Abstract:

Objective To investigate the anti-inflammatory efficacy and the underlying molecular mechanisms of Thymosin α1 （Tα1） in a mouse model of necrotizing enterocolitis （NEC）. Methods Thirty 5-day-old specific pathogen-free （SPF） C57BL/6J mice were randomly divided into five groups： normal control group （GC）， necrotizing enterocolitis （NEC） model group， Tα1 treatment group （TF）， N-acetylcysteine （NAC） treatment group， and combined Tα1 and NAC treatment group （TF+NAC）. The NEC model was established via formula feeding combined with hypoxia stimulation and lipopolysaccharide （LPS） challenge. Starting from the fourth day of modeling， mice in each treatment group received daily intraperitoneal injections for 5 consecutive days. Intestinal injury was assessed via abdominal X-ray examination， hematoxylin-eosin （HE） staining， and fluorescein isothiocyanate-dextran 70kDa （FD70） intestinal permeability assay. The levels of pro-inflammatory cytokines including interleukin-6 （IL-6）， interleukin-17A （IL-17A） and tumor necrosis factor-α （TNF-α） were measured by enzyme-linked immunosorbent assay （ELISA）. The ratio of regulatory T cells （Treg） to T helper 17 cells （Th17） was analyzed by flow cytometry， and the expression levels of TLR4/MyD88/TRAF6/TRIF signaling pathway proteins were detected by Western blot. Results Compared with the GC group， the NEC group exhibited significant growth retardation， intestinal distension， pneumatosis intestinalis， and mucosal barrier damage， accompanied by increased pro-inflammatory cytokine concentrations， an imbalanced Treg/Th17 ratio， and upregulated expression of TLR4 signaling proteins （P < 0.05）. In contrast， the treatment groups showed attenuated intestinal injury， decreased pro-inflammatory cytokine concentrations， restored Treg/Th17 homeostasis， and downregulated expression of TLR4 signaling proteins （P < 0.05）. Notably， the combined treatment of TF and NAC exerted superior efficacy in alleviating inflammatory storms and correcting immune dysregulation （P < 0.05）. Conclusion Tα1 alleviates intestinal injury in NEC mouse models via inhibiting the TLR4 signaling pathway and correcting Treg/Th17 immune imbalance. Furthermore， the synergistic effect of Tα1 and cysteine offers a promising immunomodulatory strategy for the clinical management of NEC.

Key words: necrotizing enterocolitis, thymosin α1, Treg/Th17 balance, TLR4 signaling pathway, immunomodulation