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

ABSTRACT

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.

TBI induces NETs formation, as evidenced by the upregulation of PAD4 and MPO. NETs activate the necroptosis pathway via phosphorylation of RIP1‐RIP3‐MLKL, thereby exacerbating secondary brain injury (neuronal death, BBB disruption, and neuroinflammation). Inhibition of NETs or necroptosis significantly alleviates these pathological changes, but combined inhibition shows no synergistic effect. These findings demonstrate that NETs aggravate TBI‐induced secondary injury partially through the necroptosis pathway.

1. Background

TBI is a major global public health concern affecting many countries, including China (Gao et al. 2020). Worldwide, TBI is one of the leading causes of death and disability, particularly among young and middle‐aged populations (Dewan et al. 2019). It is estimated that approximately 69 million people worldwide suffer from TBI each year, with TBI accounting for a significant proportion (GBD 2016 Traumatic Brain Injury and Spinal Cord Injury Collaborators 2019). The harm caused by TBI is particularly evident, leading to not only high mortality rates but also long‐term neurological dysfunction, placing a heavy burden on patients and their families (Jiang et al. 2019). The initial damage from TBI is only the beginning, as subsequent secondary injuries play a crucial role in patient prognosis. These types of secondary injuries mainly include brain edema, neuronal death, blood–brain barrier (BBB) disruption, and inflammatory responses (Jamjoom et al. 2021). In‐depth research into the mechanisms and intervention strategies of TBI‐induced secondary injury is crucial for improving patient prognosis.

Neutrophil extracellular traps (NETs) are reticular suprastructures released into the extracellular space by neutrophils upon activation, with core components including depolymerized chromatin DNA, histones, and granular proteins like MPO, neutrophil elastase (NE), and matrix metalloproteinase‐9 (MMP‐9). Their unique reticular structure can physically “trap” pathogens, and at the same time kill pathogens through the cationic toxicity of histones and the enzymatic activity of granular proteins, thus effectively limiting the spread of infection (Papayannopoulos 2018). However, this defensive mechanism often exhibits a pathological shift in central nervous system (CNS) diseases. In multiple sclerosis (MS), NETs can accelerate the demyelination process of nerve fibers through histone‐mediated myelin damage and MPO‐induced oxidative stress (Quan et al. 2025; Sadeghi et al. 2023). In ischemic brain injury (IBI), the DNA fragments released by NETs can activate the cGAS‐STING pathway in microglia, amplify neuroinflammation, and impede vascular recanalization (Wang et al. 2021; Sun et al. 2023; Hu et al. 2025). Furthermore, NETs can also damage the integrity of the BBB, promote the infiltration of peripheral inflammatory cells into the brain, and further aggravate neurological function damage (Tang et al. 2023; Shi et al. 2023).

The formation of NETs is the result of neutrophils undergoing “NETosis,” a specific programmed cell death process (Thiam et al. 2020). This process is initiated by the activation of NADPH oxidase (NOX), which catalyzes the production of superoxide anions to trigger a reactive oxygen species (ROS) burst. Subsequently, calcium signals activate PAD4; PAD4 catalyzes the citrullination of arginine residues in histones, disrupting the electrostatic interaction between histones and DNA, and further promoting the transformation of chromatin from a compact heterochromatin state to a loose euchromatin state. Finally, depolymerized chromatin and granular proteins are released into the extracellular space through nuclear membrane rupture and assemble to form functional NETs. Notably, the NETosis process is accompanied by the selective degradation of cytoplasmic and nuclear components, and the continuous generation of ROS is crucial for maintaining the structural stability and functional activity of NETs; if PAD4 activity is inhibited or extracellular DNA is degraded, the formation of NETs will be significantly blocked (Papayannopoulos 2018; Chen et al. 2022). Although the role of NETs in various CNS diseases has been extensively studied, their specific role in TBI still needs further investigation. Existing research primarily focuses on NETs exacerbating inflammation and tissue damage in disease processes, but there is still insufficient evidence on whether NETs can improve the prognosis of TBI through necroptosis pathways. Moreover, the exact mechanisms of NETs’ formation and their functional regulation in TBI need further study.

Necroptosis is an active and orderly cell death process that is significantly associated with inflammation and immune responses (Meier et al. 2024). Since 2005, when the process and its specific inhibitor, necrostatin‐1 (Nec‐1), were first clearly described, necroptosis has become a major focus in cell biology and CNS diseases research (Degterev et al. 2005). The signaling pathways and mechanisms of necroptosis can be roughly divided into several major steps. The first step is the signal induction phase, where many different external signals, including tumor necrosis factor (TNF), interferons (IFN), and certain pathogen components, can trigger necroptosis (Hu et al. 2022; Ye et al. 2023). These signals are then transmitted through the interaction of receptor‐interacting protein kinase 1 (RIP1) and receptor‐interacting protein kinase 3 (RIP3), forming a RIP1‐RIP3 complex, also known as the “death complex” or “necrosome.” Upon activation, RIP3 further phosphorylates and activates the mixed lineage kinase domain‐like pseudokinase (MLKL), which forms pores in the cell membrane, leading to ion imbalance and cell swelling rupture (Pasparakis and Vandenabeele 2015). In CNS diseases such as cerebral ischemia, cerebral hemorrhage, and MS, necroptosis promotes neuronal cell death, leading to severe inflammatory responses and secondary damage (Caccamo et al. 2010; Ito et al. 2016). Studies have shown that Nec‐1 can effectively inhibit the occurrence of necroptosis by specifically inhibiting RIP1 kinase activity, significantly reducing pathological damage and inflammatory responses in various disease models, enhancing tissue survival rate, and improving prognosis (Newton et al. 2016). Current studies have preliminarily revealed the potential role of necroptosis in TBI (Wang et al. 2024; Hu et al. 2022). However, the signaling pathways of necroptosis in TBI, especially its interaction with other forms of cell death, still require further investigation.

