. The first part used a two-factor experimental design to determine the appropriate incubation time of LPS by detecting the RGR. The third-passage BMEC were divided into 24 groups with six replicates in each group. The first factor was LPS concentration, which was 0(control), 0.1,1.0 and 10.0 μg/mL;the second factor was LPS incubation time(2,4, 6, 8,12 and 24 h). The optimum LPS incubation time was6 h according to the results of the first part of the experiment. The second part of the experiment was conducted using a single-factor experimental design, and the third-passage cells were divided into four groups with six replicates in each group. The cells were incubated with culture medium containing different concentrations of LPS(0 [control], 0.1, 1.0 and 10.0 μg/mL) for 6 h to select the appropriate concentration of LPS to measure the antioxidant indicators and inflammatory factors. The results showed the RGR was significantly reduced as the concentration of LPS and the incubation time increased;the interaction between concentration and incubation time was also significant. The cells treated with0.1 μg/mL of LPS for 6 h had no significant difference in the activities of glutathione peroxidase(GPx) and superoxide dismutase(SOD)(P > 0.05) compared with the cells in the control group. On the contrary,catalase(CAT) activity and malondialdehyde(MDA) content were markedly lower and higher, respectively, in the 0.1 μg/mL LPS-treated group for 6 h compared with the control group(P < 0.05). The activities of GPx, CAT and SOD in the BMEC treated with 1.0 or 10.0 μg/mL of LPS were significantly lower compared with the cells treated with 0.1 μg/mL of LPS and cells in the control group after 6 h of incubation; however, the opposite trend was detected in MDA content. There was no significant(P > 0.05)difference between the 10.0 and 1.0 μg/mL LPS-treated groups. Compared with the control group,interleukin-1, interleukin-6 and nitric oxide concentrations and the activity of inducible nitric oxide synthase in the 0.1 μg/mL LPS-treated group significantly increased(P < 0.0001), but the levels of tumour necrosis factor did not significantly change(P > 0.05). All of observed indicators were higher in the 1.0 and 10.0 μg/mL LPS-treated groups(P < 0.0001) compared with the other groups, but there was no significant(P> 0.05) difference between the 1.0 and 10.0 μg/mL LPS-treated groups. The results indicated that a concentration of 1.0 μg/mL of LPS and an incubation time of 6 h were the optimum conditions necessary to induce oxidative stress in the BMEC and establish a model for oxidative damage.展开更多
The aim of this study is to investigate the effects of leucine（Leu） and histidine（His） on the expression of both the mammalian target of rapamycin（mTOR） signaling pathway-related proteins and caseins in immortal...The aim of this study is to investigate the effects of leucine（Leu） and histidine（His） on the expression of both the mammalian target of rapamycin（mTOR） signaling pathway-related proteins and caseins in immortalized bovine mammary epithelial cells（CMEC-H）, using a single supplement through Western blotting. The Earle＇s balanced salt solution（EBSS） was set as the control group and other treatment groups, based on the EBSS, were added with different concentrations of Leu or His, respectively. The results showed that, compared with the control group, the expression of caseins and the phosphorylation of mTOR（Ser^2481）, Raptor（Ser^792）, e IF4E（Ser^209）, and e EF2（Thr^56） increased with the Leu concentrations ranging from 0.45 to 10.80 mmol/L（P〈0.01）. The P-4EBP1（Thr^37） at 10.80 mmol/L Leu, and P-RPS6（Ser^235/236） at 5.40 to 10.80 mmol/L Leu all decreased. Similarly, the His supplementation from 0.15 to 9.60 mmol/L increased the expression of αs2-casein, β-casein, κ-casein, P-mTOR（Ser^2481）, P-Raptor（Ser^792）, P-S6K1（Thr^389）, P-4EBP1（Thr^37）, P-e IF4E（Ser^209）, and P-e EF2（Thr^56）（P〈0.01） in CMEC-H, whereas the αs1-casein expression was only reduced at 9.60 mmol/L His, G protein β subunit-like protein（GβL） at 0.15 and 9.60 mmol/L His, and P-RPS6 at 4.80 to 9.60 mmol/L His. Our linear regression model assay suggested that the αs1-casein expression was positively correlated with P-mTOR（P〈0.01）, P-S6K1（P〈0.01）, and P-e EF2（P〈0.01） for the addition of Leu, while the expressions of β-casein（P〈0.01） and κ-casein（P〈0.01） were positively correlated with P-e EF2 for the addition of His. In conclusion, the milk protein synthesis was up-regulated through activation of the mTOR pathway with the addition of Leu and His in CMEC-H.展开更多
