Traumatic brain injury (TBI) is defined as the structural and functional damage to the brain induced by external mechanical forces, resulting in temporary or permanent impairment of neural tissues, cognitive functions, and states of consciousness. Known as a “silent epidemic,” TBI is one of the top causes of illness, disability, and death worldwide across all age groups. Moreover, TBI represents the primary contributor to disability and death in all trauma-related injuries worldwide1,2. Current estimates suggest that 50–60 million individuals worldwide experience TBI annually. The high incidence, disability, and mortality rates of TBI pose a substantial burden on familial care systems and public healthcare infrastructure globally1,3. Unfortunately, current therapeutic strategies for TBI primarily rely on surgical intervention, organ function support, and rehabilitation therapies, with no effective pharmacotherapies currently available4,5.

The intestine serves as the “central organ” in the human stress response. Emerging evidence highlights that TBI is associated with profound morphological and functional alterations in the gut, including increased permeability, intestinal epithelial injury, inflammatory activation, and dysbiosis of the gut microbiota. This bidirectional communication network between the brain and the intestine is termed the gut-brain axis6,7,8,9,10. Following TBI, the gut-brain axis may mediate the impairment of intestinal barrier function. Intestinal barrier dysfunction not only initiates systemic inflammatory responses and multi-organ dysfunction but also contributes to the pathogenesis of “secondary brain injury.” Targeting the intestine as a therapeutic organ for TBI patients may not only restore intestinal homeostasis but also mitigate secondary brain injury and improve clinical outcomes11,12.

Lycopene (Lyc) is a fat-soluble hydrocarbon carotenoid predominantly extracted from plants such as tomatoes, watermelons, and papayas. Considerable research interest has been garnered owing to its broad-spectrum biological activities, encompassing singlet oxygen quenching, free radical scavenging, inflammatory response attenuation, immune enhancement, tumor cell proliferation inhibition, and a remarkable safety profile13,14. Lyc exerts neuroprotective effects in the nervous system by mitigating cerebral oxidative stress and inflammatory cascades15. Within the digestive system, Lyc enhances intestinal barrier integrity, suppresses inflammatory signaling, and reduces intestinal bacterial translocation16. Regarding the brain-gut axis, Lyc ameliorates high-fat diet-induced cognitive impairment in mice by alleviating inflammation along the intestine-liver-brain axis17. Moreover, preclinical studies have demonstrated that Lyc alleviates deoxyribonucleic acid-induced memory and behavioral deficits in mice by regulating the microbiota-short chain fatty acid-intestine-brain axis balance18. Previous studies by our research team revealed that Lyc improves intestinal barrier function in mice with TBI by reducing intestinal pro-inflammatory cytokine levels, although the underlying mechanisms require further elucidation.

Under physiological intestinal homeostasis, the intestinal mucosal epithelial cells maintain an immunotolerant state, thereby preventing unwarranted inflammatory activation. However, upon pathogen recognition or cellular damage via pattern recognition receptors (PRRs), these cells elicit inflammatory cascades that mediate host defense mechanisms19. Nucleotide-binding and leucine-rich repeat receptors (NLRs) constitute a pivotal PRR subgroup. Among them, NOD, LRR, and pyrin domain-containing protein 3 (NLRP3), a canonical member of the NLR family, serves as a critical sentinel for the innate immune response and is widely expressed in immune and intestinal mucosal epithelial cells20. NLRP3 functions as a molecular scaffold for cysteine-aspartic protease 1 (Caspase-1) cleavage and activation, driving the massive expression and bioactive processing of downstream inflammatory cytokines, including interleukin-18 (IL-18) and interleukin-6 (IL-6). This cascade potentiates the inflammatory response and pyroptotic cell death21. Mechanistically, pyroptosis involves three sequential steps: NLRP3 inflammasome assembly, caspase activation, and gasdermin D (GSDMD)-mediated pore formation, accompanied by the release of pro-inflammatory cytokines. Accumulating evidence indicates that the NLRP3 inflammasome is involved in neuronal injury after TBI and is also central to the regulation of the intestinal barrier through its associated cytokines22,23.

Moreover, the NLRP3 inflammasome exacerbates intestinal injury by promoting neutrophil infiltration and pyroptosis via the Caspase-1/IL-1β axis24. More recently, emerging research has demonstrated that Lyc suppresses NLRP3 inflammasome-mediated pyroptosis in the spleen, kidneys, pancreas, and microglia under various pathological conditions25,26,27,28. However, the role of Lyc in TBI-induced intestinal pyroptosis remains unclear. Therefore, the study focused on investigating the effects of Lyc on TBI-related intestinal barrier dysfunction and pyroptosis.