NETs and necroptosis play a crucial role in exacerbating secondary brain injury after TBI. Therefore, this study aims to investigate the role of NETs in brain injury and neurological dysfunction following TBI (Gao et al. 2020); determine the impact of NET inhibition on neuronal necroptosis after TBI (Dewan et al. 2019); and explore the potential mechanisms by which NETs regulate neuronal necroptosis and influence brain injury following TBI (GBD 2016 Traumatic Brain Injury and Spinal Cord Injury Collaborators 2019). In this study, controlled cortical impact (CCI) mice were used to simulate clinical TBI. The PAD4 inhibitor Cl‐amidine and NETs‐degrading agent DNase I were employed to inhibit NETs, and the RIP1 inhibitor Nec‐1 was used to block the necroptosis pathway (Chen et al. 2022; Chen et al. 2021).

2. Materials and Methods

2.1. Animals

Male C57BL/6J mice, aged 8 weeks and weighing 20–25 g, were selected for the study. Before the experiments began, the mice were acclimated in a controlled environment with a humidity range of 50%–60%, a temperature range of 20°C–24°C, and a 12‐h light/dark cycle. They had ad libitum access to food and water for at least 1 week to ensure proper adaptation to the housing conditions.

2.2. TBI Model

The mice were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally). After achieving anesthesia, a longitudinal skin incision was made to expose the skull. A 3.5 mm × 3.5 mm bone window was created over the left parietal bone, 2 mm lateral to the sagittal suture and 2 mm posterior to the fontanelle. The bone fragment was carefully removed to avoid damaging the dura mater. These mice were then positioned in a stereotactic frame, and the controlled cortical impact (CCI) model was employed using a 3 mm flat‐headed impactor. The impact parameters were set as follows: a depth of 2 mm, velocity of 6 m/s, and a contact duration of 200 ms, resulting in an infarct volume of approximately 30%–40% (Ito et al. 2016). During the procedure, the core body temperature of the animals was maintained at 37°C ± 0.5°C using a heating pad. After the TBI procedure, anesthesia was discontinued, the incision was closed, and the mice were ventilated with 100% oxygen until they recovered spontaneous breathing. In the sham‐operated group, a similar craniotomy procedure was performed without the application of CCI.

2.3. Drug Administration

The PAD4 inhibitor Cl‐amidine (506282, Millipore) was prepared by dissolving it in DMSO (Sigma‐Aldrich), followed by dilution in saline (5% v/v) for intraperitoneal (i.p.) injection at a dose of 50 mg/kg. Administration began 10 min post‐TBI and continued daily until the animals were sacrificed. DNase I (human recombinant, Roche, 11284932001) was reconstituted in sterile water to a concentration of 10 mg/mL, further diluted in PBS, and administered intravenously (i.v.) at a dose of 10 mg/kg, starting 24 h after TBI and repeated every 12 h until sacrifice on Day 3. Necrostatin‐1 (MedChem Express) was dissolved in DMSO and administered i.p. at 50 mg/kg, as a single intraperitoneal injection 1 h after surgery.

2.4. Neurological Score Assessment

All experimental mice underwent behavioral training for 3 days prior to the controlled cortical impact (CCI) procedure, which ensured a modified neurological severity score (mNSS) between 0 and 1 at the start of the experiment. After 72 h, we measured the mNSS in mice from the sham‐operated, control, and treatment groups. The mNSS evaluation covers four areas: motor function (muscle condition and abnormal movements), sensory function (vision and touch), reflexes, and balance. Each abnormal behavior or failed reflex test scores 1 point. A total score of 0 indicates normal function, 1–6 indicates mild neurological impairment, 7–12 indicates moderate impairment, and 13–18 indicates severe impairment. See details in Table S1.

2.5. Measurement of Brain Edema

In the experiment, mice were deeply anesthetized with 1.5% isoflurane gas to ensure they did not feel any pain. They were then humanely euthanized by decapitation. Afterward, the cranial cavity was quickly opened to extract cortical tissue samples from the impacted hemisphere. The wet weight (Ww) of the fresh brain tissue was measured using a high‐precision electronic balance to ensure data accuracy. These cortical samples were then heated in a 100°C oven for 48 h to ensure complete drying. Once dried, the dry weight (Dw) was measured using the same high‐precision electronic balance. The brain water content (H₂O%) was then calculated using the following formula: $H20\%=(1-\frac{Dw}{Ww})\times 100\%$.

2.6. ELISA

To evaluate the inflammatory response in the cortex surrounding the injury site in mice, this study used the ELISA method to detect the levels of TNF‐α and IL‐1β. The experimental time point was set at 3 days after TBI. Mice were anesthetized and euthanized, after which their brains were harvested, and the cortical tissue on the injured side was isolated. Protein samples were obtained through processing; following protein quantification, operations were performed in accordance with the manufacturer's instructions of the ELISA detection kit (Boster Biological Technology, Wuhan, China). The absorbance was measured using a microplate reader, and the concentration of inflammatory factors was calculated accordingly.