Background： Bovine milk contains not only a variety of nutritional ingredients but also microRNAs （miRNAs） that are thought to be secreted by the bovine mammary epithelial cells （BMECs）. The objective of this stu...Background： Bovine milk contains not only a variety of nutritional ingredients but also microRNAs （miRNAs） that are thought to be secreted by the bovine mammary epithelial cells （BMECs）. The objective of this study was to elucidate the production of milk-related miRNAs in BMECs under the influence of lactogenic hormones. Results： According to a microarray result of milk exosomal miRNAs prior to cellular analyses, a total of 257 miRNAs were detected in a Holstein cow milk. Of these, 18 major miRNAs of interest in the milk were selected for an expression analysis in BMEC culture that was treated with or without dexamethasone, insulin, and prolactin （DIP） to induce a lactogenic differentiation. Quantitative polymerase chain reaction （qPCR） results showed that the expressions of miR-21-Sp （P = 0.005）, miR-26a （P = 0.016）, and miR-320a （P = 0.011） were lower in the DIP-treated cells than in the untreated cells. In contrast, the expression of miR-339a （P-- 0.017） in the cell culture medium were lower in the DiP-treated culture than in the untreated culture. Intriguingly, the miR-148a expression in cell culture medium was elevated by DIP treatment of BMEC culture （P = 0.018）. The medium-to-cell expression ratios of miR- 103 （P = 0.025）, miR-148a （P 〈 0.001）, and miR-223 （P = 0.013） were elevated in the DIP-treated BMECs, suggesting that the lactogenic differentiation-induced secretion of these three miRNAs in BMECs. A bioinformatic analysis showed that the miRNAs down-regulated in the BMECs were associated with the suppression of genes related to transcriptional regulation, protein phosphorylation, and tube development. Conclusion： The results suggest that the miRNAs changed by lactogenic hormones are associated with milk protein synthesis, and mammary gland development and maturation. The elevated miR-148a level in DIP-treated BMECs may be associated with its increase in milk during the lactation period of cows.展开更多
High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants.Inadequate biodegradation of Fusarium mycotoxins by rumen microflora f...High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants.Inadequate biodegradation of Fusarium mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds can lead to their circulatory transport to target tissues such as mammary gland.The bovine udder plays a pivotal role in maintaining milk yield and composition,thus,human health.However,toxic effects of Fusarium mycotoxins on bovine mammary gland are rarely studied.In this study,the bovine mammary epithelial cell line was used as an in-vitro model of bovine mammary epithelium to investigate effects of deoxynivalenol(DON),enniatin B(ENB)and beauvericin(BEA)on bovine mammary gland homeostasis.Results indicated that exposure to DON,ENB and BEA for 48 h significantly decreased cell viability in a concentration-dependent manner(P<0.001).Exposure to DON at 0.39μmol/L and BEA at 2.5μmol/L for 48 h also decreased paracellular flux of FITC-40 kDa dextran(P<0.05),whereas none of the mycotoxins affected transepithelial electrical resistance after 48 h exposure.The qPCR was performed for assessment of expression of gene coding tight junction(TJ)proteins,toll-like receptor 4(TLR4)and cytokines after 4,24 and 48 h of exposure.DON,ENB and BEA significantly upregulated the TJ protein zonula occludens-1,whereas markedly downregulated claudin 3(P<0.05).Exposure to DON at 1.35μmol/L for 4 h significantly increased expression of occludin(P<0.01).DON,ENB and BEA significant downregulated TLR4(P<0.05).In contrast,ENB markedly increased expression of cytokines interleukin-6(IL-6)(P<0.001),tumor necrosis factorα(TNF-a)(P<0.05)and transforming growth factor-β(TGF-β)(P<0.01).BEA significantly upregulated IL-6(P<0.001)and TGF-β(P=0.01),but downregulated TNF-α(P<0.001).These results suggest that DON,ENB and BEA can disrupt mammary gland homeostasis by inducing cell death as well as altering its paracellular permeability and expression of genes involved in innate immune function.展开更多
基金Supported by Natural Science Foundation of Inner Mongolia Autono-mous Region (200711020407)China Agricultural University and Inner Mongolia Agricultural University Cooperation Projects~~
文摘[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.
