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A reactive oxygen species-responsive hydrogel loaded with Apelin-13 promotes the repair of spinal cord injury by regulating macrophage M1/M2 polarization and neuroinflammation

Journal of Nanobiotechnology volume 23, Article number: 12 (2025) Cite this article

Abstract

Spinal cord injury (SCI) is a chronic condition whereby persistent aberrant macrophage activation hinders the repair process. During acute trauma, dominant M1 macrophages produce high levels of reactive oxygen species (ROS), leading to increased apoptosis in neurons, glial cells, and oligodendrocytes. This study investigated the specific effects of a ROS-responsive hydrogel loaded with Apelin-13 (Apelin-13@ROS-hydrogel) on macrophage polarization and neuroinflammation, thereby exploring its role in boosting SCI repair. Apelin-13@ROS-hydrogel was prepared, and its ROS-scavenging capacities were evaluated using DPPH, H2O2, and ·O2- assays. The effects of Apelin-13@ROS-hydrogel on macrophage polarization, inflammatory mediators and oxidative stress were assessed in LPS-pre-treated microglia BV2 cells and an SCI rat model. Apelin-13 was downregulated in SCI rats. Treatment with Apelin-13 improved functional recovery and reduced inflammatory factors and M1 markers but increased the M2 marker Arg-1. Apelin-13@ROS-hydrogel showed significantly higher ROS-scavenging capacities compared to the control hydrogel. Apelin-13@ROS-hydrogel decreased pro-inflammatory mediators and increased anti-inflammatory mediators in BV2 cells. Apelin-13@ROS-hydrogel enhanced the healing process and neurological functions, reducing inflammatory factors and M1 markers while increasing Arg-1 levels by day 28 in SCI rats. Collectively, Apelin-13 enhances SCI repair through macrophage regulation, M1/M2 polarization, and neuroinflammation. The ROS-responsive hydrogel further amplifies these effects, offering a promising therapeutic strategy for SCI.

Introduction

Spinal cord injury (SCI) is a chronic wound condition that is commonly caused by traumatic events such as motor vehicle accidents, falling from heights, contact sports and can result in a wide range of physical, sensory, and cognitive impairments due to the inability of the adult central nervous system (CNS) to recover damaged neurons and create functional connections following the acute trauma [1,2,3]. SCI pathophysiology involves a cascade of events occurring after the initial injury, including primary mechanical damage, secondary inflammatory response, and ongoing neurodegeneration [4]. While medical progress has allowed for the acute management of SCI, there are currently no viable treatments for enhancing long-term functional recovery. Therefore, devising novel therapeutic strategies to enhance spinal cord regeneration is warranted.

The chronic complication during SCI is characterized by persistent macrophage activation [5]. Inadequate macrophage activation can disrupt transitions across distinct repair phases, leading to chronic inflammation and delayed healing [6]. During acute trauma reactions, dominant M1 macrophages produce a large amount of reactive oxygen species (ROS), including hydrogen peroxide (H2O2), hydroxyl radical (OH), and superoxide radical (O2−), leading to oxidative stress and increased neuronal, glial, and oligodendrocyte apoptosis [7, 8]. M1 macrophage activation and ROS secretion can also stimulate the recruitment of additional immune cells, such as neutrophils, thereby exacerbating tissue damage [9]. Conversely, M2 macrophages promote tissue repair by reducing inflammation and encouraging axonal regeneration. However, the M1/M2 polarization sequence in SCI is disrupted, leading to M1 macrophage predominance, persistent inflammation, and delayed recovery [10, 11].

Reportedly, Apelin-13, being a peptide ligand of APJ, has anti-inflammatory, antioxidant, and neuroprotective properties in multiple CNS damage models, including ischemic stroke, SCI, and traumatic brain injury [12,13,14]. Apelin treatment mitigates the inflammatory response, including microglia and astrocyte activation and the release of pro-inflammatory cytokines, in Alzheimer’s disease (AD) patients, including TNF-α and IL-1β within brain injury animal models [15, 16]. Apelin-13 protects neurons (PC12 cells) in vitro by boosting autophagy and reducing early-stage post-SCI apoptosis [13]. However, whether apelin-13 can improve SCI recovery by resetting macrophage M1/M2 polarization following acute damage remains unknown.

Excessive ROS production is a critical factor in SCI that causes oxidative stress and increased apoptosis of neuronal and glial cells [7, 8]. ROS-responsive hydrogel has been demonstrated to be beneficial in wound repair and regeneration processes in traumatic brain injuries by scavenging overproduced ROS [17,18,19,20]. For instance, a multifunctional thermosensitive hydrogel (HPP@Cu gel) effectively removed reactive oxygen and nitrogen species, promoted macrophage re-polarization towards anti-inflammatory phenotypes, and reduced cartilage degradation and pro-inflammatory mediator release in osteoarthritis [21]. ROS-scavenging scaffolds, such as rapamycin-loaded Rapa@Gel, have been reported to enhance M2 macrophage polarization and reduce M1 macrophage presence, helping in intervertebral disk regeneration [22]. Li et al. [23] reported that a ROS-responsive hydrogel encapsulated with bone marrow-derived stem cells enhances SCI repair and regeneration [23]. Therefore, it was hypothesized that integrating a ROS scavenging scaffold with Apline-13 may effectively exert synergistic therapeutic effects on SCI.

