抗菌肽CWA对断奶仔猪肠道炎症和肠道屏障功能的作用及其机制

发布时间：2018-04-24 05:14

本文选题：抗菌肽CWA + 抗生素　；参考：《浙江大学》2016年博士论文

【摘要】：断奶造成仔猪肠道功能紊乱,导致腹泻、生长性能下降甚至死亡,严重影响养猪业的健康发展。虽然腹泻的影响因素较多,但多数研究表明肠道炎症和肠道屏障功能损伤是断奶仔猪易发生腹泻的主要内在原因。大量抗生素用于防治断奶仔猪腹泻,引发细菌耐药性、肉产品中药物残留及环境污染等严重问题；同时抗生素滥用会损害机体肠道屏障功能,破坏肠道稳态。抗菌肽是重要的先天免疫分子,因其广谱抗菌活性、破膜杀菌机制以及不易产生耐药性等特点而受到关注。最新研究表明,抗菌肽不仅具有免疫调节作用,还具有肠道上皮屏障保护功能。然而,抗菌肽应用于防治断奶仔猪腹泻的效果及其对断奶仔猪肠道炎症和肠道上皮屏障功能的作用如何还不得而知。因此,在前期获得了抗菌活性高、安全性好的抗菌肽CWA的基础上,本研究开展了抗菌肽CWA对断奶仔猪腹泻的防治效果试验,同时阐明了抗菌肽CWA对肠道炎症和肠道屏障功能的作用；进一步在小鼠上探究了抗菌肽CWA对细菌感染导致的肠道炎症和肠道屏障功能损伤的作用；最后体外细胞试验初步探讨了抗菌肽CWA缓解肠道炎症和保护肠道屏障功能的作用机制。主要研究结果如下：试验一抗菌肽CWA对断奶仔猪肠道炎症和肠道屏障功能的作用(1)抗菌肽CWA对断奶应激仔猪肠道炎症的作用选择18头健康的杜长大仔猪,21日龄断奶后分别腹腔注射3次生理盐水(对照组)和抗菌肽CWA(抗菌肽CWA组),后续饲养7天,研究抗菌肽CWA对仔猪断奶应激腹泻及其肠道炎症的作用。结果表明,抗菌肽CWA降低了断奶仔猪腹泻指数(P0.05),提高了断奶仔猪ADFI (P0.05)和ADG (P0.05)。同时,抗菌肽CWA降低了血清IgG、TNF-α、IL-6、IL-22含量(P0.05),且提高了血清中TGF-β含量(P0.05)。在肠道形态方面,抗菌肽CWA提高了空肠的绒毛高度、绒毛高度/隐窝深度比值(P0.05),提高了回肠的绒毛高度/隐窝深度比值(P0.05),降低了回肠的隐窝深度(P0.05)。在肠道炎症方面,抗菌肽CWA降低了肠道中促炎因子的基因表达,其中对空肠的效果最为显著；同时抗菌肽CWA减少了肠道嗜中性粒细胞和巨噬细胞浸润；提示抗菌肽CWA能够缓解断奶仔猪肠道炎症。在肠道炎症信号通路蛋白表达方面,抗菌肽CWA显著降低空肠组织炎症信号通路中关键蛋白NF-κB、IκB-α的磷酸化水平,提示抗菌肽CWA可能通过下调NF-κB信号通路缓解肠道炎症。(2)抗菌肽CWA对腹泻断奶仔猪肠道炎症和肠道屏障功能的作用选择108头腹泻的杜长大断奶仔猪,分别腹腔注射4次生理盐水(对照组)、恩诺沙星(抗生素组)、抗菌肽CWA(抗菌肽CWA组),研究抗菌肽CWA对腹泻断奶仔猪肠道炎症和肠道屏障功能的作用。结果表明,抗菌肽CWA和抗生素均降低了断奶仔猪的腹泻指数(P0.05)和腹泻率(P0.05),均提高了断奶仔猪ADFI (P＜0.05)和ADG (P＜0.05),且两者无显著差异。在肠道形态方面,抗菌肽CWA和抗生素对小肠绒毛形态损伤均有缓解作用,其中对空肠的作用最显著；扫描电镜和透射电镜观察发现,抗菌肽CWA和抗生素改善了空肠微绒毛数量与长度,且抗菌肽CWA的改善作用优于抗生素。在肠道炎症方面,抗菌肽CWA组和抗生素组均降低了空肠中促炎因子IL-6、IL-8、IL-22的含量(P0.05),对血清也有类似规律；同时,抗菌肽CWA和抗生素均缓解了腹泻断奶仔猪空肠嗜中性粒细胞浸润；进一步,抗菌肽CWA组和抗生素组均降低了空肠组织中炎症相关蛋白NF-κB、IκB-α、AKT的磷酸化水平及上游调控关键蛋白TLR4、MyD88的表达；且抗菌肽CWA缓解肠道炎症的作用与抗生素相当。在肠道上皮屏障功能方面,抗生素组不仅降低了空肠粘液素和防御素的表达,而且降低了空肠中紧密连接蛋白ZO-1和Occludin的表达(P0.05)；然而,抗菌肽CWA却提高了空肠中ZO-1和Occludin的表达(P0.05)；提示抗菌肽CWA可能提高肠道上皮屏障功能而抗生素却损害肠道上皮屏障功能。在肠道微生物菌群及SCFAs含量方面,与对照组相比,抗菌肽CWA组和抗生素组均降低了粪便中大肠杆菌比例(P0.05),均提高了粪便中乳酸杆菌/大肠杆菌比值(P0.05)；然而,抗菌肽CWA提高了粪便中乳酸杆菌比例(P0.05),抗生素却降低了粪便中乳酸杆菌比例(P0.05)；进一步,抗菌肽CWA提高了粪便中乙酸、丙酸及丁酸的浓度(P0.05),而抗生素降低了粪便中丁酸的浓度、丁酸/乙酸比值及丁酸/丙酸比值(P0.05)。试验二抗菌肽CWA对细菌感染小鼠肠道炎症和肠道屏障功能的作用在发现抗菌肽CWA缓解断奶仔猪肠道炎症和肠道屏障功能损伤的基础上,进一步在小鼠上研究抗菌肽CWA对细菌感染引起的肠道炎症和肠道屏障功能损伤的影响。选择36只断奶小鼠分为对照组、E.coli组、E.coli+Enro组、E.coli+CWA组,EHEC O157:H7感染后分别腹腔注射生理盐水、恩诺沙星(Enro)、抗菌肽CWA。结果表明,抗菌肽CWA和恩诺沙星均提高了染菌小鼠的存活率,缓解了染菌导致的小鼠体重下降。在肠道形态方面,抗菌肽CWA和恩诺沙星均缓解了染菌导致的小鼠空肠绒毛萎缩、隐窝增生和结肠粘膜上皮破损、杯状细胞数量增加。在炎症方面,E.coli+Enro组和E.coli+CWA组均缓解了染菌导致的小鼠血清和结肠中TNF-α、IL-6、IL-10含量升高(P0.05)；同时均缓解了染菌导致的小鼠空肠中IL-6含量的升高(P0.05)；抗菌肽CWA在缓解炎症方面与恩诺沙星无显著差异。在肠道抗菌蛋白表达方面,E.coli+Enro组和E.coli+CWA组均缓解了染菌导致的空肠中REG3y基因表达升高和结肠中Remlβ基因表达升高(P0.05)。然而,抗菌肽CWA组了小鼠空肠和结肠中TFF3基因表达水平(P0.05)。同时,E.coli+Enro组和E.coli+CWA组均缓解了染菌导致的小鼠空肠中杯状细胞数量减少、粘液层厚度降低和结肠中杯状细胞数量增加。进一步,Western blot和免疫荧光的结果表明,E.coli+Enro组和E.coli+CWA组均缓解了染菌导致的小鼠空肠MUC-2表达的降低和结肠MUC-2表达的升高(P0.05)。在肠道上皮屏障功能方面,E.coli+CWA组缓解了染菌导致的小鼠空肠中ZO-1和Occludin蛋白表达的下降(P0.05),而E.coli+Enro组仅缓解了ZO-1蛋白表达的下降(P0.05),抗菌肽CWA的效果优于抗生素。