[Effect of gut microbiota homeostasis on hematopoiesis in a neonatal rat model of necrotizing enterocolitis]

Abstract

Objectives: To study the effect of gut microbiota on hematopoiesis in a neonatal rat model of necrotizing enterocolitis (NEC).

Methods: Neonatal Sprague-Dawley rats were randomly divided into a control group and a model group (NEC group), with 6 rats in each group. Formula milk combined with hypoxia and cold stimulation was used to establish a neonatal rat model of NEC. Hematoxylin and eosin staining was used to observe the pathological changes of intestinal tissue and hematopoiesis-related organs. Routine blood tests were conducted for each group. Immunohistochemistry was used to observe the changes in specific cells in hematopoiesis-related organs. Flow cytometry was used to measure the changes in specific cells in bone marrow. 16S rDNA sequencing was used to observe the composition and abundance of gut microbiota.

Results: Compared with the control group, the NEC group had intestinal congestion and necrosis, damage, atrophy, and shedding of intestinal villi, and a significant increase in NEC histological score. Compared with the control group, the NEC group had significantly lower numbers of peripheral blood leukocytes and lymphocytes (P<0.05), nucleated cells in the spleen, thymus, and bone marrow, and small cell aggregates with basophilic nuclei in the liver (P<0.05). The NEC group had significant reductions in CD71⁺ erythroid progenitor cells in the liver, CD45⁺ lymphocytes in the spleen and bone marrow, CD3⁺ T lymphocytes in thymus, and the proportion of CD45⁺CD3^-CD43⁺SSC^hi neutrophils in bone marrow (P<0.05). There was a significant difference in the composition of gut microbiota between the NEC and control groups, and the NEC group had a significant reduction in the abundance of Ligilactobacillus and a significant increase in the abundance of Escherichia-Shigella (P<0.05), which replaced Ligilactobacillus and became the dominant flora.

Conclusions: Multi-lineage hematopoietic disorder may be observed in a neonatal rat model of NEC, which may be associated with gut microbiota dysbiosis and abnormal multiplication of the pathogenic bacteria Escherichia-Shigella.

Keywords: Gut microbiota; Hematopoietic disorder; Necrotizing enterocolitis; Neonatal rat.

图1. 两组新生大鼠肠组织外观和末端回肠的HE染色比较（×100） NEC组新生大鼠肠组织明显充血，呈黑色坏死样；NEC组新生大鼠肠组织肠绒毛破损、萎缩脱落。

图2. 两组新生大鼠造血相关器官HE染色变化 NEC组新生大鼠脾脏实质及胸腺皮质区有核细胞数量、肝脏组织中嗜碱性细胞核的小细胞聚集体数量及骨髓中有核细胞数量较对照组明显减少。

图3. 两组新生大鼠造血相关器官中特定细胞的免疫组化染色（×200） NEC组新生大鼠肝脏中CD71阳性的红系祖细胞聚集体数量、脾脏中CD45阳性的白细胞数量及胸腺中CD3阳性的T淋巴细胞数量较对照组减少。阳性表达呈棕色。

图4. 两组新生大鼠骨髓中特定细胞的流式分析结果（n=3） A：CD45⁺细胞比例。NEC组骨髓中CD45⁺白细胞比例较对照组降低；B：CD45⁺CD3⁺细胞比例。NEC组骨髓中CD45⁺CD3⁺T淋巴细胞比例较对照组降低；C：CD45⁺CD3^-CD43⁺SSC^hi细胞比例。NEC组骨髓中CD45⁺CD3^-CD43⁺SSC^hi中性粒细胞比例较对照组降低。a示与对照组比较，P<0.05。

图5. 两组肠道菌群Beta多样性分析 采用基于weighted uniFrac的距离矩阵进行主坐标分析。一个圆点表示一个样品，每组6个点代表6个样品。

图6. 门水平及属水平上两组肠道菌群相对丰度 左图：门水平上菌群相对丰度。NEC组肠道菌群中变形菌门、放线菌门的相对丰度明显增加，而厚壁菌门相对丰度明显减少。右图：属水平上菌群相对丰度。NEC组肠道菌群中利乳杆菌属相对丰度明显减少，而埃希菌-志贺菌属、葡萄球菌属相对丰度明显增加。

图7. 两组相对丰度排序前5的菌群分布情况 NEC组中埃希菌-志贺菌属取代利乳杆菌属成为优势菌属。