Materials and methods

Reagents

Lyc was purchased from MedChem Express (Monmouth Junction, USA). Sunflower oil was obtained from Arawana (Shanghai, China). Bone wax was purchased from Sanyou (Shanghai, China). Sodium pentobarbital, erythromycin ointment, absolute ethanol, paraffin, xylene, hematoxylin and eosin (H&E), hydrochloric acid, acetic acid, and neutral gum were purchased from Sinopharm (Beijing, China). Paraformaldehyde and an eco-friendly dewaxing agent were purchased from Service Bio (Wuhan, China). Tumor necrosis factor-α (TNF-α), IL-18, IL-6, intestinal fatty acid binding protein (I-FABP), d-lactic acid (D-LA), diamine oxidase (DAO), and endotoxin kit were purchased from Kelu (Wuhan, China). NLRP3 was purchased from Poly Ab (Västra Frölunda, Sweden), apoptosis-associated speck-like protein containing CARD (ASC) was purchased from AdipoGen (San Diego, USA), Caspase-1 was purchased from Cell Signaling Technology (Danvers, USA), and the GSDMD-N-terminal domain (GSDMD-N) was purchased from AFFINITY (Shanghai, China). β-actin, HRP-anti-rat IgG, and HRP-anti-mouse IgG were purchased from Proteintech (Rosemont, USA).

Animals

Normal male BALB/c mice (22–26 g, 8–10 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Within specific pathogen-free facilities, mice were housed in individually ventilated cages and provided with unlimited access to sterile food and water. The environmental conditions were controlled as follows: ambient temperature of 20–24 °C, relative humidity between 40 and 60%, and a 12 h/12 h light/dark cycle. The study was approved by the Experimental Animal Ethics Committee of Quanzhou Medical College (No. 2024065).

Mouse model of TBI

Feeney’s weight-drop contusion model, a well-known standardized stereotaxic method in neuroscience, was used to induce TBI29,30. Briefly, the mice were given an intraperitoneal injection of 3% pentobarbital sodium at a dose of 50 mg/kg to induce anesthesia. After skin sterilization and preparation, the mouse heads were positioned directly beneath the weight-drop apparatus. A craniotomy with a 5-mm diameter was conducted on the right parietal cortex, positioned between bregma and lambda. During the procedure, meticulous surgical care was taken to avoid dural injuries. A weight-drop device was then placed over the exposed dura, and a 20 g cylindrical impactor was dropped from a height of 30 cm, delivering a controlled cortical contusion to induce severe traumatic injury in the right parietal brain tissue. Subsequently, bone wax was used to seal the bone window, the scalp layers were sutured sequentially, erythromycin ointment was applied to the wound, and the mice were returned to their cages for postsurgical rearing. In the control group, the craniotomy was performed on mice without inducing a cortical contusion.

Grouping and administration

The experimental unit in this study was the individual animal. To reduce variability caused by stress, thirty mice were acclimated to the testing environment for seven days in a row. These mice were then randomly assigned to five different experimental groups (6 mice per group) using a computer-generated random number sequence: (1) Control (CON); (2) TBI; (3) TBI + low-dose Lyc (TBI + Lyc-L, 5 mg/kg/day); (4) TBI + middle-dose Lyc (TBI + Lyc-M, 10 mg/kg/day); and (5) TBI + high-dose Lyc (TBI + Lyc-H, 20 mg/kg/day). The sample size was determined in accordance with the 3R principles and minimal statistical requirements. Lyc was dissolved in sunflower oil at concentrations of 1, 2, and 4 mg/mL before use. One hour after TBI, Lyc solution or the same amount of sunflower oil (5 mL/kg/day) was given orally, and this treatment was maintained daily for 14 days. The allocator, outcome assessors, and data analysts remained blinded to the group assignments. The animals were examined twice daily by the researchers, with monitoring parameters including body weight, general behavioral status, food and water intake, and any abnormal physical signs. When the humane endpoint was reached, the animals were euthanized by cervical dislocation following anesthesia induced by an intraperitoneal injection of 3% sodium pentobarbital (50 mg/kg).

Sample collection

At the end of the administration, the mice were given an intraperitoneal dose of 3% pentobarbital sodium (50 mg/kg) for anesthesia. Blood was collected from the inferior vena cava into precooled vacuum tubes containing 0.25 M ethylenediaminetetraacetic acid. After blood collection, the intestinal tract was lifted to locate the ileocecal junction. Starting approximately 3 cm proximal to the ileocecal junction, two segments of the terminal ileum were sequentially excised toward the proximal end. The intestinal contents were removed, and the ileal tissue was rinsed with normal saline.