在肠道微生物菌群及SCFAs浓度方面,不同处理小鼠盲肠内容物中总菌、双歧杆菌、芽孢杆菌的数量无显著差异,E.coli+Enro组和Ecoli+CWA组均缓解了染菌导致的盲肠中乳酸杆菌数量的降低和大肠杆菌数量的升高(P0.05),同时Ecoli+CWA组缓解了染菌导致的盲肠中乳酸杆菌/大肠杆菌比值的下降(P0.05)。同时,Ecoli+CWA组和E.coli+Enro组均对染菌导致的SCFAs浓度的降低具有一定程度的缓解作用。以上结果进一步证明了抗菌肽CWA能够缓解肠道炎症和改善肠道屏障功能,与断奶仔猪的试验结果一致。试验三抗菌肽CWA对肠道炎症和肠遒上皮屏障功能的调控机制(1)抗菌肽CWA缓解肠道炎症的作用机制利用LPS诱导的猪巨噬细胞炎症模型发现,恩诺沙星对LPS导致的炎症反应无缓解作用,而抗菌肽CWA可以缓解LPS导致的炎症反应且呈浓度依赖效应。同时,抗菌肽CWA缓解了LPS导致的IL-6、IL-8、IL-22表达升高和NF-κB、IκB-α磷酸化水平升高(P0.05)。利用siRNA技术沉默猪巨噬细胞TLR4和MyD88后,抗菌肽CWA缓解炎症的作用受到抑制。利用FITC标记的葡聚糖和流式细胞检测技术发现,抗菌肽CWA可以增强巨噬细胞吞噬FITC-葡聚糖的能力,而使用STAT-1抑制剂后,抗菌肽CWA对巨噬细胞吞噬功能的增强作用受到抑制。以上结果表明,抗菌肽CWA缓解肠道炎症可能是通过TLR4-MyD88-NF-KB信号通路调控炎症反应和通过STAT-1途径增强巨噬细胞吞噬功能等机制发挥作用。(2)抗菌肽CWA改善肠道上皮屏障功能的作用机制利用Caco-2细胞刮伤模型发现,抗菌肽CWA提高了刮伤细胞的愈合速度,改善受损肠道上皮细胞的修复功能。利用猪空肠上皮细胞(IPEC-J2)屏障功能损伤模型发现,抗菌肽CWA缓解了LPS导致的IPEC-J2单层细胞TER值的下降,同时缓解了LPS导致的肠道上皮细胞中紧密连接蛋白ZO-1和Occludin蛋白表达的下降。单独添加抗菌肽CWA提高了IPEC-J2细胞中ZO-1和Occludin的表达,而添加Rac1抑制剂NSC 23766时,抗菌肽CWA对ZO-1和Occludin的提高作用受到抑制,且抗菌肽CWA对粘附IPEC-J2细胞的减少EHEC O157:H7作用也被抑制。以上结果表明,抗菌肽CWA能够改善受损肠道上皮细胞的修复功能,通过Racl途径调控肠道上皮细胞紧密连接蛋白表达,增强肠道上皮屏障功能。上述研究不仅为抗菌肽应用于防治断奶仔猪腹泻,实现减少抗生素在畜禽养殖中的使用提供了重要的基础数据与指导意义；而且为由肠道炎症和肠道屏障功能损伤引起人类或者动物的多种肠道疾病提供了新的防治策略。
[Abstract]:Weaning caused intestinal dysfunction in piglets, caused diarrhea, decreased growth performance or even death, which seriously affected the healthy development of pig industry. Although there were many factors affecting diarrhea, most studies showed that intestinal inflammation and intestinal barrier function injury were the main internal causes of diarrhea in weanling piglets. A large number of antibiotics were used to prevent and control weaning. Diarrhea, bacterial resistance, drug residue and environmental pollution in meat products are serious problems, and the abuse of antibiotics will damage the intestinal barrier function and destroy the intestinal homeostasis. Antimicrobial peptides are important innate immune molecules, and are affected by their broad-spectrum antimicrobial activity, membrane breaking mechanism and resistance to resistance to drug resistance. Note. The latest research shows that antibacterial peptides not only have immune regulation, but also have protective function of intestinal epithelial barrier. However, the effect of antimicrobial peptides on the prevention and treatment of weanling piglets' diarrhea and its effect on intestinal inflammation and intestinal barrier function of weanling piglets are not well known. Therefore, the antibacterial activity is high and safe in the early stage. On the basis of a good antibacterial peptide CWA, the effect of antimicrobial peptide CWA on the diarrhea of weanling piglets was tested, and the effect of antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function was clarified, and the effect of antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function damage caused by bacterial infection was further explored in mice. Finally, the mechanism of antibacterial peptide CWA in alleviating