Metformin Inhibits Lipoteichoic Acid And Staphylococcus Aureus-Induced Oxidative Stress And Inflammation Through AMPK/NRF2/NF-κB Signaling Pathway In Bovine Mammary Epithelial Cells And Mouse Mammary Gland Models

Mastitis is a common disease incidence of bovine mammary pathogenic infection;that significantly affects animal welfare,health,production capacity and eventually causes significant economic loss in the whole dairy industry worldwide.The stimulation of bovine mammary epithelial cells(bMECs)with Staphylococcus aureus(S.aureus)or Lipoteichoic acid(LTA)could trigger inflammatory reactions and influences lactation in dairy cows’mammary glands.Currently,antibiotics are often used to treat mastitis,but increasing multidrug resistance bacteria issues in dairy production.Therefore,it is critical to identify and develop an alternative safe and effective medication to replace antibiotics used in treating cows’mastitis.Metformin(1,-dimethylbiguanide hydrochloride),a natural compound found in the plant Galega officinalis,has been used as first-line therapy for type 2 diabetes(T2D);attracted attention over the last decade due to its biological functions,including anti-inflammatory,anti-oxidant and anti-carcinogenic properties.Given an interesting connection between metformin’s anti-inflammatory and antioxidant function against mastitis model induced by LTA in pbMECs or S.aureus infected mouse mammary glands is needed.However,the molecular mechanism of metformin to control mastitis remains unknown.The purposes of this thesis were firstly to use LTA-stimulated pbMECs to examine the transcriptional response variability of pbMECs to LTA and Metformin treatments using RNA-Seq analysis.Secondly,to notify the effect of metformin on the LTA-induced mastitis model using isolated pbMECs and explore Metformin Inhibits Lipoteichoic acid-induced Oxidative Stress and Inflammation through AMPK/NRF2/NF-κB Signaling Pathway in Bovine Mammary Epithelial Cells.Thirdly,to prove that metformin was beneficial in suppressing proinflammatory mediators,in part,through modulation of MAPK and NF-κB signaling pathways;and activation of PPARγ in pbMECs.Finally,to assess whether metformin could have an effective role against S.aureus-infected mastitis in the mouse model and whether the anti-inflammatory and anti-oxidant activities are involved in its mechanism of action.The following experiments and analysis were carried out in this study,and the relevant results were obtained.1.To investigate the RNA-seq analysis of metformin and lipoteichoic acidinduced different immunity and inflammatory responses in bovine mammary epithelial cells,three treatment groups were devised as follow:the control group,the cells treated with 100 μg/mL LTA for 6 h(LTA group)and the cells pretreated with metformin(3 mM)for 12 h followed by 100 μg/mL LTA exposure for 6 h(MET+LTA group).Cells in the normal control group were cultured in a normal culture medium for 12 h.Total RNA was collected from the cells using TRIzol? reagent.RNA-seq transcriptome library was prepared following the TruSeqTM RNA sample preparation Kit from Illumina(San Diego,CA)using 1 μg of total RNA.To identify differential expression genes(DEGs)between two different samples,the expression level of each transcript was calculated according to the transcripts per million reads(TPM)method.Functionalenrichment analyses,including Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG),were done to identify which DEGs were significantly enriched in GO terms and metabolic pathways at a p-value ≤0.05 compared with the wholetranscriptome background.The transcriptome analysis shows the DEGs of LTA vs.Control and LTA vs.MET comparisons indicated that 121(from a total of 154)common DEGs were found in LTA vs.Control,and 297(from a total of 330)unique DEGs in LTA vs.MET,respectively.While a total of 33 DEGs were shared by the two treatment groups,of which 25 were up-regulated,and 5 were downregulated genes following LTA treatment;however,metformin reverses the responses of these genes to LTA inducement.These DEGs primarily regulate immune and inflammation responses in vitro via NF-κB signaling pathways,cytokine-cytokine receptor interaction,IL-17 signaling pathway,TNF signaling pathway,NOD-like receptor signaling pathway and chemokine signaling pathway.In addition,GRO1,CXCL3,CXCL6,CXCL8,C3,C40,CSF1,TNFAIP2,IKBKE and IL6 genes were closely interconnected with inflammation.Collectively,the abundant DEGs were expressed differently in functions as LTA may be responsible for inflammatory responses induction in bMECs,while metformin supplementation may reverse inflammation.However,the mechanism of the molecular regulation of LTA-induced inflammation in bMECs is reversed by metformin treatment needs further investigation.2.To test whether Metformin Inhibits Lipoteichoic acid-induced Oxidative Stress and Inflammation through AMPK/NRF2/NF-κB Signaling Pathway in Bovine Mammary Epithelial Cells or not,four experimental groups were designed.The pbMECs were exposed either to 3 mM metformin for 12 h as a metformin group(MET)or exposed 100 μg/mL LTA for 6 h as LTA group(LTA).Cells pretreated with 3 mM metformin for 12 h followed by 100 μg/mL LTA exposure for 6 h were served as(MET+LTA)group.PBS was added to cells as a control group.Cell apoptosis in pbMECs pretreated with metformin doses was analyzed by a flow cytometry(Annexin V-FITC assay).The quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)was performed for the mRNA expression,while western blot and immunofluorescence analysis were used to evaluate the expression of target proteins in inflammatory and anti-inflammatory responses to metformin