Serum was separated by centrifuging at 3000 × g for 15 min at 4 ℃, and the collected serum was promptly stored at − 80 ℃ for enzyme-linked immunosorbent assay (ELISA). One segment of the ileum was flash-frozen at − 80 ℃ for western blot analysis, while the other segment was immersed in 4% paraformaldehyde at 4 ℃ overnight for post-fixation. After specimen collection, the mice were euthanized by cervical dislocation.

ELISA

Following thawing, we isolated the serum and extracted intestinal proteins. TNF-α, IL-18, IL-6, I-FABP, D-LA, DAO, and endotoxin levels were determined using ELISA kits, with all procedures strictly adhering to the manufacturers’ protocols. The absorbance was measured at 450 nm with a reference wavelength of 630 nm, and standard curves were generated based on absorbance readings of known standards.

HE staining

Ileum tissues were fixed using 4% paraformaldehyde, dehydrated through a series of ethanol concentrations, cleared in xylene, embedded in paraffin, sliced into 3 μm sections, and stained with H&E for general morphological evaluation under a light microscope (×200 magnification). The severity of intestinal mucosal injury was evaluated using Chiu’s histological grading system (Chiu score). Ileal villus height and crypt depth were measured via a double-blind approach using the ImageJ software. In each microscopic field, measurements were taken from 10 consecutive villi at ×100 magnification, and the mean values were calculated.

Western blot

Ileum tissues (50 mg) were placed into 2 mL microtubes containing 800 µL of lysis buffer (1 mmol/L phenylmethylsulfonyl fluoride) and homogenized using a tissue grinder. The samples were placed on ice for 20–25 min, transferred to 1.5 mL microtubes and centrifuged at 12,000 × g for 30 min at 4 ℃. The supernatants were transferred to sterile 1.5 mL microtubes and immediately stored at − 80 ℃.

Proteins in equal quantities were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes, which were incubated overnight at 4 ℃ with antibodies targeting NLRP3, ASC, Caspase-1, GSDMD-N, and β-actin. The membrane underwent three washes with TBST and was then incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG, which was diluted at a 1:10000 ratio in TBST with 3% bovine serum albumin. After three additional washes with TBST to terminate the reaction, the reaction was visualized by enhanced chemiluminescence. The gel imaging system was used to acquire images, and ImageJ software was applied for densitometric analysis. The relative protein expression was calculated by dividing the gray value of the target protein by that of β-actin.

Study design and participants

A randomized controlled trial was conducted in the Department of Critical Care Medicine at Quanzhou First Hospital Affiliated to Fujian Medical University from 2 August 2023 to 5 March 2025. A total of 96 patients with TBI were included in the study. The clinical trial received approval from the Ethics Committee of Quanzhou First Hospital Affiliated to Fujian Medical University (No. QYL [2023] K056) and was first registered in the China Medical Research Registration and Filing Information System on 28/03/2024 (Registration Number: MR-35-24-017311). All participants provided written informed consent prior to joining the study. The criteria for inclusion were: (1) aged ≥ 18 years, regardless of sex; (2) confirmed history of head trauma and meeting the diagnostic criteria for TBI; (3) hospital admission within 12 h post-injury; (4) first-diagnosed patients without prior treatment for TBI; and (5) planned hospitalization duration ≥ 14 days. The criteria for exclusion were: (1) patients with other preexisting neurological diseases; (2) those with severe extracranial injuries; (3) individuals with systemic diseases or organ dysfunction; (4) patients with severe gastrointestinal dysfunction or intolerance to enteral feeding (oral/nasogastric tube); (5) those with unstable vital signs; (6) pregnant or lactating women; (7) individuals who had consumed foods or medications containing probiotics, antibiotics, or steroids within 2 weeks prior to enrollment; and (8) patients with missing clinical data > 20%.

Randomization and blinding

In total, 74 patients who were eligible were part of this study. They were randomly allocated to the TBI group or TBI + Lyc group in a 1:1 ratio using a computer-generated randomization sequence. The randomization sequence was generated by a statistician who was not involved in patient recruitment or data collection. The data collectors, outcome assessors, and data analysts remained blinded to the group assignments.

