Background N^(6)-methyladenosine(m^(6)A)methylation is a key epigenetic modification that can modulate gene expression and strongly affect mammalian developmental processes.However,the genome-wide methylation of long ...Background N^(6)-methyladenosine(m^(6)A)methylation is a key epigenetic modification that can modulate gene expression and strongly affect mammalian developmental processes.However,the genome-wide methylation of long non-coding RNAs(lncRNAs)and its implications for the development of skeletal muscle remain poorly understood.Bovine skeletal muscle samples from five developmental stages were analyzed in this study to establish lncRNA methylome and transcriptomic maps.Results Globally,59.67%of lncRNAs in skeletal muscle with m^(6)A modifications,and this percentage decreased progressively during development.lncRNA expression levels were positively associated with the number of m^(6)A peaks,with lncRNAs possessing 3 or more peaks showing significantly higher expression levels than those with 1 or 2 peaks.Specific lncRNAs involved in skeletal muscle development were identified through two analytical approaches.The first approach employed weighted gene co-expression network analysis(WGCNA)of transcriptomic data to identify correlations between annotated lncRNAs and growth-related traits,resulting in 21 candidate hub lncRNAs.The intersection of these 21 hub lncRNAs with 151 differentially methylated lncRNAs(DM-lncRNAs)identified 10 shared candidate lncRNAs.The second approach integrated MeRIP-seq and RNA-seq data to identify 36 lncRNAs that were both differentially m^(6)A modified and differentially expressed(dme-lncRNAs).GO and KEGG enrichment analyses of cis-target genes associated with these dme-lncRNAs identified eight candidate lncRNAs.Combining the results from the two approaches identified 16 key m^(6)A-modified lncRNAs likely involved in skeletal muscle development.Conclusions These findings highlight the regulatory and functional significance of dynamic lncRNA methylation in skeletal muscle development.展开更多
The oviduct epithelium is the initial maternal contact site for embryos after fertilization,offering the microenviron-ment before implantation.This early gestation period is particularly sensitive to stress,which can ...The oviduct epithelium is the initial maternal contact site for embryos after fertilization,offering the microenviron-ment before implantation.This early gestation period is particularly sensitive to stress,which can cause reduced fertil-ity and reproductive disorders in mammals.Nevertheless,the local impact of elevated stress hormones on the ovi-duct epithelium has received limited attention to date,except for a few reports on polyovulatory species like mice and pigs.In this study,we focused on the effects of chronic maternal stress on cattle,given its association with infertil-ity issues in this monoovulatory species.Bovine oviduct epithelial cells(BOEC)differentiated at the air–liquid interface(ALI)were stimulated with 250 nmol/L cortisol for 1 or 3 weeks.Subsequently,they were assessed for morphology,bioelectrical properties,and gene expression related to oviduct function,glucocorticoid pathway,cortisol metabo-lism,inflammation,and apoptosis.Results revealed adverse effects of cortisol on epithelium structure,featured by deciliation,vacuole formation,and multilayering.Additionally,cortisol exposure led to an increase in transepithelial potential difference,downregulated mRNA expression of the major glucocorticoid receptor(NR3C1),upregulated the expression of cortisol-responsive genes(FKBP5,TSC22D3),and significant downregulation of oviductal glycopro-tein 1(OVGP1)and steroid receptors PGR and ESR1.The systematic comparison to a similar experiment previously performed by us in porcine oviduct epithelial cells,indicated that bovine cultures were more susceptible to elevated cortisol levels than porcine.The distinct responses between both species are likely linked to their divergence in the cortisol-induced expression changes of HSD11B2,an enzyme controlling the cellular capacity to metabolise cortisol.These findings provide insights into the species-specific reactions and reproductive consequences triggered by maternal stress.展开更多
Background Mastitis caused by Staphylococcus aureus(S.aureus)is one of the most intractable problems for the dairy industry,causing significantly reduced milk yields and early slaughter of cows worldwide.MicroRNAs(miR...Background Mastitis caused by Staphylococcus aureus(S.aureus)is one of the most intractable problems for the dairy industry,causing significantly reduced milk yields and early slaughter of cows worldwide.MicroRNAs(miRNAs)can post-transcriptionally regulate gene expression and studies in recent years have shown the importance of miRNA-associated gene regulation in S.aureus-induced mastitis.Results In this study,to investigate the role of miR-223 in mastitis,we performed experiments to overexpress and suppress miR-223 in an immortalized bovine mammary epithelial cell line(MAC-T)infected with S.aureus.Overexpression of miR-223 in MAC-T cells repressed cell apoptosis and necrosis induced by S.aureus infection,whereas suppression of miR-223 had the opposite effect.Transcriptome expression profiling with weighted gene co-expression network analysis(WGCNA)and gene set variation analysis(GSVA)showed that miR-223 affects apoptosis and inflammation-related pathways.Furthermore,differentially expressed(DE)genes were evaluated,and genes exhibiting contrasting expression trends in the miR-223 overexpressed and suppressed groups were assessed as potential target genes of miR-223.Potential target genes,including CDC25B,PTPRF,DCTN1,and DPP9,were observed to be associated with apoptosis and necroptosis.Finally,through integrative analysis of genome-wide association study(GWAS)data and the animal quantitative trait loci(QTL)database,we determined that target genes of miR-223 were significantly enriched in single-nucleotide polymorphisms(SNP)and QTLs related to somatic cell count(SCC)and mastitis.Conclusion In summary,miR-223 has an inhibitory effect on S.aureus-induced cell apoptosis and necrosis by regulating PTPRF,DCTN1,and DPP9.These genes were significantly enriched in QTL regions associated with bovine mastitis resistance,underscoring their relevance in genetic regulation of disease resilience.Our findings provide critical genetic markers for enhancing mastitis resistance,particularly S.aureus-induced mastitis,through selective breeding.This work offers valuable insights for developing cattle with improved resistance to mastitis via targeted genetic selection.展开更多
