Dihydromyricetin exerts neuroprotective effects in acute spinal cord injury by inhibiting NLRP3/Caspase-1 inflammasome through gut microbiome modeling

Abstract

Background

The gut microbiota is closely associated with spinal cord injury (SCI). Dihydromyricetin (DHM), a potent anti-inflammatory compound with neuroprotective properties, has been shown to improve outcomes in various diseases. However, the role of gut microbiota mediating the mechanism neuroprotective effects of DHM in SCI is unknown.

Methods

Male Sprague-Dawley rats were randomly divided into three groups: SHAM, SCI, and DHM group. Motor function was assessed using the cylinder rearing test, and alterations in gut microbiota composition and metabolites were analyzed via 16S rRNA sequencing. Subsequently, Western blotting and immunofluorescence staining were employed to evaluate intestinal barrier integrity, as well as changes in proteins associated with the TLR4/NF-κB pathway and NLRP3/Caspase-1. Finally, fecal microbiota transplantation experiments were conducted to elucidate the necessity of gut microbiota in mediating the anti-inflammatory effects of DHM.

Results

DHM exhibits a therapeutic effect by attenuating the severity of pathological SCI and promoting motor function recovery. Notably, DHM restored a balanced microbiota pattern by increasing the relative abundance of Actinobacteria and Bacteroidetes, while concurrently decreasing that of Proteobacteria. Furthermore, DHM promoted intestinal barrier recovery, reduced blood lipopolysaccharide (LPS) levels, and suppressed the activation of the TLR4/NF-κB pathway and reduced the activity of the NLRP3/Caspase-1 inflammasome, thereby effectively decreasing the subsequent release of inflammatory factors in spinal cord tissue. Furthermore, the results from the two FMT groups demonstrated that the gut microbiota serves as a critical target for DHM to exert its anti-inflammatory effects.

Conclusion

The results of this study demonstrate that restoring microbial balance, repairing intestinal barrier integrity, reducing serum LPS levels, and suppressing the TLR4/NF-κB pathway as well as NLRP3/Caspase-1 inflammasome activity constitute the key regulatory mechanisms underlying the neuroprotective effects of DHM following SCI, thereby opening up possibilities for a novel microbiome-directed therapeutic approach to SCI.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12950-026-00499-5.

Introduction

Spinal cord injury (SCI) is a highly destructive disease of the central nervous system [1], which imposes significant psychological and economic burdens on patients, their families, and society [2]. The pathophysiological process of SCI is highly complex and is generally categorized into two components: primary injury and secondary injury; primary injury represents an irreversible process; mitigating secondary injury is crucial for enhancing the prognosis of SCI [3]. In this pathological process, the excessive neuroinflammatory response mediated by the NLRP3 inflammasome is widely recognized as a critical factor in determining injury progression and severity [4]. The NLRP3 inflammasome—a multiprotein complex consisting of NLRP3 (NOD-like receptor family pyrin domain-containing 3), ASC (apoptosis-associated speck-like protein containing a CARD), and inflammatory Caspase-1 [5], can be activated by damage-associated molecular patterns (DAMPs) released following injury, and directly leads to the death of neurons and oligodendrocytes [4, 6]. Therefore, how to effectively inhibit the NLRP3 inflammasome at an early stage, control the neuroinflammatory response and effectively and interrupt the secondary damage is a major direction that affects the prognosis of SCI.

The gut microbiome is a complex and intricately interconnected microecosystem composed of trillions of resident intestinal bacteria. Its interactions with host energy homeostasis, metabolic regulation, glucose balance, and immune function are well established [7]. Furthermore, dysbiosis of the gut microbiome has been associated with the progression of several central nervous system disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), etc [8]. After spinal cord injury, autonomic function is impaired, and over 80% of patients develop neurogenic bowel dysfunction (NBD). Such functional abnormalities lead to significantly delayed gastrointestinal transit, disruption of gastrointestinal microbiome homeostasis, and subsequent structural and functional dysbiosis of the gut flora [9]. Imbalanced microbiota directly reduces the synthesis of short-chain fatty acids (SCFAs), and the deficiency of SCFAs further compromises intestinal barrier integrity [10]. Lipopolysaccharide (LPS) produced by pathogenic bacteria crosses the damaged intestinal barrier and enters systemic circulation, where it binds to Toll-like receptor 4 (TLR-4) and specifically recruits myeloid differentiation factor 88 (MyD88), thereby promoting nuclear translocation of nuclear factor-κB (NF-κB) and subsequent activation of the NLRP3 inflammasome [11], exacerbating neurological diseases [12]. Probiotic intervention triggers an anti-inflammatory immune response in the gut-associated lymphoid tissue of SCI mice, ultimately improving motor function [13]. Furthermore, extensive evidence suggests a significant correlation between the severity of gut microbiome dysbiosis and pathological damage following SCI, additionally, SCI patients exhibit enrichment of pro-inflammatory bacterial taxa alongside reduced levels of beneficial short-chain fatty acid (SCFA)-producing microbes [14]. Consequently, how to improve gut dysbiosis is an important direction for early and effective control of neuroinflammation.

