Screening Of Traditional Chinese Medicines Against Major Pathogenic Bacteria Of Bovine Mastitis And The Mechanism Of Action Of Esculetin In The Treatment Of Mastitis In Mice

There are many problems such as bacterial resistance and drug residues in the treatment of bovine mastitis with antibiotics.The search for alternative drugs to antibiotics has become a new research direction.Chinese herbal medicine and its active ingredients have the advantages of low toxicity and less residue,and have been used in clinical practice to some extent.The purpose of this study was to screen out 15 traditional Chinese medicines with good antibacterial effect on 3 main pathogenic bacteria of bovine mastitis in some areas of eastern Henan,and to explore the therapeutic effect of their active ingredients on mouse streptococcal mastitis and its effects.The mechanism of action is expected to provide a reference for the further development and utilization of active ingredients of traditional Chinese medicine.Methods: 1.Collect 160 clinical mastitis milk samples from 3 dairy farms in eastern Henan,culture and isolate pathogenic bacteria through specific medium,and carry out 16 S r DNA identification.The suspected Staphylococcus aureus,Streptococcus uberis,and Escherichia coli were further identified by Gram stain microscopy,specific primer amplification,and nucleic acid gel electrophoresis.After purification and culture,they were used for subsequent experiments.2.The water extracts of 15 Chinese herbal medicines were prepared by the decoction method.The diameter of the inhibition zone of the water extracts of Chinese medicines against the three main pathogenic bacteria was measured by the Oxford cup method,the minimum inhibitory concentration(MIC)was determined by the agar dilution method,and the broth dilution method was used.The minimum bactericidal concentration(MBC)was determined by the method.3.Select 4 active ingredients of Quercetin,Coumarin,Chlorogenic acid and Esculetin,and determine their minimum inhibitory concentrations against 3 main pathogenic bacteria; choose Esculetin and Coumarin with better bacteriostatic effect,the ear swelling-induced inflammation test was carried out in mice,and blank control group,model control group,Esculetin and Coumarin low(25 mg/kg),medium(50 mg/kg)and high(100 mg/kg)were set up respectively dose group and dexamethasone(10 mg/kg)treatment group,after applying xylene to the right ear of mice to induce inflammation,the ear swelling was detected and the inhibition rate was calculated.4.To construct a mouse model of mastitis,set up blank control group,inflammation model group,Esculetin low(40 mg/kg),medium(80 mg/kg),high(160 mg/kg)dose groups and dexamethasone(5 mg/kg)treatment group,mice in each treatment group were treated by intraperitoneal injection of corresponding drugs,and the blank control group was intraperitoneally injected with the same amount of normal saline.After the experiment,the mice were sacrificed,the mammary gland tissues were collected,slices were made,HE staining was used to observe the histopathological changes,and TNF-α,IL-1β,IL-6 were detected by fluorescence quantitative PCR(q PCR)and enzyme-linked immunosorbent assay(ELISA).Changes in expression levels,q PCR and Western blot analysis(Western-blot)detected the changes in the expression levels of IκBα,p65,p-p65 and p38,p-p38.Results: 1.A total of 347 strains of pathogenic bacteria were isolated and identified,including 143 strains of Staphylococcus aureus,83 strains of Streptococcus uberis,and 2 strains of Escherichia coli.2.Determination results of the diameter of the inhibition zone(mm)of traditional Chinese medicines:the traditional Chinese medicines with obvious inhibitory effect on Staphylococcus aureus are:Coptidis Rhizoma(22.01±0.85)> Violae philippica(21.66±0.72)> Houttuyniae Herba(20.72±0.38)> Dandelion root(16.89±0.29);Chinese medicines with obvious inhibitory effect on Streptococcus uberis are:Coptidis Rhizoma(25.39±0.66)> Violae philippica(23.07±0.42)> Dandelion root(20.95±0.50)> Trichosanyhes kirilowii Maxim(16.91±0.33)> Vaccariae Semen(15.80±0.70)> Andrographis Herba(15.32±0.40);And the traditional Chinese medicine with obvious inhibitory effect on Escherichia coli: Houttuyniae Herba(20.45±0.55)> Coptidis Rhizoma(20.08±0.20)> Portulacae Herba(17.22±0.55)> Radix(16.14±0.37)> Andrographis paniculata(15.89±0.37)> Vaccariae Semen(15.82±0.77)> Dandelion root(15.32±0.50);MIC(mg/m L)and MBC(mg/m L)Test results: Staphylococcus aureus is sensitive to Coptis chinensis and Violae,MIC is 62.5,MBC is ≤125;Streptococcus uberis is sensitive to Coptis and Violae,MIC is 31.25,MBC is ≤62.5;sensitive to Escherichia coli The traditional Chinese medicines used are Coptis chinensis,Radix japonica,purslane,Andrographis paniculata,and Dandelion root,with MIC of 62.5 and MBC of ≤125.3.Detection results of minimum inhibitory concentrations(mg/m L)of the four active components of Violae philippica: MIC against Staphylococcus aureus: Coumarin(3.2)< Esculetin(6.4)< Quercetin(12.8)< Chlorogenic acid(25.6);MIC against Streptococcus uberis: Esculetin(1.6)= Coumarin(1.6)< Quercetin(3.2)< Chlorogenic acid(6.4);MIC against Escherichia coli: Esculetin(3.2)< Coumarin(6.4)= Quercetin(6.4)< Chlorogenic acid(12.8).The results of the inflammation test caused by ear swelling in mice: the inhibition rates(%)of the low,medium and high dose groups of Esculetin were 37.61±9.40,63.25±11.11,70.11±16.24,respectively;the inhibition rates of the Coumarin low,medium and high dose groups were(%)22.22±7.95,29.91±7.69,33.33±13.67,respectively.4.A mouse model of Streptococcus uberis mastitis was successfully constructed.In the inflammatory model group,it was observed that the acinar wall of the breast tissue was significantly thickened and swollen,the tissue structure was destroyed,accompanied by a large number of inflammatory cell infiltration,and interstitial hemorrhage;while in the treatment group,with the increase of the dose,the mammary gland tissue of the mice increased.The structure gradually returned to normal,and the inflammatory infiltration was significantly reduced.Compared with the blank control group,the expression levels of pro-inflammatory factors TNF-α,IL-1β,IL-6 and the expression levels of pathway-related factors p65,p-p65,p38 and p-p38 in the inflammation model group were significantly increased(P<0.05).Compared with the inflammation model group,the expression levels of TNF-α,IL-1β,IL-6 and the pathway-related factors p65,p-p65,p38,The expression level of p-p38 decreased gradually with the increase of dose(P<0.05),while the expression level of IκBα increased significantly(P<0.05).Conclusion: Coptidis rhizoma,Violae philippica and Dandelion root have obvious bacteriostatic effects on the three main pathogenic bacteria of bovine mastitis in eastern Henan;40-160 mg/kg Esculetin have a therapeutic effect on mice breast inflammation induced by Streptococcus uberis in a significant dose-dependent manner.It can inhibit the inflammatory response by regulating NF-κB and MAPK signaling pathways.