Effects Of Antimicrobial Peptide Sublancin On Necrotic Enteritis Of Broilers And The Innate Immunity Of Mice

Four experiments were conducted to investigate the effects of antimicrobial peptide sublancin on necrotic enteritis of broilers and innate immunity of mice.(1)In Exp.1,on the basis of foundation that sublancin exerts antibacterial activities against Clostridium perfringens CVCC2027 in vitro,two hundred and fifty-two chickens at 1 d of age were randomly assigned to one of six treatments including an uninfected control,an infected control,three C.perfringens infected groups supplemented with purified sublancin at 2.88,5.76,or 11.52 mg/L of water;and an infected group supplemented with lincomycin at 75 mg/L of water(positive control).The experiment lasted 28 d.Necrotic enteritis was induced in the broilers by oral inoculation of C.perfringens on d 15 through 21.Thereafter,the sublancin or lincomycin were administered fresh daily for a period of 7 days.The results showed that sublancin ameliorated intestinal injury induced by C.perfringens.It was observed that sublancin decreased proinflammatory cytokines IL-1?,IL-6 and TNF-? in the ileum compared with the infected control(P<0.05).The preventive effects of sublancin and lincomycin on C.perfringens CVCC2027 infected broilers were equivalent.(2)In Exp.2,we studied the immunostimulating effects of sublancin on RAW264.7 macrophages and its molecular mechanisms.The effects of sublancin on mouse peritoneal macrophages were also studied to verify the results obtained from RAW264.7 macrophages.Sublancin significantly facilitated production of IL-1?,IL-6,TNF-?,and NO by RAW cells and mouse peritoneal macrophages(P<0.05).RT-PCR analysis revealed that sublancin up-regulated genes expression including chemokines(IL-8 and MCP-1)and costimulatory molecules(B7-1 and B7-2)(P<0.05).Moreover,the functional tests showed that sublancin enhanced the phagocytic and bactericidal activity of macrophages against methicillin-resistant Staphylococcus aureus(MRSA)ATCC43300.The MAPK and NF-?B signaling pathways were involved in the activation of RAW264.7 macrophages by sublancin.(3)In Exp.3,we studied the effects of sublancin on innate immunity in normal physiological mice,cyclophosphamide(Cy)induced immunosuppressive mice model,and MRSA infected mice respectively.For normal physiological mice,oral administration of sublancin significantly enhanced the phagocytotic activity of mouse peritoneal macrophages.In the meanwhile,the genes expression of IL-?,IL-6 and TNF-? in mouse peritoneal macrophages were up-regulated in mice treated with sublancin.In Cy-induced immunosuppressive mice,sublancin was capable to induce an early recovery of red blood cells,hemoglobin,white blood cells and platelets in peripheral blood,and improve macrophage phagocytosis.In addition,treatment with sublancin(4.0 and 8.0 mg/kg BW)significantly increased the genes expression of IL-2,IL-4 and IL-6 in spleen of immunosuppressed mice.On the basis of foundation that sublancin exerts antibacterial activities against MRSA ATCC43300 in vitro,we constructed an experimental model of MRSA-induced sublethal infection.An intraperitoneal administration of 2.0 mg/kg BW of sublancin led to a reduction in the S.aureus titer in peritoneal fluids.In an early infection phase(24 h post-infection),sublancin significantly triggered the release of cytokines TNF-?,1L-6 and MCP-1 in mice infected with MRSA.However,the amounts of TNF-?,IL-6 and MCP-1 in peritoneal fluids were significantly decreased in sublancin treated mice 72 h post-infection,similar to levels seen in mice treated with vancomycin.(4)In Exp.4,transport of sublancin was studied in the Caco-2 transwell system,in order to discover if the antimicrobial peptide can pass the intestinal barrier.A flux through the cell monolayer could not be observed for sublancin(apparent permeability coefficient(Papp)<10×10-6 cm · s-1).To further evaluate the in vivo bio-distribution of sublancin,a near-infrared fluorescent scan was used.The major organs(heart,liver,spleen,lung and kidney)were isolated and the ex vivo images were studied.The results showed that no fluorescence intensity was found in the major organs after administration of Cy7-labeled sublancin.In addition,Cy7-labeled sublancin was continuously retained in the mouse intestine 12 h after the oral administration.In contrast,most of the free Cy7 had disappeared from the intestine 12 h after administration,indicating that the free Cy7 was eliminated faster from the intestine than the Cy7 labeled sublancin.In conclusion,the antimicrobial peptide sublancin presented here directly kills bacteria and further helps resolve infections through its innate immune modularoty properties.The development of antimicrobial peptide with combined antimicrobial and immunomodulatory properties represent a new approach to treat antibiotic-resistant infections.