Introduction

Spinal cord injury (SCI) is a severe neurological condition caused by various factors like accidents, injuries, tuberculosis, and tumors. It typically presents with impairments in limb sensation and movement, loss of reflexes, paralysis, and in severe cases, death [1]. With the advancement of global economy, an increasing number of individuals are affected by SCI, profoundly impacting patients’ physical and mental well-being and imposing significant financial burdens on their families [2]. SCI progresses through two phases: the primary injury phase and the secondary injury phase. Primary SCI results from traumatic events that directly or indirectly impact the spinal cord, causing various irreversible damages that persistently harm spinal cord tissue [3]. Secondary SCI can develop within minutes to days after the primary injury and may continue for about one month, making it more complex than the initial injury. Pathophysiological changes such as oxidative stress, electrolyte disturbances, tissue edema, and cell apoptosis exacerbate spinal tissue damage under stressful microenvironmental conditions [4, 5]. Secondary injuries during SCI are critical because irreversible changes often occur at this stage, significantly affecting the patient’s prognosis. Consequently, recent research on SCI has focused on effective interventions targeting secondary injury [6, 7].

Oxidative stress response is an important pathological process in secondary SCI. Following SCI, there is a reduction in the expression of antioxidant enzymes and the activation of abnormal enzymes, leading to mitochondrial dysfunction, lipid peroxidation, DNA damage, and apoptosis [8, 9]. Moreover, peroxidation and apoptosis by-products exacerbate the production of reactive oxygen species (ROS), accelerating the progression of secondary injury and hindering neuronal regeneration [10]. Therefore, addressing oxidative stress remains a significant focus in neuroscience research, aiming to develop effective solutions to mitigate its impact.

Curculigo orchioides Gaertn is a herbaceous plant belonging to the Asphodelaceae family, widely distributed in tropical regions across Asia, Africa, Oceania, and South America [11]. In traditional Chinese medicine, its dried rhizome, known as "Xian Mao," is renowned for its efficacy in treating various conditions such as joint pain, osteoporosis, and convulsions. Curculigoside (CUR), one of the active components of Curculigo orchioides Gaertn, has demonstrated diverse pharmacological activities in several studies [12,13,14]. Research by Tian et al. highlights the antioxidant and neuroprotective properties of CUR, which play a role in reducing neuronal apoptosis and ROS production [15]. Therefore, CUR holds promise for SCI treatment, although no relevant research has yet been reported on this topic. In this study, we utilized PC12 cell models induced by hydrogen peroxide (H2O2) and SCI rat models to investigate whether CUR can mitigate oxidative stress and prevent neuronal apoptosis. Additionally, we aimed to elucidate the molecular mechanisms underlying these effects. These findings could potentially contribute to the development of pharmaceuticals for the prevention and treatment of SCI.

Materials and Methods

Materials

The following reagents and materials were used in this study: Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and trypsin from Gibco-BRL (New York, USA); penicillin and streptomycin from Hyclone (Logan, USA); CUR sourced from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); MTT assay kit purchased from Abcam (Cambridge, UK); apoptosis, Western blotting, and real-time quantitative polymerase chain reaction (RT-qPCR) assay kits, as well as H2O2 and Brusatol, obtained from Sigma-Aldrich (Saint Louis, USA); Malondialdehyde (MDA), superoxide dismutase (SOD), ROS, and glutathione (GSH) assay kits acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); and Bcl-2, Caspase-3, Bax, Nrf-2, and NQO-1 antibodies sourced from Affinity Biosciences CO (Beijing, China).

Cell culture

PC12 cells were cultured following a previously published protocol [16]. The cells were maintained in DMEM medium from Gibco-BRL (New York, USA), supplemented with 10% FBS and 1% penicillin/streptomycin from Hyclone (Logan, USA). The cell culture was incubated at 37 °C with 5% CO2. Media were refreshed every 48 h, and subculturing was performed when the cell density reached 90%.

Cell treatment

The PC12 cells were divided into five groups: control group, CUR group, H2O2 group, H2O2 + CUR group, and H2O2 + CUR + Brusatol group. CUR was used at a concentration of 3 μM, H2O2 at a concentration of 300 μM, and Brusatol at a concentration of 100 nM for the treatments.

