Mechanistic Study of Jaceosidin in Regulating Secondary Inflammation After Spinal Cord Injury in Mouse by Inhibiting PKM2 Activity

Abstract

Excessive pro-inflammatory polarization of microglia is a critical driver of secondary inflammation following spinal cord injury (SCI). Jaceosidin, a natural flavonoid with established anti-inflammatory properties, has not been extensively studied in the context of post-SCI inflammation regulation. Given the fundamental role of glycolysis in cellular energy metabolism and its crucial involvement in inflammatory processes, this study investigated the effects of Jaceosidin. We demonstrated that Jaceosidin significantly attenuated the inflammatory response in lipopolysaccharide-stimulated microglia in vitro. Subsequent in vitro and in vivo experiments revealed that Jaceosidin shifted microglial polarization away from the inflammatory state and suppressed glycolytic flux. Mechanistically, Jaceosidin directly targeted and inhibited the activity of pyruvate kinase M2 (PKM2), a key glycolytic enzyme. Intervention with Jaceosidin in a mouse SCI model resulted in reduced microglial activation at the injury site, diminished tissue damage, and significantly improved motor and autonomic nerve function recovery. In conclusion, our findings indicate that Jaceosidin mitigates microglial inflammation and promotes functional recovery after SCI by inhibiting PKM2 activity and dampening glycolysis. As a natural phytochemical derived from traditional Chinese medicine, Jaceosidin presents a promising novel therapeutic strategy for the clinical management of spinal cord injury.

Introduction

Spinal cord injury (SCI) represents a severe traumatic disorder of the central nervous system, commonly caused by traffic accidents, falls, sports injuries, or violence (Ahuja et al., 2017). Globally, an estimated 500,000 new SCI cases occur annually (World Health Organization). Driven by an aging population and increased transportation activities, SCI incidence continues to rise, imposing substantial medical and socioeconomic burdens (Quadri et al., 2020). SCI pathology typically progresses through two distinct stages (Li et al., 2020): primary injury and secondary injury. The primary injury results from direct mechanical trauma to the spinal cord, causing immediate tissue disruption, vascular compromise, and neuronal cell death. Subsequently, the secondary injury phase ensues, characterized initially by local ischemia and hypoxia. These conditions trigger glutamate excitotoxicity (Kumar et al., 2018), free radical generation, and immune cell infiltration (Nguyen et al., 2007; Shi et al., 2023). Breakdown of the blood-spinal cord barrier follows, leading to activation of microglia and macrophages and the release of pro-inflammatory mediators (Bellver-Landete et al., 2019; O'Shea et al., 2017). Collectively, these events culminate in neuronal apoptosis, glial scar formation, and irreversible motor deficits (Mothe & Tator, 2012). Microglia, the resident immune cells of the central nervous system, play a pivotal role in modulating the secondary inflammatory response after SCI (DiSabato et al., 2024). They rapidly activate following the initial mechanical insult (Bradbury & Burnside, 2019; Dias et al., 2021), releasing substantial quantities of pro-inflammatory cytokines and reactive oxygen species. This secretion recruits peripheral macrophages and neutrophils (Beck et al., 2010), establishing a vicious cycle that exacerbates the local inflammatory milieu. Consequently, the pro-inflammatory response of microglia is critically implicated in amplifying secondary spinal cord damage.

Glycolysis, a central pathway in cellular energy metabolism, has emerged as a crucial regulator of microglia-mediated neuroinflammation (Hua et al., 2024; Yu et al., 2023). Under pathological conditions such as spinal cord injury (SCI) and neurodegenerative diseases, microglia undergo significant metabolic reprogramming (Cheng et al., 2021; Sabogal-Guáqueta et al., 2023). Our prior research established a direct link between microglial M1 polarization and enhanced glycolytic flux in SCI (Ding et al., 2025). Consequently, we prioritized key glycolytic enzymes (PKM2, LDHA, GLUT3) as mechanistic targets for this investigation. Following the primary SCI, the local microenvironment rapidly becomes ischemic and hypoxic. This shift prompts microglia, infiltrating macrophages, and astrocytes to transition from oxidative phosphorylation to glycolytic metabolism (Orihuela et al., 2016; Viola et al., 2019; Yu et al., 2023). While this metabolic adaptation initially meets the heightened energy demands of activated immune cells, its sustained activation exacerbates the inflammatory cascade through multiple mechanisms.