Dihydromyricetin (DHM), a natural flavonoid phytochemical that constitutes up to 35% of vine tea [15], exhibits a broad spectrum of biological activities, including anti-inflammatory, antioxidant, and neuroprotective properties [16]. Studies demonstrate that DHM ameliorates diverse neurological disorders through distinct mechanisms: modulation of brain-derived neurotrophic factor (BDNF) to alleviate diabetic neuropathic pain and depression [17]; attenuating cerebral ischemia-reperfusion injury through ferroptosis inhibition [18]; protecting dopaminergic neurons in Parkinson’s disease models by suppressing glycogen synthase kinase-3β (GSK-3β) activity [19], and enhancement of cognitive function in Alzheimer’s disease models through inhibition of neuroinflammatory response [20], etc. Furthermore, emerging evidence indicates that DHM’s therapeutic efficacy across multiple disease models is substantially mediated by modulation of the gut microbiota [21]. For instance, in a ulcerative colitis model, DHM treatment restored intestinal microbial abundance, significantly ameliorated intestinal mucosal integrity, and suppressed NLRP3 inflammasome activation [22]. Similarly, DHM can enrich intestinal commensal bacteria and regulate lipid metabolism, thereby alleviating non-alcoholic steatohepatitis [23]. Despite this mechanistic insight, to date, the biological effects and potential role of DHM in SCI remain unclear. Given that the NLRP3 inflammasome is a well-established driver of post-SCI neuroinflammation, we hypothesize that DHM may attenuate NLRP3 inflammasome expression in the spinal cord through: (1) modulation of gut microbiota composition, and increasing SCFA levels; (2) enhancement of intestinal barrier integrity, and (3) attenuation of LPS translocation with concomitant suppression of the TLR4/NF-κB signaling pathway. This multi-targeted action could ultimately improve motor function recovery after SCI.

Materials and methods

Animal grouping and SCI modeling

All animal experiments in this study complied with animal ethics guidelines and were approved by the Animal Experiment Ethics Committee of the General Hospital of Western Theater Command. All animals were purchased from SiPeiFu (Beijing) Biotechnology Co. (Certificate of Conformity No. SCXK (Jing) 2024-0010). Study 1: Forty-two healthy male Sprague–Dawley rats, after 7 days of adaptive feeding, they were randomly divided into three groups: SHAM, SCI and DHM (SCI + DHM intervention), with 14 animals in each group. The SCI and DHM groups underwent surgical establishment of the C5 unilateral clamp spinal cord injury model. Rats were initially anesthetized with 4–5% isoflurane (RWD life science, R510-22, Shenzhen, China) and maintained under 2-2.5% isoflurane anesthesia. After removing the C5 laminae to expose the spinal cord tissues, the right side of the spinal cord was clamped with medical aneurysm clips (70 g) for 30 s. The Sham group only underwent vertebral plate removal without spinal cord tissue damage. All groups resumed normal diet post-surgery, with the DHM group receiving daily gavage intervention (100 mg/kg) for 14 days (Fig. 1A).

Fig. 1.

Evaluation of general condition, motor function, and pathological injury recovery in SCI rats. (A) Experimental design diagram. (B) Each group of rats was placed in transparent cylinders for Cylinder Rearing test. (C) Comparative analysis of the usage rate of the affected forelimb in rats up to 14 days post-operation using one-way ANOVA. The usage rate of the affected forelimb was calculated as: the total number of times the affected forelimb collided with the wall (the number of times the affected forelimb collided independently + the number of times the contralateral forelimb collided independently + the number of times both forelimbs collided together). n = 6. (D) Analysis of pathological injury recovery of rat spinal cord tissues by HE staining. (E) Quantitative analysis of the relative cavity area in spinal cord tissue across different rat groups. n = 4