Cell Viability Assay

According to the previously described method, cell viability was assessed using the MTT assay [17]. The cells were divided into 5 groups and seeded at an initial density of 1 × 104 cells per well for 24 h. Two groups were exposed to varying concentrations of H2O2 (100, 200, 300, 400, 500 μM) and CUR (1, 3, 10 μM). One group was pre-treated with different concentrations of CUR for 2 h, followed by 24 h of co-incubation with 300 μM H2O2. The last group was treated with 300 μM H2O2 and incubated for varying durations. After incubation, 10 μL of MTT solution (5 mg/mL) was added to each well and incubated for 3 h at 37 °C. The formazan crystals formed were dissolved in 100 μL of Dimethylsulfoxide (DMSO) per well [18]. The mixture was shaken at room temperature for 15 min, and absorbance was measured at 570 nm using a microplate reader (Bio-Rad, USA) to determine cell viability.

Apoptosis Assay

The apoptosis rate of cells was determined using the Annexin V/propidium iodide (PI) double-staining method and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, following established protocols [19, 20]. For Annexin V/PI staining, treated cells were collected, washed, and resuspended in binding buffer containing Annexin V-FITC and PI. After incubating the cells for 15 min, flow cytometry analysis was performed.

The TUNEL assay was performed utilizing the TUNEL apoptosis detection kit, where 4’,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei. The resulting observations were recorded using fluorescence microscopy (Olympus, Japan).

RT-qPCR Analysis

Total RNA extraction was conducted using the TRIzol reagent following treatment. RT-PCR analysis was carried out using the First-Strand cDNA Synthesis Kit in combination with the PrimeScript RT reagent kit on a SYBR green system (Toyobo, Japan Ltd., Japan). The primer sequences used are detailed in Table 1.

Table 1 Primers used for target amplification in this study
Full size table

Western Blotting Analysis

Protein concentrations were determined using the BCA protein assay kit. The proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked overnight at 4 °C with the respective primary antibodies (Bcl-2, Caspase-3, Bax, Nrf-2, NQO-1). Following this, the membrane was washed with tris buffered saline + 0.5% [v/v] Tween-20 (TBST) and incubated for 2 h at room temperature with HRP-conjugated secondary antibodies (1:1000 dilution). Glyceraldehyde phosphate dehydrogenase (GAPDH) at a 1:1000 dilution served as the internal standard. Quantitative densitometric analysis was conducted using the ImageQuant LAS 4000 mini detection system (GE Healthcare, Buckinghamshire, UK), and the results were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

Oxidative Stress Assay

For the in vitro test, intracellular ROS levels were assessed using DCFH-DA reagent following the manufacturer’s instructions. Fluorescence images were captured using a fluorescence microscope and analyzed with ImageJ software. Additionally, GSH concentration was determined using a GSH assay reagent.

In the in vivo test, oxidative stress markers including MDA, SOD, and GSH were measured using specific assay kits. Spinal tissue samples were processed according to the manufacturer’s instructions to extract the liquid supernatant for analysis. MDA levels were measured at 532 nm, he activity of SOD was assessed at 550 nm, and GSH levels were quantified at 420 nm using a microplate reader.

Animals

All animal experiments were conducted following approval from the Ethics Committee of Experimental Animal Management at Guangzhou University of Chinese Medicine (approval number: 20220803007). Sprague–Dawley (SD) male rats weighing 200-250 g were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine and housed in the animal laboratory. The rats underwent a one-week adaptation period under the following conditions: five rats per cage, ambient temperature maintained at 22–26 °C, a 12-h light–dark cycle, and humidity ranging from 55 to 68%.