Spinal cord injury (SCI) constitutes a severe neurological disorder with profound physical, psychological, and socioeconomic consequences. Despite decades of research, achieving significant neurological recovery and functional restoration remains a formidable challenge. Current treatment strategies primarily focus on acute medical stabilization (including hemodynamic management to ensure spinal cord perfusion), surgical decompression and stabilization to alleviate cord compression (Fehlings et al., 2017), and comprehensive rehabilitation. These interventions aim to prevent secondary damage, optimize residual function, and improve quality of life. While high-dose methylprednisolone sodium succinate (MPSS) was once widely used, its application is now controversial due to limited efficacy and significant risks, and it is no longer routinely recommended (Liu et al., 2019; Zhu et al., 2023). Chronic management focuses on mitigating long-term sequelae and neurorehabilitation. Although rehabilitation facilitates adaptation and improves independence, it cannot overcome the intrinsic biological barriers to regeneration within the injured spinal cord (Dietz & Fouad, 2014). Critically, no FDA-approved pharmacotherapy currently exists to directly restore neurological function following SCI. The failure of numerous promising preclinical agents in clinical trials highlights the complexity of SCI pathophysiology and the limitations of single-target therapeutic approaches (Kwon et al., 2010). Endogenous repair mechanisms are further hindered by a hostile post-injury microenvironment characterized by neuroinflammation, excitotoxicity, and other inhibitory factors (Assinck et al., 2017).Given these challenges, natural compounds with multi-target regulatory properties, low toxicity, and neuroprotective potential have emerged as a significant research focus (Ji et al., 2024; Zhao et al., 2022). Jaceosidin, a flavonoid abundant in Asteraceae plants like Artemisia argyi and Artemisia capillaris, exhibits notable anti-inflammatory, antioxidant, and immunomodulatory activities (Nam et al., 2013). Recent studies demonstrate its efficacy in inhibiting fibrosis and inflammation via the VGLL3/HMGB1/TLR4 axis in liver fibrosis models (Yao et al., 2024), inducing apoptosis and G0/G1 phase arrest while inhibiting migration in gastric cancer cells through ROS-mediated pathways (Liu et al., 2024), and offering cardioprotection against doxorubicin-induced injury via Sirt1 signaling activation (Liu et al., 2021). However, the therapeutic efficacy of Jaceosidin in spinal cord injury and its underlying mechanisms remain unexplored. Crucially, it is unknown whether Jaceosidin can modulate microglial phenotype switching by regulating immunometabolic reprogramming, representing a critical gap in knowledge.

Methods

Experimental Animals

The SCI model was built using adult C57BL/6 mice in this study. The 36 6–8 weeks old mice of 25 ± 5 g were bred under a special disease-free environment at 24 + 2 °C, 55 to 65% humidity, and 12/12 h of daylight/darkness in Hangzhou Ziyuan Laboratory Animal Technology Company. After 14 days of acclimatization without food and water restrictions, the 36 mice were randomly assigned to one of three groups using a computer-generated randomization sequence: the sham group (n = 12), the SCI group (n = 12), and the SCI + JAC group (n = 12).The Animal Ethics Committee of First Affiliated Hospital, USTC (CAS No. 2024-N (A)-134) has adopted the test procedures conducted according to the China Council for Laboratory Animals Relief. Mice were anesthetized using sevoflurane gas (Shenzhen RWD Life Technology Co., Ltd., Shenzhen, China) at a 3% induction concentration and maintained at 1%. Throughout the procedure, mice were placed on a heated pad to sustain a body temperature of 37 °C. A laminectomy was conducted at the T10-T11 level to uncover the dorsal surface of the spinal cord without harming the dura mater. Subsequently, a 1.3-mm impactor applied a vertical strike to the exposed spinal cord with a 50 kdynes force. A moderate SCI model was confirmed based on observed spasmodic tail movements and hind-limb motor function loss after the surgery. The skin and muscle layers were stitched with four silk threads. In the sham operation group, mice underwent laminectomy without spinal cord injury, with the skin directly sutured after anesthesia. Following model establishment, the mice’s skin was stitched, and antibiotics were given daily. Post-surgery, manual urination was performed thrice daily until bladder reflexes were restored. The SCI + Jaceosidin group was treated with daily intraperitoneal injections of 0.2 mL Jaceosidin solution (20 mg/kg) (Min et al., 2009) for 28 days, while the remaining groups received an equal amount of normal saline.Animals meeting any of the following criteria prior to the endpoint assessments were excluded from the study: (1) death occurring during surgery or within the first 24 h post-surgery; (2) presence of severe autophagia or infection at the surgical site; (3) failure to exhibit the expected hindlimb paralysis immediately following contusion injury (indicating model failure); (4) development of severe systemic illness unrelated to the SCI procedure.

Basso Mouse Scale (BMS) Score

Hindlimb motor function recovery was assessed using the Basso Mouse Scale (BMS), a validated scoring system ranging from 0 to 9 (Basso et al., 2006). Evaluations were performed preoperatively (day before surgery) and postoperatively on days 1, 3, 5, 7, 14, 21, and 28. Mice were placed in an open field and allowed to ambulate freely for 4 min. Hindlimb locomotor activity was observed and scored independently by three investigators blinded to group allocation.

Assessment of Residual Urine Volume

Bladder function was evaluated by measuring post-void residual urine volume. Briefly, mice received an intra-tail vein injection of 2 mL phosphate-buffered saline (PBS). After 1 h, residual urine volume was measured. This protocol was performed on separate cohorts of mice at postoperative days 1, 3, 5, 7, 14, 21, and 28. Residual urine volume data were analyzed to assess bladder functional recovery (Li et al., 2022). Assessments were conducted and recorded by three investigators blinded to group allocation.