2.7. Immunofluorescence

The mice were humanely euthanized under anesthesia and analgesia. Subsequently, a cardiac perfusion with phosphate‐buffered saline (PBS, pH 7.4) was performed, followed by a perfusion of 4% paraformaldehyde. The brains were sectioned coronally at a thickness of 8 µm using a cryostat set to −20°C, and the slices were transferred to slides pre‐coated with poly‐L‐lysine. To eliminate any residual medium or contaminants, the slides were washed thoroughly with PBS. The sections were then fixed with 4% paraformaldehyde for 15 min at room temperature to preserve cellular integrity and stabilize antigens. After fixation, the slides were treated with 0.5% Triton X‐100 for 10–15 min at room temperature to permeabilize the membranes, allowing for effective antibody penetration. The slides were subsequently incubated overnight at 4°C with primary antibodies targeting MPO (1:400, ab208670, Abcam), PAD4 (1:400, ab96758, Abcam), and NeuN (1:500, ab104224, Abcam), enabling specific binding to the target proteins. Following primary antibody incubation, the slides were washed with PBS and exposed to a secondary antibody conjugated to a fluorophore (BA1032, Boster, Wuhan, China) for 2 h at room temperature. This secondary antibody was selected for its strong compatibility with the primary antibody and its capacity to emit fluorescence upon excitation. To visualize the nuclei, the slides were incubated with DAPI (4',6‐diamidino‐2‐phenylindole) for 5–10 min. DAPI binds specifically to DNA, producing blue fluorescence signals. A blocking solution was then applied to prevent non‐specific binding and reduce background fluorescence. Finally, the slides were examined under a Nikon Fi3 biomicroscope, which was equipped with the appropriate filters for fluorescence detection. Fluorescent images were captured to evaluate the localization and expression of the target proteins, as well as the overall cell morphology.

2.8. Western Blot Analysis

Western blot analysis was conducted using protein samples extracted from the ipsilateral cortical tissue of mice. Equal amounts of protein (8 µg per lane) were loaded onto 10% SDS‐PAGE gels for separation and subsequently transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk prepared in TBST (Tris‐buffered saline with 0.1% Tween‐20) at room temperature for 1 h. They were then incubated overnight at 4°C with the following primary antibodies: PAD4 (1:1000, 214810, Abcam), MPO (1:1000, ab208670, Abcam), Bcl‐2 (1:1000, A0208, Abclonal), Bax (1:1000, A19684, Abclonal), RIP1 (1:1000, #3493, Cell Signaling Technology), RIP3 (1:1000, #95702, Cell Signaling Technology), MLKL (1:1000, #37705, Cell Signaling Technology), P‐RIP1 (1:1000, #31122, Cell Signaling Technology), P‐RIP3 (1:1000, #91702, Cell Signaling Technology), P‐MLKL (1:1000, #37333, Cell Signaling Technology), and GAPDH (1:1000, AB‐P‐R001, GOODHERE Biotech). Following the primary antibody incubation, membranes were treated with an HRP‐conjugated secondary antibody (GB23303, Servicebio, 1:10,000) for 1 h at room temperature. Chemiluminescence was used to detect the protein bands, utilizing an ECL detection reagent. GAPDH served as a loading control to normalize the data.

2.9. TUNEL Staining

Mice were anesthetized, the right ventricle severed, and a needle inserted into the left atrium. PBS buffer perfusion was then performed, followed by fixation in 4% paraformaldehyde. After removal, brain tissue was fixed in 4% paraformaldehyde for 24 h and subsequently embedded in paraffin. Coronal sections (5 µm thick) were prepared at the injury site. For cell samples, slides were immersed in 4% paraformaldehyde solution and fixed at room temperature for 15 min, followed by three PBS washes. DNA fragmentation in tissue sections or cell samples was detected using the cell death detection kit (E‐CK‐A322, Elabscience, Wuhan, China) as per the manufacturer's instructions. Briefly, samples were permeabilized with 0.2% Triton X‐100 (prepared in PBS) at room temperature for 20 min. Deparaffinized tissue sections or cell samples were then incubated with a working solution containing TdT enzyme and labeled dUTP at 37°C in the dark for 60 min. After incubation, the nuclei were counterstained with diluted DAPI solution, followed by slide mounting. For the neuronal samples, double staining with NeuN (red) and TUNEL (green) was performed. Fluorescent imaging was carried out using an inverted fluorescence microscope (e.g., Nikon Fi3 or Olympus). TUNEL‐positive cells were manually counted using ImageJ software. For tissue samples, apoptotic cells were quantified as TUNEL‐positive cells per square millimeter, with data analysis conducted in a blinded manner.

2.10. Statistical Analysis

Data are expressed as mean ± SD or SEM, as appropriate. All statistical analyses were conducted using GraphPad Prism 9.0.0 (GraphPad Software, San Diego, CA, USA). Normality was tested with the Shapiro–Wilk test, excluding the sham group. For multiple comparisons, one‐way ANOVA with Tukey's post hoc test was used, and for two‐group comparisons, unpaired Student's t‐test (two‐tailed) or Welch's t‐test was applied. Non‐parametric data, such as mNSS test results, were analyzed using the Kruskal–Wallis test with Dunn's multiple comparisons. All experiments were performed in triplicate, with the number of repeats indicated in the figure legends. Statistical significance was set at p < 0.05.