Establishment of the Rat Model of SCI

The SCI animal model was induced using Allen’s method [21]. Rats were anesthetized, secured for shaving, and a median incision was made on the back to expose the T9-11 spinous processes and lamina, using T10 as a reference point. The spinous process and lamina of T10 were removed to expose the intact spinal cord underneath. A PinPoint™ precision SCI impactor was used to strike the T10 spinal cord with a velocity of 1.2 m/s, a depth of 1.0 mm, and a contact time of 85 ms. This caused rapid congestion and tissue redness in the T10 region. Transient spasms and convulsions in the tails and hind limbs confirmed successful establishment of the SCI model. Subsequently, hemostasis was achieved and the wound was sutured, followed by iodine application to the sutured skin. In sham-operated rats, the spinal cords were exposed similarly but not subjected to the impact procedure..

Grouping and Treatment of SCI Model

Fifty mature male Sprague–Dawley (SD) rats weighing 200-250 g were randomly divided into four groups, each comprising 30 rats: (1) sham group, (2) SCI model group, (3) SCI + NS group, and (4) SCI + CUR group. One day after successful SCI modeling, the rats received oral treatments once daily for 14 days at a dose of 50 mg/kg. The SCI + NS group was administered 10 mL/kg of normal saline, while the SCI + CUR group received 50 mg/kg of CUR. On day 14 post-treatment, the rats were euthanized for analysis.

Locomotion Recovery Assay

To evaluate the mobility of rats following SCI, inked footprint analysis and the Basso-Beattie-Bresnahan (BBB) locomotion rating scale were used [22, 23]. Rats were allowed 15 min of free activity in an open field on days 3, 7, and 14 post-surgery. The BBB scale, ranging from 0 to 21 points, was used to assess hindlimb locomotion ability (with 0 indicating paralysis and 21 indicating normal mobility). Inked footprint analysis was conducted on day 14 after injury: the rats’ hind paws were coated with dye and they were guided in a straight line across white paper to capture representative images of their gait and coordination.

Tissue Preparation And Preservation

After euthanizing the rats, tissue samples were collected from the lesion site in the spinal cord. One portion of the tissue was promptly frozen in liquid nitrogen for subsequent oxidative stress assays and western blotting analysis. The remaining tissue was decalcified in 10% ethylenediamine tetraacetic acid (EDTA), dried, embedded in paraffin wax, and then sectioned at a thickness of 5 μm. These sections were used for histological staining and immunohistochemical analysis.

Histopathological Staining

The tissue sections were stained with hematoxylin and eosin (HE) for general histology and with cresyl violet for Nissl staining to assess neuronal morphology and density. Histopathological changes and lesions were observed using an inverted microscope (Olympus, Japan).

Immunofluorescence Staining and Analysis

Before antigen retrieval with 0.01 M citric acid (pH 6.0) at 95 °C for 10 min, tissue sections underwent deparaffinization with xylene and hydration through varying alcohol concentrations. Subsequently, the sections were rinsed with 0.01 mol/L phosphate-buffered saline (PBS) and incubated in a 10% goat serum antibody blocking solution for 2 h at room temperature. Following blocking, the tissues were incubated overnight at 4 °C with primary antibodies against MAP2 (1:200, Boster Biological Engineering Co.), GFAP (1:200, Boster Biological Engineering Co.), and NeuN (1:200, Boster Biological Engineering Co.). The next day, the tissue sections were incubated with Alexa Fluor 594-conjugated goat anti-rat IgG secondary antibody (1:300, Invitrogen) diluted in PBS. Nuclei were counterstained with DAPI. Finally, fluorescent images were captured using a fluorescence microscope to visualize the labeled proteins and cellular structures.

Statistical Analysis

The data were presented as mean ± standard deviation. Statistical analyses were conducted using SPSS version 16.0 software (SPSS Inc., Chicago, IL, USA). Differences among groups were assessed using either one-way analysis of variance (ANOVA) or Student’s t-test. A P value less than 0.05 was considered statistically significant.

