334 - Exosome-derived peptide IGKVDP of human milk in the protection against experimental Necrotizing Enterocolitis

Necrotizing enterocolitis is a common inflammatory disease in newborns, especially premature infants. Human milk exosomes have been shown to have the potential to prevent NEC. Our research group has previously validated the function of breast milk exosome-derived peptides in the prevention and treatment of NEC. According to the preliminary experiment, we screened out an immunoglobulin-derived peptide IGKVDP using mass spectrometry. 

Objective: The aim of this study was to investigate the effect and mechanism of IGKVDP on NEC.

Design/Methods:

1、The peptide was modified with a transmembrane sequence, which was thought to improve the capacity of transmembrane. FITC-conjugated IGKVDP were prepared to detect the intracellular localization in IEC-6.

2、 At in vivo level, we conduct NEC animal model through cold stimulation, hypoxia, and hypertonic milk gavage for 4 days, Immediately after killing, the terminal ileum was harvested and divided into two parts to assess the severity of disease and detect the inflammatory level through H&E staining and immunohistochemical techniques.

3、 At the cellular level, IEC-6 were pretreated with IGKVDP for 2h, followed by LPS for 48h. The expression levels of inflammatory cytokines were measured by Quantitative real-time PCR. Western blot was used to detect the expression of intestinal barrier protein.

4、Further mechanisms of IGKVDP were obtained using mRNA biogenic analysis.

Results:

1、 FITC-labeled peptides were enriched in the cytoplasm of IEC-6.

2、 We found that external intervention of IGKVDP can effectively improve the survival rate, abdominal distension, hematochezia in NEC models. At the same time, intestinal histological injury was improved, and NEC pathological score was decreased, along with the decreased expression of pro-inflammatory factor and the increased expression of intestinal barrier proteins.

3、LPS-induced inhibition of proliferation and migration was improved by IGKVDP and improved barrier damage in IEC-6. The pro-inflammatory factors TLR4, TNF-α, IL-1β and phosphorylation of IκBα and NF-κB were inhibited by IGKVDP.

4、 Pathway analysis of differentially expressed genes showed that there were significant differences in IκB/NF-κB signaling between NEC group and IGKVDP group. We used IGKVDP to regulate the IEC-6 treated by PMA (NF-ΚB activators) and found that the phosphorylation of NF-κB was significantly reduced, suggesting that IGKVDP could inhibit the IκBα/NF-κB signaling pathway.

Conclusion(s):

In conclusion, IGKVDP may improve the development of NEC by inhibiting the IκB/NF-κB signaling pathway. Therefore, IGKVDP is expected to be a treatment for NEC.