3. Results

3.1. Time‐Dependent Expression of PAD4 and MPO in the Cerebral Cortex Following TBI

We induced TBI in mice and analyzed their cerebral cortex at 1, 3, 5, and 7 days post‐injury. PAD4 is a crucial histone‐modifying enzyme that plays an essential role in NETs’ formation. Using PAD4 antibodies, we observed a time‐dependent increase in the expression of the NETs marker PAD4 in the ipsilateral cerebral cortex compared to sham‐operated mice, peaking at 3 days post‐injury (p < 0.001; Figure 1A). Similarly, the total content of neutrophil enzyme MPO in the cortical region significantly increased, also peaking at 3 days post‐injury (p < 0.001; Figure 1B). To further confirm these findings, we performed immunofluorescence co‐staining for MPO and PAD4 (Figure 1C). MPO is a core marker of neutrophils, and neutrophils can be efficiently localized via immunofluorescence staining (Li et al. 2025). The results showed that the expression of neutrophils in the ipsilateral cortex increased in a time‐dependent manner and reached a peak on the 3rd day after injury (p < 0.001; Figure 1D). Likewise, the co‐staining findings demonstrated that the expression of PAD4 displayed a similar trend as well (p < 0.001; Figure 1E). In summary, both Western blot and immunofluorescence analyses confirm that MPO and PAD4 expression peaks at Day 3 after injury, providing a basis for further research using this time point.

FIGURE 1.

Dynamic expression of PAD4 and neutrophils after TBI over time. (A) Western blot analysis of PAD4 expression at different time points (sham, 1, 3, 5, and 7 days). The lower panel shows the quantitative analysis of PAD4 levels normalized to β‐actin (n = 4). (B) Western blot analysis of MPO expression at different time points. The lower panel shows the quantitative analysis of MPO levels normalized to β‐actin (n = 4). (C) Immunofluorescence staining of PAD4 (red) and MPO (green) in tissue sections at various time points. DAPI staining (blue) was used to label cell nuclei. Scale bar = 50 µm. (D) Percentage of neutrophils in the tissue at different time points (n = 4). (E) Percentage of PAD4+ neutrophils at different time points. Data are expressed as mean ± SD. Significant differences are indicated (*P < 0.05, **p < 0.01, ***p < 0.001).

3.2. NETs Exacerbate Brain Injury Following TBI

To investigate the role of NETs in secondary brain injury after trauma, we administered Cl‐amidine every 24 h, starting 10 min post‐injury, for 3 consecutive days. In addition, the DNA‐degrading enzyme DNase I was injected via the tail vein every 12 h. To further assess neuronal survival, we performed NeuN and TUNEL immunofluorescence co‐staining (Figure 2A). The results showed that compared with the TBI+Vehicle group, the Cl‐amidine and DNase I treatment groups exhibited a significant reduction in neuronal apoptosis on the 3rd day after TBI (p < 0.001; Figure 2B). To further corroborate the anti‐apoptotic effects of NETs inhibition, we performed Western blot analysis to assess the relative protein expression levels of Bcl‐2 (an anti‐apoptotic marker) and Bax (a pro‐apoptotic marker), and calculated the Bcl‐2/Bax ratio as a critical indicator of apoptotic balance (Figure 2C,D). Consistent with the TUNEL staining results, the TBI+Vehicle group exhibited a significantly reduced Bcl‐2/Bax ratio compared to the sham‐operated group (p < 0.001), reflecting enhanced apoptotic signaling following TBI. In contrast, treatment with Cl‐amidine or DNase I markedly elevated the Bcl‐2/Bax ratio (p < 0.05 for Cl‐amidine; p < 0.01 for DNase I), indicating that NETs inhibition shifts the apoptotic balance toward anti‐apoptosis. Results from the TBI model showed that the brain edema content in the TBI+Vehicle group was significantly higher than in the sham‐operated group. Treatment with Cl‐amidine or DNase I significantly reduced brain edema content, although it remained higher than that in the sham‐operated group (p < 0.001; Figure 2E). To evaluate the effects of Cl‐amidine and DNase I on short‐term neurological recovery, we performed mNSS tests on the experimental groups. By the 3rd day post‐injury, mice in the TBI+Vehicle group exhibited significant neurological deficits compared to the sham‐operated group. The mNSS results (p < 0.001; Figure 2F) confirmed the successful establishment of the CCI‐induced TBI model. In contrast, treatment with Cl‐amidine or DNase I significantly reduced mNSS scores, indicating their neuroprotective effects (p < 0.001; Figure 2F). Western blot analysis of the BBB‐associated protein ZO‐1 revealed that its expression was significantly reduced in the TBI+Vehicle group. However, Cl‐amidine and DNase I treatments significantly increased ZO‐1 expression levels (p < 0.001; Figure 2G,H). Finally, ELISA analysis showed that inflammatory factors TNF‐α and IL‐1β were significantly elevated in the TBI group compared to the sham‐operated group. Treatment with Cl‐amidine or DNase I significantly reduced the levels of TNF‐α and IL‐1β (p < 0.05; Figure 2I,J).

FIGURE 2.