Based on previous findings, this study hypothesizes that integrating a ROS-responsive hydrogel with Apelin-13 might synergistically enhance SCI repair. To assess this hypothesis, the expression of Apelin-13 in SCI rats was explored, and its effects on SCI symptoms, local inflammation, and macrophage M1/M2 polarization were investigated. An Apelin-13-loaded ROS-responsive hydrogel (Apelin-13@ROS-hydrogel) was prepared, and its ROS-scavenging capacities, its effects on LPS-stimulated microglia BV2 cells, SCI symptoms, local inflammation, and macrophage polarization were evaluated. This study aims to elucidate the roles and mechanisms of Apelin-13 and Apelin-13@ROS-hydrogel in promoting SCI recovery and regeneration.

Materials and methods

The preparation of Apelin-13@ROS-hydrogel gel (PVA-TSPBA hydrogels)

The weighed polyvinyl alcohol (PVA) and 4,4’-(Diphenylsilanediyl)bis(N, N-diphenylaniline) (TSPBA) were dissolved in a PBS solution sequentially to yield a mixed prepolymer solution consisting of 9% PVA and 3% TSPBA. For the preparation of Apelin-13@ROS-responsive hydrogel, Apelin-13 (SinoBiological, Beijing, China) was dissolved in deionized H2O and mixed with 9% PVA, followed by mixing with 3% TSPBA, maintaining the final concentration of Apelin-13 at 5 mmol/L.

Scanning electron microscopy (SEM) observing the microstructure of Apelin-13@ROS-hydrogel

The ROS-responsive hydrogel and Apelin-13@ROS-hydrogel were frozen in liquid nitrogen for 20 min before being transferred to a freeze-dryer prior to a 48-h freeze-drying period. The freeze-dried sample was sprayed with gold after being affixed to the base with conductive adhesive and then observed using SEM (FEI Quanta200; Thermo Fisher Scientific, Eindhoven, Netherlands).

Degradation test

During the hydrolysis and ROS-responsive degradation test, ROS-responsive hydrogel and Apelin-13@ROS-hydrogel were pre-soaked in PBS overnight prior to swelling to equilibrium and weighing using an electronic scale (m0). The hydrogels were subsequently immersed into PBS or PBS-added 200 µM H2O2 at 37 °C, respectively. The hydrogel wet weight (mw) was collected at various points, and the solutions were replaced by fresh ones daily, respectively. The remaining weight was computed as mw/m0 × 100%.

The release rate of Apelin-13 from hydrogel

Apelin-13@ROS-hydrogel was immersed in PBS with 200 µM H2O2 at 37 °C. At different time points, the concentration of Apelin-13 in PBS was measured using the Human Apelin ELISA kit (CSB-E14334h). The release rate was calculated as (time point determined concentration×total liquid volume) / (total apelin-13 amount in the hydrogel) ×100%.

Rheological characterization

The rheological characterization of ROS-hydrogel and Apelin-13@ROS-hydrogel was performed. Briefly, 1mL of each hydrogel was taken and analyzed using a rheometer (Haake Mars 40, Thermo Scientific, Germany) with a 35 mm rotor, a 2° taper, and a corresponding cone plate at 25°C. A stress scan was first performed to measure the linear viscoelastic regions, and then the appropriate stress value was selected for frequency scanning, ranging from 0.1 to 100 rad s − 1, at a fixed strain of 1%. The storage modulus (G’) and the loss modulus (G’’) were automatically recorded.

Fourier transform infrared spectroscopy (FTIR) analysis

Fourier transform infrared spectroscopy (FTIR) analysis of ROS-hydrogel and Apelin-13@ROS-hydrogel was performed using a Thermo Scientific Nicolet iS20 spectrometer. The attenuated total reflectance (ATR) accessory was placed in the optical path of the spectrometer, and an air background scan was conducted in a dry environment. The surface of the hydrogel sample was subsequently tightly pressed against the ATR crystal surface of the ATR accessory, and the infrared spectra were recorded.

Circular dichroism (CD) spectropolarimetry analysis

CD spectropolarimetry analysis was conducted to evaluate the structural integrity of Apelin-13 before and after its release from the ROS-hydrogel using a JASCO J-1500 spectropolarimeter (Jasco, Japan) at room temperature. Apelin-13 was re-suspended in PBS (0.01 M, pH 7.4) and subjected to analysis. Apelin-13@ROS-hydrogel was immersed in PBS (v/v 1:10) with 200 µM H2O2 at 37 °C for 6 days. The extracted liquid was subjected to analysis. Far-UV CD spectra were acquired from 260 to 190 nm at a data pitch of 0.1 nm and a scan speed of 100 nm/min, using cuvettes with a 0.1 mm path length. All spectra were acquired in triplicate, and experiments were carried out in duplicates with independently prepared samples. Data were averaged, blank subtracted, and reported as mean residue ellipticity (MRE), with a sliding window average over 3 nm stretches.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) test

The DPPH scavenging capability of different hydrogels, including control-hydrogel, ROS-hydrogel, and Apelin-13@ROS-hydrogel, was determined through the incubation of 200 µL hydrogel into 200 µM DPPH ethanolic solution (BC4750, Solabio, China) in light-deprived conditions. The absorbance was measured at 517 nm.

