Mice and experimental design of NEC

C57BL/6 mice were obtained from the Experimental Animal Center of Southern Medical University (Guangzhou, China) and were bred in a specific pathogen-free facility. All animal experiments were approved by the Institutional Animal Care and Use Committee of The Sixth Affiliated Hospital, Sun Yat-sen University (protocol number: 20191028-005). NEC was induced as described in our previous report ^{5, 21} in 10-day-old mouse pups via gavage feeding (five times daily) of formula [Similac Advance infant formula (Abbott Nutrition, Columbus, OH, USA): Esbilac (PetAg, Hampshire, IL, USA) milk replacer for puppies, 2:1] containing enteric bacteria from a patient with surgical NEC (12.5 µL original stool slurry in 1 mL formula). Mice were simultaneously exposed to hypoxic conditions (5% O₂, 95% N₂) for 10 min twice per day in a modular chamber (Billups-Rothenberg, San Diego, CA, USA) for 4 days.

The experimental pups were treated with melatonin once daily 1 h prior to the NEC procedure until the end of the experiment. Each mouse was intraperitoneally (i.p.) injected with melatonin (Cat #S1204, Selleck Chemicals, Shanghai, China) at 10 mg/kg body weight in a total volume of 100 μL; the melatonin was dissolved in a vehicle consisting of < 25% ethanol in phosphate-buffered saline (PBS; Cat #18912014, Thermo Fisher Scientific, PA, USA). For exogenous IL-17 administration experiments, mice were injected with 100 ng of rIL-17A (Cat #210-17, Peprotech, Rocky Hill, NJ, USA) once daily on postnatal day (P) 9 to P12 via i.p. injection. In Treg cell depletion experiments, aCD25 mAb (Cat #102002, BioLegend, San Diego, CA USA, clone: PC61) or control IgG1 (cIgG1, Cat #401902, BioLegend) was administered to mice at 10 µg/mouse via i.p. injection on P2 and P8. rIL-17A, aCD25 mAb, and cIgG1 were dissolved in PBS. To prevent SIRT1 and AMPK activation, Ex-527 (2 mg/kg body weight; Cat #S1541, Selleck Chemicals) and Compound C (20 mg/kg body weight; Cat #S7840, Selleck Chemicals) were also dissolved in the same vehicle as melatonin, and mice were i.p. injected with 100 μL of either agent once daily on P9, P10, P11, and P12. Control mice (BF group) were left with their dams to breastfeed and vehicle-treated NEC pups (VEH group) were i.p. injected with the vehicle as a control for melatonin (MEL group), melatonin + rIL-17 (NEC + MEL+ rIL-17 group), melatonin + aCD25 mAb (MEL+ aCD25 mAb group), melatonin + Ex-527 (MEL+ Ex-527 group), and melatonin + Compound C (MEL+ CC group) treatment. These pups were monitored closely and weighed on P10, P12, and P14. Animals were euthanized on P14 or earlier if they demonstrated NEC signs.