Study 2: In Study 2, sixteen healthy male Sprague–Dawley rats, following 7 days of acclimatization, underwent gut microbiota depletion with broad-spectrum antibiotics (ABX). Rats were administered an antibiotic cocktail (containing vancomycin at 100 mg/kg, metronidazole at 200 mg/kg, ampicillin sodium at 200 mg/kg, and neomycin sulfate at 200 mg/kg) via gavage once daily for 7 consecutive days. Following microbiota depletion, rats were randomly allocated into two groups (n = 8 each): the SCI-FMT group and the DHM-FMT group. Both groups underwent identical surgical procedures to those described in Study 1, for the establishment of a C5 hemiclip compression spinal cord injury model. Following surgery, the SCI-FMT group received fecal microbiota transplantation (FMT) from SCI group donors, whereas the DHM-FMT group received FMT from DHM group donors. The transplant solution was prepared as follows: 1 g of donor feces was suspended in 10 ml of sterile PBS and thoroughly homogenized using a sterile glass rod; the suspension was then centrifuged at 800 × g for 3 min, and the resulting supernatant was collected as the FMT solution. Thereafter, both groups received 1 ml of the transplant solution per rat daily via gavage for 14 consecutive days (Fig. 5A).

Fig. 5.

Fecal microbiota transplantation confirms gut microbiota as a critical mediator of DHM-mediated anti-inflammatory effects. (A) Experimental design diagram. (B, C) Representative Western blot images and quantitative analysis of TLR4, MyD88, and p-NFκB in the spinal cord, n = 4. (D, F) Representative immunofluorescence images of NLRP3 (green) and Caspase-1 (red) in the colon of each group, scale bar = 50 μm. (E, G) Quantitative analysis of immunofluorescence intensity for NLRP3 and Caspase-1, n = 4. (H, I) Representative Western blot images and quantitative analysis of cleaved-Caspase-1 and Pro-Caspase-1 in the spinal cord, n = 4. (J) qPCR analysis showing the expression levels of IL-1β and TNF-α in the spinal cord of rats, n = 4

Behavioral tests

Behavioral tests were performed on Study 1 rats one day before surgery and on postoperative days 3, 7, and 14. The cylinder rearing test is commonly used to assess forelimb motor asymmetry. The rats were placed in a clean transparent cylinder (20 cm diameter × 30 cm height) with mirrors placed behind both sides to facilitate observation of movements in all directions (Fig. 1B). Behavior was recorded for 15 min using a video camera. The experimenter then counted from the recordings the number of times the affected forelimb (R), contralateral forelimb (L), or both forelimbs simultaneously (D) contacted the cylinder wall. The frequency of forelimb use on the injured side was calculated as: (R + ½ D)/(R + L + D).

Hematoxylin and Eosin staining (HE staining)

Spinal cord tissues were harvested from Study 1 rats at 14 days post-intervention, then subjected to fixation, dehydration, and paraffin-embedding, then sectioned at a thickness of 5 μm. The experimental steps were conducted in accordance with published literature [24]. Images were acquired using a microscope, and the cavity area was quantified using ImageJ.

Immunofluorescence

Following 14 days of intervention, spinal cord and colon tissues were harvested from Study 1 rats, and the embedded blocks were sectioned at a thickness of 5 μm, antigen-retrieved with sodium citrate solution at elevated temperatures, blocked for 20 min at room temperature using Immunostaining Rapid Closure Solution (Beyotime), and incubated overnight with the relevant primary antibody in a 4 °C environment: NLRP3 (1:300, Affinity), Caspase-1 (1:300, Affinity), Occludin (1:300, Abcam), and ZO-1 (1:300, Abcam). Sections were then incubated with the relevant secondary antibody (1:500, Bioss-0295G-BF488; 1:1000 Bioss-0295-BF594) for 1 h at room temperature in the dark, followed by 10-minute incubation with DAPI (1:1000, Beyotime) at room temperature in the dark. Each step was followed by 3 washes with PBS for 3 min each. Finally, the sections were dehydrated through an ethanol gradient and mounted with neutral resin. After applying anti-fade mounting medium, sections were imaged using a Nikon A1RHD25 confocal microscope and analyzed with NIS-Elements Viewer. Fluorescence intensity was quantified using ImageJ.

Extraction of RNA and quantitative real-time PCR (RT-qPCR) analysis

First, total RNA was extracted from the samples using an RNA extraction kit (Fastagen). RNA concentration and purity were measured using a spectrophotometer (Life Real) following the manufacturer’s instructions. RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara). Gene-specific primers (Supplementary Table) were designed and synthesized by Sangon. Finally, the amplification kit (Takara) was used to perform the above two reactions on a real-time fluorescence quantitative PCR instrument (Bio-rad), and the procedure and reaction conditions followed the kit’s instructions. Three wells were set up for each sample, and GAPDH was used as the internal reference gene for relative quantitative analysis, and the relative expression of target genes was calculated by the 2^-ΔΔCt method.