intestinal inflammation and protecting intestinal barrier function was preliminarily studied in vitro. The main results were as follows: test the effect of antibacterial peptide CWA on intestinal inflammation and intestinal barrier function of weanling piglets (1) the effect of antibacterial peptide CWA on intestinal inflammation of weanling stress piglets was selected healthy. After 21 days of weaning, 3 physiological saline (control group) and antibacterial peptide CWA (antibacterial peptide CWA group) were intraperitoneally injected after 21 days of weaning. The effect of antimicrobial peptide CWA on weanling stress diarrhea and intestinal inflammation was studied. The results showed that antimicrobial peptide CWA reduced the diarrhea index of weaned piglets (P0.05) and increased ADFI (P0.05) in weanling piglets. And ADG (P0.05). At the same time, antibacterial peptide CWA reduced serum IgG, TNF- alpha, IL-6, IL-22 content (P0.05), and increased the content of TGF- beta in serum (P0.05). In the form of intestinal tract, antimicrobial peptides CWA increase the height of the villus, the ratio of the villi height / recess depth (P0.05), increase the ratio of the villus height / recess depth of the ileum, and reduce the return of the ileum. Intestinal fossa depth (P0.05). In intestinal inflammation, antimicrobial peptide CWA reduces the gene expression of proinflammatory factors in the intestinal tract, which is the most significant for jejunum; at the same time, antimicrobial peptide CWA reduces the infiltration of neutrophils and macrophages in the intestinal tract, suggesting that antimicrobial peptide CWA can relieve intestinal inflammation in weanling piglets. In the expression of road protein, antibacterial peptide CWA significantly decreased the phosphorylation level of key protein NF- kappa B and I kappa B- alpha in the inflammatory signaling pathway of jejunum tissue, suggesting that antibacterial peptide CWA may alleviate intestinal inflammation by down regulation of NF- kappa B signaling pathway. (2) the effect of antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function of weanling piglets of diarrhoea is selected for 108 diarrhoea. The piglets were intraperitoneally injected with 4 normal saline (control group), enrofloxacin (antibiotic group) and antibacterial peptide CWA (antibacterial peptide CWA group). The effects of antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function of weanling piglets were studied. The results showed that the diarrhea index (P0.05) and diarrhea rate of weanling piglets were reduced by antimicrobial peptide CWA and antibiotics. (P0.05), both ADFI (P < 0.05) and ADG (P < 0.05) were increased in weanling piglets, and there was no significant difference between them. In intestinal morphology, antibacterial peptide CWA and antibiotics had a relieving effect on the