Protective effects of Cl‐amidine and DNase I treatment on TBI‐induced brain injury. (A) Representative immunofluorescence images showing DAPI (nuclei, blue), NeuN (neuronal marker, green), TUNEL (apoptotic cells, red), and merged channels in the pericontusional cortex. Scale bar = 50 µm (n = 4 per group). (B) Quantification of TUNEL‐positive apoptotic cells in the pericontusional cortex. (C) Western blot analysis of Bcl‐2 and Bax protein levels in the cortex of mice from Sham, TBI+Vehicle, TBI+Cl‐amidine, and TBI+DNase I groups. GAPDH was used as the loading control (n = 4 per group). (D) Quantification of the relative Bcl‐2/Bax ratio normalized to β‐actin. (E) Quantification of brain edema content (%) in sham, TBI+Vehicle, TBI+Cl‐amidine, and TBI+DNase I groups (n = 4 per group). (F) Neurological function was assessed using the mNSS test across experimental groups (n = 10 per group). (G) Western blot analysis of ZO‐1 protein levels in the cortex of mice from Sham, TBI+Vehicle, TBI+Cl‐amidine, and TBI+DNase I groups. β‐actin was used as the loading control (n = 4 per group). (H) Quantification of relative ZO‐1 protein levels normalized to β‐actin. (I and J) ELISA analysis of TNF‐α (I) and IL‐1β (J) levels in the pericontusional cortex (n = 4 per group). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with indicated groups.

3.3. Inhibiting NETs Will Reduce the Expression of Proteins in the Necroptosis Pathway

To investigate the effect of NETs on the necroptosis signaling pathway and assess whether inhibiting NETs can reduce the expression of key proteins in this pathway. To this end, we used two NETs inhibitors, Cl‐amidine and DNase I, to evaluate their regulatory effects on this signaling pathway. Western blot analysis showed that the protein expression levels of RIP1, RIP3, and MLKL were significantly higher in the TBI+Vehicle group compared to the sham‐operated group (p < 0.001; Figure 3B–D), and their phosphorylated forms (P‐RIP1, P‐RIP3, P‐MLKL) were also markedly upregulated, indicating that TBI can activate the necroptosis signaling pathway (p < 0.001; Figure 3E–G). After treatment with Cl‐amidine and DNase I, the expression levels of RIP1, RIP3, MLKL, and their phosphorylated forms decreased (p < 0.05; Figure 3B–G).

FIGURE 3.

Effects of Cl‐amidine and DNase I on necroptosis‐related protein expression after TBI. (A) Western blot analysis of RIP1, RIP3, MLKL, P‐RIP1, P‐RIP3, and P‐MLKL protein levels in the cortex of mice from Sham, TBI+Vehicle, TBI+Cl‐amidine, and TBI+DNase I groups. GAPDH was used as the loading control (n = 4 per group). (B–G) Quantification of relative protein levels of RIP1, RIP3, MLKL, P‐RIP1, P‐RIP3, and P‐MLKL, normalized to GAPDH. Data are shown as mean ± SD (n = 4 per group). Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, compared with the indicated groups; ns: not significant.

3.4. NETs Exacerbate Brain Injury After TBI Through the Necroptosis Pathway

To investigate the role of NETs in exacerbating brain injury via the necroptosis pathway after TBI, we repeated the relevant experiments and administered the necroptosis inhibitor Nec‐1 every 24 h starting 1 h post‐injury for 3 consecutive days. To further confirm that NETs exacerbate brain injury after TBI through the necroptosis pathway, we used Cl‐amidine and DNase I, each combined with Nec‐1, for treatment. Results from immunofluorescence co‐staining of the neuronal marker NeuN and TUNEL showed that Nec‐1 treatment could reduce neuronal apoptosis, and combined treatment of Nec‐1 with Cl‐amidine or DNase I also similarly alleviated neuronal apoptosis (p < 0.001; Figure 4A,B). Consistent with the TUNEL staining results, the Western blot analysis of TBI + Vehicle group showed a significantly decreased Bcl‐2/Bax ratio compared with the sham‐operated group (p < 0.001; Figure 4C,D), indicating enhanced apoptotic activation after TBI. Treatment with Nec‐1 alone significantly elevated the Bcl‐2/Bax ratio (p < 0.05), while there was no statistical difference in the Bcl‐2/Bax ratio between the Nec‐1 monotherapy group and the combined treatment groups (p > 0.05; Figure 4C,D). Subsequently, We analyzed the brain water content of mice at 3 days after TBI. The results showed that the brain edema content in the TBI + Vehicle group was significantly higher than that in the sham‐operated group and the TBI+Nec‐1 group (p < 0.001; Figure 4E). Additionally, results showed no statistical significance in the comparison between the group with single inhibition of necroptosis and the group with dual inhibition of necroptosis and NETs (Figure 4E). We used the mNSS test to evaluate the short‐term neurological function recovery. On the 3rd day post‐injury, mice in the TBI+Vehicle group exhibited significant neurological deficits compared to the sham‐operated group (p < 0.001; Figure 4F). In comparison with the TBI+Vehicle group, the group with sole inhibition of necroptosis and the dual inhibition group both exhibited decreased mNSS scores (p < 0.001; Figure 4F), while no statistical significance was found between these three groups (Figure 4F). Western blot results showed that compared with the TBI+Vehicle group, Nec‐1 treatment alleviated the reduction of ZO‐1 protein, and the combined treatment of Nec‐1 with Cl‐amidine or DNase I also alleviated the reduction of ZO‐1 protein (p < 0.001; Figure 4G,H). Finally, to evaluate inflammation, we used ELISA to measure TNF‐α and IL‐1β levels. The results showed that both inflammatory factors were significantly elevated in the TBI group compared to the sham‐operated group (p < 0.001; Figure 4I,J). The TBI+Nec‐1 group and the combined treatment group of Nec‐1 with Cl‐amidine or DNase I significantly reduced the levels of TNF‐α and IL‐1β (p < 0.05; Figure 4I,J), and there was no statistical difference among these groups (Figure 4H,I).