Western blot

Total protein was extracted from the spinal cord tissues of rats in each group using a protein extraction kit (Solarbio), and protein concentrations were quantified using a BCA assay (Solarbio). Proteins were separated by 4–12% SDS-PAGE and transferred onto PVDF membranes (Millipore). The membranes were blocked with rapid blocking buffer (BIO-RAD) for 15 min, prior to overnight incubation at 4 °C with the following primary antibodies: TLR4 (1:1000, Proteintech), MyD88 (1:3000, Abcam), NF-κB p65 (1:1000, Abmart), Caspase-1 (1:3000, Cell Signaling Technology), and GAPDH (1:5000, Proteintech). Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized using a chemiluminescence imaging system (AZURE Biosystems) and subsequently quantified using ImageJ software.

Fluorescein isothiocyanate dextran (FITC-d) assay

Intestinal permeability was assessed by measuring serum FITC-d levels [25]. Following 14 days of intervention, rats from each group in Study 1 were fasted for 4 h and subsequently administered FITC-dextran solution (4 kDa, 5 mg/100 g; Yuanye) via oral gavage. Blood samples were collected following a subsequent 4-hour fasting period, and serum fluorescence intensity was measured using a fluorescence spectrophotometer (with excitation and emission wavelengths of 485 nm and 528 nm, respectively). Fluorescence intensities were interpolated against a standard curve to determine serum FITC-d concentrations.

Enzyme-linked immunosorbent assay

Blood samples collected from Study 1 were centrifuged at 3500 rpm for 10 min to isolate serum. LPS concentration was measured using an ELISA kit (ZCiBio) following the manufacturer’s instructions.

Amplification, sequencing and analysis of 16S rRNA gene

Total genomic DNA was extracted from fecal samples of rats in each group of Study 1 using the PF Mag-Bind Stool DNA Kit (Omega Bio-tek) following the manufacturer’s instructions. DNA integrity was verified by 1% agarose gel electrophoresis, while concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Following PCR amplification of the V3-V4 region, amplicon size was verified by 2% agarose gel electrophoresis. Products were quantified using a Quantus™ Fluorometer (Promega), followed by sequencing on an Illumina PE300/PE250 platform. For data analysis, DADA2 in QIIME2 was used for sequence denoising, followed by taxonomic classification using a Naive Bayes classifier. Alpha diversity indices were calculated using mothur, with between-group differences assessed using Wilcoxon rank-sum tests. Beta diversity was evaluated through PCoA using Bray-Curtis distances. Differentially abundant taxa (phylum to genus) were identified using LEfSe analysis.

Detection of SCFAs

Gas chromatography-mass spectrometry (GC-MS) was employed to quantify short-chain fatty acid (SCFA) levels in fecal samples from rats in each group of Study 1. Samples were pretreated according to the following procedure: fecal samples were accurately weighed, suspended in pure water, vortexed, homogenized, and sonicated for extraction. After centrifugation, the supernatant was harvested, mixed with 50% sulfuric acid and methyl tert-butyl ether containing the internal standard (2-methylvaleric acid), vortexed, sonicated, and subsequently centrifuged. The final supernatant was then analyzed by GC-MS. Chromatographic separation was performed on an Agilent 7890B-5977B system equipped with an HP-FFAP capillary column. The injection volume was 1 µL, the split ratio was 5:1, and helium was used as the carrier gas with a programmed temperature gradient. Detection was conducted using electron ionization mass spectrometry. Quality control samples were analyzed every 10 study samples. Accuracy ranging from 80% to 120% and relative standard deviation (RSD) of ≤ 20% served as the criteria for system stability assessment. SCFA quantification was performed using an internal standard method with standard curves.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). For normally distributed data with homogeneous variance, one-way ANOVA was performed, followed by post hoc comparisons using either SNK-q or LSD-t tests. Non-normally distributed data were analyzed using the Kruskal-Wallis test. Statistical significance was defined as: ns = no significant difference P>0.05, *P <0.05, **P <0.01, ***P <0.001.

Results

DHM attenuates pathological spinal cord injury and promotes motor function recovery in SCI rats

To evaluate DHM’s effect on motor function post-SCI, motor recovery was assessed using the cylinder rearing test. No significant intergroup differences in affected limb use frequency were observed pre-SCI (Day 1). By post-op Day 3, the SHAM group showed minimal reduction in forelimb use, while the SCI group and DHM group demonstrated significantly impaired function versus SHAM. All groups showed progressive recovery of forelimb function, with the DHM group exhibited superior recovery compared to the SCI group at both postoperative days 7 and 14, with this difference reaching statistical significance at day 14 (P = 0.0267; Fig. 1C). These findings demonstrate DHM’s therapeutic potential for promoting motor recovery after SCI.