Treatment and management

All patients received standardized routine treatments, including dehydration for intracranial pressure reduction using dehydrating agents, endotracheal intubation and ventilator support, analgesia and sedation, infection prophylaxis, expectorant nebulization, enteral nutrition support, and rehabilitation therapy. Starting from day 1 post-enrolment, the TBI + Lyc group was administered 100 mL of tomato juice (containing approximately 10 mg Lyc per 100 mL) orally or via a nasogastric tube, whereas the TBI group was administered 100 mL of warm-boiled water using the same administration routes. Both groups received the intervention thrice daily (Lyc 30 mg/day) for 14 consecutive days.

Data collection

Data collected included demographic characteristics (sex, age, comorbidity), surgical parameters (surgery indication, start time, and duration), laboratory markers (C-reactive protein [CRP], procalcitonin [PCT]), gastrointestinal complications, intestinal function assessments (gastrointestinal function score and ultrasound), neurological deficit assessments (Helsinki CT and Glasgow Coma Scale [GCS] scores), and critical care severity indices (sequential organ failure assessment [SOFA], acute physiology and chronic health evaluation II [APACHE II], mortality risk, and length of intensive care unit [ICU] stay). Data on mortality-related indices (including total hospital stay, 28-day mortality, clinical outcomes, and Glasgow Outcome Scale [GOS] score) were also collected.

Statistical analysis

GraphPad Prism 9.0 and IBM SPSS Statistics 24.0 were utilized for statistical analyses. The Kolmogorov‒Smirnov test was used to evaluate the normality of the variables. Normally distributed continuous data were expressed as mean ± standard deviation and compared using an unpaired two-tailed t-test. One-way analysis of variance (ANOVA) was used for multiple group comparisons, and post-hoc tests were conducted for pairwise comparisons. Non-normally distributed continuous data were reported as median (interquartile range) and analyzed using the Mann–Whitney U test. Categorical variables were expressed as numbers (%) and compared using the χ2 test or Fisher’s exact test, depending on suitability. Statistical significance was set at a two-sided P < 0.05. Missing clinical data were handled using multiple imputation.

Results

Lyc improves inflammation and intestinal barrier function in mice with TBI

Figure 1 shows that Lyc improves inflammation and intestinal barrier function in mice with TBI. Compared with the CON group, ileum TNF-α, IL-18, and IL-6 levels in the TBI group, as well as serum I-FABP, D-LA, DAO, and endotoxin levels, were significantly increased. However, administration of Lyc reduced the levels of these markers in a dose-dependent manner. Compared with the TBI group, the TBI + Lyc-L, TBI + Lyc-M, and TBI + Lyc-H groups showed significant decreases in TNF-α, IL-18, IL-6, I-FABP, DAO, and endotoxin levels; the TBI + Lyc-M and TBI + Lyc-H groups exhibited reduced D-LA levels. Moreover, compared with the TBI + Lyc-L group, both the TBI + Lyc-M and TBI + Lyc-H groups exhibited further downregulation of IL-6, while the TBI + Lyc-H group showed significant reductions in TNF-α, IL-18, I-FABP, D-LA, DAO, and endotoxin levels. Furthermore, the TBI + Lyc-H group demonstrated further suppression of IL-18 when compared with the TBI + Lyc-M group (Fig. 1).

Fig. 1
Fig. 1
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Lyc improves inflammation and intestinal barrier function in mice with TBI. Levels of ileal TNF-α (A), IL-18 (B), IL-6 (C) and serum I-FABP (D), D-LA (E), DAO (F), Endotoxin (G) measured by ELISA. n = 3. Data are represented as mean ± SD. Compared with the CON group, ####P < 0.0001. Compared with the TBI group, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Compared with the TBI + Lyc-L group, &P < 0.05; &&P < 0.01; &&&P < 0.001; &&&&P < 0.0001. Compared with the TBI + Lyc-M group, ‡P < 0.01.

Lyc ameliorates ileal injury in mice with TBI

In the TBI group, the ileal villi exhibited significant shortening with a disrupted villus-to-crypt ratio. Partial villi exhibited shedding or necrosis accompanied by structural disruption of the lamina propria and submucosa, irregular cellular arrangement, and extensive inflammatory cell infiltration in the submucosa. Lyc administration ameliorated ileal injury in a dose-dependent manner, with the recovery becoming more pronounced at higher Lyc concentrations. Compared with the TBI group, the TBI + Lyc-L group showed partial restoration of villus height, reduced epithelial cell shedding, a mild increase in goblet cell number, and alleviated submucosal edema and congestion. The TBI + Lyc-M group demonstrated significant improvements in villus height and density, nearly normal vascular morphology, and the absence of submucosal bleeding or exudation. The TBI + Lyc-H group exhibited substantial villus height restoration, with a more orderly cellular arrangement than the TBI group; goblet cells were increased in number and evenly distributed, and epithelial integrity was preserved (Fig. 2A).