基金supported by the Research Project of Natural Science Foundation of Jiangsu Province (SBK2019043455)the National Natural Science Foundation of China (No. 31972589)+1 种基金China Agriculture Research System (CARS-36)Development Project of China (2017YFD0502104–3)。
文摘Background: Tea tree oil(TTO) plays an important role in antibacterial activity and alleviating the inflammatory responses. Bovine mammary epithelium and polymorphonuclear leukocytes(PMNL) can actively respond to bovine mastitis infection. However, regulatory effects of TTO extracts on the innate immune response of bovine mammary epithelial cells(BMECs) and PMNL remain not reported. Therefore, aim of the study was to evaluate the effects of TTO extracts on the m RNA levels of the genes involved in the innate immune response of BMECs and PMNL.Results: Our results demonstrated that addition of 0.025% and 0.05% TTO increased the proliferation of BMECs, and significantly enhanced(P < 0.05) the viability of BMECs exposed to Staphylococcus aureus(S. aureus). An inhibitory effect was observed against the growth of S. aureus by TTO incubation. The 0.05% TTO reduced S. aureus biofilm formation, association and invasion of S. aureus to BMECs, and changed the morphological and structural features of S. aureus. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were decreased(P < 0.001) by the incubation of TTO. Interestingly, the expression of IL-8 known for PMNL chemotactic function was elevated(P < 0.05) by 0.05%TTO treatment. Consistently, 0.05% TTO increased the migration of PMNL in S. aureus-exposed BMECs when compared with S. aureus treatment alone(P < 0.05). In addition, PMNL incubated with 0.05% TTO decreased the levels of NFKB inhibitor alpha(NFKBIA) and TNF-α.Conclusions: Our results indicate that use of TTO can relieve the BMECs pro-inflammatory response caused by S.aureus and promote the migration of PMNL to mount the innate immune responses, and it may be novel strategy for the treatment of bovine mastitis caused by S. aureus.
基金This work was supported by the National Natural Science Foundation of China(Beijing,China,grant no.32002348 and 32072931)the Project funded by China Postdoctoral Science Foundation(Beijing,China+3 种基金grant no.2019 M661316)the Heilongjiang Postdoctoral Science Foundation(Heilongjiang,ChinaGrant No.LBH-Z19090)the Personnel Foundation in Heilongjiang Bayi Agricultural University(XYB201909).
文摘Background:Bovine mammary epithelial cells after calving undergo serious metabolic challenges and oxidative stress both of which could compromise autophagy.Transcription factor EB(TFEB)-mediated autophagy is an important cytoprotective mechanism against oxidative stress.However,effects of TFEB-mediated autophagy on the oxidative stress of bovine mammary epithelial cells remain unknown.Therefore,the main aim of the study was to investigate the role of TFEB-mediated autophagy in bovine mammary epithelial cells experiencing oxidative stress.Results:H_(2)O_(2) challenge of the bovine mammary epithelial cell MAC-T increased protein abundance of LC3-II,increased number of autophagosomes and autolysosomes while decreased protein abundance of p62.Inhibition of autophagy via bafilomycin A1 aggravated H_(2)O_(2)-induced reactive oxygen species(ROS)accumulation and apoptosis in MAC-T cells.Furthermore,H_(2)O_(2) treatment triggered the translocation of TFEB into the nucleus.Knockdown of TFEB by siRNA reversed the effect of H_(2)O_(2) on protein abundance of LC3-II and p62 as well as the number of autophagosomes and autolysosomes.Overexpression of TFEB activated autophagy and attenuated H_(2)O_(2)-induced ROS accumulation.Furthermore,TFEB overexpression attenuated H_(2)O_(2)-induced apoptosis by downregulating the caspase apoptotic pathway.Conclusions:Our results indicate that activation of TFEB mediated autophagy alleviates H_(2)O_(2)-induced oxidative damage by reducing ROS accumulation and inhibiting caspase-dependent apoptosis.
基金supported by the Natural Science Foundation of Shaanxi Province(2021JM-100)the Shaanxi Key Research and Development Program(2018ZDXM-NY-046).