Background Ferroptosis is characterized by increased production of reactive oxygen species(ROS)and membrane lipid peroxidation that can exacerbate inflammatory damage.Extracellular vesicles(EVs)isolated from bovine mi...Background Ferroptosis is characterized by increased production of reactive oxygen species(ROS)and membrane lipid peroxidation that can exacerbate inflammatory damage.Extracellular vesicles(EVs)isolated from bovine milk have many biological functions,including antioxidant properties.However,the role of EVs on Klebsiella pneumoniaeinduced ferroptosis and oxidative stress in bovine mammary epithelial cells(bMECs)and murine mammary tissue is unclear.In this study,EVs were isolated from bovine colostrum,mature milk and clinical mastitis milk(defined as C-EVs,M-EVs and CM-EVs,respectively)and assessed by transmission electron microscopy,Western blot and transcriptome sequencing.Effects of EVs on K.pneumoniae-induced ferroptosis and oxidative stress in bMECs were evaluated with immunofluorescence and Western blot.Results In bMECs,infection with K.pneumoniae induced oxidative stress,decreasing protein expression of Nrf2,Keap1 and HO-1 plus SOD activity,and increasing ROS concentrations.However,protein expression of GPX4,ACSL4 and S100A4 in bMECs,all factors that regulate ferroptosis,was downregulated by K.pneumoniae.Furthermore,this bacterium compromised tight junctions in murine mammary tissue,with low expression of ZO-1 and Occludin,whereas protein expression of Nrf2 and GPX4 was also decreased in mammary tissue.Adding C-EVs,M-EVs or CMEVs reduced oxidative stress and ferroptosis in K.pneumoniae-infected bMECs in vitro and murine mammary tissues in vivo.Conclusion In conclusion,all 3 sources of milk-derived EVs alleviated oxidative stress and ferroptosis in K.pneumoniae-infected bMECs and mammary tissues.展开更多
Background In perinatal dairy cows,ketosis is a prevalent metabolic disorder that lowers milk output and per-formance.Mitochondrial dysfunction and chronic inflammation in mammary tissue are linked to elevated blood k...Background In perinatal dairy cows,ketosis is a prevalent metabolic disorder that lowers milk output and per-formance.Mitochondrial dysfunction and chronic inflammation in mammary tissue are linked to elevated blood ketone levels,particularlyβ-hydroxybutyrate(BHB).Recent research has linked cytosolic mitochondrial DNA(mtDNA)with chronic aseptic inflammation by activating the cGAS-STING pathway during metabolic disorders,while autophagy activation effectively reverses this process.However,whether it is involved in mammary gland damage during ketosis is poorly understood.Therefore,this study aimed to explore the underlying mechanisms of mtDNA-induced inflammation under BHB stress and evaluate the potential therapeutic strategy of autophagy activation in mitigating this damage.Results Our study found an increased cytoplasmic mtDNA abundance in mammary gland tissues of dairy cows with ketosis and bovine mammary epithelial cell line(MAC-T)subjected to BHB stress.Further investigations revealed the activation of the cGAS-STING pathway and inflammatory response,indicated by elevated levels of cGAS and STING,along with increased phosphorylation levels of TBK1,P65,and IκB,and higher transcript levels of pro-inflammatory factors(IL-1B,IL-6,and TNF-α)in both in vivo and in vitro experiments.Notably,STING inhibition via si-STING transfection reversed BHB-induced inflammation.Additionally,autophagy activation appeared to protect against BHB stress by facilitating the removal of cytoplasmic mtDNA and preventing cGAS-STING pathway-mediated inflammation.Conclusions The findings illustrate that elevated BHB levels lead to the release of cytoplasmic mtDNA,which in turn activates the cGAS-STING pathway and triggers an inflammatory response in the mammary glands during hyper-ketonemia.Conversely,autophagy activation has been shown to alleviate this process by promoting cytoplasmic mtDNA degradation.展开更多
The exosomes hold significant potential in disease diagnosis and therapeutic interventions.The objective of this study was to investigate the potential of aqueous two-phase systems(ATPSs)for the separation of bovine m...The exosomes hold significant potential in disease diagnosis and therapeutic interventions.The objective of this study was to investigate the potential of aqueous two-phase systems(ATPSs)for the separation of bovine milk exosomes.The milk exosome partition behaviors and bovine milk separation were investigated,and the ATPSs and bovine milk whey addition was optimized.The optimal separation conditions were identified as 16%(mass)polyethylene glycol 4000,10%(mass)dipotassium phosphate,and 1%(mass)enzymatic hydrolysis bovine milk whey.During the separation process,bovine milk exosomes were predominantly enriched in the interphase,while protein impurities were primarily found in the bottom phase.The process yielded bovine milk exosomes of 2.0×10^(11)particles per ml whey with high purity(staining rate>90%,7.01×10^(10)particles per mg protein)and high uniformity(polydispersity index<0.03).The isolated exosomes were characterized and identified by transmission electron microscopy,zeta potential and size distribution.The results demonstrated aqueous two-phase extraction possesses a robust capability for the enrichment and separation of exosomes directly from bovine milk whey,presenting a novel approach for the large-scale isolation of exosomes.展开更多