Footprint Analysis

The experimental procedure involved dipping the hind limbs of mice from different groups into red and blue ink, respectively. Subsequently, the mice were allowed to walk across a white runway to record their movement patterns. The scoring system was based on predefined criteria. A score of 0 indicated continuous limping or dragging of the hind legs, resulting in no identifiable footprints. A score of 1 was assigned when at least three toe prints were visible. A score of 2 was given when the footprints showed inward or outward rotation exceeding twice the standard angle. A score of 3 denoted the absence of noticeable dragging, with only mild inward or outward rotation observed. Finally, a score of 4 indicated normal footprints with no inward or outward rotation (Xu et al., 2024; Zeng et al., 2018). The experiments were conducted, and the results were recorded by three blinded evaluators who were unaware of the experimental conditions.

Hematoxylin–Eosin Staining

Following dehydration via a sucrose gradient, spinal cord specimens were embedded in optimal cutting temperature (OCT) compound. Serial transverse sections (8 μm thickness) were prepared on a cryostat microtome. Sections centered at the injury epicenter were selected for hematoxylin and eosin (HE) staining. Briefly, sections were rinsed in distilled water to remove residual OCT, then stained with hematoxylin to visualize nuclei. After differentiation in acid ethanol and bluing in Scott’s tap water, sections were counterstained with eosin to highlight cytoplasm. Subsequently, sections were dehydrated through a graded ethanol series, cleared in xylene, and mounted with a synthetic resin mounting medium.Images were acquired using an Olympus BX53 light microscope (Olympus Corporation, Tokyo, Japan).

Nissl Staining

For Nissl staining, tissue sections were rinsed in distilled water to remove residual OCT compound and then immersed in a 0.05% toluidine blue aqueous solution for 5 min. Sections were briefly differentiated in 90% ethanol (3–10 s), followed by dehydration through absolute ethanol and clearing in xylene. Finally, sections were mounted with a synthetic resin mounting medium and examined under an Olympus BX53 light microscope (Olympus Corporation, Tokyo, Japan) to assess the integrity of Nissl bodies.

Cell Culture

BV2 microglial cells (CL0493, RRID: CVCL_0182; Purcell Life Science and Technology Co., Ltd., Wuhan, China) were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Purcell) supplemented with 10% fetal bovine serum (FBS; Corning Inc., Corning, NY, USA) and 1% penicillin/streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂, with medium changes every 48 h. Subculturing was performed at 80% confluence, and cells were used within 10 passages.

Immunofluorescence

The BV2 microglia were grown on the cover sheets of 24-well plates. The sheets are preconditioned by 4% paraformaldehyde for 30 min, then permeabilized for 15 min with 0.5% Triton X-100. EDTA buffer was applied to extract the spinal cord tissue slices at pH 8.0 after dewaxing and hydrating. All specimens were sealed for one hour with 10% BSA (BSA), followed by incubation with a primary antibody at 4 °C for one night. The main antibodies employed were: rabbit anti-Iba-1 (1:200, Proteintech, Cat. No. 10904-1-AP), rabbit anti-NeuN (1:100, Proteintech, Cat. No. 26975-1-AP), mouse anti-PKM2 (1:400, Proteintech, Cat. No. 60268-1-Ig), rabbit anti-Arg-1 (1:1000, Proteintech, Cat. No. 66129-1-Ig), and rabbit anti-iNOS (1:1000, Abcam, Cat. No. ab178945).On the next day, the samples were incubated with secondary antibodies (CoraLite488-labeled goat anti-mouse IgG, 1:400, Proteintech SA00013-1; CoraLite594-labeled goat anti-rabbit IgG, 1:400, Proteintech SA00013-4) on the following day. Following the staining, a high-resolution panorama scanner (Pannoramic DESK P-MIDI P250, 3DHISTECH) was employed to measure the fluorescent strength and the percentage of positive regions in the three randomly selected visual fields.

Western Blotting

The BV2 microglia and the spinal cord were obtained, and the RIPA lysate buffer was applied to extract the protein. The extracted proteins were isolated from SDS-PAGE, followed by a wet transfer process on a polyvinylidene fluoride (PVDF) film. In order to reduce the non-specificity of the binding, the film was subjected to a blocking buffer for one hour at ambient temperature. After that, the film was subjected to a primary antibody at a temperature of 4 °C for one night. The main antibodies used were anti-iNOS in rabbits, Arg-1 in rabbits, anti-LDHA in rabbits, anti-PKM2 in mice, anti-GLUT3 in rabbits, and in mice against beta-actin (1:10,000 as an internal standard). The main antibody was removed the next day, and the film was washed. Then, the second antibody with horseradish peroxidase was added and incubated for 2 h at room temperature. The enhancement of the CL (ECL) agent promoted the development of the product, and the CL Gel Imaging System (FluorChem R, Proteinsimple) was used to record the protein signal. The intensity of the target band was analyzed with the ImageJ software (version 1.53). Every test was repeated three times, and the data were normalized.

