植物益生菌促进肠道屏障功能
Probiotic Lactobacillus plantarum Promotes Intestinal Barrier Function by Strengthening the Epithelium and Modulating Gut Microbiota
Frontiers in Microbiology (2018)
DOI 10.3389/fmicb.2018.01953
Abstract
Weaning disturbs the intestinal barrier function and increases the risk of infection in piglets. Probiotics exert beneficial health effects, mainly by reinforcing the intestinal epithelium and modulating the gut microbiota. However, the mechanisms of action, and especially, the specific regulatory effects of modulated microbiota by probiotics on the intestinal epithelium have not yet been elucidated. The present study aimed to decipher the protective effects of the probiotic Lactobacillus plantarum strain ZLP001 on the intestinal epithelium and microbiota as well as the effects of modulated microbiota on epithelial function. Paracellular permeability was measured with fluorescein isothiocyanate-dextran (FD-4). Gene and protein expression levels of tight junction (TJ) proteins, proinflammatory cytokines, and host defense peptides were determined by RT-qPCR, ELISA, and western blot analysis. Short-chain fatty acid (SCFA) concentrations were measured by ion chromatography. Fecal microbiota composition was assessed by high-throughput sequencing. The results showed that pretreatment with 10^8 colony forming units (CFU) mL-1 of L. plantarum ZLP001 significantly counteracted the increase in gut permeability to FD-4 induced by 10^6 CFU mL-1 enterotoxigenic Escherichia coli (ETEC). In addition, L. plantarum ZLP001 pretreatment alleviated the reduction in TJ proteins (claudin-1, occludin, and ZO-1) and downregulated proinflammatory cytokines IL-6 and IL-8, and TNF-α expression and secretion caused by ETEC. L. plantarum ZLP001 also significantly increased the expression of the host defense peptides pBD2 and PG1-5 and pBD2 secretion relative to the control. Furthermore, L. plantarum ZLP001 treatment affected piglet fecal microbiota. The abundance of butyrate-producing bacteria Anaerotruncus and Faecalibacterium was significantly increased in L. plantarum ZLP001-treated piglets, and showed a positive correlation with fecal butyric and acetic acid concentrations. In addition, the cell density of Clostridium sensu stricto 1, which may cause epithelial inflammation, was decreased after L. plantarum ZLP001 administration, while the beneficial Lactobacillus was significantly increased. Our findings suggest that L. plantarum ZLP001 fortifies the intestinal barrier by strengthening epithelial defense functions and modulating gut microbiota.
断奶扰乱了仔猪的肠道屏障功能，增加了仔猪感染的风险。益生菌具有有益的保健作用，主要通过强化肠道上皮细胞和调节肠道微生物群来发挥作用。然而，其作用机制，特别是益生菌调节的微生物群如何作用于肠道上皮细胞，我们尚不清楚。
本研究旨在了解植物乳杆菌（Lactobacillus plantarum）菌株ZLP001对肠道上皮细胞和微生物群的保护作用，以及调节后的肠道微生物群对上皮细胞功能的影响。
研究对象：猪肠上皮细胞系IPEC-J2（细胞模型）和断奶仔猪
实验方法：
细胞模型：当达到80%细胞融合时，在IPEC-J2细胞培养液中加入植物乳杆菌ZLP001（10^8 CFU/ml）进行预处理。未进行植物乳杆菌预处理的细胞做为阴性对照组。 37度培养6小时后，用PBS冲洗细胞三次，随后加入埃希氏大肠杆菌（enterotoxigenic Escherichia coli，ETEC，10^6 CFU/ml）37度培养3小时。随后用PBS冲洗细胞三次，收集细胞进行后续实验。同时收集培养基上清液。所有实验进行三次重复。
动物模型：10只断奶仔猪被分为益生菌组（饲料2g/kg加冷冻干燥的植物乳杆菌ZLP001，5x10^9CFU/g）或对照组（饲料加安慰剂，2g/kg）,每组由两公三母组成，共进行30天饲养。
检测方法：
用荧光素异硫氰酸酯-葡聚糖(FD-4)测定细胞外通透性；
采用RT-qPCR、ELISA和western blot方法检测TJ蛋白（tight junction protein）、促炎细胞因子和宿主防御肽（HDP）的基因和蛋白表达水平；
采用离子色谱法测定短链脂肪酸(SCFA)的浓度；
采用16s rDNA V3-V4区高通量测序法测定粪便微生物群组成。
研究结论：
IPEC-J2细胞受到埃希氏大肠杆菌（ETEC）的感染后，FD-4的渗透率明显升高，TJ蛋白的mRNA表达水平和蛋白表达水平都显著降低，促炎细胞因子（如IL-6， IL-8和TNFα）mRNA和蛋白水平的表达增高；
植物乳杆菌 ZLP001预处理后的细胞在受到ETEC感染时，上述基因和蛋白表达均不受影响；
以上结果显示，植物乳杆菌ZLP001可以缓解由ETEC造成的上皮组织损伤；可以通过维持TJ蛋白的丰度来增强肠道上皮对病原菌的抵抗力；
在本研究中，植物乳杆菌ZLP001本身并不影响促炎细胞因子的表达，而是抑制促炎细胞因子在ETEC诱导下的过度表达，从而发挥免疫抑制作用；
通过对断奶仔猪粪便的高通量测序分析发现，益生菌组某些菌属的丰度相对较低，这些菌属可能与疾病相关，例如消化链球菌属的Peptostreptococcaceae incertae sedi 和梭菌属的Clostridium sensu stricto 1；
本研究没有对仔猪肠道组织中的促炎细胞因子进行评估，因此无法分析其与微生物群丰度的相关性。这种关系值得进一步研究；
断奶仔猪模型中，益生菌组的柔嫩梭菌属（Faecalibacterium spp. ）和厌氧棍状菌属（Anaerotruncus spp.）丰度较对照组显著偏高；而粪便中丁酸含量和厌氧棍状菌属丰度呈正相关，粪便中乙酸浓度与柔嫩梭菌属丰度呈正相关；
短链脂肪酸，尤其是丁酸，可以刺激宿主防御肽（HDP）的表达，mRNA表达水平的结果显示植物乳杆菌可以刺激HDP的表达，从而防止细菌感染。
FIGURE 8. Suggested mechanism by which L. plantarum ZLP001 protects intestinal barrier function from weaning stress.L. plantarum ZLP001 functions not only through maintaining epithelial integrity, improving TJs, regulating the immune response, and stimulating HDPs, but also via modulating the intestinal indigenous microbiota. Modulated microbiota and alterations in certain bacterial taxa in turn enhance epithelial function.
图8. 植物乳杆菌（Lactobacillus plantarum ZLP001）保护肠道屏障功能免受断奶应激的可能机制。植物乳杆菌不仅通过维持上皮组织的完整性、改善TJ蛋白表达、调节免疫应答和刺激宿主防御肽（HDP）来发挥作用，还通过调节肠道固有菌群来发挥作用。调节后的微生物群和某些细菌类群的改变反过来又增强了上皮组织功能。
知几未来研究院正在搭建一个“疾病-肠道菌群”知识库。
你可以在公众号菜单栏“学术工具“版块直接查阅疾病和肠道菌群的相关信息；
也可以在公众号对话框回复疾病名称，如“IBD”或“肠易激综合征”，查看与疾病相关的菌群；
或回复菌种名称，如“双歧杆菌”或“Hp”，查看与细菌相关的疾病。
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