Aim To study the reaction mechanism between gatifloxacin and bovine serumalbumin (BSA) at different pHs. Methods Fluorescence spectra and UV absorbance spectra were used.Results The binding constants were determined f...Aim To study the reaction mechanism between gatifloxacin and bovine serumalbumin (BSA) at different pHs. Methods Fluorescence spectra and UV absorbance spectra were used.Results The binding constants were determined from a double reciprocal Lineweaver-Burk curves atdifferent pHs. The binding distance r under normal physiological condition was obtained according toFoster theory of non-radiative energy transfer. The binding force between gatifloxacin and BSA wasinferred by thermody-namical coordination. Conclusion The interaction between gatifloxacin and BSAseems to be strong and the main binding force is electrostatic force.展开更多
[ 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
Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set...Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.展开更多
Interaction of ioliquiritigenin（ISL）, which is the main active component of a commonly used traditional Chinese medicine（TCM） Glycyrrhiza uralensis Fisch. with bovine serum albumin（BSA） has been investigated. Th...Interaction of ioliquiritigenin（ISL）, which is the main active component of a commonly used traditional Chinese medicine（TCM） Glycyrrhiza uralensis Fisch. with bovine serum albumin（BSA） has been investigated. The quenching mechanism of fluorescence of bovine serum albumin by ISL was discussed. The binding sites number n and apparent binding constant K were measured by fluorescence quenching method. The thermodynamic parameters ΔH^0, ΔG^0, ΔS^0 at different temperatures were calculated. The distance r between donor（bovine serum albumin） and acceptor（ISL） was obtained according to F？rster theory of non-radiation energy transfer. The results of synchronous fluorescence spectra and UV-vis absorption spectra show that the conformation of bovine serum albumin has been changed.展开更多
The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell ...The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase Ⅱ stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls （87.4% vs. 64.4%, P 〈 0.05）. Fur- thermore; centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.展开更多
In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the f...In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.展开更多
[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and lig...[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.展开更多
The assays for bovine immunodeficiency virus (BIV) induced syncytium formation and BIV long terminal repeat (LTR) directed luciferase (Luc) gene expression were applied to screen and evaluate anti AIDS drugs. Frequen...The assays for bovine immunodeficiency virus (BIV) induced syncytium formation and BIV long terminal repeat (LTR) directed luciferase (Luc) gene expression were applied to screen and evaluate anti AIDS drugs. Frequency of the syncytium formation and BIV LTR directed Luc activity were in proportion to the number of input BIV infected cells. AZT inhibited the syncytium formation and the BIV LTR directed Luc gene expression level. Its inhibitory effects were dosedependent with the IC 50 being 0.24 and 0.052 mmol / L, respectively.展开更多
为了研究大型肉牛比利时蓝牛生长发育的遗传规律,筛选优异基因,试验基于Illumina Bovine SNP 50K芯片数据,采用PLINK软件对270头比利时蓝牛常染色体数据进行基因组长纯合片段(ROH)检测并基于选择信号分析,通过核苷酸多态性检测取前5%的...为了研究大型肉牛比利时蓝牛生长发育的遗传规律,筛选优异基因,试验基于Illumina Bovine SNP 50K芯片数据,采用PLINK软件对270头比利时蓝牛常染色体数据进行基因组长纯合片段(ROH)检测并基于选择信号分析,通过核苷酸多态性检测取前5%的单核苷酸多态性(single nucleotide polymorphism,SNP)位点,基于牛参考基因组(ARS-UCD1.2)对结果SNPs进行基因注释,对候选基因进行GO功能注释与KEGG信号通路富集分析,并计算染色体上ROH长度占基因组总长度的比例(FROH)。结果表明:在全部270个个体数据中共检测出1893个ROH片段,平均长度13.2311 Mb,平均FROH为0.0392;得到与生长发育相性状相关的基因有NEB、TET2、NEK11、NCKAP1、MYH15、EIF4A2、bta-miR-1248-1、DCAF8、PRORP、DOCK3、SYT15、MYEF2、ZDHHC13,与公牛生育能力相关的基因有CFAP61、DNAL1、BAG1。说明通过对比利时蓝牛生长发育性状相关分子标记的解析可以为比利时蓝牛遗传改良提供理论指导。展开更多
The quenching interaction of atomoxetine(ATX) with bovine serum albumin(BSA) was studied in vitro under optimal physiological condition(pH=7.4) by multi-spectroscopic techniques. The mechanism of ATX-BSA system was a ...The quenching interaction of atomoxetine(ATX) with bovine serum albumin(BSA) was studied in vitro under optimal physiological condition(pH=7.4) by multi-spectroscopic techniques. The mechanism of ATX-BSA system was a dynamic quenching process and was confirmed by the fluorescence spectra and lifetime measurements. The number of binding sites, binding constants and other binding characteristics were computed. Thermodynamic parameters ΔH^0 and ΔS^0 indicated that intermolecular hydrophobic forces predominantly stabilized the drug-protein system. The average binding distance between BSA and ATX was studied by F?rsters theory. UV-absorption, Fourier transform infrared spectroscopy(FT-IR), circular dichroism(CD), synchronous spectra and three-dimensional(3D) fluorescence spectral results revealed the changes in micro-environment of secondary structure of protein upon the interaction with ATX. Displacement of site probes and the effects of some common metal ions on the binding of ATX with BSA interaction were also studied.展开更多