H2O2 scavenging

The H2O2 scavenging ability of control-hydrogel, ROS-hydrogel, and Apelin-13@ROS-hydrogel was determined through a half-hour incubation of 200 µL hydrogel into 1 mL 100 µM H2O2 solution in light-deprived conditions at 37 °C. The content of H2O2 was subsequently determined by an H2O2 content measurement kit (BC3595, Solabio) as directed by the manufacturer. The absorbance value at 415 nm was measured. The scavenging ratio was computed by the following formula: (OD sample - OD water)/(OD control- OD water) × 100%.

O2 − scavenging

A total of 200 µL control-hydrogel, ROS-hydrogel and Apelin-13@ROS-hydrogel were used following the inhibition and production superoxide anion assay kit’s protocol (Nanjing Jiancheng Bioengineering Institute, China) to ascertain the·O2− scavenging ability. The absorbance value at 550 nm was measured. The scavenging ratio was computed by the following formula: (OD sample - OD water)/(OD control- OD water) × 100%.

Measurement of reactive oxygen species (ROS)

BV2 cells were plated at a density of 4 × 105cells per well in a 6-well culture plate. After different treatments, the cells were washed with PBS, followed by an incubation of 5µM dichlorofluorescein diacetate (DCFH-DA; Beyotime, green fluorescence) or dihydroethidium (DHE, Beyotime, red fluorescence) for 30 min at 37 ℃ at dark. The cells were imaged using a fluorescence microscope (Olympus).

Rat model of SCI and treatment

The SCI model was established as follows: isoflurane (3%) was used during surgery after 5%, and isoflurane was used to anesthetize Sprague-Dawley rats. The spinal cord was seen after the T9-T10 laminoid and spinous processes excision. To create a model of acute SCI, the T9 region of the spinal cord was struck with a 10 g hammer from a height of 25 mm, and the incision was sewn shut in layers. The rats in the control group underwent laminotomy but not the hard blow treatment. For apelin-13 treatment, after being struck and complete hemostasis, 50 nmol apelin-13 in 10 µl, 10 µl of Apelin-13@ROS-hydrogel was injected into the lesion site with a microsyringe at a controlled speed of 1 µL min− 1, respectively. The SCI rats that received the same amount of sterile saline or ROS-hydrogel injection were grouped as the corresponding control. For APJ knockdown, 500 pmol APJ siRNA in 10 µl was injected into the lesion site. Rats with SCI received the same scramble siRNA dose served as the corresponding control group. Thus, the experiments were divided into 9 groups: (1) sham (n = 12), (2) SCI (n = 12), (3) SCI + vehicle (n = 6), (4) SCI + Apelin-13 (n = 6), (5) SCI + Apelin-13 + scramble siRNA (n = 6), (6) SCI + Apelin-13 + APJ siRNA (n = 6), (7) SCI + ROS-hydrogel (n = 6), (8) SCI + Apelin-13@ROS-hydrogel (n = 6).

Cages were used to house and observe the rats. 0.9% of cefazolin sodium solution was intraperitoneally administered to the rats twice daily. They were also given artificial help to induce urination twice in the early morning and late afternoon.

Evaluation of functional recovery of SCI rats

The Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale and the inclined plate test were applied to assess rats’ recovery to normal spinal cord function. BBB scale ranging from 0 to 21 was performed as previously described [24]. One inclined plate with adjustable activity was pre-configured for testing inclined plates on the desktop. The vertical axis of the rats’ bodies and the long axis of the inclined plate were found to be perpendicular. Rats could board the inclined plate for 5 s after it was gently lifted, at which point the angle was measured [25].

In vivo degradation of Apelin-13@ROS-hydrogel

In vivo degradation of Apelin-13@ROS-hydrogel was evaluated using the Maestro in vivo optical imaging system (CRI, Inc., Woburn, MA, USA). FITC-labeled Apelin-13 (FITC-Apelin-13, SinoBiological) was embedded in the ROS-hydrogel to produce a fluorescence signal. Approximately 210 µL of cold FITC-Apelin-13@ROS-hydrogel solution was injected into the injured spinal cord in situ. The rats were sacrificed, and their spinal cords were separated at 15 min, 1, 3, 5, 7, 14, and 21 days after the injection of the hydrogel. The residual Apelin-13 in the hydrogel was determined by observing the fluorescence intensity. Each group consisted of three animals.

Hematoxylin–eosin (H&E) staining

On day 28 of the modeling process, rats were sacrificed under anesthesia before harvesting their spinal cords. Samples were subjected to a 6-h fixation using 2% paraformaldehyde and followed dehydration, transparent and embedded in wax. The samples were cut into 5 μm-thick cross-sections. After dewaxing, hydration, and staining sections using an H&E staining kit (Beyotime), samples were observed under an optical microscope.

Immunohistochemical staining (IHC staining)

IHC staining was carried out using an HRP-DAB staining kit (R&D, Inc., Minneapolis, MN, USA). The primary antibody was a polyclonal antibody against 8-hydroxy-2-deoxyguanosine (8-OHdG) (Bioss, Beijing, China), applied at a 1:100 dilution in PBS and incubated for 60 min. The sections were subsequently incubated with a biotinylated goat anti-rabbit secondary antibody for 30 min. The interaction between the primary and secondary antibodies facilitated the enzymatic conversion of the chromogenic substrate 3,3′-diaminobenzidine (DAB) by streptavidin HRP, resulting in a brown precipitate for visualization.

