INTRODUCTION
Spinal cord injury (SCI) is characterized by primary and secondary injury phases that contribute to severe neurological deficits. Following the initial mechanical damage, secondary injury processes including inflammation and oxidative stress further exacerbate tissue damage (Ahuja et al., 2017). During this phase, astrocytes become reactive and increase the expression of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans, leading to glial scar formation, which limits axonal regeneration (Bradbury and Burnside, 2019; Sofroniew, 2015). In addition, excessive activation of the mammalian target of rapamycin (mTOR) signaling pathway after injury has been associated with astrocyte proliferation, glial scar formation, and impaired neural repair (Li et al., 2015). To investigate sequential secondary injury and neural regeneration after SCI, pharmacological strategies targeting oxidative stress and inflammation have been proposed. Among these approaches, Naringenin, a citrus-derived flavonoid, exhibits potent antioxidant and anti-inflammatory properties and has been reported to promote neuronal survival and functional recovery in experimental models of SCI (Fakhri et al., 2022; Shi et al., 2016).
In parallel, exercise has been recognized as a noninvasive therapeutic strategy that promotes neural recovery after central nervous system injury. Exercise has been shown to contribute to neuroplasticity, increase neurotrophic factor expression, and modulate inflammatory responses, thereby partly facilitating neural repair and functional recovery, through the restoration of neural network connectivity in the injured spinal cord and brain (Leech and Hornby, 2017; Rojas Vega et al., 2008). Taken together, these findings suggest that exercise and naringenin may exert neuroprotective effects through overlapping mechanisms, including the regulation of oxidative stress, inflammation, and intracellular signaling pathways. Among these pathways, the mTOR signaling plays a critical role in astrocyte activation, glial scar formation, and axonal regeneration after SCI. However, the interaction between mTOR signaling and exercise- or naringenin-mediated neuroprotection remains unclear. Furthermore, although naringenin shows promising effects in experimental models, its bioavailability and applicability to human clinical treatment remain to be fully established.
Although most previous studies have primarily focused on pathological changes within the injury epicenter (Ahuja et al., 2017), functional recovery after SCI also relies on neuroplasticity and axonal regeneration processes in the caudal spinal cord (Bareyre et al., 2004). Axonal sprouting within the caudal spinal cord contributes to functional reorganization of descending neural pathways, and increased expression of regeneration-associated proteins such as activating transcription factor-3 (ATF-3) and growth-associated protein-43 (GAP-43) has been observed during this process (Seijffers et al., 2007; Yuan et al., 2009). Given the role of mTOR signaling in axonal regeneration, understanding its involvement in the caudal spinal cord may be critical for elucidating mechanisms of functional recovery beyond the injury site.
Collectively, naringenin treatment and exercise have been reported to promote neural regeneration and functional recovery following SCI. However, most studies have investigated these interventions independently, and research evaluating their combined effects on axonal sprouting and the regenerative microenvironment in the caudal region remains limited. Therefore, the present study investigates the combined effect of exercise and naringenin treatment on neural regeneration after SCI, with a particular focus on mTOR signaling, neuronal cell survival in the ventral region of the injury site, and sprouting of corticospinal tract (CST) fibers and regeneration-related signaling changes in the caudal region of the injured spinal cord. This approach may provide insights into the mechanisms underlying neural plasticity and functional recovery beyond the injury site.
MATERIALS AND METHODS
Experimental animals
Male Sprague-Dawley rats (4 weeks old) were used in the present study. The animals were housed under controlled environmental conditions with a temperature of 22°C–24°C and a relative humidity of approximately 60%, under a 12 hr light-dark cycle. Standard laboratory chow (Samyang Co., Korea) and water were provided ad libitum throughout the experimental period. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Jeju National University (approval No. 2022-0056) and were performed in accordance with the established guidelines for animal care and use. The animals were randomly assigned into four groups (n=10 in each group): the normal control group (Norm), the SCI+sedentary group (SCI+S), the SCI+naringenin group (SCI+Na), and the SCI+naringenin combined with exercise group (SCI+Na+Ex).