文摘Background:The biosynthesis of milk fat affects both the technological properties and organoleptic quality of milk and dairy products.MicroRNAs(miRNAs)are endogenous small non-coding RNAs that inhibit the expression of their mRNA targets and are involved in downstream signaling pathways that control several biological processes,including milk fat synthesis.miR-34b is a member of the miR-34 miRNA cluster,which is differentially expressed in the mammary gland tissue of dairy cows during lactation and dry periods.Previous studies have indicated miR-34b is a potential candidate gene that plays a decisive role in regulating milk fat synthesis;therefore,it is important to focus on miR-34b and investigate its regulatory effect on the biosynthesis of milk fat in bovine mammary epithelial cells(BMECs).Results:In this study,elevated miR-34b levels reduced milk fat synthesis,upregulated 1,999 genes,and downregulated 2,009 genes in BMECs.Moreover,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis of differentially expressed genes suggested that miR-34b may play an inhibitory role in milk fat synthesis via the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway by reducing phosphorylation levels.Notably,the mTOR activator MHY1485 rescued the inhibitory effect of miR-34b.Furthermore,we demonstrated that retinoic acid-induced protein 14(RAI14)is a target of miR-34b via TargetScan and immunofluorescence assays.RAI14 mRNA and protein levels were significantly decreased by the miR-34b mimic and increased by the miR-34b inhibitor.Moreover,the reduction in RAI14 levels led to the inhibition of the Akt/mTOR signaling pathway.Conclusions:Overall,our results identified a miR-34b-RAI14-Akt/mTOR regulatory network,while also providing a theoretical basis for the molecular breeding of dairy cows.
基金supported by the National Natural Science Foundation of China (31802035, 31872324 and 31601915)
文摘Fat is an indispensable nutrient and basic metabolite for sustaining life,and milk is particularly rich in fatty acids,including a variety of saturated and unsaturated fatty acids.MicroRNA(miRNA)and mRNA play an important role in the regulation of milk fat metabolism in mammary gland tissue.It has been shown that lipid metabolism has a complex transcriptional regulation,but the mechanism by which milk fat synthesis is regulated through miRNA–mRNA interactions is poorly understood.In this study,we performed transcriptome sequencing with bovine mammary gland tissue in the late lactation(270 and 315 days after parturition)to identify the key gene that regulating milk fat metabolism.A total of 1207 differentially coexpressed genes were selected,828 upregulated genes and 379 downregulated genes were identified.The transforming growth factor alpha(TGFA)gene was selected as the target gene,and luciferase reporter assay,Western blotting and q RT-PCR were used for further study.The results demonstrated that miR-140 was an upstream regulator of TGFA,and miR-140 could inhibit(P<0.01)unsaturated fatty acid and triglyceride(TAGs)production in bovine mammary epithelial cells(BMECs).In contrast,TGFA promoted(P<0.01)unsaturated fatty acid and TAG production.Rescue experiments further indicated the mi R-140/TGFA regulatory mechanism.Taken together,these results suggest that the mi R-140/TGFA pathway can inhibit(P<0.01)milk fat metabolism and improve milk quality by genetic means.
基金supported by the Independent Innovation in Jiangsu Province of China(CX(21)3119)the National Natural Science Foundation of China(31802035,31702095 and 31872324)+1 种基金the Jiangsu Natural Science Fund(BK20181221)Yangzhou Liangde Antibody Bio Tech.,China。
文摘Excess ammonia(NH_(3))in the circulation of dairy animals can reduce animal health and the quality of products for human consumption.To develop effective prevention and treatment methods,it is essential to examine the molecular mechanisms through which excess NH_(3) may affect the mammary gland.The present study used bovine mammary epithelial cells(BMECs)to evaluate the effects of exogenous NH_(4)Cl on the abundance of circular RNAs(circRNAs)using high-throughput sequencing.Among the identified circRNAs,circ02771 was the most significantly upregulated by exogenous NH_(4)Cl(P<0.05),with a fold change of 4.12.The results of the apoptosis and proliferation assays,transmission electron microscopy,H&E staining,and immunohistochemistry revealed that circ02771 increased apoptosis and inflammation.A double luciferase reporter assay revealed that circ02771 targeted miR-194b,and the overexpression of circ02771(pcDNA-circ02771)reduced(P<0.05)the expression of miR-194b and led to apoptosis and inflammation.Circ02771 also enhanced the expression of transforming growth factor beta-induced factor homeobox 1(TGIF1),which is a target gene of miR-194b.Overall,this study suggests that the circ02771/miR-194b/TGIF1 axis plays a role in mediating the effects of NH_(4)Cl on BMECs.Therefore,this axis provides a novel target to help control hazards within the mammary gland from high circulating NH_(4)Cl levels.
