Abstract：O157: H7, an enterohemorrhagic Escherichia coli (EHEC) strain, is a foodborne pathogen that can cause disease in livestock, poultry and humans (Homo sapiens). Magainins is a cationic peptide with antibacterial and antitumor properties, and its antibacterial activity against Gram-negative and Gram-positive bacteria has been reported in previous studies. The purpose of this study was to explore the effect of frog (Xenopus laevis) derived Magainin Ⅱ on intestinal inflammation and microflora in mice (Mus musculus) induced by EHEC O157: H7. Forty C57/BL6 male mice with similar weigh were randomly divided into 4 groups with 10 replicates in each group, CK group, O157 group, Mag Ⅱ group, O157+Mag Ⅱ group, respectively. All the groups received the same basal diet. The O157 group and O157+MagⅡgroup were orally administered 0.1 mL O157 bacterial solution with a concentration of 106 CFU/mL on 1, 5 and 9 d; CK group and MagⅡgroup were orally administered 0.1 mL PBS in the same time. After 1 h, MagⅡ and O157+MagⅡ group were intraperitoneally injected with 0.25 mL Magainin Ⅱ solution according to 5 mg/kg body weight, while CK and O157 group were intraperitoneally injected 0.25 mL sterilized saline. On the 10 d, mice were treatmented by euthanasia, and corresponding tissue samples were collected for testing. The results showed that Magainin Ⅱ treatment alone had no effect on the serum inflammatory cytokine concentration and the expression of jejunal inflammatory cytokines and barrier function related genes. EHEC O157: H7 treatment resulted in significant increase in the jejunum gene expression levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) and serum concentration of IL-1β, IL-6, TNF-α in mice (P<0.05). The serum IL-10 levels significantly decreased (P<0.05), as well as relative gene expression of jejunal tight junction proteins claudin-1, mucin-1 (MUC-1), and MUC-2 (P<0.05); Magainin Ⅱ treatment significantly improved EHEC O157: H7-induced relative gene expression of IL-1β, IL-6, TNF- α, claudin-1, MUC-1, MUC-2 in mouse jejunum and abnormal serum concentration of IL-1β, IL-6, IL-10 (P<0.05). EHEC O157: H7 infection could cause changes in intestinal microorganisms. The structure of intestinal microorganisms could be analyzed by 16S rDNA sequencing, and then the changes of intestinal microorganisms before and after Magainin Ⅱ intervention were analyzed. Sequencing results showed that at the genus level, compared with CK group, the relative abundance of Prevotella and Bacteroides in O157 group significantly increased (P<0.05). After MagaininⅡtreatment, the relative abundance of Bacteroides in O157+MagⅡ group decreased (P<0.05). The results showed that the abundance of Prevotella and Bacteroides related to the development of inflammation significantly increased after EHEC O157:H7 challenge, while the abundance of the unfavorable bacteria genus Bacteroides decreased significantly in the Magainin Ⅱ treatment group. This study showed that Magainin Ⅱ could maintain the intestinal barrier and alleviate the level of inflammation, thus playing a protective role in the intestinal injury induced by EHEC O157:H7 in mice, and the change of intestinal flora might be the related factor for Magainin Ⅱ to protect the intestinal injury. In conclusion, Magainin Ⅱ could be used as a potential drug for the treatment of intestinal diseases caused by EHEC O157:H7.
[1] 陈国伟, 邱春红, 田灵敏, 等. 2019. 食源性天然产物中多糖干预炎症性肠病的研究进展[J]. 食品科学, 40(13): 281-287. (Chen G W, Qiu C H, Tian L M, et al. 2019. Recent progress in food-derived natural polysaccharide intervention in inflammatory bowel disease[J]. Food Sci-ence, 40(13): 281-287. ) [2] 崔雅倩, 魏志涛, 曹月, 等. 2020. 丁酸钠对肾草酸钙结石大鼠肠道菌群及炎症因子的影响[J]. 四川大学学报(自然科学版), 57(04): 818-824. (Cui Y Q, Wei Z T, Cao Y, et al. 2020. Effects of sodium butyrate on intestinal flora and inflammatory factors in rats with renal calcium oxa-late stones[J]. Journal of Sichuan University (Natural Science Edition), 57(04): 818-824. ) [3] 关嘉琦, 李柏良, 焦雯姝, 等. 2020. 益生菌对促进肠道发育作用的研究进展[J]. 