Mechanism Study Of Berberine Against Bovine Mastitis

Bovine mastitis is a common disease of dairy cows that affects animal life,animal health and lactation capacity,and has become a bottleneck problem in the development of dairy farming and dairy industry.Staphylococcus aureus(S.aureus)is one of the most common subclinical mastitis-causing pathogens in dairy animals and often causes degeneration and necrosis of mammary epithelial cells,resulting in the reduction of milk production and the quality of milk products,bring huge economic losses to the dairy industry.At present,the prevention and treatment of bovine mastitis mainly relies on antibiotics.However,the long-term and increased use of antimicrobials can easily cause the emergence of anti-microbial resistance bacteria strains and antimicrobial residues in dairy products,which poses serious potential threats to public health.In particular,the national control of antibiotic use in the aquaculture industry is increasingly strict,so how to use non-antimicrobials strategies to prevent bovine mastitis seems more urgent.In addition,the reports on the prevention and treatment of mastitis mainly focus on the prevention and control based on pathogenic bacteria and its mechanism of action,and few studies on the epigenetic mechanism of the prevention and treatment of mastitis.Berberine is one of the most abundant components in many common medicinal plants.It has many pharmacological effects such as resistant pathogen microorganism,anti-inflammatory and anti-tumor.It has been used to treat and control diseases for hundreds of years as a component of many traditional Chinese medicine prescriptions in China.In order to explore the feasibility of berberine in the treatment of bovine mastitis and promote the clinical application of berberine in the prevention and treatment of bovine mastitis,this study evaluated the effect of berberine on S.aureus-induced injury models using bovine mammary epithelial cell line(MAC-T);Then,methylated RNA immunoprecipitation sequencing(Me RIP-seq)was used to detect the differential m~6A modified transcripts in MAC-T cells with berberine intervention,and to further analyze the role of m~6A modification in S.aureus-induced mastitis.Finally,mice mastitis caused by S.aureus was used to evaluate the effect of berberine on mastitis in mice.The main research results are as follows:1.Establishment of inflammatory model of bovine mammary epithelial cells infected with S.aureus and optimal conditions of berberine intervention on inflammatory injury of MAC-T cells:MAC-T cells were treated with S.aureus or berberine,and the gene and protein levels of inflammatory cytokines IL-6 and TNF-αwere detected.The results showed that when MAC-T cells were exposed to S.aureus at a multiplicity of infection(MOI)of 10for 6 h,the gene and protein expression level of IL-6 and TNF-αreached the highest.Under this condition of S.aureus-induced inflammatory damage,pretreatment of MAC-T cells with 10μg/m L berberine for 3 h significantly reduced the relative m RNA expressions of IL-6 and TNF-α,which was the optimal condition for berberine in the intervention of S.aureus-induced mastitis.2.Berberine intervention alleviates inflammatory injury of bovine mammary epithelial cells caused by S.aureus infection:Normal MAC-T cells and MAC-T cells pretreated with 10μg/m L berberine for 3 h were divided into two groups.One group of cells was treated with 10 MOI S.aureus,and the other group was treated with culture medium.The normal cell group(MEC),berberine group(BER),S.aureus-induced inflammatory group(SA)and inflammation group interfered by berberine(BSA)were obtained.The results showed that berberine intervention could improve the morphology of cells treated by S.aureus,significantly decrease the apoptosis caused by S.aureus infection(P<0.01),decrease the adhesion rate of S.aureus to MAC-T cells(P<0.01),and decrease the phosphorylation level of AKT.The secretion and m RNA expression levels of inflammatory cytokines IL-6 and TNF-αwere down-regulated(P<0.01),and the m RNA expression level of IL-10 was up-regulated(P<0.01),indicating that berberine could alleviate the inflammatory injury of bovine mammary epithelial cells caused by S.aureus.3.Transcriptome mechanism of berberine against S.aureus infection in bovine mammary epithelial cells:Anti-inflammatory cells treated with berberine(BSA group)and inflammation cells treated with S.aureus(SA group)were harvested.RNA was