Results

Establishment of PC12 Cell Injury Model using H2O2

The PC12 cell line was originally derived from rat pheochromocytoma. Due to its morphological, structural, and functional similarity to neuronal cells, it is widely used in in vitro studies as a model for neuronal cells [24]. PC12 cells exhibit oxidative stress, mitochondrial damage, and apoptosis when stimulated with H2O2 similar to the conditions observed in spinal cord cells of individuals with SCI [25]. To determine the optimal concentrations of H2O2 and CUR, cell viability was assessed using the MTT assay. Following exposure to varying concentrations of H2O2 for 24 h, a significant reduction in cell viability was observed (P < 0.05), with viability dropping to approximately 50% at an H2O2 concentration of 300 μM (Fig. 1-b). Additionally, treatment of PC12 cells with 300 μM H2O2 for different durations revealed a significant decline in cell viability after 24 h of exposure (Fig. 1-c). Therefore, a 24-h treatment with 300 μM H2O2 proved most effective in eliciting cellular responses in PC12 cells [19].

Fig. 1
figure 1

Effects of CUR on the viability of H2O2-induced PC12 cells. a Molecular structure of CUR. b Viability of PC12 cells treated with different concentrations of H2O2(100, 200, 300, 400, 500 μM) for 24 h. (c) Viability of PC12 cells cultured with 300 μM H2O2 for different durations (12, 24, 48 h). (d) Viability of PC12 cells treated with different concentrations of CUR (1, 3, 10 μM) for 24 h. (e) Viability of PC12 cells incubated with 300 μM H2O2 for 24 h after treatment with different concentrations of CUR (1, 3, 10 μM). Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared to the control group. # P < 0.05 compared to the H2O2-induced alone group. CUR, Curculigoside

Full size image

Effect of CUR on PC12 Cell Viability Induced by H2O2

To investigate the impact of CUR (molecular structure in Fig. 1-a) on PC12 cell activity following H2O2 treatment, we initially assessed its effects on normal PC12 cells. Our results clearly demonstrated that CUR had no observable effect on normal PC12 cells, regardless of concentration (Fig. 1-d). However, when PC12 cells were exposed to H2O2, their viability decreased significantly following treatment with CUR concentrations of 1, 3, and 10 μM (P < 0.05). It indicated that the protective effect of curcumin on PC12 cells was evident only at specific concentrations, showing a dose-dependent response (Fig. 1-e). Consequently, a concentration of 3 μM was selected for subsequent experiments.

Protective Effect of CUR on H2O2-induced Apoptosis in PC12 cells

Apoptosis-related tests indicated no statistically significant differences between the control group (4.46% ± 0.22) and the group treated with CUR (4.60% ± 0.23) in terms of the percentage of double-stained positive cells (P > 0.05). However, following treatment with H2O2, there was a significant increase in the proportion of double-stained positive cells (P < 0.05), which was reversed upon addition of CUR (P < 0.05). Subsequently, the proportion of positive cells increased significantly after pre-treatment with Brusatol in the H2O2 + CUR + Brusatol group, demonstrating a significant difference (P < 0.05) (Fig. 2-a, b). These findings were consistent with results obtained from the TUNEL assay (Fig. 2-c, d). Furthermore, analysis of apoptosis-related proteins in PC12 cells induced by H2O2 revealed upregulation of pro-apoptotic proteins Bax and Caspase-3, alongside downregulation of the anti-apoptotic protein Bcl-2 at both the protein and mRNA levels. However, pre-treatment with CUR effectively reversed this expression pattern (Fig. 2-e, f, g). Lastly, to explore the relationship between CUR’s anti-apoptotic and anti-oxidative properties, we utilized Brusatol, an inhibitor of Nrf-2 signaling. Interestingly, Brusatol was found to attenuate the inhibitory effect of CUR against apoptosis.

Fig. 2
figure 2figure 2figure 2

Effect of CUR on H2O2-induced apoptosis in PC12 cells. (a-b) Annexin V/PI double-staining method used to evaluate apoptosis. (c-d) Quantification of apoptosis using the TUNEL assay. (e) Gene expression levels of Bax, Caspase-3, and Bcl-2 in PC12 cells measured by RT-qPCR. (f-g) Protein expression levels of Bax, Caspase-3, and Bcl-2 in PC12 cells measured by Western blot. Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared to the control group, # P < 0.05 compared to the H2O2-induced alone group, and & P < 0.05 compared to the CUR + H2O2-induced group