食品科学, 41(21): 278-285. (Guan J Q, Li B L, Jiao W S, et al. 2020. Recent advances in understanding the role of probiotics in promoting intesti-nal development[J]. Food Science, 41(21): 278-285. ) [4] 宦海琳, 白建勇, 周维仁, 等. 2015. 抗菌肽对仔猪血清生化指标、肠黏膜形态结构及空肠上皮紧密连接蛋白基因相对表达量的影响[J]. 动物营养学报, 27(12): 3797-3804. (Huan H L, Bai J Y, Zhou W R, et al. 2015. Ef-fects of antimicrobial peptides on serum biochemical in-dices, intestinal mucosal morphology and the relative expression level of tight junction protein gene of jeju-num of piglets[J]. Chinese Journal of Animal Nutrition, 27(12): 3797-3804. ) [5] 黄则华, 曾悦, 黄春兰. 2019. 肠道菌群对黏蛋白 2 的影响及其作用机制[J]. 胃肠病学, 24(02): 111-114. (Huang Z H, Zeng Y, Huang C H. 2019. Effect and mechanism of intestinal flora on mucin 2[J]. Chinese Journal of Gastro-enterology, 24(02): 111-114. ) [6] 李方方, 苏航, 张勇, 等. 2015. 饲粮添加复合抗菌肽与包被氧化锌对断奶仔猪生长性能及血清生化指标的影响[J]. 动物营养学报, 27(09): 2811-2819. (Li F F, Su H, Zhang Y, et al. 2015. Effects of dietary anti-microbial peptides and coated zinc oxide on growth performance and serum biochemical indices of weaned piglets[J]. Chinese Journal of Animal Nutrition, 27(09): 2811-2819. ) [7] 刘颖, 许文侠, 黄彩群, 等. 2020. 幽门螺杆菌感染对慢性浅表性胃炎患者胃内菌群的影响[J]. 浙江医学, 42(14): 1496-1499, 1503, 1571-1572. (Liu Y, Xu W X, Huang C Q, et al. 2020. Effects of Helicobacter pylori infection on gastric flora in patients with chronic superficial gastritis[J]. Zhejiang Medical Journal, 42(14): 1496-1499, 1503, 1571-1572. ) [8] 孟祥升, 辛崇兴, 邵晞. 2011. 肠出血性大肠杆菌 O157:H7 研究进展[J]. 中国动物检疫, 28(11): 69-71. (Meng X S, Xin C X, Shao X. 2011. Research progress of Enterohe-morrhagic Escherichia coli O157: H7[J]. China Animal Health Inspection, 28(11): 69-71. ) [9] 商佳琪, 郭宇帆, 张梦洁, 等. 2021. 抗生素处理对高脂饲料诱导肥胖 SD 大鼠肠道菌群与血清炎性因子的影响[J]. 营养学报, 43(05): 498-503. (Shang J Q, Guo Y F, Zhang M J, et al. 2021. Changes of intestinal flora and inflammatory factors after antibiotic treatment in high-fat diet fed SD rats[J]. Acta Nutrimenta Sinica, 43(05): 498-503. ) [10] 夏溪. 2015. 猪抗菌肽 PR39 抗细菌感染和保护肠道屏障功能的作用及其机制研究[D]. 博士学位论文, 浙江大学, 导师 : 汪以真, pp. 1-133. (Xia X. 2015. Effects and mechanism of antimicrobial peptide PR39 on bacterial infection and intestinal barrier function[D]. Thesis for Ph. D., Zhejiang University, Supervisor: Wang Y Z, pp. 1-133. ) [11] 杨靖源, 蒙俊, 杨堃. 2022. 肠紧密连接蛋白与肠道屏障功能[J]. 医学综述, 28(02): 235-239. (Yang J Y, Meng J, Yang K. 2022. Intestinal tight junction protein and intes-tinal barrier function[J]. Medical Recapitulate, 28(02): 235-239. ) [12] 张英春, 张兰威, 马微. 2003. 乳酸菌对免疫系统功能的调节作用[J]. 食品研究与开发, (05): 80-82. (Zhang Y L, Zhang L W, Ma W. 2003. Regulation of immune system function by lactic acid bacteria[J]. Food Research and Development, (05): 80-82. ) [13] Ananthakrishnan A N, Luo C, Yajnik V, et al. 2017. Gut mi-crobiome function predicts response to anti-integrin bio-logic therapy in inflammatory bowel diseases[J]. Cell Host Microbe, 21(5): 603-610. [14] Berry D, Schwab C, Milinovich G, et al. 2012. Phylotype-lev-el 16S rRNA analysis reveals new bacterial indicators of health state in acute murine colitis[J]. ISME Journal, 6(11): 2091-2106. [15] Bian X, Tu P, Chi L, et al. 2017. Saccharin induced liver in-flammation in mice by altering the gut microbiota and its metabolic functions[J]. Food and Chemical Toxicolo-gy, 107(Pt B): 530-539. [16] Bloom S M, Bijanki V N, Nava G M, et al. 2011. Commensal Bacteroides species induce colitis in host-genotype-spe-cific fashion in a mouse model of inflammatory bowel disease[J]. Cell Host Microbe, 9(5): 390-403. [17] Cirioni O, Giacometti A, Ghiselli R, et al. 2002. Single-dose intraperitoneal magainins improve survival in a gram-negative-pathogen septic shock rat model[J]. Antimicro-bial Agents and Chemotherapy, 46(1): 101-104. [18] Cirioni O, Silvestri C, Ghiselli R, et al. 2008. Protective ef-fects of the combination of alpha-helical antimicrobial peptides and rifampicin in three rat models of Pseudomo-nas aeruginosa infection[J]. Journal of Antimicrobial Chemotherapy, 62(6): 1332-1338. [19] Engelberg Y, Landau M. 2020. The human LL-37(17-29) anti-microbial peptide reveals a functional supramolecular structure[J]. Nature Communications, 11(1): 1-10. [20] Hans W, Schölmerich