基金supported by the National Natural Science Foundation of China(Beijing,China,grant no.32072931 and 32002348)Natural Science Foundation of Heilongjiang Province(grant no.LH2020C085).
文摘Background:In early lactation,bovine mammary epithelial cells undergo serious metabolic challenges and oxidative stress both of which could be alleviated by activation of autophagy.Nuclear factor erythroid 2 related factor 2(NFE2L2),a master regulator of cellular redox homeostasis,plays an important role in the regulation of autophagy and oxidative stress.Thus,the objective of this study was to investigate the role of NFE2L2-mediated autophagy on oxidative stress of bovine mammary epithelial cells in response to exogenous free fatty acids(FFA).Results:Exogenous FFA induced linear and quadratic decreases in activities of glutathione peroxidase(GSH-Px),catalase(CAT),and superoxide dismutase(SOD),and increases in the contents of reactive oxygen species(ROS)and malondialdehyde(MDA).Protein abundance of LC3-phosphatidylethanolamine conjugate(LC3-Ⅱ)and the number of autophagosomes and autolysosomes decreased in a dose-dependent manner,while protein abundance of p62 increased in cells challenged with FFA.Activation of autophagy via pre-treatment with Rap attenuated the FFAinduced ROS accumulation.Importantly,FFA inhibited protein abundance of NFE2L2 and the translocation of NFE2L2 into the nucleus.Knockdown of NFE2L2 by siRNA decreased protein abundance of LC3-Ⅱ,while it increased protein abundance of p62.Furthermore,sulforaphane(SFN)pre-treatment attenuated the FFA-induced oxidative stress by activating NFE2L2-mediated autophagy.Conclusions:The data suggested that NFE2L2-mediated autophagy is an important antioxidant mechanism in bovine mammary epithelial cells experiencing increased FFA loads.
基金supported by the National Natural Science Foundation of China(Grant Nos.31872324,31802035,and 31601915)the Natural Science Foundation of the Jiangsu Higher Education Institutions of China(18KJA230003)+1 种基金Six Talent Peaks Project in Jiangsu Province(NY-093)the China Postdoctoral Science Foundation(Grant Nos.2017M621841 and 2019T120472).
文摘Cow mastitis is the most common disease that affects the dairy farming industry and causes serious harm to dairy cows and humans,and Staphylococcus(S.)aureus is one of the main pathogens that cause mastitis in dairy cows.In this study,a mastitis model was established through the infection of bovine mammary epithelial cells(BMECs)with S.aureus(bacterial concentration of 1×10^(9)/mL),and these cells and a blank group(untreated)were analyzed by flow cytometry(10000 cells,200 cells collected per second),hematoxylin and eosin(H&E)staining and immunohistochemistry.In addition,the lncRNAs(long non-coding RNAs)in the normal and S.aureus-infected BMEC group were screened by second-generation sequencing.Flow cytometry,H&E staining,and immunohistochemistry assays were performed to verify the successful construction of an S.aureus infection model in BMECs.A close relationship was found between the differential expression of lncRNAs and S.aureus mastitis.The total original sequencing reads were 627.13 M,and the average reads from each sample were approximately 104.52 M.After removing the unwanted reads,the total clean reads were 606.43 M,and the average reads from each sample were approximately 101.07 M.After S.aureus infection,30 lncRNAs were differentially expressed,and these included 21 upregulated and nine down-regulated lncRNAs.This research will not only expand our understanding of the lncRNA map in dairy cows but also help us hypothesize the function of lncRNAs in the genome and identify novel molecular markers of mastitis.
基金This study was supported by the National Natural Science Foundation of China(Grant Nos.3210273131872324).