This study was conducted to determine genetic diversity and antimicrobial susceptibility profiles of Staphylococcus aureus recovered from bovine mastiffs in Zhejiang Province, China. Out of 3178 quarter milk samples f...This study was conducted to determine genetic diversity and antimicrobial susceptibility profiles of Staphylococcus aureus recovered from bovine mastiffs in Zhejiang Province, China. Out of 3178 quarter milk samples from 846 lactating cows, among which 459 cows （54.3%） were found HMT positive, 890 quarters （28%） were found having subclinical mastitis. From 75 representative S. aureus isolates, 16 distinct types were identified by pulsed-field gel electrophoresis （PFGE）. Four major PFGE types （A, B, C, and D） accounted for 82.7% of all isolates, and type A （41.3%） was observed in multiple herds across the studied areas. Each region was found to have a predominant type： Hangzhou type A （64.1%）, Ningbo type C （34.5%） and type B （23.1%）, Jinhua type D （53.3%）, and Taizhou type C （62.5%）. Results of antimicrobial susceptibility tests showed that 90.7% of the isolates were resistant to at least one antimicrobial. Resistance to penicillin and ampicillin （77.3%）, tetracycline （60.0%）, or erythromycin （48.0%） was observed. The bacteria resistant to multiple antibiotics such as penicillin, ampicillin, tetracycline, and erythromycin were commonly found. The information obtained from this study is useful for designing specific control programs for bovine S. aureus mastitis in this region.展开更多
The binding pattern of Ethaselen to bovine serum albumin （BSA） was studied by fluorescence spectroscopic technique and Autodock 3.0.5 analysis. The result showed that the binding constant K of Ethaselen and BSA is 3...The binding pattern of Ethaselen to bovine serum albumin （BSA） was studied by fluorescence spectroscopic technique and Autodock 3.0.5 analysis. The result showed that the binding constant K of Ethaselen and BSA is 3.5×10^4L/mol and the number of binding sites n = 0.9. The binding between Ethaselen and BSA or human serum albumin （HSA） is mainly through hydrophobic interactions.展开更多
Objective：Bovine endometritis is one of the most common reproductive disorders in cattle.The aim of this study was to investigate the anti-inflammation potential of punicalagin in lipopolysaccharide（LPS）-induced bo...Objective：Bovine endometritis is one of the most common reproductive disorders in cattle.The aim of this study was to investigate the anti-inflammation potential of punicalagin in lipopolysaccharide（LPS）-induced bovine endometrial epithelial cells（bE ECs）and to uncover the underlying mechanisms.Methods：bE ECs were stimulated with different concentrations（1,10,30,50,and 100μg/ml）of LPS for 3,6,9,12,and 18 h.MTT assay was used to assess cell viability and to identify the conditions for inflammatory injury and effective concentrations of punicalagin.Quantitative real-time polymerase chain reaction（q RT-PCR）was used to assess gene expression of pro-inflammatory cytokines.Western blotting was used to assess levels of inflammation-related proteins.Results：Treatment of b EECs with 30μg/ml LPS for 12 h induced cell injury and reduced cell viability.Punicalagin（5,10,or 20μg/ml）pretreatment significantly decreased LPS-induced productions of interleukin（IL）-1β,IL-6,IL-8,and tumor necrosis factor-α（TNF-α）in bE ECs.Molecular research showed that punicalagin inhibited the activation of the upstream mediator nuclear factor-κB（NF-κB）by suppressing the production of inhibitorκBα（IκBα）and phosphorylation of p65.Results also indicated that punicalagin can suppress the phosphorylation of mitogen-activated protein kinases（MAPKs）including p38,c-Jun N-terminal kinase（JNK）,and extracellular signal-regulated kinase（ERK）.Conclusions：Punicalagin may attenuate LPS-induced inflammatory injury and provide a potential option for the treatment of dairy cows with Escherichia coli endometritis.展开更多
Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. The resistant strains make the disease difficult to cure. The aim of this study was to characterize the genetic nature of the antimicro...Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. The resistant strains make the disease difficult to cure. The aim of this study was to characterize the genetic nature of the antimicrobial resistance in S. aureus cultured from bovine mastitis in Northwest China in 2014. A total of 44 S. aureus were isolated for antimicrobial resistance and resistance-related genes. Antimicrobial resistance was determined by disc diffusion and the corresponding resistance genes were detected by PCR. Phenotype indicated that S. aureus isolates were resistant to penicillin （84.09%）, erythromycin （20.45%）, tetracycline （15.91%）, gentamicin （9.09%）, tobramycin （6.82%）, kanamycin （6.82%） and methicillin （2.27%）. 9.09% of the S. aureus isolates were classified as multidrug resistant. In addition, genotypes showed that the isolates were resistant to rifampicin （100%, rpoB）, penicillin （95.45%, blaZ）, tetracycline （22.73%, tetK, tetM, alone or in combination）, erythromycin （22.73%, ermB or ermC）, gentamicin/tobramycin/kanamycin （2.27%, aacA-aphD）, methicillin （2.27%, mecA） and vancomycin （2.27%, vanA）. Resistance to tetracycline was attributed to the genes tetK and tetM （r=0.558, P〈0.001）. This study noted high-level geno- and phenotypic antimicrobial resistance in S. aureus isolates from bovine mastitis cases in Northwest China.展开更多
基金supported by the National Key R&D Program of China(2023YFD1300103)the Science and Technology Plan Project of Yantai City(2023ZDCX024)+5 种基金the National Natural Science Foundation of China(32372852)the Science Fund for Distinguished Young Scholars of Shaanxi Province(2024JC-JCQN-30)Shaanxi Provincial Innovation Leadership Program in Sciences and Technologies for Young and Middle-aged Scientists(2023SR205)the China Agriculture Research System-beef(CARS-37)the Innovation Team of Cattle Industry in Technological System of Shandong Modern Agriculture Industry(SDAIT-09-03)the Key Research and Development Project in Shandong Province(Competitive Innovation Platform)(2022CXPT010).
