The Therapeutic Effects Of Tea Tree Oil Nanemulsion Combined With Caffeic Acid Against Escherichia Coli-Induced Mastitis In Rats

Bovine mastitis is one of the important diseases that affect the dairy industry,causing significant economic losses.With the widespread use of antibiotics,bacterial resistance and antibiotic residue problems have become increasingly serious.Therefore,there is an urgent need for new green antimicrobial agents.E coli is one of the main pathogens that cause bovine mastitis,often inducing acute inflammatory responses in the mammary gland of cows.The research team has prepared tea tree oil nanomicelles(TNM)with good antibacterial activity and demonstrated that caffeic acid(CA)can effectively inhibit LPS-induced inflammatory and oxidative stress responses in mammary epithelial cells,as well as the M1 polarization of macrophages.Therefore,this study explores the in vitro antibacterial activity of TNM combined with CA,the therapeutic effect on E.coli-induced rat mastitis,and its mechanism of action,in order to provide new ideas and references for the prevention and control of E.coli-type bovine mastitis..Experiment 1:Investigating the in vitro inhibitory activity of TNM and CA against E coli.The minimum inhibitory concentration(MIC)of TNM and CA against seven strains of E.coli isolated from bovine mastitis and the standard strain ATCC25922 were determined using the broth microdilution method.The minimum bactericidal concentration(MBC)of TNM and CA were determined using the agar diffusion method.The effect of TNM and CA on bacterial growth curves was analyzed by OD600 measurement using an enzyme-linked immunosorbent assay.The antibacterial activity of TNM combined with CA was evaluated using a checkerboard dilution method.The results showed that the MIC of TNM against HM 28-4 and GM 3-2 was 1.25 mg/mL,with an MBC of 2.5 mg/mL,while the MIC against the other strains was 0.625 mg/mL,with an MBC of 1.25 mg/mL.The MIC of CA against HM 28-4 and GM 3-2 was 2.5 mg/mL,with an MBC of 5 mg/mL,while the MIC against the other strains was 1.25 mg/mL,with an MBC of 2.5 mg/mL.The growth of the tested E.coli strains was inhibited at 1/2 MIC of TNM and CA,and completely inhibited at 1 ×MIC and 2xMIC of TNM and CA.The combined antibacterial effect of TNM and CA was synergistic against YD7281 and GM 10-1,and additive against ATCC25922 and the other five clinical isolates.Experiment 2:Investigating the therapeutic effect of different doses(1,2,4 mg/kg)of TNM on YD7281-induced mastitis in rats.Forty-eight female Wistar rats,5-7 days postpartum,were randomly divided into 6 groups:Control,YD7281,YD7281+TNM1,YD7281+TNM2,YD7281+TNM4,and TNM4.Except for the Control and TNM4 groups,all groups were intraualmarily inoculated with 100 μL of 1.0×107 CFU/IL bacterial suspension in the fourth pair of mammary glands,and treated with TNM(1,2,4 mg/kg)twice at 6 and 12 h after infection,respectively.The TNM4 group was intramammarily infused with 4 mg/kg TNM(200 μL).The Control and YD7281 groups were intramammarily infused with the same volume of sterile saline.After 24 h of infection,the rats were anesthetized and samples were collected to observe mammary gland lesions and analyze organ indices.Blood routine indexes were detected,and HE staining was used to observe pathological changes in mammary gland tissues.Immunohistochemical staining was used to detect the expression of MPO in mammary gland tissues.Chemiluminescence was used to detect the activity of MPO,SOD,GSH-Px,TNOS,iNOS,and MDA,NO in serum and mammary gland tissues.ELISA was used to detect the contents of IL-1β,TNF-α,IL-10,NAGase,LPS in serum and mammary gland tissues.The results showed that TNM at medium and high doses significantly reduced inflammatory damage in mammary gland tissues.TNM at medium and high doses significantly reduced the bacterial load in mammary gland tissues and decreased the levels of,NAGase IL-1β,TNF-α,LPS,NO,and MDA in serum and mammary gland tissues,as well as the activities of MPO,iNOS,and TNOS.TNM at medium and high doses significantly increased the activities of SOD and GSH-Px,as well as the content of IL-10.A dose of 2 mg/kg