Mechanism Of Antimicrobial Peptide Analogue JH-3 Against Salmonella

With the extensive use of traditional antibiotics,many clinically resistant strains and super bacteria gradually increased highlight the need for alternatives to antibiotics.Antimicrobial peptides(AMPs),a family of defensive small peptide produced by organisms against the invasion of foreign microorganisms,are important part of the body’s innate immune system.Compared with traditional antibiotics,AMPs have the advantages of small molecular weight,good water solubility,good thermal stability,unique antibacterial mechanism,broad-spectrum antibacterial activity against clinically resistant bacteria,and not easy to induce bacterial resistance.In this study,we used the Salmonella CVCC541 as a model strain and antibacterial peptide P3 analogue JH-3 isolated from the bovine hemoglobin alpha subunit as the research object to validate the antibacterial effects of JH-3 against Salmonella CVCC541 in vitro and in vivo through the following researches:The minimum inhibitory concentration(MIC)of antimicrobial peptide JH-3 against Salmonella CVCC541 was 100 μg/m L through double agar gel immune diffusion test and gradient dilution.The antimicrobial kinetics study revealed that the antimicrobial peptide JH-3 had good bactericidal activity within 5 h,which shows dose-dependent effect.During treatment with the antimicrobial peptide JH-3,increased bacterial permeability.The morphology of the bacteria was observed by scanning electron microscopy and transmission electron microscopy.It was found that bacterial cells was shrinking and membrane integrity was destroyed,the bacteria cell membrane permeability were increased,the leakage of cell contents happen to bacteria.These results revealed that the antimicrobial peptide JH-3 has high bactericidal activity in vitro.The establishment of BALB/c mouse model infected by Salmonella CVCC541.In this study,20 g of BALB/c female mice were selected and randomly divided into five groups containing 10 animals each,which was injected intraperitoneally with Salmonella CVCC541 in 1ⅹ107 CFU/mouse,1ⅹ106 CFU/mouse,1ⅹ105 CFU/mouse,1ⅹ104 CFU/mouse and PBS control.The results suggested the severity of clinical symptoms in mice was positively correlated with the dose and duration of infection.In 1ⅹ107 CFU/mouse Salmonella CVCC541 infected group,all mice died within 4 days after infection.In 1ⅹ106 CFU/mouse Salmonella CVCC541 infected group,all mice died within 1 week after infection.The mortality rate of mice was 20% for 1ⅹ105CFU/mouse and 1ⅹ104 CFU/mouse showed no death.mice infected by Salmonella CVCC541 behaved loose coat and hunched back,sluggish,anorexic,and tendency to gather together.The necropsy results showed the severity of pathological changes were also positively correlated with the dose of infection.The major lesion occurred in the intestine.The mice had attenuated bowel wall,severe hemorrhage in the intestinal tract,especially in jejunum and ileum.Meanwhile,we found the amount of neutrophils increased,and the amounts of macrophages and T lymphocytes decreased significantly in the spleen of mice infected with Salmonella CVCC541.Based on the above results,1ⅹ106 CFU/mouse was selected as the optimal infection dose established in the mouse model,which laid the foundation for the subsequent therapeutic trial of the antimicrobial peptide JH-3.20 g of BALB/c female mice were selected for exploring the therapeutic effect of antibacterial peptide JH-3 on Salmonella CVCC541 infected mice.A total of 80 mices were randomly assigned to 8 groups containing 10 animals each: PBS-negative control group,CVCC541 infection group,CVCC541+Ciprofloxacin(40 mg/kg,2 h)treatment group,CVCC541+JH-3(40 mg/kg,2 h)treatment group,CVCC541+JH-3(10 mg/kg,2 h)treatment group,CVCC541+ Ciprofloxacin(40 mg/kg,P 3 d)treatment group,CVCC541+JH-3(10 mg/kg,P 3 d)treatment group,CVCC541+JH-3(40 mg/kg,P 3 d)treatment group.JH-3(40 mg/kg or 10 mg/kg)were used to treat CVCC541-infected mice intraperitoneally at 2 h or 3 days postinfection.Our results showed that treatment with 40 mg/kg JH-3 at 2 h postinfection had a better therapeutic effect than those at 3 days and could significantly protect mice from a lethal dose of Salmonella CVCC541.In addition,the clinical symptoms,bacterial burden in blood and organs,intestinal pathological changes were decreased,all these parameters were close to normal status at 2 h postinfection(groups).This study confirmed the therapeutic effect of the antimicrobial peptide JH-3 against Salmonella CVCC541 infection for the first time and determined the therapeutic effect of different doses and different treatment times of JH-3,laying the foundation for the research of new antimicrobial agents.In order to explore the mechanism of antimicrobial peptide JH-3 against Salmonella CVCC541 infection,RAW264.7 was infected with Salmonella CVCC541 at MOI 10 to establish infection model.The effect of the final concentration 100 μg/m L of antimicrobial peptide JH-3 on the cells was observed by transmission electron microscopy.It was found that the antibacterial peptide JH-3 showed uniform cytoplasm and complete cell morphology.We further explore the anti-infection mechanism of antimicrobial peptide JH-3,the molecular mechanisms of anti-inflammatory and anti-apoptosis of JH-3.The RAW264.7 cells was pretreated with antibacterial peptide JH-3 for 1 h,and then infected with Salmonella CVCC541 with MOI 10.After 12 hours,the cell supernatant and total cellular protein were collected.The inflammatory cytokines were detected by ELISA and the cellular RNA and total protein were extracted for fluorescence quantitative RT-PCR and WB analysis.The results showed that the antibacterial peptide JH-3 could reduce the production of IL-2,IL-6 and TNF-α induced by Salmonella CVCC541 infection by inhibiting the p38 MAPK signaling pathway,indicating that the antibacterial peptide JH-3 has good anti-inflammatory effect.Confocal laser scanning and flow cytometry tests further confirmed that the antimicrobial peptide JH-3 can also effectively inhibit the activation of caspase-3 and caspase-8,thereby reducing Salmonella CVCC541 induced macrophage apoptosis.