Necroptosis Inhibition Preserves Diaphragm Function in Experimental Sepsis

Diaphragm dysfunction in sepsis remains a critical challenge in intensive care, yet its underlying mechanisms are poorly understood. This study investigates the role of necroptosis, a recently recognized form of programmed cell death, in sepsis-induced diaphragm dysfunction. A cecal ligation and puncture model in C57BL/6 mice was employed to induce sepsis. Diaphragm function was assessed through ultrasound imaging and pulmonary function testing. Necroptosis markers [receptor-interacting protein kinase (Ripk)-1, Ripk3, and mixed-lineage kinase domain-like protein (Mlkl)] and inflammatory cytokines [Il-6, tumor necrosis factor (Tnf)-α] were quantified using real-time quantitative RT-PCR, Western blot analysis, and enzyme-linked immunosorbent assay. The effect of the necroptosis inhibitor necrostatin-1 (Nec-1) was evaluated in vivo and in vitro. Septic mice exhibited significant diaphragm dysfunction correlated with an elevated expression of necroptosis markers and inflammatory cytokines in diaphragm tissue. Nec-1 treatment not only suppressed nec-roptosis but also markedly improved diaphragm function and respiratory parameters. In vitro, peri-toneal lavage fluid from septic mice induced necroptosis in C2C12 myotubes, an effect mitigated by Nec-1. The findings unveil necroptosis as a key player in sepsis-induced diaphragm dysfunction. A novel mechanism is proposed in which Tnf-α, produced by activated peritoneal macrophages, triggers diaphragm necroptosis. This study not only advances the understanding of the pathophysiology of sepsis but also identifies necroptosis inhibition as a promising therapeutic strategy for preserving diaphragm function in sepsis. (Am J Pathol 2025, 195: 2373-2386; https://doi.org/10.1016/j. ajpath.2025.08.013) Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, remains a significant global health challenge associated with high morbidity and mortality rates. 1 Among the various complications associated with sepsis, diaphragm dysfunction has emerged as a crucial factor contributing to poor outcomes in critically ill patients. The impairment of diaphragm function can lead to prolonged mechanical ventilation, increased intensive care unit stay, and higher mortality rates. 1-3 The pathophysiology of sepsis-induced diaphragm dysfunction is complex and multifaceted. While previous research has focused on the roles of inflammation, oxidative stress, and proteolysis in muscle wasting, the underlying cellular mechanisms remain incompletely understood. Recent evidence has suggested that pathways for programmed cell death may play a crucial role in sepsis-induced muscle injury. 4-6 Among these, necroptosis, a regulated form of necrotic cell death, has gained attention due to its involvement in various inflammatory conditions. 7,8 Necroptosis is mediated by a signaling cascade involving receptor-interacting protein kinase (Ripk)-1, Ripk3, and mixed-lineage kinase domain-like protein (Mlkl). Unlike apoptosis, necroptosis results in cell membrane rupture and the release of damage-associated molecular patterns, potentially amplifying the inflammatory response. While necroptosis has been implicated in multiple-organ