Immunoblotting

Protein was extracted from the tissues or cells of the rat’s spinal cord and its concentration was determined using bicinchoninic acid (BCA) reagents (Thermo, Rockford, IL, USA). Proteins were electroblotted to a PVDF membrane after being placed onto a 10–15% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA). The membrane was subjected to a 1-h incubation using 5% skim milk (Bio-Rad) in Tris-buffered saline (TBS) containing 0.05% Tween 20 to prevent non-specific bindings. The membrane was subsequently incubated with an anti-Apelin-13 antibody (DF13350, Affinity Biosciences, Changzhou, China), anti-APJ antibody (20341-1-AP, Proteintech, Wuhan, China), anti-Arg1 antibody (16001-1-AP, Proteintech), anti-CD16 antibody (DF007, Affinity Biosciences) and anti-GAPDH antibody (AF7021, Affinity Biosciences). The membrane was then subjected to an overnight incubation at 4 °C. After washing with PBS thrice, secondary antibodies conjugated with HRP (Proteintech) were applied to the membranes for 1 h at room temperature. The signals were identified using an ECL reagent (Beyotime, Shanghai, China) and an automatic chemiluminescent imaging system (Tanon-5200, Shanghai, China). ImageJ was used to calculate band densities.

Quantitative real-time PCR (qRT-PCR)

Trizol Reagent (Invitrogen) was used to isolate total RNA from target tissue samples or cell lines following various treatments. A Biometra Optical Thermocycler was used for qRT-PCR (Analytik Jena, Goettingen, Germany). RNA (500 ng) was reversely transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). SYBR qRCR PreMix (Takara, Kyoto, Japan) was applied to perform qRT-PCR as directed by the manufacturer. The 2−∆∆CT method was used to calculate the expression levels of the target gene. The internal reference utilized was GAPDH.

Immunofluorescence staining (IF staining)

The BV2 cells were fixed in paraformaldehyde for 1 h after LPS and hydrogel treatments. After a 20-min permeabilization at room temperature (RT) using PBS with 0.5% Triton X 100 (PBST), cells were rinsed thrice, 5 min each time and incubated with 1% bovine serum albumin (BSA) for 30 min at room temperature. An incubation buffer without an antibody was used as a negative control, and primary antibodies against Arg-1, or iNOS (Proteintech, dilution is 1:100 to 1:200) were added overnight at 4 °C. The temperature was recovered for 20 min, followed by rinsing thrice with PBST for 5 min per rinse. The samples were subjected to a 1-h incubation at 37 °C with the corresponding secondary antibody labeled with FITC (green) at dark (Beyotime), followed by rinsing thrice with PBST for 5 min per rinse. DAPI was applied to stain cell nuclei. The procedures for IF staining in the tissue section are as follows: the hydrated section underwent antigen retrieval, blocking non-specific binding sites. The section was subsequently incubated with primary antibodies against CD68, CD16/32, or Arg-1 (Proteintech, dilution is 1:100 to 1:200) overnight at 4℃. The temperature was recovered for 20 min, followed by rinsing the samples thrice with PBST for 5 min per rinse. The samples were subjected to a 1-h incubation at 37 °C with the corresponding secondary antibody labeled with Cy3 (red) or FITC (green) at dark (Beyotime), followed by rinsing thrice with PBST for 5 min per rinse. DAPI was applied to stain cell nuclei, and a fluorescent microscope was used to capture corresponding pictures.

Flow cytometry analysis

After LPS and hydrogel treatments, the BV2 cells were fixed in fixation buffer for 30 min and permeabilized in permeabilization wash buffer (R&D, USA) for 10 min at room temperature. Then, cells were incubated with CoraLite ® Pluss 488-labeled antibody against iNOS (CL488-18985, Proteintech) or FITC-labeled antibody against Arg-1 (IC5868F, R&D) for 30 min at room temperature at dark. After washed with wash buffer three times, cells were resuspended in cell staining buffer and subjected to flow cytometry (Novocyte, CA, USA) analysis immediately.

Myeloperoxidase (MPO) activity assay

A previously described method was used to assess MPO activity within the tissue samples [26]. Tissues were homogenized in 0.5% hexadecyltrimethylammonium bromide mixed in 50 mM potassium phosphate buffer (pH = 6.0) before being centrifuged. The supernatants were added to a PBS buffer with o-dianisidine dihydrochloride and H2O2. The absorbance was measured at 460 nm using a spectrophotometer.

Cell line and cell cultivation

Microglia BV2 cells were procured from the Procell (Wuhan, China) and cultivated at 37 °C in 5% CO2 into DMEM high-glucose (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). BV2 cells were stimulated with 100 ng/ml LPS (Gibco, USA) for 48 h to attenuate the activation of microglial cells.