Quantitative Polymerase Chain Reaction (q-PCR)

According to the reagent guidelines, a Trizol process (Shanghai Sangon Biotech Corporation) was employed to extract the whole RNA from BV2 cells. The RNA concentration and purity were determined using an ultra-micro-spectrophotometer (Thermo Fisher Scientific, USA) (A260/A280 would range from 1.8 to 2.1). The first-strand cDNA (Nanjing Vazyme Biotech Company, Inc., Cat. No. R122-01) was synthesized with 1 μg of RNA and AceQ SYBR Green pre-mixed agent (Nanjing Vazyme, Cat. No. Q111-02). The real-time quantitative PCR response system employed AceQ SYBR Green pre-mixed agent (Nanjing Vazyme, Cat. No. Q111-02) and amplified with a LightCycler480 II fluorescence PCR apparatus (Roche, Switzerland). Target genes are iNOS, Arg-1, LDHA, PKM2, associated with glycolysis, GLUT3, and endogenous standard gene β-actin. The specific primer sequences for every gene are described in Table 1. The process was as follows: predenaturalization was performed at 95 °C for five minutes, then at 95 °C for 10 s, then at 60 °C for 30 s. Relative gene expression levels were computed by the 2^−ΔCt method. The results showed that iNOS, Arg-1, LDHA, PKM2, GLUT3, and β-actin were detected by this method.

Table 1.

Primer sequences

Primer name	Sequence	Base count (bp)
iNOS	5′-ACA TCG ACC CGT CCA CAG TAT-3′	177
	3′-CAG AGG GGT AGG CTT GTC TC-5′
Arg-1	5′-TGT CCC TAA TGA CAG CTC CTT-3′	204
	3′-GCA TCC ACC CAA ATG ACA CAT-5′
LDHA	5′-GCA GTT CGG CTA TAA CAC TGG-3′	82
	3′-GCG GTG GTT CCA TGT TTG ATT G-5′
PKM2	5′-CGC CTG GAC ATT GAC TCT G-3′	135
	3′-GAA ATT CAG CCG AGC CAC ATT-5′
GLUT3	5′-TCATCA ATG CAC CTG AGA CAA TC-3′	84
	3′-GTC CCT CAC TTG GTA GGT CTT-5′
β-actin	5′-GTG ACG TTG ACA TCC GTA AAG A-3′	245
	3′-GCC GGA CTC ATC GTA CTC C-5′

Small Interfering RNA Transfection

In this study, the small interfering RNA (siRNA) targeting the gene was obtained by chemical synthesis. Anhui General Biotechnology Co., Ltd. (Chuzhou, China) completed the sequence design and synthesis verification. The steps for preparing the transfection complex are as follows: Gently mix five μL of Lipo3000 liposome transfection reagent (Thermo Fisher Scientific, USA, Cat. No. L3000001) with 125 μL of Opti-MEM serum-free medium (Thermo Fisher Scientific, Cat. No. 31985070), and let it stand for 5 min. Separately, dissolve five μL of siRNA in 125 μL of Opti-MEM containing 10 μL of P3000 enhancer, and vortex for 30 s. After mixing the above two solutions in equal volumes, incubate them at 37 °C for 15 min to form a stable transfection complex. The prepared complexes were uniformly added drop-by-drop to the pre-cultured BV2 cells in a six-well plate and then placed in an incubator at 37 °C with 5% CO₂ for a further 48-h incubation. The mRNA expression levels of target genes were detected by quantitative real-time PCR (qPCR), and the corresponding protein expression levels were analyzed by Western blot to evaluate the knockdown efficiency of siRNA systematically. The experiment set up three biological replicates, and the data were normalized based on the non-transfected group.

Cytotoxicity Assay

BV2 microglial cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well. Following cell adhesion, treatment solutions containing Jaceosidin at concentrations ranging from 0 to 160 μM were added to the wells. The plates were then incubated in a humidified atmosphere at 37 °C with 5% CO₂ for 24 h. After removal of the drug-containing medium, 10 μL of CCK-8 reagent (Beyotime Biotechnology Co., Ltd., Cat. No. C0038) was added to each well, and the plates were further incubated in the dark for 30 min. The absorbance was subsequently measured at 450 nm using a multi-functional microplate reader (Thermo Fisher Scientific, model Multiskan MK3), and cell viability was expressed as a percentage relative to the non-drug-treated control group.

In vitro Inflammation Model

BV2 cells were seeded in 6-well dishes and stimulated with lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, USA; Cat. No. L3129) at a concentration of 1 μg/mL for various durations, up to 24 h. After treatment, protein lysates were prepared and subjected to Western blot analysis.

Analysis of Cellular Bioenergetics

To evaluate the energy metabolism characteristics of BV2 microglia, the Seahorse XFe24 Cell Energy Metabolism Analysis System was employed to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). The experimental procedure was as follows: BV2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. The study was divided into four experimental groups (n = 3): the control group (treated with PBS), the LPS group (10 mg/L LPS), the LPS + 10 μM Jaceosidin group, and the LPS + 20 μM Jaceosidin group. The treatment duration was 4 days. Cells from each group were harvested and seeded onto Seahorse XF24 assay plates at a density of 1 × 10⁴ cells per well.