文摘Mastitis is a common inflammatory cow mammary infection;that causes significant economic loss in dairy industry.Given the interesting connection between metformin’s anti-inflammatory function and mastitis model induced by LTA in pbMECs,our objective was to prove that metformin was beneficial in suppressing proinflammatory response induced by LTA through modulation of mitogen-activated protein kinase(MAPK)and nuclear factor kappa B(NF-κB)signaling pathways and activation of peroxisome proliferator-activated receptor-γ(PPARγ)in pbMECs.The proliferation of cells and mRNA expression were measured using EdU assay and quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR).Immunoblotting and immunofluorescence analysis were conducted to evaluate the expression of target proteins in inflammatory and anti-inflammatory responses to metformin and LTA.Finally,pbMECs were allowed to treat with the PPAR antagonist GW9662,and inflammatory markers were detected in the cells.Our results showed that LTA concentration at 100μg/mL significantly stimulated the MAPK14,IL-6 and IL-1βmRNA expressions compared to the control cells(P<0.05)in dose-dependent tests for LTA.Metformin suppressed the phosphorylation expressions of MAPK(ERK1/2,p38,and JNK)in LTA-stimulated pbMECs.Metformin also reduced the protein expression of NF-κB,interleukin-8(IL-8),interleukin-1β(IL-1β)and interleukin-6(IL-6)in pbMECs pretreated with LTA.Metformin administration activated PPARγphosphorylation by up-regulating the expression of PPARγin LTA-stimulated pbMECs.Treatment with GW9662 resulted in increased IL-6 expression,which was reversed by metformin.These findings collectively indicated that metformin act to attenuate LTA-stimulated inflammatory response in pbMECs by suppressing MAPK and NF-κB activation via a mechanism partially dependent on PPARγactivation.These results suggested that metformin could function as an anti-inflammatory drug in the treatment of mastitis.
基金supported by the Development Project of China(2017YFD0502104-3)the China Agriculture Research System(CARS-36)the National Natural Science Foundation of China(No.31972589)
文摘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 m RNA 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.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA26040304)。
文摘Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-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 b MECs 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.
基金supported by China Postdoctoral Science Foundation funded project(2019M662971)The Basic Scientific Research Operating Expenses of Higher Education Institutions of Heilongjiang Province(No.2020-KYYWF-0283).
文摘Short-chain fatty acids are important nutrients that regulate milk fat synthesis.They regulate milk syn-thesis via the sterol regulatory element binding protein 1(SREBP1)pathway;however,the details are still unknown.Here,the regulation and mechanism of sodium acetate(SA)in milk fat synthesis in bovine mammary epithelial cells(BMECs)were assessed.BMECs were treated with SA supplementation(SAþ)or without SA supplementation(SA-),and milk fat synthesis and activation of the SREBP1 pathway were increased(P=0.0045;P=0.0042)by SAþand decreased(P=0.0068;P=0.0031)by SA-,respectively.Overexpression or inhibition of SREBP1 demonstrated that SA promoted milk fat synthesis(P=0.0045)via the SREBP1 pathway.Overexpression or inhibition of TATA element modulatory factor 1(TMF1)demon-strated that TMF1 suppressed activation of the SREBP1 pathway(P=0.0001)and milk fat synthesis(P=0.0022)activated by SAþ.Overexpression or inhibition of TMF1 and SREBP1 showed that TMF1 suppressed milk fat synthesis(P=0.0073)through the SREBP1 pathway.Coimmunoprecipitation analysis revealed that TMF1 interacted with SREBP1 in the cytoplasm and suppressed the nuclear localization of SREBP1(P=0.0066).The absence or presence of SA demonstrated that SA inhibited the expression of TMF1(P=0.0002)and the interaction between TMF1 and SREBP1(P=0.0001).Collectively,our research sug-gested that TMF1 was a new negative regulator of milk fat synthesis.In BMECs,SA promoted the SREBP1 pathway and milk fat synthesis by suppressing TMF1.This study enhances the current understanding of the regulation of milk fat synthesis and provides new scientific data for the regulation of milk fat synthesis.