FIGURE 4.

Effects of Nec‐1, Cl‐amidine, and DNase I on brain injury and inflammation following TBI. (A) Representative immunofluorescence images showing DAPI (nuclei, blue), NeuN (neuronal marker, green), TUNEL (apoptotic cells, red), and merged channels in the pericontusional cortex. Scale bar = 50 µm (n = 4 per group). (B) Quantification of TUNEL‐positive apoptotic cells in the pericontusional cortex. (C) Western blot analysis of Bcl‐2 and Bax protein levels in the cortex of mice from Sham, TBI+Vehicle, TBI+Nec‐1, TBI+Nec‐1+Cl‐amidine, and TBI+Nec‐1+DNase I groups. GAPDH was used as the loading control (n = 4 per group). (D) Quantification of the relative Bcl‐2/Bax ratio normalized to β‐actin. (E) Brain edema content (%) in the Sham, TBI+Vehicle, TBI+Nec‐1, TBI+Nec‐1+Cl‐amidine, and TBI+Nec‐1+DNase I groups (n = 4 per group). (F) Neurological function assessed by the mNSS test across experimental groups (n = 10 per group). (G) Western blot analysis of ZO‐1 protein expression in experimental groups, with β‐actin as a loading control (n = 4 per group). (H) Quantification of ZO‐1 protein levels normalized to β‐actin. (I and J) ELISA analysis of TNF‐α (I) and IL‐1β (J) levels in the pericontusional cortex. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the indicated groups; ns: not significant.

4. Discussion

TBI is a major global cause of neurofunctional impairment and death, and its secondary injury—including neuroinflammation, disruption of the BBB, and cell death—represents a key bottleneck for improving prognosis. Among these secondary injury types, the inflammatory response is particularly complex and destructive. Various cells involved in the inflammatory response and the signaling molecules they release interact, continuously amplifying the extent of secondary damage (Simon et al. 2017). Despite some progress in the pathophysiology and treatment of TBI, many unresolved issues remain. Currently, research primarily focuses on early intervention in pathological processes, BBB protection, and anti‐inflammatory treatments (Donnelly and Popovich 2008). Neutrophils, serving as core effector cells of the body's innate immunity, assume a dual role in the maintenance of CNS homeostasis and pathological responses, and exert an irreplaceable function, especially in the immune defense against acute injuries (such as trauma and ischemia) and infectious diseases (Kolaczkowska and Kubes 2013). When the CNS is exposed to stresses such as mechanical injury, pathogen invasion, or ischemia‐hypoxia, the permeability of the BBB increases significantly (Shafqat et al. 2023). Neutrophils in the peripheral circulation rapidly migrate across the membrane to the affected area, release various cytokines and chemicals, phagocytose bacteria, and clear necrotic tissue to fight infection and promote tissue repair (Kolaczkowska and Kubes 2013; Shafqat et al. 2023). However, excessive activation and inappropriate presence of neutrophils may lead to secondary tissue damage and chronic inflammation (Jenne and Kubes 2013). The physiological functions of neutrophils are achieved through a series of complex mechanisms (Kolaczkowska and Kubes 2013). They capture and eliminate pathogens directly through phagocytosis and release active substances, including proteases and antimicrobial peptides, through degranulation (Kolaczkowska and Kubes 2013). In addition to relying on classical phagocytosis and degranulation for their immune effects, neutrophils use the formation of NETs as an important supplementary mechanism to resist pathogens (Papayannopoulos 2018).