Full size image

In Vitro Antioxidative Effects of CUR

The results obtained using the DCFH-DA probe revealed that H2O2 stimulation significantly increased the fluorescence intensity of intracellular ROS compared to the control group (P < 0.05). However, pre-treatment with CUR resulted in a significant reduction in ROS fluorescence intensity following H2O2 stimulation (P < 0.05). Conversely, when PC12 cells pre-treated with CUR were co-treated with Brusatol, the fluorescence intensity of ROS increased significantly (P < 0.05) (Fig. 3-a, b). Similarly, the level of GSH was significantly elevated after H2O2 stimulation (P < 0.05), but it decreased significantly with CUR treatment (P < 0.05). Importantly, intervention with Brusatol reversed this decrease (P < 0.05) (Fig. 3-c). These findings provide compelling evidence of CUR’s protective activity against oxidative stress in PC12 cells.

Fig. 3
figure 3

Inhibition of H2O2-induced oxidative stress by CUR in PC12 cells. (a-b) ROS levels were measured using H2DCF-DA and analyzed for fluorescence intensity. (c) GSH levels were measured using a GSH assay kit. Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared to the control group, # P < 0.05 compared to the H2O2-induced alone group, and & P < 0.05 compared to the CUR + H2O2-induced group. CUR, Curculigoside; ROS, reactive oxygen species; GSH, glutathione

Full size image

CUR Inhibited H2O2-induced Apoptosis of PC12 Cells through the Nrf-2/NQO-1 Signaling Pathway

To investigate the involvement of the Nrf-2/NQO-1 signaling pathway in the antioxidative stress effects of CUR, Western blotting was employed to assess the levels of Nrf-2 and NQO-1 proteins. As depicted in Fig. 4, Nrf-2 and NQO-1 protein expression significantly decreased in H2O2-stimulated cells (P < 0.05), but this reduction was considerably reversed following CUR treatment(P < 0.05). Furthermore, there was a significant decrease in the protein levels of Nrf-2 and NQO-1 in the H2O2 + CUR + Brusatol group (P < 0.05). In conclusion, CUR’s antioxidant and anti-apoptotic effects are mediated through the Nrf-2/NQO-1 signaling pathway.

Fig. 4
figure 4

Protective effect of CUR on PC12 cells against apoptosis and oxidative stress induced by H2O2 via the Nrf-2/NQO-1 signaling pathway. Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared to the control group, # P < 0.05 compared to the H2O2-induced alone group, and & P < 0.05 compared to the CUR + H2O2-induced group. CUR, Curculigoside

Full size image

CUR Promoted Functional Recovery in SCI rats

A rat model of SCI (Fig. 5-a) was used to assess the impact of CUR on hind limb movement using the BBB locomotion rating scale and inked footprint analysis. Footprint analysis revealed that rats in the sham group exhibited normal gait, while those in the SCI and SCI + NS groups displayed impaired gait. However, following 14 days of CUR intervention, SCI rats demonstrated partial improvement in the gait of their right hind limb (Fig. 5-b). These findings were further confirmed by the results of the BBB scale (Fig. 5-f). No significant differences were observed between the SCI and SCI + NS groups in terms of functional scores. Nevertheless, after CUR intervention, the BBB scores exhibited an upward trend, with significant differences observed on the 7th and 14th days. These results suggest that CUR could promote the recovery of motor function in the rat model of SCI.

Fig. 5
figure 5

Effect of CUR on tissue repair and motor function recovery following spinal cord injury in rats within 14 days. (a) Hindlimb status of SCI rats. (b) Changes in hindlimb motor function evaluated by inked footprint analysis in SCI rats. (c) Results of HE staining of spinal cord tissue from SCI rats. (d) Results of Nissl staining of spinal cord tissue from SCI rats. (e) Quantification of surviving neurons in spinal cord tissue. (f) Motor function scores of rats in each group. Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared to the control group, # P < 0.05 compared to the SCI + NS group (n = 5). CUR, Curculigoside; SCI, spinal cord injury