SCI and naringenin extract administration
Spinal cord contusion injury was induced in rats under inhalation anesthesia with isoflurane (1.5%–2.5%) using an anesthesia system (Jeungdo Bio and Plant, Korea). After a midline skin incision, the spinal cord was exposed at the T9–10 level by laminectomy without damaging the dura mater. A moderate contusion injury was then produced by dropping a 10-g weight from a height of 2.5 cm onto the exposed dorsal surface of the spinal cord using the New York University Impactor System (NYU Impactor, USA) as previously described (Yu and Seo, 2024). Naringenin (purity 95%, molecular weight 272.25; Sigma-Aldrich, USA) was administered in a volume of 20 μL immediately after SCI (Fakhri et al., 2022). The compound was locally injected at the lesion site using a microsyringe. Following the procedure, the muscles and skin were sutured, and the animals were placed on a heating pad to maintain body temperature (37°C) until recovery from anesthesia. Twenty-four hr after the final exercise session, all animals were euthanized by CO2 inhalation for tissue collection. The thoracic spinal cord (T5–12) and cerebrum were rapidly dissected and stored at −80°C until further analysis.
Low-intensity treadmill exercise program
Two weeks after SCI, treadmill exercise training was initiated. The animals performed treadmill running 5 days per week for 4 consecutive weeks. The exercise protocol was adapted from Kim et al. (2025). Training was conducted on a motorized treadmill (CAST-10R, CASSCALE, Korea) at a speed of 6–8 m/min with a 0° incline.
Immunofluorescence staining
Injured spinal cord tissues were embedded in frozen section compound (Leica Biosystems Richmond Inc., USA) and sectioned using a cryostat (CM1860, Leica, Germany). Cross sections of the spinal cord were cut at a thickness of 30 μm. Double immunofluorescence staining was performed to detect neurofilament-200 (NF-200) and GFAP in the spinal cord. The sections were fixed in 4% paraformaldehyde containing 4% sucrose in phosphate-buffered saline (PBS) for 40 min, permeabilized with 0.5% Nonidet P-40 in PBS, and blocked with 2.5% bovine serum albumin (BSA; Sigma-Aldrich) for 4 hr. The sections were then incubated with the following primary antibodies: anti-NF-200 (rabbit polyclonal, 1:1,000; Sigma-Aldrich), neuronal nuclei (NeuN, rabbit monoclonal, 1:200; Cell Signaling Technology, USA) and GFAP (rabbit and mouse monoclonal, 1:200; Cell Signaling Technology). After primary antibody incubation, the sections were incubated with rhodamine-conjugated goat anti-rabbit IgG (1:400; Molecular Probes, USA) and fluorescein-conjugated goat anti-mouse IgG (1:400; Molecular Probes) for 1 hr at room temperature. Cell nuclei were counterstained with Hoechst 33258 (2.5 μg/μL; Sigma-Aldrich) for 10 min prior to the final wash. Finally, the sections were mounted with gelatin mounting medium and observed using a fluorescence microscope (E-600, Nikon, Japan). Digital images were captured and processed using Adobe Photoshop software (CS6, Adobe Systems, USA).
Western blot analysis
Tissue samples were rinsed with PBS and lysed in Triton lysis buffer. Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto a polyvinylidene difluoride membrane for 2 hr. The membranes were blocked with 5% BSA (Sigma-Aldrich) in tris-buffered saline containing 0.1% Tween-20 at room temperature for 1 hr, followed by incubation with primary antibodies at 4°C for 24 hr. The primary antibodies used were anti-NF-200 (rabbit polyclonal, 1:1,000; Sigma-Aldrich), anti-NeuN (mouse monoclonal, 1:1,000; Cell Signaling Technology), anti-GFAP (rabbit monoclonal, 1:1,000; Cell Signaling Technology), anti-phosphoinositide 3-kinase (PI3K, mouse monoclonal, 1:250; Santa Cruz Biotechnology, USA), anti-phosphorylated protein kinase B (p-AKT, rabbit polyclonal, 1:250; Cell Signaling Technology), anti-phosphorylated mTOR (p-mTOR, mouse monoclonal, 1:250; Santa Cruz Biotechnology), anti-GAP-43 (mouse monoclonal, 1:200; Santa Cruz Biotechnology), anti-ATF-3 (rabbit polyclonal, 1:400; Santa Cruz Biotechnology), anti-interleukin-6 (IL-6, rabbit polyclonal, 1:1,000; GeneTex, USA), anti-cellular fos proto-oncogene protein (c-Fos, rabbit monoclonal, 1:1,000; Thermo Fisher Scientific, USA), and anti-β-actin (mouse monoclonal, 1:1,000; Santa Cruz Biotechnology). After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; GeneTex) for 1 hr at room temperature. Protein bands were detected using Westar enhanced chemiluminescence detection reagent (Cyanagen, Italy) and visualized with a ChemiDoc imaging system (Bio-Rad, USA).