ECAR measurement: Following two washes with a detection buffer pre-equilibrated to pH 7.4 ± 0.1, sequential injections of 10 mmol/L glucose (to stimulate glycolysis), 1 μmol/L oligomycin (to inhibit ATP synthase), and 50 mmol/L 2-deoxyglucose (2-DG, to block glycolysis) were performed. Baseline glycolytic capacity and maximal glycolytic flux are monitored in real time.

OCR measurement: Parallel samples were sequentially exposed to 1 μmol/L oligomycin (to uncouple ATP synthesis), 2 μmol/L FCCP (to maximize mitochondrial respiration), and 2 μmol/L rotenone/antimycin A (Rot/AA, to inhibit complexes I and III), in order to assess basal respiration, ATP production, and maximal respiratory capacity. Data were normalized and analyzed using Wave software to reflect dynamic changes in cellular energy metabolism under different treatment conditions.

Enzyme-Linked Immunosorbent Assay (ELISA)

The spinal cord tissue from the injured area was collected, rinsed with saline, and homogenized thoroughly in a mortar. After centrifugation at 10,000 r/min for 10 min, the supernatant was collected to measure the levels of IL-6, TNF-α, and PGE2 using an ELISA kit from Proteintech, according to the manufacturer’s instructions.

Simulation of Molecular Docking

Molecular docking simulations were conducted using AutoDock Vina (v.1.1.2) and PyMOL (v.2.4.1) to evaluate the binding affinity between JAC and its potential molecular targets. Generally, lower binding energy values reflect more favorable and stable docking conformations. A binding energy threshold of −4.25 kcal/mol or lower is considered indicative of docking activity, while values equal to or below −5 kcal/mol suggest strong docking potential. The three-dimensional structure of the PKM2 protein used in the docking process was retrieved from the Protein Data Bank (PDB), and the 3D structure of JAC was sourced from the PubChem database.

Statistical Analysis

Statistical evaluation was carried out using GraphPad Prism 9 for Windows (San Diego, California, version 23.0). All statistical procedures were executed in a double-blind fashion. The results are expressed as mean values ± standard error of the mean (SEM), and were adjusted to account for external variability. To compare differences between two independent groups, independent-sample t-tests were employed. When assessing variations across three groups, one-way ANOVA was conducted, followed by LSD post hoc analysis or Dunnett’s T3 method (in cases where homogeneity of variances could not be assumed). For comparisons involving four or more groups, two-way ANOVA was used in combination with Tukey’s multiple comparisons test for normally distributed data, while the nonparametric Mann–Whitney U test was applied for non-normal distributions. Additionally, for between-group comparisons of BMS scores across multiple time points, two-way repeated-measures ANOVA and the least significant difference (LSD) test were utilized. A p-value less than 0.05 was regarded as statistically significant.

Results

Jaceosidin Can Alleviate Secondary Inflammation in the Spinal Cord Injury Area of Mice

To elucidate the anti-inflammatory effects of Jaceosidin in spinal cord injury (SCI), this study conducted a systematic evaluation using both in vitro and in vivo models. First, the CCK-8 assay was used to assess the impact of various concentrations of Jaceosidin on microglial cell viability. The results showed that concentrations below 20 μM did not significantly inhibit cell viability (Fig. 1C), indicating minimal cytotoxicity within this range. Subsequent experiments utilized an SCI mouse model, which was divided into three groups: a sham operation group, an SCI group, and an SCI + Jaceosidin group, to evaluate the neuroprotective and anti-inflammatory effects of the compound. Immunofluorescence staining performed on the 14th day post-surgery revealed an increased number of neurons in the Jaceosidin treatment group, demonstrating enhanced neuronal survival and repair capacity compared to the SCI group. The expression of Iba-1, a marker of microglial activation, was significantly reduced in the Jaceosidin group, suggesting that the compound effectively suppresses the abnormal activation of inflammation-related glial cells in the injured area (Fig. 1E). To quantitatively evaluate changes in inflammatory factors, ELISA was performed on spinal cord tissue homogenates. The results indicated that the protein levels of the pro-inflammatory cytokines IL-6 and TNF-α were significantly lower in the Jaceosidin group compared to the SCI group (Fig. 1F), confirming its anti-inflammatory effect through the inhibition of inflammatory mediator release.

Fig. 1.