The in vitro cytotoxicity and polarization ability determination of ROS-hydrogel and Apelin-13@ROS-hydrogel

BV2 cells were suspended in a complete culture medium and seeded in a 24-well plate overnight. The ROS-hydrogel and Apelin-13@ROS-hydrogel (low dose 100 µl and high dose 200 µl) were subsequently placed on the upper transwell chamber of the 24-well plate. Cell proliferation was detected by adding the CCK-8 (Beyotime) solution to culture plates. Following a 2-h incubation period, 100 µl of the supernatant mixed solution was transferred onto a 96-well plate, and the optical density of each well was determined by a microreader (SpectraMax M5, USA) at 490 nm. 100 ng/ml LPS was added to the BV2 cells in a 24-well plate and simultaneously incubated with 200 µl ROS-hydrogel and Apelin-13@ROS-hydrogel to achieve the polarization of BV2 cells. The BV2 cells were collected for qRT-PCR or IF staining assays for TNF-α, IL-1β, iNOS, IFN-γ, IL-10, IL-4, and TGF-β1 expression.

Statistical analysis

All data were presented in terms of mean ± standard deviation (SD). Comparisons among groups were performed using ANOVA followed by Tukey’s test or Dunnett’s test and t-test with GraphPad. A P-value of less than 0.05 was considered statistically significant.

Results

Apelin-13 is downregulated in SCI rats

Prior to examining the specific effects of Apelin-13 on SCI wound healing and macrophage polarization at the injury sites, SCI models were established in rats and Apelin-13 levels were evaluated. Spinal cord samples from rats sacrificed after 28 days of modeling were collected and analyzed for histopathological characteristics using H&E staining to validate the model. In the modeling group, the injured sites were atrophic, with necrotic and cystic holes in the central gray matter and peripheral white matter (Fig. 1A). On day 1 after modeling, the BBB scores of SCI mice were significantly lower than that of the control group and remained so (Fig. 1B). Consistently, the limb motor function was measured by inclined-plane angle assay and the rats in the SCI group were dramatically impaired since day 1 after modeling. They lasted until the end of the modeling (Fig. 1C). Since day 1 after modeling, Apelin-13 levels in the spinal cord tissues of model rats have declined moderately. Since day 14 after modeling, they have decreased dramatically relative to the control group (Fig. 1D). Therefore, Apelin-13 has been significantly downregulated in the injured sites of the SCI model since day 14 after modeling.

Fig. 1
figure 1

Apelin-13 is downregulated in spinal cord injury (SCI) rats SCI models were established as described. (A) After 28 days of modeling, spinal cord tissues were collected from model rats sacrificed under anesthesia and examined for histopathological characteristics using H&E staining. (B-C) The Basso-Beattie-Bresnahan (BBB) Locomotor Rating Scale and inclined-plane test were used to evaluate the behavioral outcomes and motor ability of rats from different groups. (D) The protein levels of Apelin-13 in spinal cord tissues were determined using Immunoblotting. n = 6 or 3, ** p < 0.01

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Apelin-13 promotes functional recovery in rats with SCI

Concerning the specific functions of Apelin-13 on SCI, the rats were randomly allocated into five groups: sham surgery, SCI modeling plus vehicle treatment, SCI modeling plus Apelin-13 treatment, SCI modeling plus Apelin-13 treatment and scramble siRNA transduction, and SCI modeling plus Apelin-13 treatment and APJ siRNA transduction. At day 28 after modeling and treatment, atrophic injured sites, necrotic central gray matter and peripheral white matter with cystic cavities in SCI rats were partially recovered by the Apelin-13 treatment; however, after APJ silencing by APJ siRNA transduction, the histopathological characteristics improvements by Apelin-13 treatment were partially abolished (Fig. 2A). The BBB scores of all the modeling groups were dramatically decreased since day 1 after modeling and continued to be lower than the control group; Apelin-13 treatment significantly increased BBB scores compared with the SCI plus vehicle group, whereas APJ silencing by APJ siRNA transduction also significantly lowered BBB scores compared with the Apelin-13 or Apelin-13 plus scramble siRNA group (Fig. 2B). Consistently, the motor ability of rats were significantly impaired in all the modeling groups compared with the sham surgery group; Apelin-13 treatment significantly improved the behavioral outcomes and motility of rats since day 21 after the modeling and treatment, whereas APJ silencing by APJ siRNA transduction partially abolished the benefits entailed by Apelin-13 treatment (Fig. 2C). In the injury sites of SCI rats, Apelin-13 and APJ protein levels were significantly downregulated compared with the sham surgery group; in Apelin-13 treatment and Apelin-13 plus scramble siRNA groups, Apelin-13 and APJ protein levels were significantly elevated compared with the SCI plus vehicle group, but decreased by APJ silencing (Fig. 2D).

Fig. 2
figure 2

Apelin-13 promotes functional recovery in rats with SCI Rats were randomly allocated into five groups: sham surgery, SCI modeling plus vehicle treatment, SCI modeling plus Apelin-13 treatment, SCI modeling plus Apelin-13 treatment and scramble siRNA transduction, and SCI modeling plus Apelin-13 treatment and APJ siRNA transduction. (A) After 28 days of modeling and treatment, spinal cord tissues were collected from model rats sacrificed under anesthesia and examined for histopathological characteristics using H&E staining. (B-C) The BBB Locomotor Rating Scale and inclined-plane test were used to evaluate the behavioral outcomes and motor ability of rats from different groups at days 0 (pre-surgery), 1, 5, 7, 10, 14, 21, and 28. (D) After 28 days of modeling and treatment, the protein levels of Apelin-13 and APJ in spinal cord tissues were determined using Immunoblotting. n = 6 or 3, ** p < 0.01, vs. sham group; ## p < 0.01, vs. SCI + vehicle group; && p < 0.01, vs. SCI + Apelin-13 group

