514 - The role of human Lactoferrin-derived peptide LFDP1 in suppression of ACSL4/LPCAT3 signaling relieves necrotizing enterocolitis in vitro and in vivo

Poster Number: 514
Publication Number: 514.426

Boshi Yu, Department of Pediatrics, Women's Hospital of Nanjing Medical University, Nanjing, Jiangsu, China (People's Republic); shuping Han, Maternity Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu, China (People's Republic); Zhangbin Yu, Maternity Hospital Affiliated to Nanjing Medical University, Nanjin, Jiangsu, China (People's Republic)

Background: A new peptide, LFDP1, derived from the human lactoferrin, which is highly expressed in preterm-human breast milk-derived exosomes.

Objective: Its role in necrotizing  enterocolitis(NEC) has not been disclosed till now. In the present study, we aim to assess the functions of LFDP1 in alleviating intestinal injury in the NEC rat models and cellular viability, proliferation, migration and apoptosis of FHC and IEC6 cells.

Design/Methods: Cellular viability was assessed by Cell Counting Kit-8, the EdU Kit was used to evaluate cellular proliferation, cell migration was determined by scratch-wound healing assay and apoptosic rate of FHC and IEC6 cells was examined using FITC Annexin-V staining followed by flow cytometry. Newborn Sprague–Dawley rats were randomized into four groups. Intestinal  tract  samples were stained with hematoxylin and eosin (HE) for microscopic evaluation and protein expression of tissues were immunohistochemically determined. Gene expression analysis and identification of differentially expressed genes in FHC cells treated with LFDP1 were evaluated by RNA sequencing. To identify protein-peptide interactions, pulldown assays were performed. The protein expression of FHC and IEC6 cells were evaluated by western blotting.

Results: LFDP1 could facilitate the proliferation, stimulate migration and suppress apoptosis of LPS-induced FHC and IEC6 NEC cells model. Administration of LFDP1 (5mg/kg) along with formula protected the villous integrity from injury in the NEC rat model. Furthermore, the neonatal rats with NEC showed a significant reduction in protein expression of TLR4 and zonula occludens 1 which was restored in LFDP1 treated animals. RNA sequencing showed the biosynthesis of unsaturated fatty acids was significantly enriched. Biotin-LFDP1 pull-down followed by silver staining exhibited the LPCAT3-interacting proteins. LPS-induced FHC and IEC6 NEC cells model showed an elevated protein expression of LPCAT3 and ACSL4 and a decreased protein expression of GPX4.That expression pattern was validated in human NEC intestinal samples.Conclusion(s): LFDP1 facilitated the proliferation, stimulated migration and suppressed apoptosis of LPS-induced NEC cells model and protected the villous integrity from injury in the NEC rat model. Preliminary studies on the mechanism showed LFDP1 relieves necrotizing enterocolitis via ACSL4-LPCAT3 pathway which had a close relationship with ferroptosis. Therefore, the next research direction can be the relationship of NEC and ferroptosis. Furthermore, we could study the effects of other breast milk active ingredients on NEC via different mechanisms of action.