Dysbiosis of the gut microbiota induces brain inflammation in neonatal necrotizing enterocolitis via the gut-brain axis

Abstract Background Necrotizing enterocolitis (NEC) is the most common fatal gastrointestinal emergency in premature infants. The intestinal damage caused by NEC can lead to bacterial and endotoxin translocation into the bloodstream, triggering systemic sepsis and significantly increasing the risk of mortality in infants with NEC. Even survivors often experience adverse outcomes such as neurological dysfunction. However, the underlying mechanisms related long-term neurological dysfunction in NEC remain incompletely understood. Neonatal intestinal colonization is affected by multiple factors, among which breastfeeding is a key factor in the composition of the intestinal microbiota in early life, helping to regulate the balance of the neonatal gut microbiota. Therefore, this study aims to investigate the impact of gut microbiota on brain inflammation in NEC. Methods Fecal samples from 18 infants with NEC and 29 healthy newborns were collected for 16S rRNA sequencing analysis to investigate differences in gut microbiota composition. 6 SD pregnant rats were randomly divided into 2 groups (normal water-fed group and antibiotic-fed group). A suspension containing 0.2 g/L ampicillin, 0.2 g/L metronidazole, 0.2 g/L neomycin, and 0.1 g/L vancomycin was provided for lactating female rats to drink freely to establish a pseudo-sterile rat model. The newborn pups were randomly divided into 2 groups: control group (normal breast milk feeding) and NEC group. A NEC model was established in 4-day-old pups through 4 consecutive days of hypoxia, cold stimulation, formula feeding, and lipopolysaccharide intervention. Western blot analysis was used to detect the expression levels of intestinal barrier proteins (ZO-1 and Occludin). Real-time quantitative polymerase chain reaction (qPCR) was used to detect changes in pro-inflammatory factors (IL-1β, TNF-α and IL-6) and immune regulatory markers (T-bet and GATA3) in the ileum and hippocampus. Results 16S rRNA sequencing results indicated that there were significant differences in intestinal microbiota composition between infants with NEC and the control group, especially a significant increase in Staphylococcus aureus and Enterococcus. The qPCR results of intestinal tissue and hippocampus showed elevated mRNA expression of IL-1β, TNF-α, and IL-6 in NEC group. Western blot results showed that the expression levels of tight junction proteins ZO-1 and Occludin in the intestinal of infants with NEC were significantly reduced. The bacterial culture results of blood and hippocampal homogenate from NEC rat showed that Staphylococcus aureus can cross the intestinal barrier into the bloodstream and penetrate the blood-brain barrier to enter brain tissue. The mRNA expression of IL-1β, TNF-α, and IL-6 in intestines and hippocampus was significantly reduced in NEC of the antibiotic-fed group. Compared with the normal water-fed NEC group, the transcription levels of T-bet and GATA3 were significantly reduced in antibiotic-fed group, indicated that the gut microbiota mediated the significant inflammatory activation observed in the normal feeding group. Conclusion NEC brain inflammation is closely related to the gut microbiota, and antibiotic intervention in lactating mother rats can reduce intestinal and hippocampal inflammatory responses in offspring with NEC. (Acknowledgements: This study was supported by National Natural Science Foundation of China，Grant No. 32370139 to Hongying Fan)