J, Gross V, et al. 2000. The role of the resident intestinal flora in acute and chronic dextran sul-fate sodium-induced colitis in mice[J]. European Jour-nal of Gastroenterology & Hepatology, 12(3): 267-273. [21] Huo X, Wang Z, Xiao X, et al. 2021. Oral administration of nanopeptide CMCS-20H conspicuously boosts immuni-ty and precautionary effect against bacterial infection in fish[J]. Frontiers in Immunology, 12: 811616. [22] Iacob S, Iacob D G, Luminos L M. 2018. Intestinal microbio-ta as a host defense mechanism to infectious threats[J]. Frontiers in Immunology, 9: 3328. [23] Kamada N, Seo S U, Chen G Y, et al. 2013. Role of the gut microbiota in immunity and inflammatory disease[J]. Nature Reviews Immunology, 13(5): 321-335. [24] Lee Y S, Kim T Y, Kim Y, et al. 2018. Microbiota-derived lac-tate accelerates intestinal stem-cell-mediated epithelial development[J]. Cell Host Microbe, 24(6): 833-846. [25] Li M C, He S H. 2004. IL-10 and its related cytokines for treatment of inflammatory bowel disease[J]. World Jour-nal of Gastroenterolog, 10(5): 620-625. [26] Liu Q, Yu Z, Tian F, et al. 2020. Surface components and me-tabolites of probiotics for regulation of intestinal epithe-lial barrier[J]. Microbial Cell Factories, 19(1): 23. [27] Lozupone C A, Stombaugh J I, Gordon J I, et al. 2012. Diver-sity, stability and resilience of the human gut microbiota[J]. Nature, 489(7415): 220-230. [28] Madsen K L, Doyle J S, Jewell L D, et al. 1999. Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice[J]. Gastroenterology, 116(5): 1107-1114. [29] Maloy W L, Kari U P. 1995. Structure-activity studies on magainins and other host defense peptides[J]. Biopoly-mers, 37(2): 105-122. [30] Mazmanian S K, Round J L, Kasper D L. 2008. A microbial symbiosis factor prevents intestinal inflammatory dis-ease[J]. Nature, 453(7195): 620-625. [31] Mccoy A J, Liu H, Falla T J, et al. 2001. Identification of Pro-teus mirabilis mutants with increased sensitivity to anti-microbial peptides[J]. Antimicrobial Agents and Chemo-therapy, 45(7): 2030-2037. [32] Patel R, Dupont H L. 2015. New approaches for bacteriothera-py: Prebiotics, new-generation probiotics, and synbiotics[J]. Clinical Infectious Diseases, 60(Suppl 2): S108-S121. [33] Riley L W, Remis R S, Helgerson S D, et al. 1983. Hemor-rhagic colitis associated with a rare Escherichia coli sero-type[J]. The New England Journal of Medicine, 308(12): 681-685. [34] Round J L, Mazmanian S K. 2010. Inducible Foxp3+ regulato-ry T-cell development by a commensal bacterium of the intestinal microbiota[J]. Proceedings of the National Academy of Sciences of the USA, 107(27): 12204-12209. [35] Ruseler-Van Embden J G, Van Der Helm R, Van Lieshout L M. 1989. Degradation of intestinal glycoproteins by Bacteroides vulgatus[J]. FEMS Microbiology Letters, 49(1): 37-41. [36] Shiba T, Aiba Y, Ishikawa H, et al. 2003. The suppressive ef-fect of bifidobacteria on Bacteroides vulgatus, a putative pathogenic microbe in inflammatory bowel disease[J]. Microbiology and Immunology, 47(6): 371-378. [37] Wang J, Ji H, Wang S, et al. 2018. Probiotic Lactobacillus plantarum promotes intestinal barrier function by strengthening the epithelium and modulating gut micro-biota[J]. Frontiers in Microbiology, 9: 1953. [38] Yi H, Hu W, Chen S, et al. 2017. Cathelicidin-WA improves intestinal epithelial barrier function and enhances host defense against enterohemorrhagic Escherichia coli O157: H7 infection[J]. The Journal of Immunology, 198(4): 1696-1705. [39] Yue B, Yu Z L, Lv C, et al. 2020. Regulation of the intestinal microbiota: An emerging therapeutic strategy for inflam-matory bowel disease[J]. World Journal of Gastroenter-ology, 26(30): 4378-4393. [40] Zasloff M. 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two ac-tive forms, and partial cDNA sequence of a precursor[J]. Proceedings of the National Academy of Sciences of the USA, 84(15): 5449-5453. [41] Zhong C, Zhang F, Yao J, et al. 2021. New antimicrobial pep-tides with repeating unit against multidrug-resistant bac-teria[J]. ACS Infectious Diseases, 7(6): 1619-1637.