文摘Background N^(6)-methyladenosine(m^(6)A)methylation is a key epigenetic modification that can modulate gene expression and strongly affect mammalian developmental processes.However,the genome-wide methylation of long non-coding RNAs(lncRNAs)and its implications for the development of skeletal muscle remain poorly understood.Bovine skeletal muscle samples from five developmental stages were analyzed in this study to establish lncRNA methylome and transcriptomic maps.Results Globally,59.67%of lncRNAs in skeletal muscle with m^(6)A modifications,and this percentage decreased progressively during development.lncRNA expression levels were positively associated with the number of m^(6)A peaks,with lncRNAs possessing 3 or more peaks showing significantly higher expression levels than those with 1 or 2 peaks.Specific lncRNAs involved in skeletal muscle development were identified through two analytical approaches.The first approach employed weighted gene co-expression network analysis(WGCNA)of transcriptomic data to identify correlations between annotated lncRNAs and growth-related traits,resulting in 21 candidate hub lncRNAs.The intersection of these 21 hub lncRNAs with 151 differentially methylated lncRNAs(DM-lncRNAs)identified 10 shared candidate lncRNAs.The second approach integrated MeRIP-seq and RNA-seq data to identify 36 lncRNAs that were both differentially m^(6)A modified and differentially expressed(dme-lncRNAs).GO and KEGG enrichment analyses of cis-target genes associated with these dme-lncRNAs identified eight candidate lncRNAs.Combining the results from the two approaches identified 16 key m^(6)A-modified lncRNAs likely involved in skeletal muscle development.Conclusions These findings highlight the regulatory and functional significance of dynamic lncRNA methylation in skeletal muscle development.
基金German research Foundation(DFG,grant numbers:CH2321/1–1 and SCHO1231/7–1)JH has received a scholarship from the Chinese Scholarship Council(CSC No.:201908350115).
文摘The oviduct epithelium is the initial maternal contact site for embryos after fertilization,offering the microenviron-ment before implantation.This early gestation period is particularly sensitive to stress,which can cause reduced fertil-ity and reproductive disorders in mammals.Nevertheless,the local impact of elevated stress hormones on the ovi-duct epithelium has received limited attention to date,except for a few reports on polyovulatory species like mice and pigs.In this study,we focused on the effects of chronic maternal stress on cattle,given its association with infertil-ity issues in this monoovulatory species.Bovine oviduct epithelial cells(BOEC)differentiated at the air–liquid interface(ALI)were stimulated with 250 nmol/L cortisol for 1 or 3 weeks.Subsequently,they were assessed for morphology,bioelectrical properties,and gene expression related to oviduct function,glucocorticoid pathway,cortisol metabo-lism,inflammation,and apoptosis.Results revealed adverse effects of cortisol on epithelium structure,featured by deciliation,vacuole formation,and multilayering.Additionally,cortisol exposure led to an increase in transepithelial potential difference,downregulated mRNA expression of the major glucocorticoid receptor(NR3C1),upregulated the expression of cortisol-responsive genes(FKBP5,TSC22D3),and significant downregulation of oviductal glycopro-tein 1(OVGP1)and steroid receptors PGR and ESR1.The systematic comparison to a similar experiment previously performed by us in porcine oviduct epithelial cells,indicated that bovine cultures were more susceptible to elevated cortisol levels than porcine.The distinct responses between both species are likely linked to their divergence in the cortisol-induced expression changes of HSD11B2,an enzyme controlling the cellular capacity to metabolise cortisol.These findings provide insights into the species-specific reactions and reproductive consequences triggered by maternal stress.
基金supported by the National Key Research and Development Program of China(Grant No.2023YFF1000902,2021YFD1200903)the National Science Foundation for Young Scientists of China(Grant No.32302706)the Beijing Dairy Industry Innovation Team(Grant No.BAIC06).
