植物乳杆菌CCFM639缓解铝毒性作用及机制研究

发布时间：2018-04-04 20:43

本文选题：益生菌　切入点：乳酸菌　出处：《江南大学》2017年博士论文

【摘要】：铝(Al,aluminum)不是人体必需的金属元素并且没有任何生理功能,但它却广泛存在于自然界中(地壳中含量最多的金属元素),并在日常生活中被广泛应用。铝离子可以蓄积在哺乳动物的大多数组织并造成损伤和病变,其中对神经系统的损伤被广泛研究。传统螯合剂疗法虽然可以促进铝离子的排出,但具有不可避免的副作用,安全性存在缺陷。乳酸菌被公认为安全的食品级微生物,因已有报道表明乳酸菌可以吸附多种毒性金属,维持肠道微生态平衡,并通过肠道菌群-脑肠轴影响神经系统和行为表现,表明其具备缓解铝离子毒性的潜力,而且目前国内外还没有利用乳酸菌缓解铝离子毒性的相关报道。因此本研究探索乳酸菌在缓解铝离子毒性效应中的效果及相关机制,用以深层次开发乳酸菌的新益生功能,以期挖掘更具应用价值的益生功能乳酸菌,开发出新的解决铝毒性问题的膳食策略和应用。主要研究结果如下:建立了体外缓解铝离子毒性潜力菌株的筛选模型,具体特性如下:高效的铝离子吸附能力;优良的铝离子耐受能力和胃肠道的耐受能力;优秀的抗氧化能力,可缓解铝离子暴露带来的氧化损伤。依据以上评价指标对30株乳酸菌菌株进行筛选,发现植物乳杆菌CCFM639在初始铝离子浓度为5mg/L和50mg/L的条件下,其均体现出最强的吸附能力,分别为64.54%和26.83%;而且该菌株的铝离子耐受能力特别突出,可以达到甚至超过2480 mg/L的最小抑菌浓度,即MIC,显著高于该试验的其他菌株测试能力。该菌株在模拟胃液和肠液中的存活率分别为90.69%和91.86%,说明其可以在肠道内保持了较高的活性;同时,对抗氧化四个方面(羟自由基清除率、DPPH清除率、抗脂质过氧化能力和还原能力)的能力进行了测试,结合主成分分析进一步综合评价得出CCFM639具有较好的抗氧化能力。综上,选择植物乳杆菌CCFM639进行后续研究。利用傅里叶红外光谱、超薄切片透射电镜、扫描电镜联用能量色散X射线光谱和全基因组序列解析,确定了CCFM639菌体表面的多种功能基团参与了金属铝离子的吸附过程,多个功能基因参与缓解铝离子毒性作用。通过急性和慢性铝离子暴露动物模型的综合效果评价,证明植物乳杆菌CCFM639菌株在铝离子暴露动物模型中具有缓解铝离子毒性作用。在急性实验中,该菌株明显增加存活率,促进粪便铝离子的排出、降低铝离子在血液、组织和器官中的蓄积、缓解氧化应激状态,进而减轻肝脏的病理病变和降低异常肝细胞的数量。在慢性实验中,该菌株降低铝离子在血液、组织和器官中的蓄积和损伤、调节组织器官中微量元素的水平,减轻氧化应激和生理生化指标、缓解组织病理损伤,以及完全避免了铝离子暴露导致的罗非鱼死亡。肠道是植物乳酸杆菌能够直接发挥作用的器官,同时也是铝离子的主要吸收部位,因此进一步研究了该菌株对肠道屏障及肠道功能的调控作用,以解析其缓解铝毒性的具体机制。在体外肠道模型(Batch Culture Fermentation Model)中,铝离子暴露显著降低了双歧杆菌和乳杆菌的数量,促进了拟杆菌、梭状芽孢杆菌和肠杆菌的数量。而植物乳杆菌CCFM639对部分有益菌群有显著提升作用,其中对双歧杆菌和乳杆菌的作用最明显。通过对体外肠道模型代谢产物水平的分析结果显示,CCFM639可显著提高代谢产物中丁酸盐和乳酸盐水平。在HT-29细胞模型和慢性铝离子暴露小鼠模型中,铝离子暴露破坏肠道屏障,增加了肠道通透性,损伤过程涉及氧化应激,促炎反应和紧密连接蛋白(TJs)的损伤。而灌胃CCFM639显著缓解氧化应激和促炎因子水平,对TJs的损伤也有一定恢复作用,显著增加血清内毒素水平,增加肠道通透性。在慢性铝离子暴露罗非鱼模型中,在门水平,铝离子暴露显著增加Bacteroidetes的丰度,而减少Firmicutes的丰度。在属水平,铝离子暴露明显降低Plesiomonas和Deefgea的丰度,大大增加Flavobacterium,Enterovibrio,Porphyromonadaceae和Comamonadaceae的丰度。饲料添加CCFM639可显著增加罗非鱼粪便中的Lactobacilli含量,提高Deefgea的丰度,而降低Comamonadaceae的丰度。以上结果表明,植物乳杆菌CCFM639可调节罗非鱼肠道菌群的结构和组成,明显提高肠道菌群中的有益菌群含量,同时有效降低有害菌群含量。通过慢性铝离子暴露小鼠模型,分析了植物乳杆菌CCFM639与铝离子暴露导致的行为异常之间的关联性。慢性铝离子暴露小鼠行为学(水迷宫、新物体识别和开放旷场)测试的结果表明铝离子暴露可导致小鼠的学习记忆能力下降、对物体辨识度发生障碍以及忧虑行为,而CCFM639能够显著缓解和改善这些不利影响。此外,慢性铝离子暴露组小鼠脑GSH水平以及SOD、GPx和CAT活性大大降低,促炎细胞因子水平和脑淀粉样蛋白Aβ1-40和Aβ1-42含量显著升高,小鼠脑组织中微量元素水平被影响以及脑紧密连接蛋白被破坏。灌胃植物乳杆菌CCFM639可显著恢复这些指标,说明该菌能够缓解慢性铝离子暴露造成的脑损伤和行为异常,其可能原因是植物乳杆菌CCFM639可调节肠道屏障、肠道菌群及其代谢产物水平,进而通过脑肠轴而最终达到保护效果。
[Abstract]:Aluminum (Al, aluminum) is not essential metal elements without any physiological function, but it is widespread in nature (the content of metal elements in the crust, and the largest) are widely used in daily life. The aluminum ion can accumulate in most tissues of mammals and cause damage to the nervous system and the lesions. The injury has been extensively studied. Although traditional chelation therapy can promote the excretion of aluminum ions, but the side effect is inevitable, the defects of security. Lactic acid bacteria are recognized as food grade microorganism safety, as has been reported that lactic acid bacteria can absorb a variety of toxic metals, maintain the intestinal micro ecological balance, and through the intestinal bacteria group of brain gut axis affects the nervous system and behavior, show that it has the potential to ease aluminum ion toxicity, and there is no remission of aluminium toxicity by lactic acid bacteria Reported. The present study explored the lactic acid bacteria in alleviating