Full size image

CUR Inhibited Pathological Changes in Spinal Cord Tissue of SCI Rats

This study aimed to investigate the effect of CUR on the histopathology of SCI in rodents. Results from HE staining indicated that spinal cord tissue from rats in the sham group exhibited normal characteristics, including abundant cells, normal morphology, and dense distribution. In contrast, the SCI group and SCI + NS group showed abnormal spinal cord tissue morphology, characterized by a significant decrease in cell numbers and the presence of multiple large cavities. Following CUR intervention, most areas of the spinal cord tissue returned to normal, with only small vacancies remaining in some regions. Moreover, the number of neural cells with normal morphology increased significantly (Fig. 5-c, e). Nissl staining further demonstrated an increase in the number of Nissl bodies and a reduction in the number of cavities in the CUR-treated group compared to the untreated group (Fig. 5-d). Immunofluorescence staining revealed a significant decrease in MAP2 and NeuN-positive cells, along with a significant increase in GFAP-positive cells in the SCI group and SCI + NS group compared to the sham group. Importantly, these changes were reversed upon administration of CUR (Fig. 6).

Fig. 6
figure 6

Effect of CUR on astrocyte activation and neuronal cell apoptosis. (a-c) Immunofluorescence staining of MAP2, GFAP, and NeuN in spinal tissue samples. Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared to the SCI group, # P < 0.05 compared to the SCI + NS group (n = 5). CUR, Curculigoside; SCI, spinal cord injury

Full size image

Effects of CUR on Apoptosis-Related Proteins Bcl-2, Caspase-3, and Bax in Spinal Cord Tissues of SCI Rats

To investigate the effect of CUR on apoptotic cells in the spinal cord tissue of rats with SCI, the expression of apoptosis-related proteins Bax, Caspase-3, and Bcl-2 in spinal tissue samples was evaluated. Compared to the sham operation group, the expression of Bax and Caspase-3 proteins was found to be upregulated (P < 0.05), while the expression of Bcl-2 protein was downregulated (P < 0.05) in both the SCI and SCI + NS groups. However, intervention with CUR significantly reversed these alterations (Fig. 7). These findings indicated that CUR effectively mitigated apoptosis in SCI.

Fig. 7
figure 7

Effect of CUR on apoptosis in a rat SCI model. (a-d) Western blot analysis of protein expression levels of Bax, Bcl-2, and Caspase-3 in spinal tissue samples. Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared to the SCI group, # P < 0.05 compared to the SCI + NS group (n = 5). CUR, Curculigoside; SCI, spinal cord injury

Full size image

Effect of CUR on Oxidative Stress in Spinal Cord Tissues of SCI Rats

To evaluate the effect of CUR on oxidative stress in spinal cord tissues of rats with SCI, SOD, MDA, and GSH assay kits were used to analyze the supernatant of spinal cord tissue samples. Compared to the sham operation group, the levels of SOD and GSH were significantly reduced (P < 0.05), while the level of MDA was elevated (P < 0.05) in both the SCI and SCI + NS groups. However, following intervention with CUR, the levels of SOD, MDA, and GSH were significantly restored (P < 0.05). These findings highlighted that CUR effectively mitigated oxidative stress in SCI (Fig. 8).

Fig. 8
figure 8

CUR decreased oxidative stress in a rat SCI model. (a) The level of SOD was measured by a SOD assay kit. (b) The level of MDA was measured by an MDA assay kit. (c) The level of GSH was measured by a GSH assay kit. Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared with the SCI group, # P < 0.05 compared with the SCI + NS group (n = 5). CUR, Curculigoside; MDA, Malondialdehyde; SOD; superoxide dismutase; ROS, reactive oxygen species; GSH, glutathione

Full size image

CUR Protected the Spinal Cord of SCI Rats via the Nrf-2/NQO-1 Signaling Pathway

To investigate whether the protective effect of CUR is mediated through the Nrf-2/NQO-1 signaling pathway, the levels of Nrf-2 and NQO-1 proteins in spinal tissue samples were analyzed. As depicted in Fig. 9, the protein levels of Nrf-2 and NQO-1 were significantly reduced in both the SCI and SCI + NS groups (P < 0.05). However, following intervention with CUR, there was a significant increase in the protein levels of Nrf-2 and NQO-1 (P < 0.05). These results strongly suggest that the Nrf-2/NQO-1 signaling pathway might play a crucial role in mediating the protective effects of CUR.