Compared with the CON group, the TBI group showed a significantly elevated ileal Chiu score, accompanied by marked reductions in ileal villus height and crypt depth. However, Lyc significantly reduced the ileal Chiu score and increased both villus height and crypt depth after TBI, demonstrating a clear dose-dependent trend. Compared with the TBI group or TBI + Lyc-L group, the ileal Chiu scores in the TBI + Lyc-M and TBI + Lyc-H groups were significantly decreased (Fig. 2B). In addition, the TBI + Lyc-H group showed a significantly greater ileal villus height and crypt depth than the TBI group (Fig. 2C, D).

Fig. 2
Fig. 2
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Lyc ameliorates ileal injury in mice with TBI. (A) HE staining showing ileal tissue changes (×100 and ×200); (B) Chiu score; (C) Villus height; (D) Crypt depth. n = 3. Data are represented as mean ± SD. Compared with the CON group, #P < 0.05; ####P < 0.0001. Compared with the TBI group, *P < 0.05; **P < 0.01. Compared with the TBI + Lyc-L group, &P < 0.05; &&P < 0.01.

Lyc downregulates NLRP3, ASC, Caspase-1, and GSDMD-N expression in ileum

Figure 3 shows that Lyc downregulates the expression of NLRP3, ASC, Caspase-1, and GSDMD-N in the ileum. Compared with the CON group, the expression of NLRP3, ASC, Caspase-1, and GSDMD-N in the TBI group were significantly upregulated. However, Lyc significantly attenuated these levels following TBI in a dose-dependent manner. Compared with the TBI group, the TBI + Lyc-L, TBI + Lyc-M, and TBI + Lyc-H groups showed significant downregulation of NLRP3, ASC, Caspase-1, and GSDMD-N. Moreover, compared with the TBI + Lyc-L group, both the TBI + Lyc-M and TBI + Lyc-H groups exhibited further downregulation of GSDMD-N, while the TBI + Lyc-H group showed significant reduction in NLRP3 and Caspase-1 expression. Furthermore, the TBI + Lyc-H group demonstrated further suppression of NLRP3 and Caspase-1 compared with the TBI + Lyc-M group (Fig. 3).

Fig. 3
Fig. 3
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Lyc downregulates NLRP3, ASC, Caspase-1, and GSDMD-N expression in ileum. (A) Western blot detection of protein expression of NLRP3, ASC, Caspase-1, and GSDMD-N; Relative protein expression of NLRP3 (B), ASC (C), Caspase-1 (D), and GSDMD-N (E). n = 3. Data are represented as mean ± SD. Compared with the CON group, ###P < 0.001; ####P < 0.0001. Compared with the TBI group, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Compared with the TBI + Lyc-L group, &P < 0.05; &&&P < 0.001. Compared with the TBI + Lyc-M group, †P < 0.05; ‡P < 0.01.

Patients

Figure 4 shows a CONSORT flow diagram. Between 2 August 2023 and 5 March 2025, 74 eligible patients were enrolled in the study and randomly allocated to the TBI and TBI + Lyc groups. However, the final analysis excluded two patients from the TBI group and four from the TBI + Lyc group because they withdrew from treatment. Moreover, one patient in the TBI + Lyc group was excluded because of incomplete data. Consequently, the final analysis included 35 participants in the TBI group and 32 in the TBI + Lyc group, respectively. The study strictly adhered to the manufacturer’s protocol and followed the established standard operating procedures and ethical guidelines (Fig. 4).

Fig. 4
Fig. 4
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Flow chart showing patient enrollment, randomization, and analysis.

Baseline data

The population had a mean age of 59.04 years with a standard deviation of 13.99, and 77.6% were male. Baseline characteristics were well balanced between the groups (Table 1).

Table 1 Comparison of baseline characteristics in the TBI group and the TBI + Lyc group. CRP C-reactive protein, PCT procalcitonin, GCS Glasgow coma Scale, SOFA sequential organ failure assessment, APACHE II, acute physiology and chronic health evaluation II.
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Prognostic indicators

Table 2 compares the prognostic indicators between the two groups. Regarding inflammatory markers, CRP and PCT levels were significantly decreased in the TBI + Lyc group relative to the TBI group.

