The role of gut microbiota in mediating tissue injury through the promotion of proteolysis and heme bioavailability in the small intestine

Background: Excessive meat intake is considered to be associated with multiple intestinal injuries. However, the specific mechanism is still controversial.
Results: It was found that pork diet (typical meat diet) only induced small intestinal injury in specific-pathogen free (SPF) mice but not in germ-free (GF) mice, highlighting the indispensable role of gut microbiota in intestinal health. ELISA and RT-qPCR indicated that the intestinal tissue in GF mice had lower levels of heme and Hmox1 compared with the SPF mice, suggesting the low bioavailability of myoglobin without symbiotic microbes. High throughput sequencing showed that dietary myoglobin and heme effected intestinal flora diversity, with Lactobacillus, Muribaculaceae and Bifidobacterium being enriched by myoglobin. Additionally, the enrichment of protease and peptidase by myoglobin demonstrated the regulated proteolytic capacity of small intestinal microbiota. In contrast with hemoglobin, the degradation of myoglobin and subsequent heme release must be initiated by gastric digestion and ultimately enhanced by gut microbes under a simulated condition. Peptidomics revealed the peptide-shortening role of gut microbes in digestion. Both the inhibition of gastric digestion by esomeprazole and clearance of gut microbes by amoxicillin hindered heme absorption in mice that were orally administered with myoglobin. Moreover, the incubation of human intestinal epithelial cells (INT 407) with microbial hydrolysate of myoglobin and peptide-heme mixture induced the accumulation of intracellular iron and cell death which were found to be dependent on heme carrier and PEPT1. Inhibitor screening indicated that although utilization and metabolism of free heme was independent of endocytosis, F-actin-mediated endocytosis inhibited the bioavailability of peptide-heme mixture and downregulated ferritin.
Conclusions: These findings describe the direct role of small intestinal microbes in host digestion and give a new insight into the underlying mechanism of bioavailability of dietary myoglobin.

研究背景：过量肉类摄入被认为与多种肠道损伤存在关联，但其具体致病机制仍存在争议。
研究结果：研究发现，典型肉类饮食（猪肉饮食）仅在无特定病原体（specific-pathogen free, SPF）小鼠中诱导小肠损伤，而在无菌（germ-free, GF）小鼠中未引发此类损伤，这凸显了肠道菌群对肠道健康的不可或缺作用。酶联免疫吸附实验（ELISA）与实时定量聚合酶链式反应（RT-qPCR）结果显示，相较于SPF小鼠，GF小鼠的肠道组织中血红素与血红素加氧酶1（Hmox1）水平更低，表明在缺乏共生微生物的情况下，肌红蛋白的生物利用度较低。高通量测序结果表明，膳食肌红蛋白与血红素可影响肠道菌群多样性，其中乳杆菌属（Lactobacillus）、鼠杆菌科（Muribaculaceae）与双歧杆菌属（Bifidobacterium）可被肌红蛋白富集。此外，肌红蛋白可富集蛋白酶与肽酶，这证实小肠菌群的蛋白水解能力受到调控。与血红蛋白不同，肌红蛋白的降解及后续血红素释放必须先经过胃部消化启动，最终在模拟实验条件下由肠道菌群进一步增强。肽组学（peptidomics）分析揭示了肠道菌群在消化过程中缩短肽链的作用。无论是通过埃索美拉唑（esomeprazole）抑制胃部消化，还是通过阿莫西林（amoxicillin）清除肠道菌群，均会阻碍经口给予肌红蛋白的小鼠对血红素的吸收。此外，将人肠道上皮细胞（INT 407）与肌红蛋白的微生物水解物及肽-血红素混合物共孵育，可诱导细胞内铁积累与细胞死亡，且该过程依赖于血红素载体与肽转运体1（PEPT1）。抑制剂筛选实验显示，尽管游离血红素的利用与代谢不依赖于内吞作用，但F-肌动蛋白（F-actin）介导的内吞作用会抑制肽-血红素混合物的生物利用度，并下调铁蛋白（ferritin）的表达。
研究结论：本研究揭示了小肠菌群在宿主消化过程中的直接作用，并为膳食肌红蛋白的生物利用度相关潜在机制提供了全新视角。