To further assess DHM’s neuroprotective effects, HE staining was performed on paraffin-embedded spinal cord sections. Compared with SHAM controls, SCI animals exhibited severe tissue damage characterized by disrupted cytoarchitecture and prominent cavitation. DHM treatment markedly preserved tissue integrity, showing significantly attenuated vacuolation and near-normal morphology (Fig. 1D). Quantitative analysis revealed significantly reduced vacuolar area percentage in DHM-treated versus SCI animals (P < 0.001; Fig. 1E). These findings demonstrate DHM’s capacity to attenuate SCI-induced pathological changes.

DHM modulate gut microbiome disruption after SCI

Following the collection of fecal samples from rats in each group, 16S rDNA sequencing was performed to analyze the composition of the gut microbiome and assess the potential of DHM to ameliorate microbiome dysbiosis post-SCI. As shown in Fig. 2A, DHM treatment significantly enhanced α-diversity, with marked increases in Chao1, ACE, and Shannon indices, while β-diversity demonstrated substantial community-level alterations (Fig. 2B). Subsequent analysis revealed that, at the genus level, SCI reduced Allobaculum (P = 0.043) and increased Prevotella abundances (P = 0.034) compared to the SHAM group (Fig. 2C, D). In contrast, DHM treatment significantly elevated Ruminococcus (P<0.001)、Clostridium (P = 0.00208) and Paraeggerthlla (P = 0.026; Fig. 2C, E). At the phylum level, although not statistically significant, the DHM group showed trends toward increased Actinobacteria and Bacteroidetes abundances with concurrent decreases in Proteobacteria (Supplementary Fig. 1A-C). We subsequently analyzed short-chain fatty acid (SCFA) levels in rat fecal samples. As shown in Fig. 2F, compared with the SHAM group, the SCI group exhibited decreased levels of all SCFA subtypes. Following DHM intervention, butyric acid levels were significantly elevated (P = 0.0434). Although the differences did not reach statistical significance for hexanoic acid, isovaleric acid, and acetic acid, an increasing trend was observed. These results demonstrate that DHM treatment modulates the gut microbiome composition in SCI rats, increased butyric acid levels in vivo.

Fig. 2.

DHM can modulate the gut microbiota. (A) Alpha diversity images of Chao 1, ACE, and Shannon indices for each group. n=5. (B) PCoA (Principal Coordinates Analysis) images for each group. n=5. (C) Abundance of microbiota at the genus level for each group. n=5. (D) Microbiota at the genus level that show statistically significant differences between the SHAM and SCI groups. n=5. (E) Microbiome at the genus level that show statistically significant differences between the SCI and DHM groups. n=5. (F) The content of butyric acid, hexanoic acid, isovaleric acid, and acetic acid in fecal samples of rats in each group, n=4

DHM improves intestinal barrier and reduces blood LPS levels

Tight junction proteins are important for maintaining intestinal integrity [26]. The expression levels of two tight junction proteins, Occludin and ZO-1, in the colon of each group were examined by immunofluorescence and qPCR. The results showed that Occludin and ZO-1 protein levels were reduced in the SCI group compared with the SHAM group (P<0.001, P<0.001); and the DHM intervention dramatically increased the expression of these two proteins (P = 0.0039, 0.0085; Fig. 3A-B, D-E). qPCR results followed the same trend as immunofluorescence, and the DHM intervention reversed the reduction of Occludin and ZO-1 proteins caused by SCI (P = 0.0017, 0.0019; Fig. 3C, F). Furthermore, serum FITC-d levels were markedly elevated in the SCI group relative to the SHAM group (P < 0.001; Fig. 3G). By contrast, DHM treatment significantly reduced serum FITC-d levels compared with the SCI group (P = 0.014; Fig. 3G), suggesting that SCI compromises intestinal barrier function, which is restored by DHM intervention.

Fig. 3.

DHM enhanced intestinal barrier integrity and reduced serum LPS levels. (A, D) Representative immunofluorescence images of Occludin (green) and ZO-1 (red) in the colon of each group, scale bar = 50 μm. (B, E) Quantitative analysis of immunofluorescence intensity for Occludin and ZO-1, n = 4. (C, F) qPCR analysis showing the expression levels of Occludin and ZO-1 in the colon of rats, n = 4. (G) Serum FITC-Dextran levels in each group of rats, n = 4. (H) ELISA analysis showing serum LPS levels in rats, n = 4

To evaluate systemic endotoxin levels following SCI and DHM treatment, we quantified serum LPS concentrations using ELISA on day 14 post-injury. SCI induction resulted in significantly elevated circulating LPS levels compared to SHAM (P<0.001); DHM treatment effectively attenuated this increase, demonstrating a significant reduction in serum LPS concentrations relative to untreated SCI animals (P = 0.0239, Fig. 3H). These findings suggest that DHM exerts dual protective effects by both modulating gut microbiota composition and enhancing intestinal barrier integrity, thereby limiting bacterial endotoxin translocation into systemic circulation after SCI.