Fig. 9
figure 9

The therapeutic role of CUR in SCI involves modulation of the Nrf-2/NQO-1 signaling pathway. Protein expression levels of Nrf-2 and NQO-1 in spinal tissue samples were analyzed using Western blotting. Data are presented as mean ± standard error of the mean (SEM). ⋆P < 0.05 compared to the SCI group, # P < 0.05 compared to the SCI + NS group (n = 5). CUR, Curculigoside; SCI, spinal cord injury

Full size image

Discussion

Since ancient times, SCI has been recognized as a severe neurological condition [26, 27]. According to data from the Global Burden of Disease (GBD) released in 2019, approximately 0.93 million new cases of SCI are reported annually, resulting in a total of 27 million individuals living with SCI [28]. Despite significant advancements in SCI research, there remains a lack of universally recognized and fully effective treatments [1]. Pharmacological treatment is among the strategies aimed at protecting neurons, inhibiting nerve cell apoptosis, and promoting nerve cell regeneration [29]. Numerous studies have highlighted the importance of drugs in countering oxidative stress and inhibiting apoptosis [30,31,32,33,34]. CUR is known for its antioxidant properties. For instance, Liu et al. [35] demonstrated that CUR promoted osteoclastogenesis through its antioxidant effects, while Xu et al. [36] showed that CUR protected against cerebral ischemia by combating oxidative stress. However, its effects specifically on SCI have remained obscure.

In this study, we utilized H2O2-induced PC12 cells to investigate the anti-apoptotic and antioxidant properties of CUR, consistent with findings from previous research [37]. Initially, in vitro treatment with CUR significantly enhanced the viability of PC12 cells and reduced the incidence of apoptosis. Analysis of apoptosis-related proteins revealed decreased expression of the key apoptotic proteins Bax and Caspase-3, alongside increased expression of the anti-apoptotic protein Bcl-2. Markers of oxidative stress indicated elevated levels of GSH and reduced levels of ROS expression. Subsequently, we established an SCI rat model and conducted in vivo experiments to confirm the effects of CUR. Additionally, in an in vitro model using H2O2-induced PC12 cells, treatment with Brusatol increased the protein levels of Nrf-2 and NQO-1, suggesting that the molecular mechanisms underlying the antioxidative and anti-apoptotic effects of CUR may involve the Nrf-2/NQO-1 signaling pathway.

Apoptosis, a programmed cell death process mediated by cysteine aspartate-specific proteases (caspases), is prominently observed in SCI [38]. There are two classical apoptotic pathways, and Caspase-3, a pivotal protease in apoptosis, initiates both pathways [33, 39]. Additionally, the Bcl-2/Bax family of proteins plays a critical role, where Bcl-2 inhibits apoptosis while Bax promotes it [40]. The interaction between these proteins forms heterodimers that reduce the release of cytochrome c, effectively inhibiting Caspase-mediated cell apoptosis [41]. Consequently, the ratio of Bcl-2 to Bax proteins has been explored as an effective measure to halt the apoptotic process in neuronal cells [42]. In this study, we assessed apoptosis-related proteins and employed the Annexin V/PI and TUNEL methods to confirm that H2O2 induces cell apoptosis, consistent with prior research [43,44,45,46]. Treatment with CUR reversed these outcomes both in vitro and in vivo, underscoring its anti-apoptotic effects. Previous studies have indicated that the neuroprotective effect of CUR may involve concentration-dependent reductions in apoptosis and necrosis [15], which was consistent with the findings of this study.