文摘Background Mastitis caused by Staphylococcus aureus(S.aureus)is one of the most intractable problems for the dairy industry,causing significantly reduced milk yields and early slaughter of cows worldwide.MicroRNAs(miRNAs)can post-transcriptionally regulate gene expression and studies in recent years have shown the importance of miRNA-associated gene regulation in S.aureus-induced mastitis.Results In this study,to investigate the role of miR-223 in mastitis,we performed experiments to overexpress and suppress miR-223 in an immortalized bovine mammary epithelial cell line(MAC-T)infected with S.aureus.Overexpression of miR-223 in MAC-T cells repressed cell apoptosis and necrosis induced by S.aureus infection,whereas suppression of miR-223 had the opposite effect.Transcriptome expression profiling with weighted gene co-expression network analysis(WGCNA)and gene set variation analysis(GSVA)showed that miR-223 affects apoptosis and inflammation-related pathways.Furthermore,differentially expressed(DE)genes were evaluated,and genes exhibiting contrasting expression trends in the miR-223 overexpressed and suppressed groups were assessed as potential target genes of miR-223.Potential target genes,including CDC25B,PTPRF,DCTN1,and DPP9,were observed to be associated with apoptosis and necroptosis.Finally,through integrative analysis of genome-wide association study(GWAS)data and the animal quantitative trait loci(QTL)database,we determined that target genes of miR-223 were significantly enriched in single-nucleotide polymorphisms(SNP)and QTLs related to somatic cell count(SCC)and mastitis.Conclusion In summary,miR-223 has an inhibitory effect on S.aureus-induced cell apoptosis and necrosis by regulating PTPRF,DCTN1,and DPP9.These genes were significantly enriched in QTL regions associated with bovine mastitis resistance,underscoring their relevance in genetic regulation of disease resilience.Our findings provide critical genetic markers for enhancing mastitis resistance,particularly S.aureus-induced mastitis,through selective breeding.This work offers valuable insights for developing cattle with improved resistance to mastitis via targeted genetic selection.
基金supported by the National Natural Science Foundation of China(32273082 and U21A20262).
文摘Background Ferroptosis is characterized by increased production of reactive oxygen species(ROS)and membrane lipid peroxidation that can exacerbate inflammatory damage.Extracellular vesicles(EVs)isolated from bovine milk have many biological functions,including antioxidant properties.However,the role of EVs on Klebsiella pneumoniaeinduced ferroptosis and oxidative stress in bovine mammary epithelial cells(bMECs)and murine mammary tissue is unclear.In this study,EVs were isolated from bovine colostrum,mature milk and clinical mastitis milk(defined as C-EVs,M-EVs and CM-EVs,respectively)and assessed by transmission electron microscopy,Western blot and transcriptome sequencing.Effects of EVs on K.pneumoniae-induced ferroptosis and oxidative stress in bMECs were evaluated with immunofluorescence and Western blot.Results In bMECs,infection with K.pneumoniae induced oxidative stress,decreasing protein expression of Nrf2,Keap1 and HO-1 plus SOD activity,and increasing ROS concentrations.However,protein expression of GPX4,ACSL4 and S100A4 in bMECs,all factors that regulate ferroptosis,was downregulated by K.pneumoniae.Furthermore,this bacterium compromised tight junctions in murine mammary tissue,with low expression of ZO-1 and Occludin,whereas protein expression of Nrf2 and GPX4 was also decreased in mammary tissue.Adding C-EVs,M-EVs or CMEVs reduced oxidative stress and ferroptosis in K.pneumoniae-infected bMECs in vitro and murine mammary tissues in vivo.Conclusion In conclusion,all 3 sources of milk-derived EVs alleviated oxidative stress and ferroptosis in K.pneumoniae-infected bMECs and mammary tissues.
基金supported by the National Natural Science Foundation of China(32125038)the National Key R&B Program of China(2023YFD1801100)China Agriculture Research System(CARS-36).
文摘Background In perinatal dairy cows,ketosis is a prevalent metabolic disorder that lowers milk output and per-formance.Mitochondrial dysfunction and chronic inflammation in mammary tissue are linked to elevated blood ketone levels,particularlyβ-hydroxybutyrate(BHB).Recent research has linked cytosolic mitochondrial DNA(mtDNA)with chronic aseptic inflammation by activating the cGAS-STING pathway during metabolic disorders,while autophagy activation effectively reverses this process.However,whether it is involved in mammary gland damage during ketosis is poorly understood.Therefore,this study aimed to explore the underlying mechanisms of mtDNA-induced inflammation under BHB stress and evaluate the potential therapeutic strategy of autophagy activation in mitigating this damage.Results Our study found an increased cytoplasmic mtDNA abundance in mammary gland tissues of dairy cows with ketosis and bovine mammary epithelial cell line(MAC-T)subjected to BHB stress.Further investigations revealed the activation of the cGAS-STING pathway and inflammatory response,indicated by elevated levels of cGAS and STING,along with increased phosphorylation levels of TBK1,P65,and IκB,and higher transcript levels of pro-inflammatory factors(IL-1B,IL-6,and TNF-α)in both in vivo and in vitro experiments.Notably,STING inhibition via si-STING transfection reversed BHB-induced inflammation.Additionally,autophagy activation appeared to protect against BHB stress by facilitating the removal of cytoplasmic mtDNA and preventing cGAS-STING pathway-mediated inflammation.Conclusions The findings illustrate that elevated BHB levels lead to the release of cytoplasmic mtDNA,which in turn activates the cGAS-STING pathway and triggers an inflammatory response in the mammary glands during hyper-ketonemia.Conversely,autophagy activation has been shown to alleviate this process by promoting cytoplasmic mtDNA degradation.