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Apelin-13 reduces neuroinflammation by recovering macrophage polarization sequence interruption at damaged sites in SCI rats

Regarding the neuroinflammation and macrophage polarization states in the injured sites of model rats; at day 28 after SCI, the levels of pro-inflammatory cytokines, anti-inflammatory cytokines, MPO activity, and M1/M2 macrophage markers were determined. Regarding the cytokines, the mRNA levels of pro-inflammatory mediators TNF-α, IL-1β, IL-2, and iNOS showed to be dramatically up-regulated, while anti-inflammatory mediator TGF-β1, IL-4 and IL-10 showed to be down-regulated in the injured sites of SCI rats; Apelin-13 stimulation reduced pro-inflammatory mediators and elevated anti-inflammatory mediator, whereas APJ silencing significantly attenuated the effects of Apelin-13 treatment on cytokines (Fig. 3A). Similarly, MPO activity was significantly elevated in SCI rats, and was partially decreased by Apelin-13 treatment, and increased after APJ silencing (Fig. 3B). Regarding macrophage/microglia polarization, at day 28 after SCI, the contents of M1 macrophage marker CD16/32 increased, and M2 macrophage marker Arg1 decreased at the injury site of SCI rats; Apelin-13 treatment increased the levels of Arg-1 and decreased CD16/32, whereas APJ silencing exerted opposite effects on macrophage polarization markers (Fig. 3C-D). Immunoblotting revealed similar results in that at day 28 after SCI, the protein levels of M1 macrophage marker CD16 increased, and M2 macrophage marker Arg1 decreased at the injury site of SCI rats; Apelin-13 treatment increased the levels of Arg-1 and decreased CD16, whereas APJ silencing exerted opposite effects on macrophage polarization markers (Fig. 3E). On day 28 of the acute injury phase of SCI, pro-inflammatory cytokines remained elevated and macrophage M1 polarization predominated, according to these findings; Apelin-13 decreased pro-inflammatory cytokines and promoted macrophage M2 polarization, whereas APJ silencing attenuated the effects of Apelin-13 treatment.

Fig. 3
figure 3

Apelin-13 reduces neuroinflammation by recovering macrophage polarization sequence interruption at damaged sites in SCI rats (A) At day 28 after SCI, the mRNA expression levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-2 and iNOS and anti-inflammatory cytokines TGF-β1, IL-4 and IL-10 were analyzed using qRT-PCR. (B) At day 28 after SCI, MPO activity at the injured site of SCI rats was detected using an MPO fluorescence assay kit. (C-D) On day 28 after SCI, the contents of M1 macrophage marker CD16/32 and M2 macrophage marker Arg1 at the injured site of SCI rats were detected using Immunofluorescent staining (IF staining). (E) On day 28 after SCI, the protein levels of M1 macrophage marker CD16 and M2 macrophage marker Arg1 at the injured site of SCI rats were detected using Immunoblotting. n = 6 or 3, ** p < 0.01, vs. sham group; ## p < 0.01, vs. SCI + vehicle group; && p < 0.01, vs. SCI + Apelin-13 + scramble siRNA group

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The preparation process and characteristics of the ROS-scavenging hydrogel loaded with Apelin-13 (Apelin-13@ROS-hydrogel)

After confirming the improvement by Apelin-13 treatment, Apelin-13@ROS-hydrogel was prepared and verified with the characteristics. The appearance of the hydrogels is depicted in Fig. 4A: (A) hydrogel in the liquid state, (B) hydrogel in the solidified state, and (C) the injectable properties of ROS-responsive hydrogel and Apelin-13@ROS-hydrogel. Figure 4B depicts representative SEM images for ROS-responsive hydrogel and Apelin-13@ROS-hydrogel (1000×/2500×); both hydrogels were composed of interconnected pores, forming a sponge-like 3D porous structure that interconnects. The pore walls of Apelin-13@ROS-hydrogel are composed of original fibers and spherical protrusions, indicating that Apelin-13 is encapsulated within the ROS-responsive hydrogel (Fig. 4B). Regarding the hydrolysis and ROS response degradation abilities, the degradation rates of ROS-responsive hydrogel and Apelin-13@ROS-hydrogel by 200 µM H2O2/PBS or pure PBS were evaluated. The ROS-responsive hydrogel and Apelin-13@ROS-hydrogel degraded faster in H2O2/PBS than in PBS, with both ROS-hydrogel and Apelin-13@ROS-hydrogel dissolving between day 8 and day 10 (Fig. 4C), indicating the quicker degradation of the hydrogel or scaffold treated with ROS-responsive bio-polymer upon H2O2 stimulation.