Oxidative stress, characterized by an imbalance between elevated levels of ROS and antioxidant defenses, represents a significant pathological process in SCI [47]. Normally, ROS are present in spinal cord tissue and are regulated by antioxidant enzymes. However, disruption of this balance, due to the spinal cord’s high metabolic rate and relatively weak antioxidant capacity, can lead to damage to organelles and cytoskeletal structures caused by excessive oxygen free radicals [48]. Furthermore, polyunsaturated fatty acids in cell membranes can bind ROS, forming highly reactive peroxyl radicals that initiate a chain reaction with other polyunsaturated fatty acids within the cell or in neighboring cells, leading to a progressive and continuous oxidative damage. Unfortunately, spinal cord tissue contains a high concentration of fatty acids, making it particularly susceptible to oxidative stress[2]. A key aspect in evaluating oxidative stress is the assessment of essential indicators such as GSH (an initial antioxidant defense), SOD (an enzyme that catalyzes the dismutation of superoxide radicals), and MDA (a final product of lipid peroxidation) [49, 50]. In this study, CUR treatment demonstrated enhanced antioxidative stress capacity in both in vivo and in vitro models. It suggests that CUR’s antioxidative stress properties may play a beneficial role in neurological disorders. There has been emerging research indicating that CUR reduces and alleviates oxidative stress in brain tissue of mice with Alzheimer’s disease [51], which supports the findings of the current study.

To investigate the molecular mechanisms underlying the antioxidant and anti-apoptotic effects of CUR, we conducted an in vitro study using Brusatol to inhibit the Nrf-2/NQO-1 signaling pathway. Our findings revealed that Brusatol attenuated the efficacy of CUR. Brusatol is a specific inhibitor of the Nrf-2/NQO-1 pathway, which is crucial for the effectiveness of CUR [52]. Among the various pathways involved in oxidative stress, the Nrf-2/NQO-1 signaling pathway is particularly pivotal [53, 54]. Under normal physiological conditions, Nrf-2, a redox-sensitive transcription factor, remains inhibited in the cytoplasm through its interaction with Kelch-like ECH-associated protein-1 (Keap1). However, during oxidative stress, Nrf-2 dissociates from Keap1 and translocates into the nucleus, where it binds to the antioxidant response element (ARE) and activates the expression of downstream antioxidant enzymes such as HO-1 and NQO1, thereby exerting antioxidant effects [55, 56]. Our study demonstrated a significant decrease in the protein levels of Nrf-2 and NQO-1 in PC12 cells upon the induction of H2O2. However, treatment with CUR led to a significant increase in the protein levels of Nrf-2 and NQO-1. This effect was consistent in both in vivo and in vitro models. These findings strongly suggest that CUR exerts its antioxidant and anti-apoptotic effects through modulation of the Nrf-2/NQO-1 signaling pathway, consistent with previous findings by Du et al. [57].

Following SCI, the glial scar formation contributes significantly to the progression of injury [58]. During the secondary injury phase, activated astrocytes in spinal cord tissues transform into reactive astrocytes, which release chondroitin sulfate and chondroitin sulfate proteoglycans, forming a barrier that impedes neuronal reconstruction [26, 59]. GFAP serves as a marker protein for astrocytes and correlates with cellular reactivity, while NeuN is a marker for mature and stable neuronal cells, and MAP2 is crucial for neuronal development, maturation, plasticity, and memory formation [60,61,62]. In the SCI group, there was a significant decrease in NeuN- and MAP2-positive cells, alongside an increase in GFAP-positive cells. In contrast, CUR intervention showed promising results in inhibiting glial scar formation and promoting neuronal reconstruction.

In this study, we also examined the spinal tissue and hind limb motor function in rats with SCI. In the SCI animal model, severe damage to the spinal tissue was evident, accompanied by nearly complete loss of motor function, consistent with established standards [63]. Following treatment with CUR, a notable improvement in hind limb motor function was observed within 14 days, along with evidence of partial repair in the spinal tissue. These findings highlight the therapeutic potential of CUR in animal models of SCI.

Conclusion

In summary, CUR was demonstrated to play a crucial role in inhibiting apoptosis and oxidative stress induced by H2O2 in PC12 cells in vitro. Moreover, it enhanced spinal cord tissue and hind limb function through its antioxidative properties in vivo. This therapeutic effect was mediated by CUR’s suppression of oxidative stress via the Nrf2/NQO1 signaling pathway, which ultimately reduced neuronal cell apoptosis. These findings present promising implications for the development of drugs targeting SCI and advancing SCI treatment strategies.