The Effects And Mechanisms Of Antimicrobial Peptide Cecropin A On Alleviating The Inflammatory Bowel Disease

In the modern pig industry,piglet diarrhea is one of the focus problems for researchers.The main causes of piglet diarrhea are pathogenic microbial infection,inmature gastrointestinal digestion system,leaky intestinal barrier,imperfect immune function,weaning stress and so on.Antimicrobial peptides(AMPs)are a class of polypeptide molecules with antimicrobial,fungal and antiviral activities.They are considered as potential antibiotic substitutes because of their broad-spectrum antimicrobial activity and unlikely to have drug resistance for bacterias.Studies have shown that some antimicrobial peptides not only have antimicrobial activity,but also have the functions of regulating immunity,repairing injury,enhancing the cell barrier function of epithelial tissues such as skin and intestine.By establishing DSS-induced enteritis mice model and E.coli infection cell model,this study explored the antimicrobial activity,metabolic time,optimal therapeutic dose and related mechanisms of cecropin A.The main test results are as follows:Experiment 1: Seven candidate antimicrobial peptides were screened out according to the database of antimicrobial peptides.After artificial synthesis,their antimicrobial activities against enteritis-related pathogenic bacteria such as Escherichia coli,Salmonella,Pseudomonas aeruginosa and Staphylococcus aureus were tested.MTT assay was used to detect the effects of different concentrations of antimicrobial peptides on cell viability.Cecropin A has a good bacteriostatic activity against Gram-negative bacteria MIC and MBC of 3.125-6.25μg/m L,and has no negative effect on cell viability in the lower concentration range of 3.125-12.5μg/m L.The other antimicrobial peptides decreased cell viability in a dose-dependent manner.Experiment 2: In order to investigate the retention time and metabolism of cecropin A in vivo,free FITC and cecropin A labeled with FITC were injected into nude mice.The fluorescence intensity of mice was measured and compared by small animal imaging instrument at 15 min,1 h,2 h and 4 h.The results showed that FITC-cecropin A and FITC could be absorbed by abdominal cavity and distributed with blood in 15 minutes.The fluorescence intensity of cecropin A mice gradually decreased from whole body to abdomen within 1-2 hours,and disappeared at 2 hours,while that of FITC mice was still distributed throughout the whole body.These results indicate that cecropin A can act as a macromolecule in vivo before being excreted from the body.Experiment 3: In order to explore the appropriate dosage of cecropin A,36 mice were divided into control group,5,15,30 mg/kg dosage group.The control group was injected with saline intraperitoneally.The experimental group was injected with different dosages of cecropin A according to the body weight of mice for 5 days.The results showed that 5 and 15 mg/kg cecropin A had no negative effects on the main organs such as heart,liver,spleen and kidney of mice,but promoted the height of ileum villi and the depth of colonic crypt(P < 0.05),had no significant effects on serum lactate dehydrogenase(LDH),aspartate transaminase(AST)and alkaline phosphatase(AKP).Intraperitoneal injection of 30 mg/kg cecropin A could significantly increase liver index(P < 0.05).The results of H&E staining showed that the hepatic structure of mice in 30mg/kg group were changed,some hepatic cells appeared vacuole morphology,and the boundary was scattered and the structure was irregular.Serum indexes showed that the contents of AST and AKP tend to be increased(0.05 < P < 0.1).The results suggested that cecropin A could not negatively affect the organs of mice and promote intestinal development,while high dose cecropin A could negatively affect the liver of mice and could not promote intestinal development.Experiment 4: In order to explore the therapeutic effect of cecropin A on inflammatory bowel disease(IBD)and screen the appropriate dose,36 mice were divided into control group,5 mg/kg cecropin A group,15 mg/kg cecropin A group and DSS group based on experiment 3.Firstly,lethal IBD model was induced by adding 4% DSS(5 days)to the drinking water of mice.After induction,cecropin A was injected intraperitoneally at 5 mg/kg and 15 mg/kg in the antimicrobial peptide treatment group,while saline was injected into DSS group and control group as control group.Weight,diarrhea rate,bleeding rate and survival rate were measured daily.The mental state of mice was also observed.The results showed that there was no significant difference in survival rate and body weight between 5 mg/kg cecropin A group and DSS group(P > 0.05),while intraperitoneal injection of 15 mg/kg cecropin A could significantly improve the survival rate of mice(P < 0.01),and both of 5 and 15 mg/kg ccecropin A could significantly improve the length,weight,diarrhea,hemorrhage and disease index of colon(P < 0.05).H&E staining and intestinal morphology analysis showed that 15 mg/kg cecropin A could significantly improve the morphology and structure of intestinal epithelium.The results showed that 15mg/kg