The Intestinal Immune Regulation And Barrier Protection Function Of Animal Derived Peptides And The Preliminary Mechanism

It is well known that antibiotics had been used in the livestock feeding process for a long time and high dose, which would cause problems such as super drug-resistant bacteria, food safety concerns and environmental pollution, so seeking for the substitutes for antibiotics is very important. Recent studies have shown that the function of antimicrobial peptides were not only confined to the antibacterial, but the more important is to exert immune regulation and barrier protection function. Therefore, for the purpose of developing new antibiotics substitutes, and we do researches aimed at alleviating intestinal inflammation and improving the physical barrier function of intestinal tract. We set up the DSS induced ulcer colitis like mice model and explore the intestinal immune regulation functions and epithelium barrier protecton effects of C-BF and pBD2 on model mice, and we further studied the underlied mechanism of C-BF and pBD2 exerting immune regulation and intestinal barrier protection function in vitro experiments, the main results were as below:1. The effects of antimicrobial peptides on DSS induced intestinal inflammationThis experiment was conducted through the establishment of dextran sodium sulfate (DSS) induced ulcerative colitis model in mice, by apparent symptom, the inflammation of whole body and colon inflammation levels to study the influence of antimicrobial peptide C-BF and pBD2 on DSS induced mice intestinal inflammation:(1) The set up of DSS induced colitis model and the effects of antimicrobial peptides on the apparent symptoms:we determine the best concentration of DSS for the inducing of ulcerative colitis in mice was 3%(m/v) DSS, from disease activity index score we found the above two kinds of antibacterial peptides injection can obviously relieve DSS caused rise of disease activity index score, the results provided the basis for subsequent studies at the molecular level;(2) The effects of antimicrobial peptides on the inflammation status of the whole body: as aspect to the rate of main immune cells in whole blood, the results showed that DSS significantly reduced the rate of macrophage, B lymphocyte and T lymphocyte (p< 0.05), after the treatment of antimicrobial peptide C-BF and pBD2, compared with DSS group, the ratio of main immune cells could be improved in different degree, it suggested that antimicrobial peptides could recover the innate and adaptive immune function to certain degree; as aspect to the secretion of inflammatory cytokines in serum, compared with the control group, DSS treatment group significantly elevated the concentration of IL-1β,TNF-α, IL-6, IL-8 and IL-10 in serum (p< 0.05), after the treatment of antimicrobial peptides C-BF and pBD2, compared with DSS group, the concentration of IL-1β, TNF-α, IL-6, IL-8 and IL-10 in serum were significantly decreased (p< 0.05), the results suggested that antimicrobial peptides could alleviate the inflammation status of the whole body through reducing the secretion of inflammation cytokines.(3) The effects of antimicrobial peptides on the inflammation status of intestinal tissues: as aspect to the infiltration degree of inflammatory cells in colon, through immunohistochemical method, we found that compared with normal control group, the infiltration degree in DSS trea tment group of neutrophils and macrophages increased significantly (p<0.05), and the treatment of antimicrobial peptides C-BF and pBD2 significantly reduced the DSS caused inflammatory cells infiltration in colon (p< 0.05); as aspect to the expression abundance of mCRAMP in colon:the mCRAMP expression levels in colon were tested, we found DSS treatment group significantly elevated the mCRAMP expression abundance in colon, and the treatment of antimicrobial peptide C-BF and pBD2 mCRAMP expression abundance is significantly lower compared with DSS group; as aspect to the inflammatory related enzyme activity in colon:The concentration of inflammatory related enzymes in colon tissues, INOS and cox-2, were also determined, the results showed that compared with normal group, DSS treatment group secreted significantly higher level of INOS and cox-2 in colon tissues (p<0.05), and the peptides C-BF and pBD2 treated group significantly reduced the DSS caused the increase of the concentration of colonic inflammatory related enzyme (p<0.05).2. The preliminary mechanism of antimicrobial peptides alleivating inflammatory statusFrom the above results, we found antimicrobial peptides C-BF and pBD2 could alleviate DSS induced apparent symptoms and intestinal inflammation in mice, on this basis, we further explored the preliminary mechanism of antimicrobial peptides alleivating inflammatory status:(1) The study of signaling pathway which antimicrobial peptides alleivating DSS induced inflammation:results showed that DSS could significantly cause the increase of phosphorylation level of inflammatory factors such as NF-κB (p65) and c-jun (p<0.05), and antimicrobial peptideS C-BF and pBD2 treatment groups can significantly reduce the NF-κB (p65) phosphorylation levels (p<0.05), but had no obvious effect on c-jun phosphorylation levels; as aspect to the expression level of inflammatory cytokines in RAW264.7 cells:With in vitro inflammatory cell model, we found that antimicrobial peptides C-BF and pBD2 could significantly reduce the LPS induced increase of the concentration of TNF-α in RAW264.7 cells, and improved the abnormally elevated concentration of PGE2 and NO (p<0.05), and compared with LPS group, antimicrobial peptides treatment groups could significantly reduce gene expression levels of inflammatory cytokines such as TNF-α, IL-1β, IL-6 and IFN-y (p< 0.05), as aspect to the signaling pathway alleviating inflammation in RAW264.7 cells:the NF-κB related signaling pathways were detected, we found that with the treatment of antimicrobial peptides C-BF and pBD2, the phosphorylation level of inflammatory signal factors such as AKT, IkB-α, NF-κB signal factors were decresed compared with LPS treated group;(2) The effects of antimicrobial peptides on phagocytosis function of 3D/42 cells and the possible mechanism:as aspect to the effects of antimicrobial peptides to the phagocytosis function of 3D/42 cells, through the determination of the effects of different dose (12.5,25,50 μg/ml) of antimicrobial peptides on the phagocytosis function of 3D/42 cells by FACS, we found antimicrobial peptides