DHM inhibits NLRP3/Caspase-1 inflammasome signaling in spinal cord

The TLR4 complex functions as the primary recognition receptor for LPS, thereby playing a pivotal role in mediating LPS-induced inflammatory responses. Activation of the TLR4/NF-κB signaling pathway and subsequent assembly of its downstream NLRP3/caspase-1 inflammasome trigger a neuroinflammatory cascade that critically contributes to secondary spinal cord injury pathogenesis. Accordingly, we quantified spinal cord expression of relevant inflammatory markers. Western blot analysis revealed significantly elevated levels of TLR4, MyD88, and phosphorylated NF-κB (p-NF-κB) in the SCI group relative to the SHAM group (P = 0.0028, 0.0094, 0.0057). DHM treatment markedly attenuated their expression (P = 0.0332, 0.0468, and 0.0079; Fig. 4A-B), findings that were corroborated by qPCR analysis (P < 0.001, < 0.001, and 0.0018; Supplementary Fig. 2A-C). Immunofluorescence results showed that compared with the SHAM group, the mean fluorescence intensity of NLRP3 and Caspase-1 in the spinal cord was markedly enhanced in the SCI group (P < 0.001, < 0.001), and the relative expression of Cleaved-Caspase-1 was elevated (P < 0.001), indicating activation of the NLRP3/Caspase-1 inflammasome following SCI. DHM treatment effectively suppressed this upregulation (P = 0.0218, 0.033, 0.0056; Fig. 4C-D, F-G, I-J), demonstrating its anti-inflammatory potential. At the transcriptional level, qPCR analysis confirmed increased NLRP3 expression and Caspase-1 activation in SCI animals (P<0.001,<0.001), both of which were significantly attenuated by DHM treatment (P<0.001, = 0.0015; Fig. 4E, H). Furthermore, we observed that DHM intervention suppressed SCI-induced elevations of downstream inflammatory cytokines IL-1β and TNF-α (P = 0.0019, <0.001; Fig. 4K). In summary, DHM intervention attenuates TLR4/NF-κB pathway activation while concurrently suppressing NLRP3/Caspase-1 inflammasome activity, thereby attenuating spinal cord inflammatory responses.

Fig. 4.

DHM reduced inflammatory levels in the spinal cord. (A, B) Representative Western blot images and quantitative analysis of TLR4, MyD88, and p-NFκB in the spinal cord, n = 4. (C, F) Immunofluorescence images of NLRP3 (green) and Caspase-1 (red) in the spinal cord of each group, scale bar = 50 μm. (D, G) Quantitative analysis of immunofluorescence intensity for NLRP3 and Caspase-1, n = 4. (E, H) qPCR analysis showing the expression levels of NLRP3 and Caspase-1 in the spinal cord of rats, n = 4. (I, J) Representative Western blot images and quantitative analysis of cleaved-Caspase-1 and Pro-Caspase-1 in the spinal cord, n = 4. (K) qPCR analysis showing the expression levels of IL-1β and TNF-α in the spinal cord of rats, n = 4

The gut microbiota serves as a critical pathway for DHM-mediated anti-inflammatory effects