Fig. 4
figure 4

The preparation process and characteristics of the ROS-responsive hydrogel loaded with Apelin-13 (Apelin-13@ROS-hydrogel) (A) ROS-responsive hydrogel (ROS-hydrogel) and Apelin-13@ROS-hydrogel: ((A)) hydrogel in its liquid state; ((B)) hydrogel in its solidified state; ((C)) injectable properties of ROS- hydrogel and Apelin-13@ROS-hydrogel. (B) Representative SEM images for ROS-hydrogel and Apelin-13@ROS-hydrogel (1000×/2500×). (C) Hydrolysis and ROS response degradation abilities: degradation rates of ROS-hydrogel and Apelin-13@ROS-hydrogel by 200 µM H2O2/PBS or pure PBS. (D) Cumulative release properties of Apelin-13 from Apelin-13@ROS-hydrogel hydrogel treated with 200 µM H2O2/PBS. (E-G) The DPPH-, H2O2-, and ·O2−-scavenging capacities of control hydrogel, ROS-hydrogel, and Apelin-13@ROS-hydrogel. n = 3, ** p < 0.01

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The curve of Apelin-13 released from the hydrogel was subsequently detected in the H2O2 solution; Apelin-13 was slowly and sustainedly released as opposed to a fast release (Fig. 4D). Consistent with the rate of hydrogel degradation, 84.47 ± 9.06% of Apelin-13 was released within 9 days in high-concentration H2O2 solution, and 83.23 + 4.797% of Apelin-13 was released within 15 days (Fig. 4D), indicating that the hydrogel composite material functions as an in-situ gel system with sustained drug release capability. DPPH, H2O2, and ·O2− were cleared to further compare both hydrogels’ antioxidant and free radical scavenging characteristics. In comparison with the control hydrogel, the ROS-hydrogel and Apelin-13@ROS-hydrogel exhibited certain antioxidant capacities, and the DPPH clearance, H2O2 clearance, and ·O2− clearance capacities of Apelin-13@ROS-hydrogel were significantly higher than those of the control hydrogel and ROS-responsive hydrogel (Fig. 4E-G).

Characterization of ROS-hydrogel and Apelin-13@ROS-hydrogel

The rheological characteristics of ROS-hydrogel and Apelin-13@ROS-hydrogel were characterized by measuring the storage modulus (G’) and loss modulus (G’’). The results demonstrated that G’ was greater than G”, indicating that the hydrogels possess the properties of an elastic solid. With the increase in angular frequency, G’ remained greater than G”, suggesting that the hydrogels could stably exist in gel form (Fig. 5A). FTIR analysis of ROS-hydrogel and Apelin-13@ROS-hydrogel revealed a stretching vibration peak for -OH at 3400 cm− 1, a stretching vibration peak for C-H at 2929 cm− 1, a stretching vibration peak for C-O at 1087 cm− 1, a characteristic peak for C-N at 1421 cm− 1, and a characteristic peak for B-O at 1335 cm− 1. These findings indicate significant crosslinking between PVA and TSPBA, with no disappearance of other characteristic peaks (Fig. 5B). CD spectropolarimetry analysis of Apelin-13 and Apelin-13@ROS-hydrogel released Apelin-13 showed a strong negative band at 200 nm, consistent with a random coil conformation and matched the literature reported [27], indicating that the released Apelin-13 retained its biological activity (Fig. 5C).

Fig. 5
figure 5

Characterization of ROS-hydrogel and Apelin-13@ROS-hydrogel (A) The rheological characters of ROS-hydrogel and Apelin-13@ROS-hydrogel were characterized; storage modulus (G’) and loss modulus (G’’) were shown. (B) FTIR analysis of ROS-hydrogel and Apelin-13@ROS-hydrogel. (C) Circular dichroism (CD) spectropolarimetry analysis of Apelin-13 and Apelin-13@ROS-hydrogel released Apelin-13

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The anti-inflammatory and anti-oxidative effects of Apelin-13@ROS-hydrogel in vitro

After confirming the characteristics and antioxidant capacity of both hydrogels, their specific functions were investigated in LPS-stimulated microglia cells in vitro. Firstly, Microglia BV2 cells were incubated with low-dose/high-dose ROS-responsive hydrogel or Apelin-13@ROS-hydrogel, and assessed for cytotoxicity. A CCK-8 assay revealed that low-dose/high-dose ROS-responsive hydrogel or Apelin-13@ROS-hydrogel treatment induced no decrease in cell viability, indicating that both hydrogels exhibit no cytotoxicity to microglia cells (Fig. 6A). Therefore, high doses of ROS-responsive hydrogel and Apelin-13@ROS-hydrogel were used in following investigations. Under LPS stimulation, the mRNA levels of pro-inflammatory mediators TNF-α, IL-1β, iNOS, and IFN-γ and anti-inflammatory mediators TGF-β1, IL-10, and IL-4 were monitored. ROS-responsive hydrogel or Apelin-13@ROS-hydrogel markedly reduced TNF-α, IL-1β, iNOS, and IFN-γ mRNA levels, but increased TGF-β1, IL-10, and IL-4 levels, with Apelin-13@ROS-hydrogel exhibiting pronounced effects on these cytokines (Fig. 6B). Regarding the ROS clearance effect of the hydrogels, the fluorescent intensity of DHE and DCFH-DA was evaluated. Figure 6C-D shows that LPS significantly enhanced the fluorescent intensity representing DHE and DCFH-DA levels, which was attenuated by ROS-hydrogel or Apelin-13@ROS-hydrogel, with Apelin-13@ROS-hydrogel decreasing the fluorescent intensity more. The contents of M1 macrophage marker iNOS and M2 macrophage marker Arg1 were examined concerning macrophage polarization. Both hydrogels decreased the levels of iNOS and increased Arg1 levels, with Apelin-13@ROS-hydrogel exerting stronger effects on these markers (Fig. 6E-F). Flow cytometry further confirmed the levels of iNOS and Arg1 in response to LPS stimulation and treatment by ROS-hydrogel or Apelin-13@ROS-hydrogel (Fig.S1). These findings suggest that both hydrogels promote M2 polarization of macrophages and exert anti-inflammatory effects; Apelin-13@ROS-hydrogel exerts more pronounced functions on LPS-stimulated BV2 cells.