In this study, the primary outcome measures included markers of neuronal integrity (NF-200, NeuN), axonal regeneration (GAP-43, ATF-3), astrocyte activation (GFAP), inflammatory response (IL-6), and neural activity (c-Fos). In addition, activation of the PI3K/AKT/mTOR signaling pathway was analyzed to investigate the underlying molecular mechanisms associated with neural repair.
Statistical analysis
All data are expressed as the mean±standard error of the mean. Statistical comparisons were carried out using one-way analysis of variance followed by Duncan post hoc test, based on the statistical procedures used in previous studies (Yu and Seo, 2024). A significance threshold was set at P<0.05. Data processing and graphical representations were performed using Prism 6 software (GraphPad Software, USA).
RESULTS
Region-specific expressions of GFAP, NF-200, and NeuN after SCI
Western blot analysis was conducted to investigate the effects of the combined treatment of exercise and naringenin on glial scar formation and axonal outgrowth following SCI. As shown in Fig. 1, in the dorsal region of the injury site 4 weeks after SCI, GFAP expression was significantly increased in the SCI+Na (P<0.001) and SCI+Na+Ex (P<0.001) groups compared to the SCI+S group. Increased GFAP expression in the dorsal region of the injury site may reflect enhanced astrocytic activation and glial scar formation, which could contribute to the reduction of cavity formation after SCI. In addition, the expression levels of NF-200 in dorsal region of the injury site were significantly increased in the SCI+Na+Ex group (P<0.001) compared with the SCI+S group, indicating enhanced axonal integrity or axonal regeneration. Furthermore, NeuN expression in the ventral region of the injury site was significantly increased in the SCI+Na+Ex group (P<0.001) compared with the SCI+S group, suggesting that exercise combined with naringenin may improve neuronal survival after SCI.
Change in cavity through astrocyte activation after SCI
To evaluate astrocyte proliferation to form a glial scar following SCI. The spinal cord was stained with Hoechst and anti-GFAP antibody, and we performed quantification of histological findings. As shown in Fig. 2, spared spinal tissue was significantly increased in the SCI+Na (P<0.05) and SCI+Na+Ex (P<0.05) groups compared to the SCI+S group. In the enlarged images, intensity of GFAP-stained astrocytes was further increased SCI+Na (P<0.05) and SCI+Na+Ex (P<0.05) groups compared to the SCI+S group. In addition, the number of astrocytes infiltrating the cavity was significantly higher in the SCI+Na+Ex group (P<0.05) than in the SCI+S and SCI+Na groups.
Motor neuron survival in the ventral region after SCI
To evaluate neuronal survival following SCI, motor neuron stained with anti-NeuN antibody in the ventral horn around the injury site was analyzed. As shown in Fig. 3, the number of motor neurons was significantly preserved in the SCI+Na+Ex group (P<0.05) compared with the SCI+S group, suggesting that the combined treatment of naringenin and exercise may have beneficial effects on motor neuron survival after SCI.
Sprouting of CST fibers through reducing of the PI3K/AKT/mTOR signaling pathway after SCI
Western blot and immunofluorescence analyses were conducted to investigate changes in the PI3K/AKT/mTOR signaling pathway and sprouting growth of CST fibers following SCI. As shown in Fig. 4A, PI3K expression and AKT/mTOR phosphorylation levels in the region 10 mm caudal to the injury epicenter after SCI were significantly decreased in the SCI+Na and SCI+Na+Ex groups compared with the SCI+S group (P<0.001 for p-AKT; P<0.01 and P<0.001 for p-mTOR). Consistent with these findings, immunofluorescence staining showed that the intensity of NF-200–positive axons in the CST region of the spinal cord was significantly increased in the SCI+Na+Ex group compared with the SCI+S group (P<0.05) (Fig. 4B).
Changes in regenerative and inflammatory proteins after SCI
To evaluate changes in regeneration- and inflammation-related proteins at the injury site following SCI, the expression levels of GAP-43, ATF-3, IL-6, and c-Fos were analyzed (Fig. 5). GAP-43 expression was significantly increased in the SCI+Na+Ex group compared with the SCI+S group (P<0.001). ATF-3 expression was significantly increased in the SCI+Na and SCI+Na+Ex groups compared with the SCI+S group (P<0.001). In addition, IL-6 expression was significantly increased in the SCI+Na group compared with the SCI+S group (P<0.05) and further increased in the SCI+Na+Ex group compared with the SCI+S group (P<0.001). Furthermore, c-Fos expression was significantly increased in the SCI+Na+Ex group compared with the SCI+S group (P<0.01).