and LTA.The findings found that the gene expression of COX2 significantly increased in the cells challenged with LTA compared to control cells.Metformin attenuated LTA-induced expression of inflammatory genes NF-κB p65,TNFα,COX2,and IL-1β,as well as the nuclear localization and phosphorylation of NF-κBp65 protein;however,it increased the transcription of Nrf2 and Nrf2-targeted anti-oxidative genes HO-1 and Gpx1,also the nuclear localization of HO-1 protein.Metformin-induced activation of Nrf2 is AMPKdependent;as metformin pretreated pbMECs activated AMPK signaling via the upregulation of phosphorylated AMPK levels,cells pretreatment with metformin also reversed the translocation of Nrf2 that was LTA inhibited.Our results indicate that metformin exerts antiinflammation and oxidative stress through the regulation of AMPK/Nrf2/NF-κB signaling pathway,which highlights the role of AMPK as a potential therapeutic strategy for the treatment of bovine mastitis.3.Since studies reported that metformin could function as an anti-inflammation in both AMPK-dependent and-independent ways;we try to underscore the regulation of PPAR y in the modulation of inflammation by metformin.Therefore,the study of metformin alleviates LTA-induced inflammatory response through PPARy/MAPK/NF-κB signaling pathway in bovine mammary epithelial cells.We used pbMECs with the same treatment groups as in the chapter.3.The proliferation of pbMECs was assessed using the EdU assay.The qRT-PCR was used to measure mRNA expression,while 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.The results showed that LTA concentration at 100μg/mL significantly stimulated the MAPK14,IL-6 and IL-1β mRNA expressions compared to the control cells(P<0.05)in dose-dependent tests for LTA.Metformin at a dosage of 3 mM inhibited the impact of LTA-mediated cell proliferation.Pre-treatment with metformin enhanced the intensity of LTA-inhibited cells staining in the EdU test.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 PPARy phosphorylation by up-regulating the expression of PPARγin LTA-stimulated pbMECs.Treatment with GW9662 increased IL-6 expression,which was reversed by metformin.These findings collectively indicated that metformin could attenuate LTA-stimulated inflammatory response in pbMECs by suppressing MAPK and NF-κB activation via a partially dependent mechanism on PPARy activation.These results suggested an anti-inflammatory function of metformin drug that could treat bovine mastitis.4.Finally,to test if Metformin attenuates Oxidative stress and inflammation in S.aureus infected mammary gland of Mouse.A total of 24 ICR mice were randomized into four groups.Six mice were first injected with metformin and then challenged with S.aureus after 24 h,serving as the(SA+Met)group.Six mice not treated were the blank control.Six mice were only infected with S.aureus(SA-infected group).Six mice were inoculated with Phosphate buffer saline(PBS)as PBS group.For the challenge of S.aureus,50 μl of bacterial suspensions(1×108 CFU/ml)were injected into the mice in the SA+Met and SAinfection groups through intramammary infusion.All mice were euthanized by cervical dislocation after treatments,and the mammary gland samples were aseptically removed for qRT-PCR and Western blot.The breast tissue samples were also used to make paraffin sections for pathological tests.In addition,population analysis of WBCs was tested in mice in vivo.The results showed histological examination of the mouse mammary gland from the S.aureus-induced mastitis model revealed a considerable change in alveolar hemorrhage and infiltration of inflammatory cells,while these inflammatory signs were significantly reduced by metformin treatment.In the mouse mastitis model,S.aureus stimulation promoted upregulation of IL-1β,TNF-α and IL-6 gene and protein expressions(P<0.05).However,metformin-treated mice significantly downregulated the mentioned inflammatory key elements(P<0.05).S.aureus-stimulated mastitis in mouse mammary glands downregulated phosphorylation of AMPKa compared with the control group(P<0.05).However,pretreatment with metformin reversed the reaction.Pretreatment with metformin upregulated the transcription expression of Nrf2,which was downregulated by S.aureus inducement.These results indicated that metformin played an anti-inflammatory and anti-oxidant role by preventing the mastitis model induced by S.aureus in mouse mammary glands.These findings allow for the development of therapeutic approaches for the treatment of mouse mammary mastitis using metformin.The above four chapters concluded that,Firstly,the transcriptome analysis indicated abundant DEGs were expressed differently in functions as LTA may be responsible for inflammatory responses induction in bMECs,while metformin supplementation may reverse inflammation.However,the mechanism by which the molecular regulation of LTA-induced inflammation in bMECs is reversed by metformin treatment needs further investigation.Secondly,metformin exerts anti-inflammation and oxidative stress through the regulation of AMPK/Nrf2/NF-κB signaling pathway,which highlights the role of AMPK as a potential therapeutic strategy for the treatment of bovine mastitis.Thirdly,metformin could attenuate LTA-stimulated inflammatory response in pbMECs by suppressing MAPK and NF-κB activation via a mechanism that is partially dependent on PPARy activation.Finally,metformin played an anti-inflammatory and anti-oxidant role by preventing the mastitis model induced by S.aureus in mouse mammary glands.Collectively,these findings provide evidence for the development of novel therapeutic approaches for treating bovine mastitis using metformin.