extracted,and RNA sequencing(RNA-seq)was used to explore the transcriptome characteristics of bovine mammary epithelial cells in the two groups.873 differentially expressed m RNAs(DEGs)were obtained,including 93 up-regulated genes and 780 down-regulated genes.Functional annotation and enrichment analysis showed these 873 DEGs were mainly enriched in KEGG pathways such as apoptosis,cell proliferation and inflammation,among which FOS,IL6,TNF,PTGS2,CXCL1,NFKBIA,JUNB and MDM4 were involved in various signaling pathways involved in bovine mastitis.4.Epigenetic mechanism of berberine against S.aureus infection in bovine mammary epithelial cells:BSA and SA cell samples were analyzed using Me RIP-seq to explore the m~6A methylation characteristics in the process of berberine alleviating the inflammatory response of MAC-T cells caused by S.aureus.The results showed that 1)there were 460differentially expressed m~6A peak between BSA and SA group.Among them,these differentially expressed m~6A peak were annotated to 493 differentially methylated genes(DMGs),including 409 up-regulated genes and 81 down-regulated genes.2)Functional annotation and enrichment analysis showed that up-methylated genes were mainly enriched in metabolism,neurotrophic factor regulation and adhesion signal regulation pathways,while down-methylated genes were mainly enriched in tumor and inflammation related pathways.Differentially methylated genes NFKBIA,BRCA2,ATF2,HRH1,NO66,ORC1,YTHDF2,TIAM2,MBD3,ITGB8,TSC1 and Sox4 are involved in the process of berberine intervention in S.aureus-induced mastitis,which can be used as candidate genes for anti-mastitis research.3)Combined RNA-seq and Me RIP-seq data analysis showed that the expression level of m~6A methylated gene was negatively correlated with the level of differentially expressed genes.Among them,m~6A methylation up-regulated genes and differentially down-regulated genes accounted for the majority of significantly different genes between the two groups,indicating that m~6A modification affects m RNA expression and participates in the regulation of berberine to alleviate the inflammatory process of bovine mammary epithelial cells.Me RIP-q PCR and q RT-PCR were used to verify the Me RIP-seq and RNA-seq data,respectively,and the quantitative results were consistent with the sequencing results,indicating that the Me RIP-seq and RNA-seq sequencing data in this study were credible.5.Immune mechanism of berberine against S.aureus-induced mastitis in mice:12 female mice after delivery 5-7 days old were divided into two groups,which were given 25 mg/kg berberine for 7 days by intragastric administration;12 female mice after delivery 5-7 days old were both divided into two groups,which were fed normally.10~4CFU S.aureus were inoculated into the fourth mammary glands via the teat duct in one group,and the other group was given sterile normal saline.The control group(CON),berberine group(BER),S.aureus-caused mice mastitis group(SA)and mice mastitis intervention group with berberine(BSA)were obtained,with 6 mice in each group.The anticoagulant blood and mammary tissue of mice were collected to detect the inflammatory effect of berberine intervention on mice mastitis caused by S.aureus.It was found that compared with S.aureus-induced mastitis in mice,the berberine intervention group had more complete breast structure,less severe breast lesions,and the m RNA expression of IL-6 and TNF-αin breast tissue decreased significantly(P<0.01).The number of neutrophils in peripheral blood was significantly decreased(P<0.05),while the number of lymphocytes was significantly increased(P<0.05).These results indicated that berberine could alleviate the breast inflammation damage caused by S.aureus in mice.In conclusion,berberine intervention can alleviate the inflammation injury of bovine mammary epithelial cells caused by S.aureus.M~6A methylation modification affects m RNA expression and participates in the intervention process of berberine on S.aureus-induced mastitis.Several DEGs and differentially methylated genes(DMGs)related to anti-mastitis by berberine were identified.These results provide support for the analysis of the apparent mechanism of berberine in the intervention of bovine mastitis caused by S.aureus.Berberine also has a certain alleviating effect on the mice mastitis caused by S.aureus,which provide technical support for the clinical application of berberine in the prevention and treatment of bovine mastitis.