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。屏障功能的丧失（肠漏）导致这些分子进入血液，慢性低度炎症和心血管疾病[1,2]。一些膳食蛋白质到达结肠，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，这已被证明在体外降低屏障功能[3,4]，但在体内没有。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利用性对与肠漏相关的细菌代谢物产生的影响[5,6]。然而，结果是不一致的，并且通常使用水解蛋白，这些蛋白不能到达结肠。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，根据蛋白质来源的不同，蛋白水解细菌衍生代谢物的产生存在高度显著的差异（图1a）。我们已经确定了在体外对高水平蛋白质的反应中，微生物群组成和代谢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的影响在体内是高度性别依赖的（图1e）。猪是人类营养研究的有价值的、易驾驭的临床前模型，因为它们具有胃肠道生理学、免疫学和肠道微生物学的许多特征。因此，从猪的研究结果将高度转化为人类医疗保健。本博士将确定特定的益生元和益生菌是否能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。目的1：通过接种人粪便的体外连续培养肠道模型系统，确定与肠道通透性增加相关的细菌衍生代谢物（如苯酚、氨）的产生是否与非水解混合来源（如乳清、牛奶、大豆、豌豆、鱼）蛋白质可用性的增加一致（图1f）。目的2：利用上述体外系统鉴定对降低这些与屏障功能降低相关的细菌代谢物有最显著影响的益生元和益生菌（如GOS、FOS、菊粉、乳酸双歧杆菌、干酪乳杆菌）。目的3：结合人结肠细胞系和定量荧光免疫组织学，探讨上述模型上清液是否对紧密细胞连接（TCJ）相关蛋白（如ZO-1、E-cadherin）表达有直接影响。目标4：确定目标2中确定的益生元和/或益生菌的益处是否可以通过猪模型在体内观察到（图2a）。细菌种群动态、代谢物产生和tcj相关蛋白表达（和免疫）将分别使用16S测序、气相色谱/质谱和定量荧光免疫组织学进行评估。定量免疫相关蛋白的表达将实现荧光组织学。目的5：在统计分析中使用“性别”作为一个因素，通过性别平衡处理和对照组，确定是否存在上述所有性别二态效应。目标6：统计方法将用于产生肠道屏障功能，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况。这将产生与膳食蛋白质增加、微生物群改变相关的生物学过程的机制理解，并确定性别差异是否发生。

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.