morphological damage of small intestinal villi, and the most significant effect was on jejunum. The scanning electron microscopy and transmission electron microscopy showed that antibacterial peptide CWA and antibiotics improved jejunum. The number and length of microvilli, and the improvement of antimicrobial peptide CWA is better than that of antibiotics. In the field of intestinal inflammation, the CWA and IL-22 in the jejunum, the content of IL-6, IL-8, IL-22 (P0.05) in the jejunum are reduced, and the serum also has a similar rule. At the same time, the antibacterial peptide CWA and antibiotics all alleviate the neutrophils of the weanling piglets of diarrhea. Further, both the antibacterial peptide CWA group and the antibiotic group reduced the inflammation related protein NF- kappa B, the phosphorylation level of I kappa B- alpha, AKT and the expression of the key protein TLR4, MyD88, and the effect of antimicrobial peptide CWA on intestinal inflammation was equivalent to that of antibiotics. In the intestinal epithelial barrier function, antibiotic group is not only The expression of mucin and defensins in jejunum decreased and the expression of close connexin ZO-1 and Occludin in jejunum (P0.05) was reduced; however, antimicrobial peptide CWA increased the expression of ZO-1 and Occludin in jejunum (P0.05), suggesting that antimicrobial peptide CWA may improve intestinal barrier function while antibiotics damage intestinal epithelial barrier function. In terms of intestinal microbial flora and SCFAs content, compared with the control group, both the antibacterial peptide CWA group and the antibiotic group reduced the proportion of Escherichia coli in the feces (P0.05), and increased the ratio of lactobacilli / Escherichia coli (P0.05) in the feces. However, the antimicrobial peptide CWA increased the proportion of Lactobacillus in the feces (P0.05), but the antibiotic decreased the lactic acid in the feces. Bacilli ratio (P0.05); further, antibacterial peptide CWA increased the concentration of acetic acid, propionic acid and butyric acid (P0.05) in feces, while antibiotics reduced the concentration of butyric acid in feces, the ratio of butyric acid / acetic acid and the ratio of butyric acid / propionic acid (P0.05). The effect of two antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function in bacteria infected rats was found in the discovery of antimicrobial peptides On the basis of CWA alleviating intestinal inflammation and intestinal barrier function damage in weanling piglets, the effect of antibacterial peptide CWA on intestinal inflammation and intestinal barrier function damage caused by bacterial infection was further studied in mice. 36 weanling mice were divided into control group, E.coli group, E.coli+Enro group, E.coli+CWA group, EHEC O157:H7 infection and abdominal injection respectively. The results