NETs are crucial in the pathology of TBI, as their formation exacerbates brain damage and significantly impairs neurological recovery. NET formation arises from the execution of NETosis in neutrophils, a process triggered by NOX activation and the subsequent induction of a ROS burst (Azzouz and Palaniyar 2024). PAD4 is a calcium‐dependent enzyme that deiminates arginine residues in histones, converting them into citrulline (Wang et al. 2009). This process regulates chromatin decondensation and the formation of NETs. Subsequently, NETosis induces the release of extracellular DNA, histones, and granzymes through the formation of NETs, thereby enhancing the innate immune response, which in turn leads to an excessive inflammatory environment (Schiuma et al. 2022; Li et al. 2010). This inflammatory cascade aggravates secondary brain injury, leading to increased brain edema and enhanced neuronal death. Notably, the sustained generation of ROS during NETosis is not merely a “passive byproduct” but is also closely associated with the dysregulation of antioxidant pathways in neutrophils. Nuclear factor erythroid 2‐related factor 2 (Nrf2) acts as the key transcription factor governing cellular antioxidant responses. Through binding to the antioxidant response elements, ARE, of downstream target genes including heme oxygenase‐1 (HO‐1) and NAD(P)H quinone oxidoreductase 1 (NQO1), it promotes ROS clearance and thereby suppresses NETosis at its origin (Zhou et al. 2018; Shi et al. 2025). Recent studies by Shi et al. in TBI models have further revealed the upstream regulatory pathway between NETs and Nrf2: following TBI, S100A8/A9, also known as calprotectin, secreted by neutrophils can inhibit AMPK phosphorylation, block Nrf2 nuclear translocation, thereby impair its antioxidant activity and ultimately leading to ROS accumulation and PAD4‐mediated NET formation (Shi et al. 2025). Additionally, NETs compromise the BBB, allowing inflammatory factors like TNF‐αf and IL‐1β to infiltrate brain tissue, thereby intensifying the inflammatory response (Liu et al. 2016; Leshner et al. 2012; Rohrbach et al. 2012). Triggering receptor expressed on myeloid cells 1 (TREM1) is an inflammatory receptor expressed on endothelial cells, neutrophils and microglia, and serves as a key downstream target in NETs‐mediated inflammatory responses (Wu et al. 2021; Wang et al. 2025). Post‐TBI, NETs promote the expression of adhesion molecules such as intercellular adhesion molecule‐1 (ICAM‐1) in endothelial cells (Wang et al. 2025). This enhances neutrophil adhesion to and infiltration across the endothelium, while aggravating endothelial damage. In experimental SAH models, TREM1 engages and recruits its downstream signaling effector spleen tyrosine kinase (SYK) (Wu et al. 2021). Such recruitment triggers the activation of both the Card9‐NF‐κB cascade and the PAD4‐NETs axis, thereby facilitating microglial polarization toward a proinflammatory phenotype characterized by increased expression of CD68, CD16 and CD86, and augmenting NET formation (Wu et al. 2021). Administration of TREM1 inhibitory peptide (LP17) to block TREM1 signaling results in the suppression of the above pathways, accompanied by diminished microglial proinflammatory transition and reduced NET generation, which ultimately mitigates the magnitude of neuroinflammation (Wu et al. 2021). Conversely, NET stimulation of endothelial cells provokes the intracellular accumulation of membrane‐localized TREM1 and the assembly of a stable complex between TREM1 and NF‐κB. This complex acts in synergy to amplify TLR4‐mediated inflammatory responses, stimulates endothelial secretion of endothelin‐1 (ET‐1), a potent vasoconstrictor, and inhibits the synthesis of nitric oxide (NO), a key vasodilator (Wang et al. 2025). The combined effects give rise to cerebral vasospasm (CVS), a critical pathological trigger for delayed cerebral ischemia following TBI. In addition, NETs induced after TBI can also upregulate the expression of Z‐DNA binding protein 1 (ZBP1) in endothelial cells (Zhu et al. 2025). ZBP1 interacts directly with ferroptosis suppressor protein 1 (FSP1) via its RIP homotypic interaction motif (RHIM) domain, and subsequently promotes the downregulation of FSP1. Such FSP1 deficiency directly initiates ferroptotic cell death in endothelial cells, culminating in endothelial disruption (Zhu et al. 2025). This is accompanied by decreased expression of the tight junction proteins ZO‐1 and Occludin in the BBB, as well as a marked increase in barrier permeability. These combined mechanisms contribute to the worsening of secondary injury following TBI. To explore PAD4 changes in the cortex after TBI, we first determined the peak expression time of PAD4, finding that its expression in neutrophils peaked at 72 h post‐TBI. We hypothesized that 72 h is the optimal time point for evaluating the therapeutic effects in the TBI mouse model. PAD4 catalyzes histone citrullination to facilitate the formation of NETs, whereas DNase I mitigates the inflammatory effects of NETs through degrading extracellular DNA, which is a key component of NETs. Inhibiting NETs, through agents such as the Cl‐amidine or DNase I, significantly mitigates TBI‐induced pathological changes, including reduced brain edema, preserved neuronal survival, restored BBB function, and decreased inflammatory factor release. This suggests that NETs are not only key players in TBI pathology but also potential therapeutic targets. In the acute phase of TBI, strategies aimed at inhibiting NETs may offer new neuroprotective approaches by blocking inflammation and reducing tissue damage (Li et al. 2010; Fattahi and Ward 2017).

This study focused on the crosstalk regulation between NETs and necroptosis in TBI. NETosis and necroptosis share significant similarities in terms of molecular mechanisms, particularly in ROS generation, inflammation, and signaling pathways. ROS is pivotal in NETosis formation by driving NETs release, and it also mediates necroptosis by transmitting cell death signals (Azzouz and Palaniyar 2024; Voronina et al. 2024; Ferrada et al. 2020). Additionally, NETs trigger the inflammatory response through the cGAS‐STING pathway, which has been shown to worsen acute lung injury (Zhao et al. 2023). The cGAS‐STING pathway not only plays a crucial role in NETosis but is also closely linked to necroptosis, regulating cell death and inflammation (Yang et al. 2022). Studies indicate that extracellular DNA released by NETs induces necroptosis in alveolar epithelial cells via this pathway, further exacerbating lung injury (Sha et al. 2024).