基金supported by the National Natural Science Foundation of China(22378350).
文摘The exosomes hold significant potential in disease diagnosis and therapeutic interventions.The objective of this study was to investigate the potential of aqueous two-phase systems(ATPSs)for the separation of bovine milk exosomes.The milk exosome partition behaviors and bovine milk separation were investigated,and the ATPSs and bovine milk whey addition was optimized.The optimal separation conditions were identified as 16%(mass)polyethylene glycol 4000,10%(mass)dipotassium phosphate,and 1%(mass)enzymatic hydrolysis bovine milk whey.During the separation process,bovine milk exosomes were predominantly enriched in the interphase,while protein impurities were primarily found in the bottom phase.The process yielded bovine milk exosomes of 2.0×10^(11)particles per ml whey with high purity(staining rate>90%,7.01×10^(10)particles per mg protein)and high uniformity(polydispersity index<0.03).The isolated exosomes were characterized and identified by transmission electron microscopy,zeta potential and size distribution.The results demonstrated aqueous two-phase extraction possesses a robust capability for the enrichment and separation of exosomes directly from bovine milk whey,presenting a novel approach for the large-scale isolation of exosomes.
文摘Aim To study the reaction mechanism between gatifloxacin and bovine serumalbumin (BSA) at different pHs. Methods Fluorescence spectra and UV absorbance spectra were used.Results The binding constants were determined from a double reciprocal Lineweaver-Burk curves atdifferent pHs. The binding distance r under normal physiological condition was obtained according toFoster theory of non-radiative energy transfer. The binding force between gatifloxacin and BSA wasinferred by thermody-namical coordination. Conclusion The interaction between gatifloxacin and BSAseems to be strong and the main binding force is electrostatic force.
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.
文摘Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.
基金National Natural Science Foundation of China(Grant No.81560699)Scientific and Technological Project of the Science and Technology Department of Guangdong Province(Grant No.2014A020209026)+1 种基金Social Development Research and Technology Transfer Program(Grant No.2015AD002)Outstanding Young Scientific Personnel Training Plan of Shihezi University(Grant No.2015-ZRKXJQ08)
文摘Interaction of ioliquiritigenin（ISL）, which is the main active component of a commonly used traditional Chinese medicine（TCM） Glycyrrhiza uralensis Fisch. with bovine serum albumin（BSA） has been investigated. The quenching mechanism of fluorescence of bovine serum albumin by ISL was discussed. The binding sites number n and apparent binding constant K were measured by fluorescence quenching method. The thermodynamic parameters ΔH^0, ΔG^0, ΔS^0 at different temperatures were calculated. The distance r between donor（bovine serum albumin） and acceptor（ISL） was obtained according to F？rster theory of non-radiation energy transfer. The results of synchronous fluorescence spectra and UV-vis absorption spectra show that the conformation of bovine serum albumin has been changed.
基金This work was supported by the National High Technology Research and Development Program of China(863 Program)(No.2001 AA213081).
文摘The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch2-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase Ⅱ stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls （87.4% vs. 64.4%, P 〈 0.05）. Fur- thermore; centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.
基金The General Foundation of Tianjin Science Committee for Applied Basic Research (08JCZDJC21000)Chinese Ministry of Education (30770081)
文摘In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.
文摘[Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.
文摘The assays for bovine immunodeficiency virus (BIV) induced syncytium formation and BIV long terminal repeat (LTR) directed luciferase (Luc) gene expression were applied to screen and evaluate anti AIDS drugs. Frequency of the syncytium formation and BIV LTR directed Luc activity were in proportion to the number of input BIV infected cells. AZT inhibited the syncytium formation and the BIV LTR directed Luc gene expression level. Its inhibitory effects were dosedependent with the IC 50 being 0.24 and 0.052 mmol / L, respectively.
基金Karnatak University, Dharwad, India, for providing UGC-UPE fellowshipUGC, New Delhi for the award of BSR Faculty Fellowship (F No.18-1/2011) to Prof. S.T. Nandibewoor
文摘The quenching interaction of atomoxetine(ATX) with bovine serum albumin(BSA) was studied in vitro under optimal physiological condition(pH=7.4) by multi-spectroscopic techniques. The mechanism of ATX-BSA system was a dynamic quenching process and was confirmed by the fluorescence spectra and lifetime measurements. The number of binding sites, binding constants and other binding characteristics were computed. Thermodynamic parameters ΔH^0 and ΔS^0 indicated that intermolecular hydrophobic forces predominantly stabilized the drug-protein system. The average binding distance between BSA and ATX was studied by F?rsters theory. UV-absorption, Fourier transform infrared spectroscopy(FT-IR), circular dichroism(CD), synchronous spectra and three-dimensional(3D) fluorescence spectral results revealed the changes in micro-environment of secondary structure of protein upon the interaction with ATX. Displacement of site probes and the effects of some common metal ions on the binding of ATX with BSA interaction were also studied.
