Structural characterization of PAMK and its protective effect against thymic necroptosis in goslings

PAMK are heterogeneous glycans with immunomodulatory potential, but their structural basis and mechanisms in regulating thymic injury remain unclear. Structural characterization revealed that PAMK is mainly composed of arabinose, galactose, rhamnose, galacturonic acid, and glucose, featuring a dominant backbone consisting of →4)-α-D-GalpA-(1→, →4)-β-D-Galp-(1→, →2)-α-L-Rhap-(1→, and →3,4)-α-D-GalpA-(1→ with highly branched side chains. This complex structure provides the biochemical foundation for its bioactivity. To explore its immunoprotective effects, we established a CTX-induced thymic necroptosis model in goslings. CTX markedly disrupted cytokine profiles and activated necroptosis signaling, accompanied by downregulation of novel-miR-2 and upregulation of TRADD and MLKL. Dietary PAMK supplementation significantly restored cytokine homeostasis and inhibited necroptosis pathway activation. Mechanistically, PAMK upregulated novel-miR-2, which negatively regulated TRADD and MLKL expression, thereby attenuating necroptosis signaling. Overexpression of novel-miR-2 suppressed TRADD/MLKL-mediated necroptosis, whereas its inhibition produced the opposite effect. Furthermore, PAMK mitigated oxidative stress and necroptotic cell death induced by TRADD and MLKL overexpression. Collectively, our findings demonstrate that the defined structural features of PAMK underpin its ability to protect against CTX-induced thymic necroptosis by upregulating novel-miR-2 and suppressing the TRADD/MLKL axis. This work links PAMK's composition to its immunoprotective function and highlights its potential as a safe immunoregulatory agent in poultry.