Regarding the digestive system, the TBI + Lyc group had a significantly lower incidence of gastrointestinal complications. Specifically, Lyc treatment significantly increased the proportion of patients without gastrointestinal dysfunction, reduced the proportion of patients with grade II gastrointestinal dysfunction, and significantly decreased the ultrasound-based intestinal function scores. However, there were no notable differences in the percentage of patients experiencing grade I, III, or IV gastrointestinal dysfunction between the two groups.

Regarding the nervous system, the TBI + Lyc group exhibited a significantly higher GCS score than the TBI group, whereas the Helsinki CT score showed no significant difference between the two groups. Concerning hospital stay, the TBI + Lyc group had notably shorter ICU and total hospital stays compared to the TBI group.

Regarding clinical outcomes, no significant differences were found in SOFA score, APACHE II score, mortality risk, 28-day mortality, clinical outcomes, or GOS score between the two groups (Table 2).

Table 2 Comparison of prognostic indicators in the TBI group and the TBI + Lyc group. CRP C-reactive protein, PCT procalcitonin, GCS Glasgow coma Scale, SOFA sequential organ failure assessment, APACHE II, acute physiology and chronic health evaluation II, GOS Glasgow outcome Scale.
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Discussion

TBI is one of the most prevalent neurological disorders, leading to high mortality rates and long-term disabilities globally. Despite extensive studies, no pharmacological therapy has been shown to improve the clinical outcomes in patients with TBI31. The multifaceted nature and heterogeneity of TBI present challenges in the development of effective therapeutic strategies. TBI-induced central nervous system tissue damage initiates an acute, overwhelming, and prolonged inflammatory cascade that extends beyond the brain and involves peripheral organs, including the cardiovascular, pulmonary, renal, gastrointestinal, and endocrine systems. In addition, patients with TBI typically experience hypermetabolic and hyperinflammatory stress, with the intestines often being among the first organs affected. Intestinal oxidative stress, ischemia-hypoxia, and subsequent ischemia-reperfusion injury disrupt the intestinal barrier function. This barrier dysfunction allows translocation of endogenous pathogens and pro-inflammatory substances into the circulatory system, subsequently triggering or exacerbating a systemic inflammatory cascade that affects the nervous system and other organs32,33. Consequently, interventions targeting intestinal inflammation and barrier protection have emerged as promising therapeutic strategies for TBI management.

Previous studies have demonstrated that TNF-α and interleukins increase significantly within hours after TBI, a phenomenon closely associated with secondary brain injury34,35. Moreover, such persistent inflammatory stimulation can impair intestinal barrier function, which in turn generates additional pro-inflammatory cytokines, thereby further establishing a vicious cycle36. Notably, emerging evidence has demonstrated that Lyc effectively suppresses the production of pro-inflammatory cytokines, such as TNF-α and IL-1β37. In line with these results, our research demonstrated a notable increase in ileum TNF-α, IL-18, and IL-6 levels in mice with TBI. The levels of these cytokines were reduced by Lyc treatment in a dose-dependent manner, with a more significant reduction at higher doses. These results not only reaffirmed the association between TBI and elevated inflammatory markers but also demonstrated the potent anti-inflammatory effect of Lyc in the context of TBI. Accumulating evidence has shown that Lyc confers renoprotection in DEHP-exposed mice by inhibiting renal pyroptosis and reducing pro-inflammatory cytokine release27. Moreover, experimental validation confirmed that Lyc alleviates pyroptosis in hippocampal microglia due to chronic stress by inhibiting the cathepsin B/NLRP3 signaling pathway activation28. As pyroptosis serves as a key signaling pathway linking inflammation and apoptosis, combined with prior evidence and current findings, we hypothesized that Lyc may mitigate post-TBI inflammatory cascades by inhibiting pyroptosis, thereby protecting brain and intestinal functions. Subsequent experiments validated this hypothesis.