was selected for further studies of TNM.Experiment 3:Evaluate the therapeutic effect of TNM(2 mg/kg)combined with CA(5 mg/kg)on YD7281-induced rat mastitis model,and compare it with the positive drug gentamicin sulfate(GS,sensitive antibiotic).Forty-eight female Wistar rats,5-7 days after delivery,were randomly divided into six groups:Control group,YD7281 group,YD7281+CA5 group,YD7281+TNM2 group,YD7281+CA5+TNM2 group,and YD7281+GS group.Except for the Control group,all groups were instilled with 100 μL of 1.0×107 CFU/mL bacterial solution in the 4th pair of mammary glands,and were treated with drugs twice at 6 and 12 hours after bacterial challenge.The YD7281+CA5 group was instilled with 5 mg/kg CA,the YD7281+TNM2 group was instilled with 2 mg/kg TNM,the YD7281+CA5+TNM2 group was instilled with 5 mg/kg CA and 2 mg/kg TNM,and the YD7281+GS group was instilled with 5mg/kg GS(200 μL).The Control group and YD7281 group were instilled with sterile saline of equal volume.After 24 hours of bacterial challenge,the rats were anesthetized and samples were collected.Changes in inflammatory cytokines and oxidative stress markers in serum and mammary tissue were detected;flow cytometry was used to detect lymphocyte subgroups in the inguinal lymph nodes;Western blot was used to detect key proteins of TLR4/NF-κB/NLRP3 and Nrf2 signaling pathways in rat mammary tissue.The results showed that TNM combined with CA significantly reduced the bacterial load in mammary tissue;significantly reduced NAGase,LPS,NO,CCL2,CXCL2,MIF,IL-1β,TNF-α,and MDA levels in serum and mammary tissue,as well as MPO,iNOS,and TNOS activities;significantly increased SOD,GSH-Px activity,and IL-10 content;significantly reduced TLR4,Myd88,NLRP3,ASC,and Cleaved-Caspasel protein expression in mammary tissue,inhibited phosphorylation of IκBα and p65;significantly reduced Keap-1 protein expression,increased Nrf2 protein expression;and significantly increased the Bcl-2/Bax ratio.Experiment 4:Exploring the protective mechanism of TNM and CA combined treatment on YD7281-induced inflammatory injury in mouse mammary epithelial cells(EpH4-Ev).The LDH release assay was used to establish the EpH4-Ev inflammatory injury model stimulated by YD7281 at MOI=5 for 4 hours.Different concentrations of TNM and CA were tested based on this model to select 50 μg/mL CA and 25 μg/mL TNM as the drug concentrations for subsequent studies.EpH4-Ev cells were divided into Control group,YD7281 group,YD7281+CA50 group,YD7281+TNM25 group,YD7281+CA50+TNM25 group,and CA50+TNM25 group.Except for the Control and YD7281 groups,all other groups were treated with drugs 12 hours in advance.The YD7281+CA50 group was treated with 50 μg/mL CA,the YD7281+TNM25 group was treated with 25 μg/mL TNM,and the YD7281+CA50+TNM25 group and CA50+TNM25 group were treated with 50μg/mL CA and 25 μg/mL TNM.After drug treatment,except for the Control and CA50+TNM25 groups,all other groups were stimulated with YD7281 at MOI=5 for 4 hours.Cell supernatant and cells were collected after treatment to detect changes in LDH release,inflammatory cytokines,and oxidative stress markers in the supernatant,as well as key protein expression in the TLR4/NF-κB/NLRP3 and Nrf2 signaling pathways in cells by Western blot analysis.Cell apoptosis was measured by flow cytometry.The results showed that TNM combined with CA significantly reduced LDH release,NAGase,NO and MDA content,iNOS,CCL2,CXCL2,MIF,IL-1β,and TNF-αexpression,while increasing SOD,GSH-Px activity,and IL-10 expression.Additionally,TNM combined with CA significantly reduced TLR4,Myd88,NLRP3,ASC,and CleavedCaspase1 protein expression in cells,inhibited the phosphorylation of IκBα and p65,and decreased Keap-1 protein expression while increasing Nrf2 protein expression.Furthermore,TNM combined with CA significantly reduced the cell apoptosis rate and increased the Bcl2/Bax ratio.In conclusion,TNM combined with CA can synergistically inhibit YD7281 proliferation and LPS release,reduce TLR4/NF-κB/NLRP3 signaling pathway activity,enhance Nrf2 signaling pathway activity,and weaken inflammation and oxidative stress reactions,thus alleviating mammary tissue damage.