DISCUSSION
The present study investigated whether treadmill exercise combined with naringenin administration could enhance neuronal survival and regenerative responses following SCI. The major finding of this study is that the combined intervention attenuated the extent of tissue loss and neural cell death, while promoting CST sprouting after SCI compared with the SCI+S group. In particular, increased GFAP and NF-200 expression was observed in the dorsal lesion area, together with relatively preserved NeuN-stained motor neurons in the ventral region of the injured spinal cord. These molecular changes were consistent with the histological findings, suggesting that treadmill exercise combined with naringenin contributed to maintaining the structural integrity of the injured spinal cord.
In the present study, one notable observation was the difference in tissue morphology between groups. The SCI+S group exhibited severe tissue disorganization without a clearly defined cavity boundary, whereas the combined treatment group showed a more distinct lesion margin. In addition, a greater accumulation of GFAP-positive astrocytes was observed within and around the cavity in the SCI+Na+Ex group. Reactive astrocytes are known to play a dual role following SCI. Although glial scar formation has traditionally been considered inhibitory to axonal regeneration, early astrocytic activation is essential for isolating the injury site and limiting secondary tissue damage (Sofroniew, 2015). Previous studies have demonstrated that reactive astrocytes form a barrier that restricts inflammatory cell infiltration and prevents the spread of secondary degeneration (Anderson et al., 2016; Sofroniew, 2015). Therefore, increased astrocytic activation in the combined treatment group may contribute to stabilizing the lesion environment and protecting surrounding neural tissue.
This stabilization of the lesion microenvironment was associated with the preservation of motor neurons in the ventral horn of the injured spinal cord. The combined intervention group showed greater survival of NeuN-positive neurons compared with the SCI+S group. Motor neurons are particularly vulnerable to secondary injury processes following SCI, including excitotoxicity, oxidative stress, and inflammatory signaling (Ahuja et al., 2017; Chen et al., 2023). Thus, astrocyte-mediated containment of the lesion site may help preserve neuronal integrity by limiting the spread of secondary degeneration. In addition to structural preservation, the combined intervention modulated key intracellular signaling pathways associated with neural repair in the injured spinal cord. The PI3K/AKT/mTOR signaling pathway was significantly regulated in the caudal spinal cord. Although mTOR signaling is essential for neuronal growth and metabolism, dysregulated activation of the pathway following SCI can impair regenerative responses (Kanno et al., 2012).
In the present study, p-AKT and p-mTOR levels in the region 10 mm caudal to the injury epicenter after SCI were reduced toward near-normal levels in the combined treatment group, suggesting that treadmill exercise with naringenin may help maintain a balanced signaling environment favorable for neural repair. The regenerative environment was further supported by increased expression of GAP-43 and ATF-3, which are well-established markers of axonal elongation and neuronal responses in both peripheral and central nerve system injury. In addition, elevated c-Fos and IL-6 expression may reflect enhanced neuronal activity and adaptive molecular signaling in the caudal region of the injured spinal cord. The coordinated up-regulation of these markers suggests that activity-dependent neural remodeling processes were enhanced following the combined intervention.
Exercise is widely recognized as a potent modulator of neural plasticity after central nerve system injury (Bilchak et al., 2021). Activity-dependent stimulation induced by exercise promotes axonal sprouting, synaptic remodeling, and neuronal survival in the injured spinal cord and brain (Molteni et al., 2004). Previous studies have also reported that treadmill training enhances spinal neuronal plasticity and functional recovery after SCI by activating activity-dependent neural circuits (Smith and Knikou, 2016; Sun et al., 2013). Complementarily, naringenin possesses antioxidant, anti-inflammatory, and antiapoptotic properties that may attenuate secondary injury processes such as oxidative stress and inflammatory cascades (Nouri et al., 2019; Salehi et al., 2019). Therefore, the combined treatment may create a complementary therapeutic environment in which exercise promotes neuronal activation and plasticity, while naringenin stabilizes the injury microenvironment by reducing oxidative and inflammatory damage.
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