of physiological saline, enrofloxacin (Enro) and antimicrobial peptide CWA. showed that both antimicrobial peptide CWA and enrofloxacin increased the survival rate of the infected mice and alleviated the loss of weight of mice caused by the infection. In the form of intestinal tract, both the antibacterial peptide CWA and enrofloxacin both alleviated the atrophy of the jejunum villus, the recess hyperplasia and the colonic mucosa caused by the infected bacteria. The skin was damaged and the number of goblet cells increased. In inflammation, both E.coli+Enro and E.coli+CWA groups relieved the increase of TNF- alpha, IL-6 and IL-10 (P0.05) in the serum and colon of mice infected with bacteria, and alleviated the increase of IL-6 content in the jejunum caused by the infection (P0.05), and the antimicrobial peptide CWA was not associated with enrofloxacin in inflammation. Significant differences. In the expression of intestinal antibacterial protein, both E.coli+Enro and E.coli+CWA groups relieved the increase of REG3y gene expression in the jejunum and the increase of Reml beta gene expression in the colon (P0.05). However, the expression level of TFF3 gene in the jejunum and colon of mice (P0.05) was found in the group of antibacterial peptide CWA (P0.05). Both E.coli+Enro and E.coli+CWA groups were both. The decrease in the number of goblet cells in the jejunum, the decrease in the thickness of the mucous layer and the increase in the number of goblet cells in the colon were alleviated. Further, the results of Western blot and immunofluorescence showed that both E.coli+Enro and E.coli+CWA groups alleviated the decrease of MUC-2 expression in jejunum and the increase of MUC-2 expression in colon (P0.05). In the intestinal epithelial barrier function, the E.coli+CWA group relieved the decrease of ZO-1 and Occludin protein expression in the jejunum of the infected mice (P0.05), while the E.coli+Enro group only alleviated the decrease of the expression of ZO-1 protein (P0.05), and the effect of the antibacterial peptide CWA was better than that of the antibiotic. In the intestinal tract microbial flora and the concentration of SCFAs, the mice were treated differently. There was no significant difference in the number of total bacteria, bifidobacteria and Bacillus in intestinal contents. Both E.coli+Enro and Ecoli+CWA groups alleviated the decrease of lactobacilli in the cecum and the increase of Escherichia coli (P0.05), and the Ecoli+CWA group alleviated the decrease of the ratio of lactobacilli / Escherichia coli in the cecum caused by dyed bacteria (P0.05 At the same time, both the Ecoli+CWA group and the E.coli+Enro group had some alleviating effect on the decrease of the SCFAs concentration caused by the infected bacteria. The above results further demonstrated that the antibacterial peptide CWA could alleviate intestinal