Jaceosidin alleviates secondary inflammation in the spinal cord injury area. A The two-dimensional molecular configuration of jaceosidin. B The three-dimensional structural representation of jaceosidin. C Microglial cells were subjected to varying concentrations of jaceosidin and assessed using the Cell Counting Kit-8 (CCK-8) assay. D Development of the spinal cord injury (SCI) mouse model. Image created using Adobe Illustrator 2023. E Fourteen days following SCI, immunofluorescence staining was employed to evaluate the expression of ionized calcium-binding adapter molecule 1 (Iba-1, red—CoraLite594) and neuronal nuclear antigen (NeuN, green—CoraLite488) at the injury site in SCI mice.F The concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in the spinal cord tissues of each experimental group were measured.All data are presented as mean ± SEM, *p < 0.05, **p < 0.01, **p < 0.001, and ***p < 0.0001 (n = 3 per group)

Jaceosidin Can Alleviate Inflammation in an In Vitro Model of SCI

Building on previous findings that Jaceosidin can inhibit secondary inflammation following spinal cord injury (SCI), this study utilized an LPS-induced BV2 microglial cell inflammation model to investigate its effects on neuroinflammatory phenotype regulation, specifically M1/M2 polarization. LPS-activated BV2 cells were treated with 10 μM and 20 μM concentrations of Jaceosidin, which had been previously validated as non-cytotoxic, and its anti-inflammatory mechanisms were analyzed through multidimensional experimental approaches. Western blot analysis revealed that Jaceosidin significantly reduced the expression of the pro-inflammatory M1 marker iNOS while increasing the expression of the anti-inflammatory M2 marker Arg-1 in a dose-dependent manner (Fig. 2A, B). Immunofluorescence staining confirmed a significant decrease in iNOS fluorescence and an increase in Arg-1 fluorescence in Jaceosidin-treated cells (Fig. 2C, D), consistent with the observed changes in protein expression levels. Furthermore, real-time quantitative reverse transcription PCR (qRT-PCR) demonstrated that Jaceosidin dose-dependently suppressed the mRNA levels of M1-related inflammatory factors while enhancing those of M2 markers (Fig. 2E).

Fig. 2.

Jaceosidin can alleviate the excessive polarization in the microglial inflammation model. A, B Western blotting was utilized to assess the protein expression of iNOS and Arg-1 in microglia under various treatment conditions, followed by statistical analysis. C, D Immunofluorescence staining was conducted to detect the immunoreactivity of iNOS (green—CoraLite488) and Arg-1 (red—CoraLite594) in microglia treated with jaceosidin, along with corresponding statistical evaluation. E Quantitative polymerase chain reaction (q-PCR) was performed to measure the mRNA expression levels of Arg-1 and iNOS in microglia under different experimental conditions.All data are presented as mean ± SEM, *p < 0.05, **p < 0.01, ** *p < 0.001, and * ***p < 0.0001 (n = 3 per group)

Jaceosidin Regulates Glycolysis in Lipopolysaccharide-Treated Microglia

Recent studies suggest that glycolysis, beyond its central role in energy metabolism, also contributes to the inflammatory response through the regulation of immune cell metabolic reprogramming. This study investigated whether the anti-inflammatory effects of Jaceosidin involve the modulation of energy metabolism, with a specific focus on glycolytic alterations in an LPS-induced BV2 microglial cell inflammation model. Glycolytic function assays demonstrated that Jaceosidin significantly increased the oxygen consumption rate (OCR) and decreased the extracellular acidification rate (ECAR) in LPS-activated BV2 cells (Fig. 3A, B). Western blot analysis revealed a dose-dependent reduction in the expression of PKM2, a key glycolytic enzyme, following Jaceosidin treatment (Fig. 3C, D). These findings were supported by real-time quantitative reverse transcription PCR (qRT-PCR), which showed significant downregulation of PKM2 downstream target genes in the Jaceosidin-treated group (Fig. 3E). Immunofluorescence staining further confirmed reduced PKM2 expression in Jaceosidin-treated cells (Fig. 3F, G), reinforcing its inhibitory effect on PKM2 activity. Molecular docking experiments demonstrated that Jaceosidin exhibits strong binding affinity to the active site of the PKM2 protein (Fig. 3H). Taken together, these results indicate that Jaceosidin reverses the pro-inflammatory phenotype of microglia by directly targeting PKM2 and disrupting the glycolysis-inflammation signaling axis.

Fig. 3.

Jaceosidin regulates the glycolytic level of microglia after inflammatory polarization. A Assess the extracellular acidification rate (ECAR) in microglia across various conditions to reflect glycolytic flux and capacity. B Determine the oxygen consumption rate (OCR) in microglia under different experimental conditions as a marker of oxidative phosphorylation (OXPHOS) activity. C, D Conduct Western blotting and subsequent statistical analysis to evaluate the expression levels of PKM2, LDHA, and GLUT3 in microglia exposed to different treatments. E Use quantitative polymerase chain reaction (qPCR) to analyze the mRNA expression of PKM2, GLUT3, and LDHA in microglia under varying conditions. F, G Immunofluorescence staining was performed to detect PKM2 immunoreactivity (red—CoraLite594) in microglia treated with Jaceosidin, followed by statistical evaluation. H Perform molecular docking analysis to investigate the interaction between Jaceosidin and PKM2. All data are presented as mean ± SEM, *p < 0.05, **p < 0.01, **p < 0.001 and ***p < 0.0001 (n = 3 per group)