the toxic effects of aluminum ion effect and related mechanism for deep development of new probiotic lactic acid bacteria function, in order to excavate the probiotic lactic acid bacteria has higher application value, the development and application of new dietary strategies to solve the problems. The main research of aluminum toxicity the results are as follows: to establish the screening model in vitro to alleviate Al toxicity of potential strains, the specific characteristics are as follows: aluminum ion efficient adsorption capacity; excellent aluminum tolerance ability and gastrointestinal tolerance; excellent antioxidation ability, can alleviate the oxidative damage caused by aluminum ion exposure. Based on the above evaluation index of 30 strains of lactic acid strain screening, discovery of Lactobacillus plantarum CCFM639 in the initial concentration of aluminium ions for the 5mg/L and 50mg/L conditions, which are reflected in the strongest adsorption capacity, respectively 64.54% and 26.83%; and the Aluminum ion tolerance ability was particularly prominent, minimum inhibitory concentration, even more than 2480 mg/L or MIC, significantly higher than that of other strains tested ability of the test. The survival rate of the strains in simulated gastric and intestinal fluids were 90.69% and 91.86%, which can keep high activity in the intestine; at the same time. Four aspects of anti oxidative (hydroxyl radical scavenging rate and DPPH clearance rate, anti lipid peroxidation and reduction ability) were tested for their ability, combined with principal component analysis to comprehensive evaluation results show that CCFM639 has good anti oxidation ability. Therefore, selection of Lactobacillus plantarum CCFM639 for further research. By Fourier transform infrared spectroscopy, ultra thin microscope, scanning electron microscopy combined with energy dispersive X ray spectra and genome sequence analysis, to determine the various functional groups on CCFM639 cell surface in aluminum ion The adsorption process, multiple functional genes involved in alleviating aluminum ion toxicity. Comprehensive evaluation of the animal model of acute and chronic exposure by aluminum ion, that Lactobacillus plantarum strain CCFM639 exposed to aluminum ion in the animal model with ease of aluminum ion toxicity. In acute experiments, the strains significantly increased the survival rate, promote discharge waste aluminum ions, reducing the accumulation of aluminum ions in the blood, tissues and organs, alleviate oxidative stress and reduce the pathological lesions in the liver and reduce the number of abnormal liver cells. In chronic experiment, the strain reducing aluminum ions in the blood, tissues and organs in the accumulation and damage regulation of trace elements the organ level, reduce stress and physiological and biochemical indexes of oxidation, relieve pathological damage, and completely avoid the aluminum ion exposure leads to death. The intestinal Lactobacillus is tilapia rod Bacteria can directly play the role of the organ, but also the main absorption site of aluminum ion, so further study of the role of the strain on the intestinal barrier and intestinal function, to analyze the mechanism of aluminum toxicity in vitro. Alleviate intestine model (Batch Culture Fermentation Model), aluminum ion exposure significantly decreased the number of Bifidobacterium and Lactobacillus, promoted the number of Bacteroides, Clostridium and Enterobacter. And Lactobacillus plantarum CCFM639 of probiotics has significantly improved the effect of Bifidobacterium and Lactobacillus bacteria plays the most important role. Through the analysis of the model in vitro intestinal metabolite levels showed that CCFM639 significantly increase in butyrate and lactate levels of metabolites. In HT-29 cell model and chronic aluminum ion exposure in mice, aluminum ion exposure increases the damage of intestinal barrier, intestinal pass Permeability damage process involving oxidative stress, inflammatory reaction and tight junction protein (TJs) and gastric perfusion injury. CCFM639 significantly alleviated oxidative stress and pro-inflammatory cytokines, damage of TJs has a certain recovery effect, significantly increased the serum endotoxin level and increased intestinal permeability. In chronic aluminum ion exposure model of tilapia in at the gate level, aluminum ion exposure significantly increased the abundance of Bacteroidetes, and reduce the abundance of Firmicutes. At the genus level, the aluminum ion exposure significantly decreased abundance of Plesiomonas and Deefgea, Enterovibrio, Flavobacterium increased significantly, the abundance of Porphyromonadaceae and Comamonadaceae. Feed additive CCFM639 can significantly increase the Lactobacilli content of tilapia in the stool, improve Deefgea abundance, decreased the abundance of Comamonadaceae. The above results showed that Lactobacillus plantarum CCFM639 can regulate the structure of intestinal microflora of tilapia and Composition, increase the content of beneficial bacteria in the intestinal microbiota, and effectively reduce the harmful bacteria content. A mouse model of chronic aluminum exposure by ion, analyses the relationship between Lactobacillus plantarum CCFM639 and aluminum ion exposure lead to abnormal behavior between chronic exposure of aluminum ions. Mice behavior (water maze, object recognition and new open field test) results show that aluminium ion exposure can lead to decreased learning and memory ability of mice, the identification of obstacles and concerns of object behavior, while CCFM639 can significantly alleviate and improve these adverse effects. In addition, chronic aluminum exposed mice brain GSH levels and SOD, GPx and CAT activity significantly reduced, proinflammatory the levels of cytokines and cerebral amyloid A beta 1-40 and beta 1-42 A content increased significantly, the levels of trace elements in mouse brain and brain is affected by the tight junction protein was damaged. Intragastric administration of Lactobacillus Coli CCFM639 can significantly restore these indexes, indicating that the bacteria can alleviate the chronic exposure of aluminum ions and brain damage caused by abnormal behavior, the possible reason is Lactobacillus plantarum CCFM639 can regulate the intestinal barrier, intestinal microflora and its metabolite levels, and then through the brain gut axis and the end to achieve the protective effect.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS201.3

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