Limb Ischemia-Reperfusion Injury in Rats is partially mediated by enhancing Necroptosis via Phosphorylation of RIPK1 and MLKL

Limb ischemia-reperfusion injury (L-IRI) causes severe skeletal muscle damage and functional impairment through activation of regulated cell-death and inflammatory pathways. Necroptosis is a regulated form of necrotic cell death mediated through the receptor-interacting protein kinase 1 (RIPK1)-RIPK3-mixed lineage kinase domain-like protein (MLKL) signaling axis. Prior experimental studies have shown that Necrostatin-1 (Nec-1) attenuates acute skeletal muscle ischemia-reperfusion injury in rats. However, how Nec-1-sensitive protection relates to RIPK1/MLKL-associated signaling, circulating muscle injury markers, inflammatory responses, histological damage, and functional recovery during the early postischemic period remains incompletely defined. In this study, we used a rat model of unilateral hindlimb ischemia involving 3 hours of ischemia followed by reperfusion for up to 7 days to evaluate these interrelated responses. Nec-1 was administered before ischemia and after reperfusion. L-IRI resulted in elevated plasma creatine kinase and lactate dehydrogenase levels, severe histopathological injury, increased TUNEL staining, impaired grip strength, and reduced Modified Tarlov scores. These changes were accompanied by increased phosphorylation of RIPK1 and MLKL, activation of inflammatory signaling pathways, and altered expression of injury-associated inflammatory mediators. Nec-1 treatment reduced RIPK1/MLKL-associated signaling, inflammatory mediator expression, circulating muscle injury markers, and histological injury while preserving muscle architecture and improving functional recovery. These findings support and extend previous evidence that Nec-1-sensitive necroptosis-associated signaling contributes to early skeletal muscle injury following L-IRI. Pharmacologic targeting of regulated necrotic and inflammatory pathways warrants further investigation as a potential adjunct strategy for limiting acute skeletal muscle injury after limb reperfusion.