Jaceosidin Alleviates LPS Induced Inflammation in BV2 Cells by Regulating PKM2

Previous studies have demonstrated that PKM2 activity is significantly elevated in the LPS-induced inflammation model of BV2 microglial cells, suggesting a potential role in microglial inflammatory regulation. To test this hypothesis, we employed siRNA to knock down PKM2 expression in BV2 cells, with the aim of systematically investigating the causal relationship between PKM2 and the inflammatory phenotype. Transfection with PKM2-specific siRNA (siPKM2) significantly reduced both PKM2 protein and mRNA levels in BV2 cells, confirming effective gene silencing (Fig. 4A–C). Western blot analysis further revealed that PKM2 knockdown in the LPS-induced inflammation model significantly suppressed the expression of the pro-inflammatory M1 marker iNOS and enhanced the expression of the anti-inflammatory M2 marker Arg-1 (Fig. 4D, E). Real-time quantitative reverse transcription PCR (qRT-PCR) results corroborated these findings, showing decreased mRNA levels of M1-related genes and increased expression of M2-associated markers in the PKM2 knockdown group (Fig. 4F). Immunofluorescence staining of siPKM2-transfected BV2 cells confirmed a significant reduction in the fluorescence intensity of inducible nitric oxide synthase (iNOS), a hallmark of the pro-inflammatory M1 phenotype, along with a corresponding increase in arginase-1 (Arg-1) fluorescence, a marker of the anti-inflammatory M2 phenotype (Fig. 4G, H). In subsequent experiments, a PKM2 overexpression plasmid was transfected into BV2 cells. Transfection resulted in a significant increase in both PKM2 protein and mRNA levels in the overexpression group (Fig. 4I–K). Notably, following PKM2 overexpression, Jaceosidin no longer significantly suppressed iNOS protein expression or enhanced Arg-1 expression (Fig. 4L, M). Subsequent immunofluorescence assays further validated the Western blot (WB) results (Fig. 4N, O). Collectively, these findings indicate that Jaceosidin alleviates lipopolysaccharide (LPS)-induced neuroinflammation in microglia by modulating PKM2.

Fig. 4.

Jaceosidin alleviates LPS-induced inflammation in BV2 cells by regulating PKM2. A, B Western blotting and subsequent statistical evaluation were conducted using an anti-PKM2 antibody following the transfection of microglia with PKM2-targeting small interfering RNA (siRNA). C The knockdown efficiency of PKM2 was assessed through quantitative polymerase chain reaction (q-PCR). D, E Protein expression levels of Arg-1 and iNOS in microglia under various treatment conditions were analyzed by Western blotting, followed by statistical interpretation. F Quantitative polymerase chain reaction (q-PCR) was employed to measure the mRNA expression levels of Arg-1 and iNOS in microglia exposed to different experimental conditions. G, H Immunofluorescence staining was used to detect the immunoreactivity of iNOS (green—CoraLite488) and Arg-1 (red—CoraLite594) in treated microglia, accompanied by statistical analysis. I–K Assessment of PKM2 overexpression efficiency was carried out. L, M The expression levels of iNOS and Arg-1 proteins under different treatment regimens were evaluated and subjected to statistical analysis. N, O Immunofluorescence staining was applied to detect the immunoreactivity of iNOS and Arg-1 in microglial cells, followed by statistical evaluation.All data are presented as mean ± SEM, *p < 0.05, * * p < 0.01, **p < 0.001 and ***p < 0.0001 (n = 3 per group).

Jaceosidin Can Reduce the Activity of PKM2 in the Spinal Cord Injury Area, Alleviate Tissue Damage After Spinal Cord Injury, and Promote Functional Recovery

To investigate the role of PKM2 in spinal cord repair, Jaceosidin was administered intraperitoneally to injured mice daily following surgery. On day 14, immunofluorescence analysis revealed that, compared with the SCI group, the expression of Arg-1 near the injury site was significantly upregulated in the SCI + Jaceosidin group (Fig. 5A, B). In contrast, the fluorescence intensities of iNOS and PKM2 were markedly reduced (Fig. 5C–F). Histological evaluation demonstrated a smaller lesion size in Jaceosidin-treated SCI mice (Fig. 5G). Functional assessments indicated enhanced recovery in the treatment group, as evidenced by higher Basso Mouse Scale (BMS) scores, decreased residual urine volume (Fig. 5H, I), and improved gait parameters, including increased stride length and altered stride width in footprint tests (Fig. 5J–L). These findings suggest that Jaceosidin administration promotes both tissue preservation and functional recovery following spinal cord injury.

Fig. 5.

