Antagonistic Dffect Of Metformin On E.coli Induced Mammary Epithelial Cell Inflammation And Its Bacteriostatic Mechanism

Mastitis of dairy cows has caused huge economic losses to dairy farming industry.The main reason is that pathogenic microorganisms invade mammary tissue,leading to inflammation and tissue damage.Studies have shown that inflammation of the mammary glands caused by E.coli has a big impact on milk production and milk quality in cows.Antibiotics are still one of the most effective treatments for mastitis in cattle.However,pathogen resistance and drug residues in dairy products have been an important issue for the dairy industry and public health.Therefore,it is of great significance to find natural active,non-antibiotic substances and other substitutes to treat dairy mastitis.This study revealed the mechanism of breast cell inflammation caused by pasture Escherichia coli and the bacteriostatic effect of metformin from both cellular and bacterial perspectives.At the cellular level,transcriptome sequencing was used to explore the anti-inflammatory molecular pathway of metformin by constructing the inflammation model of breast epithelial cells of pathogenic bacteria.Meanwhile,a LPS-induced in vitro mastitis model was established.Based on the sequencing results,the antagonistic mechanism of metformin on the inflammation of dairy mammary epithelial cells was detected by fluorescence quantification,immunofluorescence,Western-Blot,ELISA,immunoprecipitation and other methods.At the bacterial level,the inhibitory effect of metformin on the proliferation of Escherichia coli and its biofilm formation was explored.To study the biological activity of metformin in antibacterial aspect.1.The anti-inflammatory effect of metforminIn this study,bovine mammary epithelial cells were pretreated with metformin and infected with E.coli.Transcriptome sequencing revealed that metformin inhibited cellular inflammatory response by altering MAPK signaling pathway.Subsequently,by establishing a model of LPS-induced inflammation,it was found that the addition of metformin could effectively inhibit LPS-induced pro-inflammatory genes(TNF,IL1B,IL6,CXCL8,MYD88,and TLR4)and related proteins(IL-1β,TNF-α,IL-1β,TNF-α and other proteins.NLRP3,Caspasel,ASC)were expressed with activation of AMPK signaling pathway.Compared with LPS group,the phosphorylation levels of p65 and IκBa were decreased in MET+LPS(ML)group after metformin pretreatment,and the transfer of NF-κB in the nucleus was significantly reduced.Metformin inhibits LPS-induced NF-κB binding activity in the TNFA and IL1B promoter regions and reduces the transcription level of pro-inflammatory factors.In addition,LPS-stimulated increased H3K14 acetylation levels were inhibited by metformin preconditioning.These results suggest that metformin preconditioning can mediate AMPK/NF-κB/NLRP3 signaling pathway and histone H3K14 deacetylation and metabolic changes,and inhibit the LPS-induced inflammatory response.2.Inhibitory effect of metformin on proliferation of Escherichia coliIn this study,the inhibitory curve of metformin against E.coli,Klebsiella pneumoniae and Staphylococcus aureus was established by detecting the minimum inhibitory concentration(MIC)of metformin against E.coli isolated from pasture.The results showed that the inhibitory effect of metformin against E.coli,Klebsiella pneumoniae was poor at the same concentration,while the inhibitory effect against E.coli was good.Subsequently,the results were verified by standard E.coli ATCC25922,and the MIC of metformin on the isolated bacteria was consistent with that of standard bacteria(10mg/mL).Transcriptome sequencing results showed that metformin affected the activity of Escherichia coli by affecting the formation,oxidative phosphorylation,TCA cycle,pyruvate metabolism,and expression of ABC transporter-related functional genes.The expressions of flhD,fimH,motA,ompF,luxS,fliA and yqe genes were detected by fluorescence quantitative PCR.Compared with the control group,the expression levels of related genes were reduced by metformin treatment.In addition,the checkerboard test showed that the combined effect of metformin and antibiotics was synergistic with cephalothiophene,Lincomycin and penicillin(FIC<0.5),indicating that metformin can be used as an antibacterial synergistic agent to improve the antibacterial effect of antibiotics.Finally,abdominal infection model of BLAB/C mice was established to verify the effect of metformin on E.coli infection.The results showed that metformin significantly improved the survival rate of mice,and the survival rate of mice treated with 75mg/kg metformin increased to 75%.HE staining of liver and spleen sections showed that,compared with the control group,there was no obvious congestion and inflammatory cell infiltration in the liver and spleen of mice treated with metformin.These results indicate that metformin can inhibit the proliferation of E.coli.3.Inhibition of Escherichia coli biofilm by metforminMetformin also inhibited the formation of E.coli biofilms.The count of viable bacteria in biofilm showed that the number of viable bacteria in biofilm decreased significantly with the increase of metformin concentration.Crystal violet staining showed that metformin inhibited the formation of biofilm on the tube wall.It was verified that metformin destroyed the structure of biofilm and killed LIVE bacteria on biofilm by LIVE/DEAD bacterial fluorescence staining.The results were also verified by scanning electron microscopy.It is speculated that metformin may inhibit biofilm formation by inhibiting the expression of genes related to biofilm formation.In conclusion,metformin may inhibit E.coli induced inflammatory response in dairy mammary epithelial cells through AMPK/NF-κB/NLRP3 signaling pathway and histone H3K14 deacetylation and metabolism.In addition,by affecting the formation of Escherichia coli membrane,protein transport and other influences on the proliferation of Escherichia coli,and has an inhibitory effect on the formation of Escherichia coli biofilm.In addition,metformin can be used as an antibacterial synergist to enhance the antibacterial action of antibiotics and reduce bacterial resistance.The results of this study provide theoretical basis and data support for the development of metformin as a low drug resistance,non-antibiotic drug for clinical effective against mammary germ infection of dairy cows.