Study on the Function and Mechanism of Neutrophil Extracellular Traps in Regulating Necroptosis Following Traumatic Brain Injury

Purpose: Traumatic brain injury (TBI) remains a major global public health challenge with high morbidity and mortality, and secondary injury characterized by neuroinflammation, brain edema, and neuronal cell death is a critical determinant of patient prognosis. Neutrophil extracellular traps (NETs) and Necroptosis are involved in TBI pathology, but their crosstalk remains unclear. Here, we used NETs inhibitors (Cl-amidine and DNase I) and the Necroptosis inhibitor Necrostatin-1 (Nec-1) to investigate the roles of NETs and Necroptosis in neuronal injury following TBI.

Method: Male C57BL/6J mice were used to establish a TBI model via controlled cortical impact (CCI). Cl-amidine, DNase I, and Necrostatin-1 were administered to explore the mechanism by which NETs regulate Necroptosis and exacerbate TBI-induced secondary injury. The modified neurological severity score (mNSS) assessment, brain edema measurement, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence staining, and TUNEL staining were performed in this study. Mice were sacrificed at 1, 3, 5, and 7 days post-TBI, with Day 3 post-TBI designated as the key time point for primary analyses due to the peak expression of NETs markers: myeloperoxidase (MPO) and peptidyl arginine deiminase 4 (PAD4).

Finding: Our results showed that TBI induced a time-dependent upregulation of MPO and PAD4 in the ipsilateral cortex. Inhibition of NETs or blockade of Necroptosis significantly reduced neuronal Apoptosis, alleviated brain edema, improved mNSS scores, preserved blood-brain barrier integrity, and decreased levels of pro-inflammatory cytokines (TNF-α, IL-1β). Western blot analysis revealed that TBI markedly upregulated the expression of RIP1, RIP3, MLKL, and their phosphorylated forms, while NETs inhibition downregulated these necroptosis-related proteins. Notably, combined inhibition of NETs and Necroptosis did not exert synergistic protective effects on TBI-induced brain injury.

Conclusion: NETs exacerbate TBI-induced secondary brain injury partially by activating the Necroptosis pathway. Inhibition of NETs exerts neuroprotective effects. Targeting NETs may serve as a promising therapeutic strategy to improve prognosis in TBI patients.