cecropin A is a better therapeutic dose for DSSinduced IBD.Experiment 5: In order to explore the similarities and differences between cecropin A and antibiotics,36 mice were divided into control group,DSS group,DSS + cecropin A group and DSS + gentamicin group.DSS group,DSS + cecropin A group and DSS + gentamicin group were treated with 2.5% DSS in drinking water for 5 days to induce moderate IBD model,and then changed to normal water after 5 days.Meanwhile,saline was injected intraperitoneally in DSS group,and 15 mg/kg cecropin A or 5 mg/kg gentamicin were injected intraperitoneally in DSS+Cecropin A and DSS+gentamicin groups respectively(for 3 days).During the period,mice were weighed every day;diarrhea and bleeding scores were counted.The results showed that cecropin A and gentamicin could significantly improve the weight loss,diarrhea index,hemorrhage and disease score in IBD mice(P < 0.05).In addition,cecropin A significantly improved colon length compared with gentamicin(P < 0.05).H&E staining and intestinal morphology analysis showed that the ileal and colonic epithelial structures of 2.5% DSS mice were severely damaged.Cecropin A and gentamicin could improve the intestinal epithelial morphology and increase the expression of tight junction protein.The activation of tumor necrosis factor-α(TNF-α),interlukin-1β(IL-1β),interlukin-6(IL-6)and MAPK(p38,c-jun)and NF-κB(p65)signaling pathways in colon tissues were detected by ELISA and western blotting.The results showed that both cecropin A and gentamicin could reduce the expression of pro-inflammatory factors(P < 0.05)and the phosphorylation of inflammatory-related signaling pathway proteins(P < 0.05).In addition,the intestinal epithelial structure of cecropin A recovered more completely than that of gentamicin group,and the expression of claudin-1 and occludin increased significantly(P < 0.05).Experiment 6: In order to explore the similarities and differences between cecropin A and gentamicin in regulating intestinal flora in mice,the cecum contents of control group,DSS group,DSS+cecropin A and DSS+gentamicin treatment group were collected,and the effects of IBD and different therapeutic drugs on intestinal flora were analyzed by 16 S r RNA gene sequencing combined with bioinformatics.The results showed that cecropin A and gentamicin significantly reduced the relative abundance of Bacteroideae and Enterobacteriaceae in cecum contents of mice with enteritis,inhibited the expression of inflammation(P < 0.05),and alleviated enteritis.In addition,different drugs have different regulatory effects on intestinal flora.Cecropin A significantly increased the relative abundance of Lactobacillus,while gentamicin significantly increased the relative abundance of Clostridium and Desulfovibrionaceae(P < 0.05).Experiment 7: In order to explore whether cecropin A can directly regulate the barrier function of intestinal epithelial cells,we selected IPEC-J2 as a cell model to study the potential mechanism of cecropin A in alleviating IBD.After 48 hours of treatment with 12.5 mg/m L cecropin A,E.coli(W25K)adhesion was significantly reduced.The results of qPCR showed that the expression of TNF-a,IL-6 and IL-8 decreased significantly(P < 0.05).Cells were seeded on transwell plate and treated with cecropin A for 24,48 and 72 hours respectively.The results showed that the concentration of cecropin A at 12.5 mg/m L for 48 and 72 hours could significantly increase the single layer cell resistance(TER)and decrease the permeability of FITC-dextran.Western blotting results showed that cecropin A could upregulate the expression of tight junction protein ZO-1,occludin and claudin-1.Cellular immunofluorescence showed that the tight junction proteins and cytoskeleton F-actin aggregated into the cell membrane in cecropin A treatment group.At the same time,the phosphorylation of MEK and ERK(1/2)was inhibited,and the expression of CDX2 was increased downstream.In order to prove that inhibiting MEK/ERK signaling pathway can regulate intestinal epithelial cell barrier function,DMSO+cecropin A and PD184352+cecropin A groups were set up and treated for 24 and 48 hours respectively.The results showed that after completely inhibiting ERK phosphorylation,the TER value of monolayer cells was significantly increased(P<0.05)and the expression of tight junction protein was significantly increased compared with DMSO and cecropin A groups.High(P < 0.05)and tight junction protein and F-actin aggregated into cell membrane,indicating that inhibition of ERK can regulate the expression and distribution of tight junction proteincytoskeleton,thereby regulating the function of intestinal barrier.In conclusion,cecropin A has good bactericidal effect.The survival time in mice is not more than 1 hour,but long-term high-dose continuous intraperitoneal injection may damage the liver.A moderate amount of cecropin A can effectively alleviate the intestinal inflammation induced by DSS.In addition to bactericidal effect,cecropin A can enhance intestinal epithelial cell barrier function by inhibiting MER/ERK signaling pathway,thereby reducing the adhesion and invasion of pathogenic microorganisms.