Can probiotics & prebiotics reduce the impact of high protein diets on gut barrier function by modulating the microbiota in sex-dependent manners?

肠壁提供物理屏障，防止细菌和消化的有害产物进入血液。屏障功能受损（漏肠）导致这些分子进入血液，慢性低度炎症和心血管疾病[1，2]。一些饮食蛋白质到达结肠，在那里它可以驱动蛋白水解细菌的扩张，导致更高浓度的代谢终产物，包括苯酚和氨，这已被证明会降低体外屏障功能[3，[4]但不是在体内。一些体外研究表明，特定的益生菌和益生元可以减少高蛋白可用性对与肠漏相关的细菌代谢产物产生的影响[5，6]。然而，结果一直不一致，并且经常使用不能到达结肠的水解蛋白质。此外，通常使用单一来源的蛋白质。然而，我们的初步数据显示，取决于蛋白质的来源，蛋白水解细菌衍生的代谢产物产生存在高度显著的差异（图1a）。我们已经确定了响应于高水平蛋白质的体外微生物群组成和代谢产物产生的显著性别差异（图1b-d）。我们还独立证明了益生元和益生菌对免疫力的影响在体内具有高度的性别依赖性[7]（图1 e）。猪是人类营养研究的有价值的、易处理的临床前模型，因为它们具有许多胃肠道生理学、免疫学和肠道微生物学特征。因此，这项猪研究的结果将高度可用于人类医疗保健。本博士学位将确定特定的益生元和益生菌是否可以以性别依赖的方式减少高蛋白饮食对肠道屏障功能的负面影响。确定细菌衍生代谢产物的产生是否与肠道通透性增加有关（如苯酚，氨）的增加与非水解混合源的增加一致（例如乳清、牛奶、大豆、豌豆、鱼）蛋白质利用率，使用经验证的连续培养物在接种有人类粪便的体外肠道模型系统中进行（图1f）。目标二：识别益生元和益生菌（例如GOS、FOS、菊粉、乳酸双歧杆菌、干酪乳杆菌），其对使用上述体外系统减少与屏障功能降低相关的这些细菌代谢物具有最显著的影响。探索上述模型的上清液是否对紧密细胞连接（TCJ）相关蛋白表达有直接影响目的4：确定目的2中鉴定的益生元和/或益生菌的益处是否可以使用人的猪模型在体内观察到（图2a）。将分别使用16 S测序、气相色谱/质谱和定量荧光免疫组织学评估细菌种群动力学、代谢产物产生和TCJ相关蛋白表达（和免疫）。免疫相关蛋白表达的定量将使用荧光组织学来实现。目的5：通过使用性别平衡的治疗组和对照组以及在统计分析期间使用“性别”作为因素来确定上述所有研究中是否存在性别二态性效应。统计方法将用于生成肠屏障功能、肠道微生物群、免疫、宿主代谢和宿主-微生物共代谢的全球概述。这将产生对与膳食蛋白质增加、微生物群改变相关的生物学过程的机械理解，并确定是否发生性别差异。

Gut walls provide physical barriers preventing harmful products of bacteria and digestion from entering the blood. Loss of barrier function (leaky-gut) leads to passage of these molecules into the blood, chronic low-grade inflammation and cardiovascular disease [1, 2].Some dietary protein reaches the colon where it could drive the expansion of proteolytic bacteria, resulting in higher concentrations of metabolic end-products, including phenol and ammonia, which have been shown to reduce barrier function in vitro [3, 4] but not in vivo. Some in vitro studies demonstrate that specific probiotics and prebiotics can reduce the effects of high-protein availability on the production of bacterial metabolites associated with leaky gut [5, 6]. However, the results have been inconsistent and often use hydrolysed proteins which do not reach the colon. In addition, protein from single sources is often used. However, we have preliminary data showing highly significant differences in proteolytic bacteria-derived metabolite production depending on the source of the protein (Figure 1a). We have identified significant sex-based differences in microbiota composition and metabolite production in vitro in response to high levels of protein (Figure 1b-d). We have also independently demonstrated that the effect of both prebiotics and probiotics on immunity are highly sex-dependent in vivo [7] (Figure 1e).Pigs are valuable, tractable, preclinical models for human nutrition studies since they share many characteristics of gastrointestinal physiology, immunology and gut microbiology. Thus, results from this pig study will be highly translatable for human healthcare. This PhD will determine whether specific prebiotics and probiotics can reduce the negative impacts of high protein diets on intestinal barrier function in a sex-dependent manner.ObjectivesObjective 1: Determine if production of bacterial-derived metabolites linked with increased gut permeability (eg phenol, ammonia) are increased in line with increased non-hydrolysed mixed-source (eg whey, milk, soya, pea, fish) protein availability using verified continuous culture in vitro gut model systems inoculated with human faeces (Figure 1f). Objective 2: Identify prebiotics and probiotics (eg GOS, FOS, inulin, Bifidobacteria lactis, Lactobacillus casei) which have the most significant impact on reducing these bacterial metabolites associated with reduced barrier function using the in vitro system above.Objective 3: Explore if supernatants from the models above have a direct impact on the expression of tight cell junction (TCJ)-associated protein expression (eg ZO-1, E-cadherin) using a combination of human colonic cell lines and quantitative fluorescence immunohistology.Objective 4: Determine if the benefits of the prebiotic and/or probiotic identified in objective 2 can be observed in vivo using a pig model for humans (Figure 2a). Bacterial population dynamics, metabolite production and TCJ-associated protein expression (and immunity) will be assessed using 16S sequencing, gas-chromatography/mass spectroscopy and quantitative fluorescence immunohistology respectively. Quantification of the expression of immune-associated proteins will be achieved using fluorescence histology.Objective 5: Determine if there are sexually dimorphic effects in all the above using sex-balanced treatment and control groups throughout and by using 'sex' as a factor during statistical analyses.Objective 6: Statistical approaches will be used to generate a global overview of intestinal barrier function, the gut microbiota, immunity, host metabolism and host-microbe co-metabolism. This will generate mechanistic understanding of the biological processes associated with increased dietary protein, microbiota modification and determine whether sexual disparity occurs.