To validate whether the gut microbiota serves as a key mediator of DHM-induced anti-inflammatory effects, we transplanted fecal samples from the SCI and DHM groups into gut microbiota-depleted spinal cord–injured rats for two weeks (Fig. 5A). Thereafter, Western blot and qPCR were employed to quantify relevant inflammatory markers in spinal cord tissues from both groups, thereby establishing the causal role of the microbiota. As shown in Fig. 5B–C, the DHM-FMT group, which received feces from DHM-treated donors, exhibited significantly attenuated protein expression of TLR4, MyD88, and p-NF-κB relative to the SCI-FMT group, which received feces from SCI donors (P = 0.0279, 0.0225, 0.0141; Fig. 5B–C). These findings were further corroborated by qPCR analysis (P = 0.0027, < 0.001, < 0.001; Supplementary Fig. 2D-F). Together, these findings demonstrate that DHM-modulated gut microbiota effectively suppresses TLR4/NF-κB pathway activation. Further investigation of the NLRP3/Caspase-1 inflammasome and its downstream effector molecules revealed that immunofluorescence staining revealed that the expression levels of NLRP3 and Caspase-1 proteins in the spinal cord tissue of DHM-FMT-treated rats were significantly reduced (P = 0.031, < 0.001; Fig. 5D–G). These findings were subsequently validated by qPCR analysis, demonstrating significant downregulation of NLRP3 and Caspase-1 at the transcriptional level (P = 0.0016, < 0.001; Supplementary Fig. 2G–H). Consistent with these observations, the relative abundance of Cleaved-Caspase-1 was significantly decreased (P = 0.0241; Fig. 5H–I), suggesting suppressed activation of the NLRP3/Caspase-1 inflammasome. Accordingly, the secretion levels of the pro-inflammatory cytokines IL-1β and TNF-α were concomitantly attenuated (P < 0.001, < 0.001; Fig. 5J). Collectively, transplantation of DHM-preconditioned gut microbiota recapitulates the anti-inflammatory effects of DHM in vivo, implicating the gut microbiota as a critical target for DHM-mediated neuroprotection.

Discussion

This study employed an animal model of SCI to evaluate the therapeutic efficacy of DHM on SCI and represents the first exploration of the role played by gut microbiota in DHM treatment for SCI. DHM, as a safe and non-invasive dietary intervention, effectively facilitates the healing process of spinal cord injuries and markedly enhances motor function recovery post-SCI. Subsequent research indicated that DHM effectively restores gut microbiota balance in SCI rats, enhanced SCFA production, mitigates intestinal barrier disruption post-SCI, and decreases circulating LPS levels. Building upon this, DHM suppresses TLR4/NF-κB pathway activation, attenuates NLRP3/Caspase-1 inflammasome activity, and thereby alleviates neuroinflammation following spinal cord injury. Moreover, FMT experiments in microbiota-depleted rats demonstrated that the gut microbiota serves as a critical mediator of DHM-mediated anti-inflammatory effects.

Extensive evidence indicates that SCI induces significant alterations in gut microbiome composition and abundance, with these dysbiotic changes being strongly associated with neuroinflammatory progression [12]. 16 S rRNA sequencing revealed that SCI induces marked alterations in gut microbiota composition. Relative to the SHAM group, SCI rats exhibited depleted Allobaculum populations and enrichment of Prevotella. Allobaculum possesses anti-inflammatory properties [27] and butyrate-producing capacity [28]. Accordingly, intestinal butyrate levels were significantly reduced in the SCI group, consistent with Allobaculum depletion. Conversely, Prevotella promotes inflammation; its colonization aggravates intestinal inflammation and induces systemic autoimmunity in murine models [29]. Furthermore, as a Gram-negative bacterium, Prevotella is capable of producing substantial amounts of LPS [30]. This gut microbiota dysbiosis, along with diminished SCFA production, likely contributes to the exacerbated impairment of intestinal barrier function following SCI. Consistently, SCI led to a significant reduction in the intestinal expression of tight junction proteins (ZO-1 and Occludin) and an increase in serum FITC-d levels, confirming intestinal barrier disruption. The concurrent elevation of peripheral blood LPS levels provided further evidence for this loss of intestinal barrier integrity post-SCI.

DHM, a well-characterized phytochemical with established safety profiles, demonstrates potent anti-inflammatory, microbiota-modulating, and neuroprotective properties [31, 32]. Our results reveal that DHM administration significantly enriches beneficial gut microbiota in SCI rats, particularly increasing the abundance of Ruminococcus, Clostridium, and Parabacteroides. These microbial genera play crucial physiological roles: Ruminococcus and Clostridium are recognized as primary bacterial producers of butyrate [33]. Accordingly, DHM treatment increased the abundance of these butyrate-producing genera, which was associated with a significant rise in intestinal butyrate concentration. Within the central nervous system, butyrate exerts anti-neuroinflammatory effects through mechanisms including the inhibition of histone deacetylase (HDAC) activity [34] and the modulation of microglial polarization [35]. In the intestinal lumen, butyrate plays a crucial role in maintaining barrier integrity. It serves as a preferred energy source for colonocytes, thereby strengthening the physical barrier [36], and upregulates the expression of tight junction proteins to enhance mechanical integrity [37]. In line with this protective mechanism, DHM intervention significantly upregulated the intestinal protein expression of Occludin and ZO-1, while reducing serum FITC-d leakage and peripheral blood LPS levels compared to the SCI group. These findings collectively demonstrate that DHM effectively mitigates intestinal barrier damage following SCI. In summary, DHM treatment facilitates the restoration of a beneficial gut microbiota structure following SCI, thereby promoting host health.