C-BF and pBD2 could enhance the phagocytosis of 3D/42 cells, and antimicrobial peptide C-BF showed a dose dependent manner, as aspect to the underlied mechanism of antimicrobial peptides on the phagocytosis function of 3D/42 cells:the effects of TLR2/4 were interfered to see whether antimicrobial peptides C-BF and pBD2 could promote phagocytosis of macrophage through TLR2 and TLR4 signaling pathways, we found that after the interference of TLR2/4, the enhancing effect of antimicrobial peptides on the phagocytosis function of 3D/42 cells was weakened;(3) The synergistic effects of antimicrobial peptides on inducing the polarization of macrophages:through the study of synergistic effects of antimicrobial peptides on inducing the polarization of macrophages, we found that peptides C-BF and pBD2 have synergistic effects to promote the differentiation of macrophages to M2 type, and the effect of C-BF is superior to the effect of pBD2.3. The intestinal barrier protection function of antimicrobial peptidesIn this study, we study the intestinal barrier protection function of antimicrobial peptides through the aspects of intestinal morphology, electrophysiological changes in the intestine, colon epithelial apoptosis status and tight junction protein expression:(1) The effects of antimicrobial peptides on the morphology of intestine in DSS induced mice:as aspect to the morphology of colon, compared with control group, the colon epithelial of DSS group suffered serious damage, and the structure of intestinal villus showed desultorily, there were no clear boundary and it was difficult to distinguish between the intestinal epithelium mucosa layer structure, the submucosa also showed serious edema at the same time, while the antimicrobial peptides C-BF and pBD2 obviously improved intestinal villus structure of mice compared with DSS group, the boundary between intestinal villus were more clearer, with relatively complete mucous layer structure and reduced submucosa edema;(2) The effects of antimicrobial peptides on the electrophysiological changes in colon:as aspect to the permeability changes in colon, the results showed that compared with control group, the FITC-dextran concentration in serum in DSS induced mice increased significantly (p < 0.05), while after treatment of C-BF, the concentration of FITC-dextran in serum is significantly lower compared with DSS group (p< 0.05), and pBD2 treated group had a decreased trend; as aspect to the transmembrane epithelial resistance value:the DSS treated mice showed significantly lower value of 40Ω while other treatment groups were kept stable in the range of over 40Ω(3) The effects of antimicrobial peptides on the colon epithelial apoptosis status and tight junction protein expression:as aspect to the apoptosis status in colon epithelial, compared with control group, the apoptosis index of DSS treated group increased significantly (p< 0.05), while the C-BF and pBD2 treated groups significantly decreased the cell apoptosis index in colonic epithelium (p< 0.05). as aspect to the expression level of tight junction proteins and mucins, The expression abundance of ZO-1 in colon of DSS treated group decreased significantly compared with control group (p< 0.05), while the C-BF and pBD2 treated groups significantly increased the expression abundance of ZO-1 than that of DSS treated alone. Compared with control group, the gene relative expression level of tight junction related proteins such as ZO-1, ZO-2, Claudin-1, Occludin and mucins related proteins such as Mucinl and Mucin2 in colon decreased significantly in DSS treated group (p< 0.05), while C-BF and pBD2 treated group increased the gene level of tight junction proteins significantly (p< 0.05).4. The preliminary mechanism of antimicrobial peptides exerting intestinal barrier protection functionFrom the results we found which antimicrobial peptides C-BF and pBD2 had protection effects to ntestinal barrier function, we further did studies on the preliminary mechanism which antimicrobial peptides exerting barrier protection function through the DSS induced Caco-2 cell damaged model in vitro:(1) The effects of antimicrobial peptides on the trans epithelial resistance value of Caco-2 monolayer cells:as aspect to the trans epithelial resistance value of Caco-2 monolayer cells:we found that compared with control group, the trans epithelial resistance value in Caco-2 monolayer cells of DSS treated alone group was significantly lower, we also found that with antimicrobial peptides C-BF and pBD2 pretreated groups, the trans epithelial resistance value had no significant difference compared with control group;(2) The effects of antimicrobial peptides on the tight junction structure and tight junction expression level:as aspect to the tight junction structure between Caco-2 cells, The tight junction structure between Caco-2 cells were observed by transmission electron microscope, we found that under the stimulation of DSS, the tight junction structure between Caco-2 monolayer cells were largely destroyed, with opened tight junction structures and the boundary was not clear, while antimicrobial peptides C-BF and pBD2 pretreated groups obviously improved the tight junction structure and the boundary was clear; as aspect to the expression level of tight junction proteins:compared with control group, the DSS treated alone group could significantly lowered the gene and protein expression level of tight junction proteins such as ZO-1, Claudin-1 and Occludin (p< 0.05), while pretreated with antimicrobial peptides C-BF and pBD2 improved the trends to different extent;(3) The effect of antimicrobial peptides on the morphology of single Caco-2 cell:as aspect to the morphology of single Caco-2 cell, we observed the morphological changes of intestinal cell microvilli by scanning electron microscopy (sem), from the results, we could conclude that the morphology of intestinal cell microvilli in DSS treated group were largely damaged by DSS, while C-BF and pBD2 pretreated groups significantly improved the surface of microvilli.In conclusion, the study discovered that antimicrobial peptides C-BF and pBD2 had the function of attenuating inflammation and improving physical barrier function in intestinal tract through DSS induced ulcerative colitis model, we further revealed the internal mechanism of how they exerted thess functions, the results suggested that this two antibacterial peptides had prospects to be an alternative of antibiotics in treating enteritis of human and animals.