基金Project (No. 2005C12015) supported by the Science and Technology Department of Zhejiang Province, China
文摘This study was conducted to determine genetic diversity and antimicrobial susceptibility profiles of Staphylococcus aureus recovered from bovine mastiffs in Zhejiang Province, China. Out of 3178 quarter milk samples from 846 lactating cows, among which 459 cows （54.3%） were found HMT positive, 890 quarters （28%） were found having subclinical mastitis. From 75 representative S. aureus isolates, 16 distinct types were identified by pulsed-field gel electrophoresis （PFGE）. Four major PFGE types （A, B, C, and D） accounted for 82.7% of all isolates, and type A （41.3%） was observed in multiple herds across the studied areas. Each region was found to have a predominant type： Hangzhou type A （64.1%）, Ningbo type C （34.5%） and type B （23.1%）, Jinhua type D （53.3%）, and Taizhou type C （62.5%）. Results of antimicrobial susceptibility tests showed that 90.7% of the isolates were resistant to at least one antimicrobial. Resistance to penicillin and ampicillin （77.3%）, tetracycline （60.0%）, or erythromycin （48.0%） was observed. The bacteria resistant to multiple antibiotics such as penicillin, ampicillin, tetracycline, and erythromycin were commonly found. The information obtained from this study is useful for designing specific control programs for bovine S. aureus mastitis in this region.
基金National Natural Science Foundation of China (Grant No. 30472036)Beijing Natural Science Foundation (Grant No. 7021001)
文摘The binding pattern of Ethaselen to bovine serum albumin （BSA） was studied by fluorescence spectroscopic technique and Autodock 3.0.5 analysis. The result showed that the binding constant K of Ethaselen and BSA is 3.5×10^4L/mol and the number of binding sites n = 0.9. The binding between Ethaselen and BSA or human serum albumin （HSA） is mainly through hydrophobic interactions.
基金supported by the National Key Technology R&D Program of China(No.2013BAD10B04)the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions(No.CIT&TCD20130324),China
文摘Objective：Bovine endometritis is one of the most common reproductive disorders in cattle.The aim of this study was to investigate the anti-inflammation potential of punicalagin in lipopolysaccharide（LPS）-induced bovine endometrial epithelial cells（bE ECs）and to uncover the underlying mechanisms.Methods：bE ECs were stimulated with different concentrations（1,10,30,50,and 100μg/ml）of LPS for 3,6,9,12,and 18 h.MTT assay was used to assess cell viability and to identify the conditions for inflammatory injury and effective concentrations of punicalagin.Quantitative real-time polymerase chain reaction（q RT-PCR）was used to assess gene expression of pro-inflammatory cytokines.Western blotting was used to assess levels of inflammation-related proteins.Results：Treatment of b EECs with 30μg/ml LPS for 12 h induced cell injury and reduced cell viability.Punicalagin（5,10,or 20μg/ml）pretreatment significantly decreased LPS-induced productions of interleukin（IL）-1β,IL-6,IL-8,and tumor necrosis factor-α（TNF-α）in bE ECs.Molecular research showed that punicalagin inhibited the activation of the upstream mediator nuclear factor-κB（NF-κB）by suppressing the production of inhibitorκBα（IκBα）and phosphorylation of p65.Results also indicated that punicalagin can suppress the phosphorylation of mitogen-activated protein kinases（MAPKs）including p38,c-Jun N-terminal kinase（JNK）,and extracellular signal-regulated kinase（ERK）.Conclusions：Punicalagin may attenuate LPS-induced inflammatory injury and provide a potential option for the treatment of dairy cows with Escherichia coli endometritis.
基金supported by the Central Public-Interest Scientific Institution Basal Research Fund,China (1610322015007)the Key Technology R&D Program of China during the 12th Five-Year Plan period (2012BAD12B03)the Natural Science Foundation of Gansu Province,China (145RJYA311)
文摘Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. The resistant strains make the disease difficult to cure. The aim of this study was to characterize the genetic nature of the antimicrobial resistance in S. aureus cultured from bovine mastitis in Northwest China in 2014. A total of 44 S. aureus were isolated for antimicrobial resistance and resistance-related genes. Antimicrobial resistance was determined by disc diffusion and the corresponding resistance genes were detected by PCR. Phenotype indicated that S. aureus isolates were resistant to penicillin （84.09%）, erythromycin （20.45%）, tetracycline （15.91%）, gentamicin （9.09%）, tobramycin （6.82%）, kanamycin （6.82%） and methicillin （2.27%）. 9.09% of the S. aureus isolates were classified as multidrug resistant. In addition, genotypes showed that the isolates were resistant to rifampicin （100%, rpoB）, penicillin （95.45%, blaZ）, tetracycline （22.73%, tetK, tetM, alone or in combination）, erythromycin （22.73%, ermB or ermC）, gentamicin/tobramycin/kanamycin （2.27%, aacA-aphD）, methicillin （2.27%, mecA） and vancomycin （2.27%, vanA）. Resistance to tetracycline was attributed to the genes tetK and tetM （r=0.558, P〈0.001）. This study noted high-level geno- and phenotypic antimicrobial resistance in S. aureus isolates from bovine mastitis cases in Northwest China.