The detection of intestinal barrier function can identify occult intestinal barrier dysfunction and serve as a predictor of patient prognosis and recovery. Commonly used clinical indicators for assessing intestinal barrier function include I-FABP, D-LA, DAO, and endotoxin. Under physiological conditions, serum levels of these biomarkers are extremely low. However, when intestinal barrier injury occurs with increased mucosal permeability, the aforementioned indicators are translocated into the circulatory system. Thus, alterations in serum levels can effectively represent changes in the permeability of the intestinal mucosa and the severity of its injury38,39. Our findings demonstrated that the serum levels of I-FABP, D-LA, DAO, and endotoxin were significantly elevated in mice with TBI. Notably, medium- and high-dose Lyc treatment significantly reduced the levels of these biomarkers, indicating that Lyc ameliorates post-TBI intestinal barrier dysfunction in mice. Qin et al.40 reported a close association between inflammatory response and intestinal barrier dysfunction, showing that polydatin alleviated TBI-induced intestinal injury in rats by reducing pro-inflammatory cytokine release, which is similar to our findings. The villus epithelial cells in the intestinal mucosa are essential for nutrient absorption. Intestinal villus height is governed by the number of epithelial cells, and crypt depth signifies the production rate of these cells. Therefore, intestinal villus height and crypt depth serve as morphological indicators of intestinal structural and functional integrity41. The results showed that the ileal villus architecture in mice with TBI was markedly disrupted, accompanied by significant reductions in villus height and crypt depth. Combined with the intestinal barrier function indices, these findings confirm that TBI causes structural and functional barrier injuries in the intestinal mucosa. Notably, Lyc treatment significantly improved ileal villus morphology in mice with TBI, with dose-dependent increases in villus height and crypt depth, indicating that Lyc mitigated pathological injury in the ileal tissues of TBI mice. This discovery supports prior studies demonstrating that Lyc reduces pathological injuries in the small intestinal tissue of methotrexate-treated rats42.

Pyroptosis is a type of programmed cell death that relies on lysosomes and inflammation, driven by inflammasomes, and involves the release of significant quantities of pro-inflammatory cytokines26. TNF-α and IL-6 are essential in forming the NLRP3 inflammasome, which is central to the inflammatory response triggered by tissue injury43. As a key initiator of the pyroptotic pathway, NLRP3 activates the classical Caspase-1-dependent signaling cascade. Activated Caspase-1 cleaves GSDMD, generating active GSDMD-N21. GSDMD-N polymers translocate to the cell membrane, recruiting and activating pro-inflammatory cytokines, such as IL-18 and IL-1β. Concurrently, GSDMD-N fragments downregulate the expression of intestinal epithelial junction proteins, compromise membrane integrity, promote epithelial cell death, and disrupt the intestinal barrier function44,45. This study revealed that TNF-α, IL-18, and IL-6 were significantly upregulated in mice with TBI, driving the upregulation of key pyroptotic molecules (NLRP3, ASC, Caspase-1, and GSDMD-N) with NLRP3 serving as the initiator. This cascade activated a positive feedback loop of pro-inflammatory cytokine release (TNF-α, IL-18, and IL-6), indicating that TBI triggers the pyroptotic pathway and exacerbates cytokine storm. Notably, Lyc intervention significantly downregulated the expression of all above-mentioned inflammatory cytokines and pyroptotic key molecules, confirming that Lyc protects the intestinal barrier in mice with TBI by inhibiting TNF-α, IL-18, and IL-6 release via the NLRP3/Caspase-1/GSDMD signaling axis.

In addition to the inflammatory and pyroptosis pathways that are the primary focus of this study, Lyc’s potent antioxidative stress capacity likely represents another key mechanism underlying its protective effects. Under physiological conditions, intestinal epithelium maintains cellular redox homeostasis through multiple antioxidant enzyme systems. However, in pathological states such as TBI, excessive oxidative stress exacerbates intestinal barrier damage and epithelial cell death46. Substantial evidence confirms that dietary Lyc supplementation effectively mitigates oxidative stress and improves inflammatory status, as demonstrated by significantly reduced inflammatory markers in a rat model of colitis47. More profoundly, studies have revealed that Lyc counteracts deoxynivalenol-induced intestinal toxicity by maintaining mitochondrial homeostasis and protects intestinal epithelium from oxidative damage via modulation of the Keap1/Nrf2 signaling pathway48,49. Notably, the Nrf2 signaling pathway has been identified as a potential therapeutic target against oxidative stress-induced neuropathological damage in TBI50. Therefore, we reasonably hypothesize that in our TBI mouse model, Lyc’s ameliorative effect on ileal histopathological damage is likely partially mediated through Nrf2 pathway activation and oxidative stress antagonism. Future investigations should further elucidate the specific role of Nrf2 in Lyc-mediated protection of the intestinal barrier post-TBI.