Our findings that NETs inhibition reduces necroptosis‐related protein expression and pro‐inflammatory cytokine TNF‐α levels raise the possibility that NETs may modulate necroptosis through the cGAS‐STING/TNF‐α pathway, yet these findings require further direct validation. Previous studies have established that extracellular DNA, a core component of NETs, can act as a damage‐associated molecular pattern (DAMP) to activate the cGAS‐STING pathway in microglia and endothelial cells (Wang et al. 2021; Zhao et al. 2023; Bhatia et al. 2025). Activation of this pathway not only amplifies neuroinflammation by promoting type I interferon production but also induces the release of pro‐inflammatory cytokines such as TNF‐α (Luo et al. 2025; Willemsen et al. 2021)—a well‐characterized trigger of necroptosis that initiates the RIP1‐RIP3‐MLKL signaling cascade (Xu et al. 2024). Moreover, in neurons following TBI, extracellular DNA secreted by NETs interacts with neuronal surface receptors, which facilitates the association between Neural Precursor Cell Expressed Developmentally Down‐Regulated Protein 8 (NEDD8) expressed in neural precursor cells and the ubiquitin ligase TRIM56 (Zhang et al. 2025). This complex subsequently mediates K63‐linked ubiquitination of STING, triggering activation of the NF‐κB pathway and resultant massive secretion of the proinflammatory cytokines TNF‐α and IL‐1β (Zhang et al. 2025; Fang et al. 2017). Thus, we hypothesized that NETs may serve as a key upstream signal, activating the RIP1‐RIP3‐MLKL cascade through the aforementioned inflammatory pathways and ultimately leading to the occurrence of necroptosis. In our study, we observed that inhibition of NETs via Cl‐amidine or DNase I concurrently reduced TNF‐α levels and key necroptosis‐related protein expression including RIP1, RIP3, MLKL, and their phosphorylated forms in the cerebral cortex after CCI, which aligns with the upstream role of the cGAS‐STING/TNF‐α axis in regulating cell death. These observations collectively provide indirect support for the potential involvement of this pathway in mediating NETs‐induced necroptosis after TBI. Moreover, compared with the solo application of the necroptosis inhibitor Nec‐1, the combined inhibition of necroptosis and NETs did not lead to further improvement in brain injury indicators, also suggesting a regulatory association between NETs and the necroptosis pathway. This finding aligned with existing literature, which demonstrates that NETs contribute to cell death and tissue damage in various inflammatory diseases by amplifying inflammation (Islam and Takeyama 2023). Both approaches effectively prevent excessive activation of the necroptosis pathway. These results suggest that inhibiting NETs’ formation not only reduces inflammatory factor release but may also alleviate secondary brain injury by blocking downstream cell death signals. NETs may aggravate the pathological progression of TBI via activation of the necroptosis pathway, whereas suppression of NETs may partially mitigate necroptosis by regulating the cGAS‐STING/TNF‐α pathway. This suggests that the necroptosis pathway serves as a key mediator underlying the regulatory effect of NETs on secondary brain injury after TBI. However, it is critical to acknowledge the speculative nature of this hypothesis, as direct molecular evidence remains lacking. Future investigations are warranted to characterize critical signatures of cGAS‐STING pathway activation in the injured cortex, including cGAS phosphorylation, STING and its downstream molecule IRF3, and to validate whether blockade of the cGAS‐STING pathway or neutralization of TNF‐α is sufficient to abrogate the pro‐necroptotic effect mediated by NETs.

This study is the first to uncover the interaction between NETs and the necroptotic pathway in TBI, indicating that targeting these pathways may offer new therapeutic strategies. Several limitations still warrant further investigation: First, reverse validation involving inhibition and overexpression is typically used in downstream mechanism research. However, the absence of significant synergistic effects following the dual inhibition we employed can also demonstrate that NETs may participate in post‐TBI neural injury partially through the necroptosis pathway. Although this dual inhibition method is less robust in terms of evidential strength compared to reverse validation, it is still applied in research on mechanisms related to many molecules that lack specific agonists or present difficulties in overexpression. Second, this study employed a mouse TBI model, which, while capable of simulating key pathological features of TBI, does not fully replicate the complexity of human TBI, particularly the neurodegenerative changes during the chronic phase. Future research should validate these mechanisms in human TBI patient samples or other animal models. Lastly, the study primarily focused on the acute phase of TBI (within 3 days) and did not assess the impact of NETs inhibition on long‐term neurological recovery, including motor coordination, spatial memory, cognition, and behavior. Extending the observation period in future studies is essential to explore the role of NETs and necroptosis in the chronic phase.

5. Conclusion

The present study explores the role of NETs in the regulation of necroptosis in TBI and reveals for the first time that NETs aggravate secondary brain injury partly via the necroptosis pathway. The results showed that NETs amplify the inflammatory response, impair the BBB, and trigger neuronal apoptosis, thus exacerbating cerebral edema and neurofunctional deficits. Inhibiting NETs or necroptosis can both significantly alleviate secondary brain injury, but the combined treatment shows no synergistic effect, suggesting that there are complex interactions between the pathways. This study addresses a research gap in understanding NETs' role in TBI pathology and offers a new perspective on their interaction with necroptosis. This extends the role of NETs from merely amplifying inflammation to activating cell death signals, paving the way for research on the relationship between NETs and other forms of cell death. Future studies need to further clarify the molecular pathway connections between NETs and necroptosis, optimize spatiotemporally specific combined treatment strategies, and provide new insights for the immunomodulatory therapy of TBI.

Author Contributions

A.L., T.W.P., and J.F. contributed equally to this work. T.C. designed and formulated the review theme and revised and finalized the manuscript. A.L. and T.W.P. searched and reviewed the literature and drafted the manuscript. J.F., H.Q., and L.B.S. discussed and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82072168), the Natural Science Foundation of Jiangsu Province (No. BK20211044), the top talent support program for young and middle‐aged people of Wuxi health committee (BJ2023111), and the Wuxi Science and Technology Development Fund (K20231051).

Ethics Statement

All animal procedures were approved and supervised by the Animal Ethics Committee of Anhui Medical University (No.20211012).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Table: brb371275‐sup‐0001‐TableS1.docx

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.