inflammation and improve the intestinal barrier function, which was consistent with the test results of weanling piglets. Test three antibacterial peptide CWA for intestinal inflammation and intestinal epithelium screen. Regulatory mechanism of barrier function (1) the mechanism of antibacterial peptide CWA alleviating intestinal inflammation using LPS induced macrophage inflammation model, enrofloxacin has no relieving effect on LPS induced inflammatory response, and antimicrobial peptide CWA can alleviate the inflammatory response caused by LPS and be dependent on the concentration dependent effect. At the same time, antimicrobial peptide CWA relieves IL-6 caused by LPS. IL-8, IL-22 expression increased and NF- kappa B, I kappa B- alpha phosphorylation level increased (P0.05). After siRNA technology was used to silence the TLR4 and MyD88 of pig macrophages, the effect of antimicrobial peptide CWA on inflammation was inhibited. The effect of antibacterial peptide CWA on macrophage phagocytosis was inhibited after the use of STAT-1 inhibitors. The above results suggest that antimicrobial peptide CWA may play a role in regulating inflammatory response through the TLR4-MyD88-NF-KB signaling pathway and enhancing macrophage phagocytosis through the STAT-1 pathway. (2) improvement of antimicrobial peptide CWA The mechanism of intestinal epithelial barrier function using Caco-2 cell scraping model shows that antibacterial peptide CWA improves the healing speed of the scraped cells and improves the repair function of the damaged intestinal epithelial cells. Using the porcine jejunum epithelial cell (IPEC-J2) barrier function damage model, the antibacterial peptide CWA alleviates the TER value of IPEC-J2 monolayer cells caused by LPS. The decrease of the expression of close connexin ZO-1 and Occludin protein in intestinal epithelial cells caused by LPS was reduced. The expression of ZO-1 and Occludin in IPEC-J2 cells was enhanced by the addition of antimicrobial peptide CWA alone. When the Rac1 inhibitor NSC 23766 was added, the effect of antimicrobial peptide CWA on ZO-1 and Occludin was inhibited. The effect of the decrease of EHEC O157:H7 with IPEC-J2 cells was also suppressed. The results showed that the antibacterial peptide CWA could improve the repair function of the damaged intestinal epithelial cells, regulate the expression of close connexin in the intestinal epithelial cells by Racl pathway and enhance the intestinal epithelial barrier function. It provides important basic data and guidance for reducing the use of antibiotics in livestock and poultry breeding, and provides a new prevention strategy for many kinds of intestinal diseases caused by intestinal inflammation and intestinal barrier function damage.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.28

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