基金supported by the National Natural Science Foundation of China (Project No. 31160466)
文摘Lipopolysaccharide(LPS) was selected as a stimulus to investigate its effect on cell viability and oxidative stress in bovine mammary epithelial cells(BMEC) by detecting the cell relative growth rate(RGR),antioxidant indicators and inflammatory factors. This information was used to provide the theoretical basis for the establishment of a LPS-induced oxidative damage model. The experiment was divided into two parts. The first part used a two-factor experimental design to determine the appropriate incubation time of LPS by detecting the RGR. The third-passage BMEC were divided into 24 groups with six replicates in each group. The first factor was LPS concentration, which was 0(control), 0.1,1.0 and 10.0 μg/mL;the second factor was LPS incubation time(2,4, 6, 8,12 and 24 h). The optimum LPS incubation time was6 h according to the results of the first part of the experiment. The second part of the experiment was conducted using a single-factor experimental design, and the third-passage cells were divided into four groups with six replicates in each group. The cells were incubated with culture medium containing different concentrations of LPS(0 [control], 0.1, 1.0 and 10.0 μg/mL) for 6 h to select the appropriate concentration of LPS to measure the antioxidant indicators and inflammatory factors. The results showed the RGR was significantly reduced as the concentration of LPS and the incubation time increased;the interaction between concentration and incubation time was also significant. The cells treated with0.1 μg/mL of LPS for 6 h had no significant difference in the activities of glutathione peroxidase(GPx) and superoxide dismutase(SOD)(P > 0.05) compared with the cells in the control group. On the contrary,catalase(CAT) activity and malondialdehyde(MDA) content were markedly lower and higher, respectively, in the 0.1 μg/mL LPS-treated group for 6 h compared with the control group(P < 0.05). The activities of GPx, CAT and SOD in the BMEC treated with 1.0 or 10.0 μg/mL of LPS were significantly lower compared with the cells treated with 0.1 μg/mL of LPS and cells in the control group after 6 h of incubation; however, the opposite trend was detected in MDA content. There was no significant(P > 0.05)difference between the 10.0 and 1.0 μg/mL LPS-treated groups. Compared with the control group,interleukin-1, interleukin-6 and nitric oxide concentrations and the activity of inducible nitric oxide synthase in the 0.1 μg/mL LPS-treated group significantly increased(P < 0.0001), but the levels of tumour necrosis factor did not significantly change(P > 0.05). All of observed indicators were higher in the 1.0 and 10.0 μg/mL LPS-treated groups(P < 0.0001) compared with the other groups, but there was no significant(P> 0.05) difference between the 1.0 and 10.0 μg/mL LPS-treated groups. The results indicated that a concentration of 1.0 μg/mL of LPS and an incubation time of 6 h were the optimum conditions necessary to induce oxidative stress in the BMEC and establish a model for oxidative damage.
基金Project supported by the National Basic Research Program(973)of China(No.2011CB100805)the Modern Agro-Industry Technology Research System of China(No.nycytx-04-01)the Agricultural Science and Technology Innovation Program(No.ASTIP-IAS12),China
文摘The aim of this study is to investigate the effects of leucine（Leu） and histidine（His） on the expression of both the mammalian target of rapamycin（mTOR） signaling pathway-related proteins and caseins in immortalized bovine mammary epithelial cells（CMEC-H）, using a single supplement through Western blotting. The Earle＇s balanced salt solution（EBSS） was set as the control group and other treatment groups, based on the EBSS, were added with different concentrations of Leu or His, respectively. The results showed that, compared with the control group, the expression of caseins and the phosphorylation of mTOR（Ser^2481）, Raptor（Ser^792）, e IF4E（Ser^209）, and e EF2（Thr^56） increased with the Leu concentrations ranging from 0.45 to 10.80 mmol/L（P〈0.01）. The P-4EBP1（Thr^37） at 10.80 mmol/L Leu, and P-RPS6（Ser^235/236） at 5.40 to 10.80 mmol/L Leu all decreased. Similarly, the His supplementation from 0.15 to 9.60 mmol/L increased the expression of αs2-casein, β-casein, κ-casein, P-mTOR（Ser^2481）, P-Raptor（Ser^792）, P-S6K1（Thr^389）, P-4EBP1（Thr^37）, P-e IF4E（Ser^209）, and P-e EF2（Thr^56）（P〈0.01） in CMEC-H, whereas the αs1-casein expression was only reduced at 9.60 mmol/L His, G protein β subunit-like protein（GβL） at 0.15 and 9.60 mmol/L His, and P-RPS6 at 4.80 to 9.60 mmol/L His. Our linear regression model assay suggested that the αs1-casein expression was positively correlated with P-mTOR（P〈0.01）, P-S6K1（P〈0.01）, and P-e EF2（P〈0.01） for the addition of Leu, while the expressions of β-casein（P〈0.01） and κ-casein（P〈0.01） were positively correlated with P-e EF2 for the addition of His. In conclusion, the milk protein synthesis was up-regulated through activation of the mTOR pathway with the addition of Leu and His in CMEC-H.