Jaceosidin promotes the recovery of spinal cord injury in mice. A, B Immunofluorescence staining was utilized to visualize the immunoreactivity of ionized calcium-binding adapter molecule 1 (Iba-1, red—CoraLite594) and Arg-1 (green—CoraLite488). Quantitative results demonstrated that, compared to the spinal cord injury (SCI) group, the SCI + JAC group exhibited a marked increase in Arg-1 expression near the injury site. C, D The expression of iNOS and Iba-1 (red—CoraLite594) was assessed using immunofluorescence staining. Quantitative analysis revealed that the SCI + JAC group showed a significant decrease in iNOS levels at the injury site compared to the SCI group. E, F PKM2 (red—CoraLite594) and Iba-1 (green—CoraLite488) immunostaining was performed using immunofluorescence techniques. The findings revealed that the fluorescence intensity of both PKM2 and Iba-1 surrounding the spinal cord lesion was notably lower in the SCI + Jaceosidin group than in the SCI group. G Histological evaluation of spinal cord sections from rats 14 days after SCI (n = 3 per group) was conducted using Hematoxylin–Eosin (HE) staining and Nissl staining. H, I Locomotor function was assessed using the Basso Mouse Scale (BMS), and urinary retention was recorded at multiple time points (1, 3, 5, 7, 14, 21, and 28 days) post-injury. J–L Gait analysis, including walking distance and stride width, was performed. The SCI + Jaceosidin group exhibited significantly longer step lengths and narrower stride widths compared to the SCI group.All data are presented as mean ± SEM, *p < 0.05, * * p < 0.01, ** *p < 0.001, and * ***p < 0.0001 (n = 3 per group)

Discussion

Spinal cord injury (SCI) represents a significant health challenge due to the limited availability of effective therapeutic options. A comprehensive understanding of its pathological mechanisms is essential for the development of novel treatment strategies. Microglial activation is a hallmark of the secondary inflammatory response following SCI (Devanney et al., 2020), and microglia-mediated inflammation has a substantial impact on functional recovery (Al Mamun et al., 2020). M1-type microglia, typically activated by lipopolysaccharide (LPS), release pro-inflammatory mediators that are detrimental to neuronal survival (David & Kroner, 2011). In contrast, M2-type microglia contribute to recovery by clearing cellular debris through phagocytosis and secreting neurotrophic and protective factors (Gensel et al., 2009; Lourbopoulos et al., 2015). Therefore, M2-type microglia promote neuronal and functional recovery, whereas M1-type microglia hinder it (Han et al., 2018).

Traditional Chinese herbal medicines, recognized for their robust biological activities and minimal side effects, have been widely used in clinical settings and serve as a valuable source of potential therapeutic agents. This study demonstrates that Jaceosidin significantly attenuates the secondary inflammatory response in mice following spinal cord injury (SCI) and enhances motor function recovery by inhibiting PKM2, a key enzyme in glycolysis. Following Jaceosidin administration, the expression of pro-inflammatory factors in the injured spinal cord is markedly reduced, and both excessive microglial activation and glycolytic metabolism are suppressed. Mechanistically, Jaceosidin specifically binds to the active site of PKM2, inhibiting the nuclear translocation of its monomeric form and blocking the final step of glycolysis. This action reverses Warburg effect-driven metabolic reprogramming, thereby reducing M1-type microglial polarization and the release of inflammatory mediators. Previous studies have shown that PKM2 enhances glycolytic flux, promoting the release of pro-inflammatory cytokines and immune cell infiltration, thus establishing a "metabolism-inflammation" feedback loop in inflammatory diseases (Wang et al., 2022). The role of natural compounds in modulating the PKM2-mediated glycolysis-immune network in SCI remains largely unexplored. This study reveals that Jaceosidin mitigates the neuroinflammatory cascade following SCI by inhibiting PKM2-dependent glycolysis. These findings are consistent with prior research indicating that PKM2 exacerbates inflammation via metabolic reprogramming. Importantly, this study highlights the therapeutic potential of the natural flavonoid Jaceosidin in modulating the pathological processes of SCI by targeting the glycolysis-immune interaction network, providing novel experimental evidence for the development of neuroprotective strategies based on metabolic regulation.

This study has several limitations. The effects of Jaceosidin were validated exclusively in a mouse model, without further evaluation in more complex SCI models or larger animals. Additionally, the downstream effector molecules of PKM2 and their interactions with other metabolic pathways, such as oxidative phosphorylation, remain to be elucidated. Future studies could employ single-cell sequencing to investigate the specificity of Jaceosidin’s regulatory effects on various immune cell subsets within the spinal cord microenvironment and explore its potential synergistic interactions with existing anti-inflammatory therapies.

In conclusion, Jaceosidin alleviates secondary inflammation following spinal cord injury by inhibiting PKM2 activity. These findings provide a novel theoretical framework and identify potential therapeutic candidates for the development of effective SCI treatment strategies.

Author Contributions

RF conceived the study idea, revised the manuscript, and provided financial support. BD, ZF collected the data and wrote the initial draft. RC, XW contributed to the data collection and analysis. All authors approved the final draft of the manuscript. All authors are accountable for all aspects of the work in ensuring related questions ’ accuracy or integrity. Any parts of the work are appropriately investigated and resolved. RF is the guarantor. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.

Funding

This study was supported by a grant from the Basic and Clinical Collaborative Research Promotion Initiative of the Third Affiliated Hospital of Anhui Medical University (2023sfy017).

Data Availability

No datasets were generated or analyzed during the current study.

Declarations

Competing interests

The authors declare no competing interests.