In neuroinflammation following SCI, the NLRP3 inflammasome is recognized as one of the most critical and central inflammatory regulatory platforms [4, 38], situated at the nexus of multiple upstream signals (e.g.: NF-κB, ROS, etc.) [39] and downstream effects (e.g.: microglia polarization, etc.) [40], functioning as a central node in the inflammatory cascade response. LPS activates NLRP3 inflammasome through multiple pathways [41], and is frequently utilized to develop neuroinflammatory models within the central nervous system [42]. One classical pathway involves LPS binding to its receptor TLR4, triggering MyD88 recruitment and facilitating the nuclear translocation of NF-κB. This, in turn, regulates the transcription of pro-inflammatory genes and promotes the assembly of the NLRP3 inflammasome with Caspase-1 [11]. Our findings indicate that DHM intervention significantly ameliorated the post-SCI inflammatory microenvironment, underscoring its therapeutic potential for SCI-associated neuroinflammation. Specifically, DHM administration restored intestinal barrier integrity, attenuated LPS translocation, suppressed TLR4/NF-κB pathway activation, and consequently diminished NLRP3/Caspase-1 inflammasome activity, as well as levels of the downstream inflammatory cytokines IL-1β and TNF-α. FMT experiments established that the gut microbiota is an essential mediator of the anti-inflammatory effects elicited by DHM. Notably, transplantation of the gut microbiota from DHM-treated donor mice into antibiotic-pretreated SCI recipient rats successfully transferred the anti-inflammatory phenotype. This modulation of inflammation is crucial for neuroprotection after SCI, as it mitigates secondary damage and thereby enhances neurological recovery. Furthermore, the behavioral outcomes of this study, as well as the HE staining results of the spinal cord, provided additional verification for the aforementioned conclusions. Rats in the DHM group showed improved forelimb utilization during cylinder rearing, while spinal cord lesions were less severe, with more intact histomorphology and a reduced cavity area ratio compared to the SCI group. In summary, this study unveiled the multifaceted efficacy of DHM in SCI: on one hand, DHM modulate the structure of the gut microbiome, prompting a shift towards a state conducive to organismal recovery; on the other hand, DHM exerts a significant neuroprotective effect by effectively reducing LPS translocation and suppressing the activation of the TLR-4/NF-κB inflammatory signaling pathway, thereby attenuating the activity of the NLRP3 inflammasome post-SCI (Fig. 6).

Fig. 6.

The therapeutic mechanism of DHM. DHM can improve the gastrointestinal microbiome dysregulation in SCI rats, inhibit the activation of the NLRP3/Caspase-1 inflammasome, and reduce the level of inflammatory response in the spinal cord after SCI. Consequently, it aids in the recovery of spinal cord damage and improves motor performance

Although this study achieved the anticipated results, several limitations should be acknowledged. The current work only preliminarily investigated the impact of DHM on the intestinal microecology following spinal cord injury, confirming its anti-inflammatory effects through microbiota modulation. However, whether this compound directly regulates the NLRP3 inflammasome in the SCI model remains to be further elucidated.

Conclusion

In this study, we substantiated the efficacy and safety of DHM intervention for treating SCI through animal experiments and preliminarily elucidated the underlying mechanism by which DHM promotes motor function recovery in SCI. The results indicated that DHM could ameliorate gut microbiome dysbiosis in SCI rats, increase intestinal butyrate production, restore gut barrier integrity, reduce LPS translocation, suppress TLR4/NF-κB pathway activation, and decrease NLRP3/Caspase-1 inflammasome activity, attenuates secondary inflammatory responses in the spinal cord after SCI, and ultimately enhances locomotor recovery in SCI rats. These coordinated mechanisms collectively promote tissue repair and significantly improve motor function recovery following SCI.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

The authors’ responsibilities were as follows. XM and CK designed the experiments.YY, CK, RP, SH, XH, XG, YY and YY collected samples and performed the experiments. YY, CK and RP, analyzed the data. YY, CK, RP and XM prepared the manuscript and had primary responsibility for final content. All authors read and approved the final manuscript. None of the authors reported a conflict of interest.

Funding

Prof. Xin Ma is the guarantors of this work and had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. This research was supported by the Chengdu Municipal Health Commission Medical Research Project (No. 2024060) and Sichuan Provincial Administration of Traditional Chinese Medicine Research Project (NO. 25MSZX488).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethical approval

All animal experiments in this study were conducted in accordance with animal ethics guidelines and were approved by the Animal Experiment Ethics Committee of the General Hospital of Western Theater Command. The approval number is 24ky103-1.

Competing interests

The authors declare no competing interests.