基金supported by the Japan Society for the Promotion of Science (JSPS KAKENHI 25660220) to S.M.
文摘Background： Bovine milk contains not only a variety of nutritional ingredients but also microRNAs （miRNAs） that are thought to be secreted by the bovine mammary epithelial cells （BMECs）. The objective of this study was to elucidate the production of milk-related miRNAs in BMECs under the influence of lactogenic hormones. Results： According to a microarray result of milk exosomal miRNAs prior to cellular analyses, a total of 257 miRNAs were detected in a Holstein cow milk. Of these, 18 major miRNAs of interest in the milk were selected for an expression analysis in BMEC culture that was treated with or without dexamethasone, insulin, and prolactin （DIP） to induce a lactogenic differentiation. Quantitative polymerase chain reaction （qPCR） results showed that the expressions of miR-21-Sp （P = 0.005）, miR-26a （P = 0.016）, and miR-320a （P = 0.011） were lower in the DIP-treated cells than in the untreated cells. In contrast, the expression of miR-339a （P-- 0.017） in the cell culture medium were lower in the DiP-treated culture than in the untreated culture. Intriguingly, the miR-148a expression in cell culture medium was elevated by DIP treatment of BMEC culture （P = 0.018）. The medium-to-cell expression ratios of miR- 103 （P = 0.025）, miR-148a （P 〈 0.001）, and miR-223 （P = 0.013） were elevated in the DIP-treated BMECs, suggesting that the lactogenic differentiation-induced secretion of these three miRNAs in BMECs. A bioinformatic analysis showed that the miRNAs down-regulated in the BMECs were associated with the suppression of genes related to transcriptional regulation, protein phosphorylation, and tube development. Conclusion： The results suggest that the miRNAs changed by lactogenic hormones are associated with milk protein synthesis, and mammary gland development and maturation. The elevated miR-148a level in DIP-treated BMECs may be associated with its increase in milk during the lactation period of cows.
基金The authors acknowledge the financial contributions from the Natural Sciences and Engineering Research Council[401550]Alltech(United States)[054247]to this study.
文摘High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants.Inadequate biodegradation of Fusarium mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds can lead to their circulatory transport to target tissues such as mammary gland.The bovine udder plays a pivotal role in maintaining milk yield and composition,thus,human health.However,toxic effects of Fusarium mycotoxins on bovine mammary gland are rarely studied.In this study,the bovine mammary epithelial cell line was used as an in-vitro model of bovine mammary epithelium to investigate effects of deoxynivalenol(DON),enniatin B(ENB)and beauvericin(BEA)on bovine mammary gland homeostasis.Results indicated that exposure to DON,ENB and BEA for 48 h significantly decreased cell viability in a concentration-dependent manner(P<0.001).Exposure to DON at 0.39μmol/L and BEA at 2.5μmol/L for 48 h also decreased paracellular flux of FITC-40 kDa dextran(P<0.05),whereas none of the mycotoxins affected transepithelial electrical resistance after 48 h exposure.The qPCR was performed for assessment of expression of gene coding tight junction(TJ)proteins,toll-like receptor 4(TLR4)and cytokines after 4,24 and 48 h of exposure.DON,ENB and BEA significantly upregulated the TJ protein zonula occludens-1,whereas markedly downregulated claudin 3(P<0.05).Exposure to DON at 1.35μmol/L for 4 h significantly increased expression of occludin(P<0.01).DON,ENB and BEA significant downregulated TLR4(P<0.05).In contrast,ENB markedly increased expression of cytokines interleukin-6(IL-6)(P<0.001),tumor necrosis factorα(TNF-a)(P<0.05)and transforming growth factor-β(TGF-β)(P<0.01).BEA significantly upregulated IL-6(P<0.001)and TGF-β(P=0.01),but downregulated TNF-α(P<0.001).These results suggest that DON,ENB and BEA can disrupt mammary gland homeostasis by inducing cell death as well as altering its paracellular permeability and expression of genes involved in innate immune function.