Furthermore, any discussion of intestinal function must consider the gut microbiota-gut-brain axis. Previous research has demonstrated that Lyc alleviates D-galactose-induced intestinal injury and memory deficits in mice by rebalancing the “microbiota-short-chain fatty acid-gut-brain axis"18. Another study in a constipation model found that Lyc pretreatment increased short-chain fatty acid production and restored gut microbial homeostasis, thereby enhancing intestinal barrier integrity51. Integrating these findings with our results—that Lyc improved intestinal barrier function and histomorphology—we speculate that Lyc’s modulation of gut microbiota and their metabolites (e.g., short-chain fatty acids) following TBI may represent another potential mechanism through which it alleviates intestinal damage and subsequently exerts neuroprotective effects via the gut-brain axis. Gut dysbiosis may exacerbate systemic inflammation and neurological impairment after TBI, while Lyc intervention could potentially help restore a healthy gut microenvironment, providing novel perspectives for understanding its pleiotropic effects.

Animal experiments have shown that Lyc has a protective effect on the intestinal barrier function in mice with TBI. However, its clinical efficacy in humans remains unclear, prompting us to extend this study to patients with TBI. Specifically, regarding inflammation control, the CRP and PCT levels in the TBI + Lyc group were notably lower compared to the TBI group, demonstrating Lyc’s anti-inflammatory properties in humans. This finding aligns with the core conclusions from our previous animal experiments, thereby corroborating reports that a 4-week tomato juice consumption in volunteers can lower concentrations of atherosclerosis-related inflammatory molecules52. Regarding the digestive system, while prior studies have shown that Lyc improves intestinal symptoms in patients with ulcerative colitis53, this study further revealed that Lyc significantly reduced the incidence of post-TBI digestive system complications, increased the proportion of patients without acute gastrointestinal dysfunction, decreased the proportion of patients with grade II acute gastrointestinal dysfunction, and lowered the ultrasonic intestinal function scores. Collectively, these findings validate the gastroprotective effect of Lyc, which may potentially alleviate patient discomfort and reduce the medical costs associated with TBI management. In the nervous system, Lyc was found to improve neurological function in patients with TBI, as evidenced by an increased GCS score, which is consistent with previous studies54,55. In addition, although Lyc did not significantly affect the mortality risk or clinical outcomes, it notably shortened both the ICU and total hospital length of stay, providing substantial benefits to patients’ families and society. Notably, animal experiments have demonstrated a dose-dependent biological effect of Lyc, suggesting that optimizing Lyc intake in patients with TBI may influence clinical outcomes in future studies.

This study has several limitations. First, the bioavailability of Lyc is influenced by factors such as individual gut microbiota and dietary fat intake, which may lead to variations in therapeutic efficacy. As plasma Lyc concentrations were not measured, the study could not directly correlate intervention adherence and absorption differences with clinical outcomes. Future research should incorporate this pharmacokinetic parameter to more accurately assess Lyc’s biological effects. Second, differences in participants’ dietary backgrounds prior to enrollment may represent potential confounding factors. Although intake of other Lyc-rich foods was restricted during the trial, the influence of baseline variations could not be entirely eliminated. Measuring baseline plasma Lyc levels would help clarify this issue. Third, caution is warranted when extrapolating mechanistic findings from animal studies to humans. Although both animal models and clinical data support the protective role of Lyc, species-specific differences in intestinal physiology and metabolism necessitate further validation of the NLRP3/Caspase-1/GSDMD pathway—identified as central in mice—through larger, multi-center studies utilizing human tissue samples where possible. Additionally, the clinical follow-up period focused on short-term outcomes, limiting the ability to characterize long-term trajectories of intestinal barrier function recovery. Extending follow-up to 3–6 months post-injury would provide a more comprehensive understanding of Lyc’s long-term benefits. Finally, the study was limited by a relatively small and homogeneous sample, which did not adequately include mild TBI patients or special populations such as children and the elderly. Moreover, although the use of tomato juice as the Lyc source in the clinical component enhanced feasibility and patient compliance, its Lyc content may vary due to factors such as tomato variety, processing methods, and storage conditions. Future studies employing standardized Lyc supplements with expanded sample sizes and greater population diversity would allow for more precise evaluation of its efficacy and generalizability.

Conclusion

In summary, animal studies have revealed that Lyc ameliorates intestinal barrier dysfunction in mice with TBI by inhibiting ileal pyroptosis through the NLRP3/Caspase-1/GSDMD pathway and reducing pro-inflammatory cytokine levels. Concurrently, clinical investigations have confirmed that Lyc reduces inflammatory markers and improves gastrointestinal and neurological functions in patients with TBI, highlighting its promising clinical potential in preventing TBI-related intestinal dysfunction and complications. However, the low bioavailability of Lyc in humans warrants further investigation to optimize its delivery. Further research is required to establish the effective plasma levels of Lyc in both animal models and humans to support its clinical application in TBI treatment.