Fig. 6
figure 6

The anti-inflammatory and anti-oxidative effects of Apelin-13@ROS-hydrogel in vitro BV2 cells were suspended in a complete culture medium and seeded in a 24-well plate overnight. Then, the ROS-hydrogel and Apelin-13@ROS-hydrogel (low dose 100 µl and high dose 200 µl) were placed on the upper transwell chamber of the 24-well plate and incubated for 48 h, and examined for cell viability using the CCK-8 assay (A). For polarization determination, 100 ng/ml LPS were added to the BV2 cells in 24-well plate and simultaneously incubated with upper transwell’s hydrogel for 48 h. The mRNA expression levels of pro-inflammatory cytokines TNF-α, IL-1β, iNOS, and IFN-γ, and the mRNA expression levels of anti-inflammatory cytokines TGF-β1, IL-10, and IL-4 using qRT-PCR (B); the contents of ROS using DHE and DCFH-DA staining (C-D); M1 macrophage marker iNOS using IF staining (E); the contents of M2 macrophage marker Arg1 using IF staining (F). n = 3, ** p < 0.01, vs. control group; ## p < 0.01, vs. LPS group; && p < 0.01, vs. LPS + ROS-hydrogel group

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The anti-inflammatory and anti-oxidative effects of Apelin-13@ROS-hydrogel in vivo

The specific functions of both hydrogels were investigated in SCI rats. H&E staining confirmed that both ROS-hydrogel and Apelin-13@ROS-hydrogel-treated groups exhibited significant improvement in tissue morphology with fewer neuronal injuries sustained than in the normal control. Furthermore, the healing state of SCI rats after Apelin-13@ROS-hydrogel treatment exhibited better therapeutic effects (Fig. 7A), indicating that both hydrogels, particularly Apelin-13@ROS-hydrogel, inhibited acute inflammatory response and promoted the regenerative effect. The BBB scores of the ROS-hydrogel and Apelin-13@ROS-hydrogel groups significantly improved, with a better effect observed in the Apelin-13@ROS-hydrogel group compared to the ROS-hydrogel group; the BBB score was significantly affected by hydrogel type and treating time, and the combined effects of hydrogel type and treatment time also exerted a significant impact on the BBB score (Fig. 7B). In the inclined plane test, a significant improvement in the slope angle was observed in both the ROS-hydrogel and Apelin-13@ROS-hydrogel groups, with better improvement in the Apelin-13@ROS-hydrogel group; (Fig. 7C). To study the sustained release of Apelin-13 at the injury site, FITC-labeled Apelin-13 was used instead of natural Apelin-13 and embedded into the ROS-hydrogel. Fluorescence imaging of the spinal cord was performed at different time intervals using the Maestro in vivo optical imaging system after administration of the FITC-Apelin-13@ROS-hydrogel. The results showed that the fluorescence of the FITC-Apelin-13@ROS-hydrogel gradually decreased over time, but even on day 21, strong fluorescence was still detected at the injury site, indicating sustained release of Apelin-13 at the delivery site (Fig. 7D). These results are consistent with the in vitro release study findings. After treatment with ROS-hydrogel and Apelin-13@ROS-hydrogel, Apelin-13 and APJ protein levels in the injured spinal cord tissues were significantly increased in comparison with the SCI group (Fig. 7E).

Fig. 7
figure 7

Apelin-13@ROS-hydrogel administration improves rats’ function recovery after SCI Rats were randomly allocated into four groups: Sham, SCI modeling, SCI plus ROS- responsive hydrogel (ROS-hydrogel), and SCI plus Apelin-13@ROS-hydrogel. After modeling, rats in the ROS-hydrogel and SCI plus Apelin-13@ROS-hydrogel groups were immediately injected with ROS-hydrogel or Apelin-13@ROS-hydrogel into the lesion space. (A) After 28 days of modeling and treatment, spinal cord tissues were collected from model rats sacrificed under anesthesia and examined for histopathological characteristics using H&E staining. (B-C) The BBB Locomotor Rating Scale and inclined-plane test were used to evaluate the behavioral outcomes and motor ability of rats from different groups at days 0 (pre-surgery), 1, 5, 7, 10, 14, 21, and 28. (D) The apelin-13-loaded hydrogel was synthesized using apelin-13 labeled by FITC and used for in vivo degradation experiments. (E) After 28 days of modeling and treatment, the protein levels of Apelin-13 and APJ in spinal cord tissues were determined using Immunoblotting. n = 6 or 3, ** p < 0.01, vs. sham group; ## p < 0.01, vs. SCI group; && p < 0.01, vs. SCI + ROS-hydrogel group

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Regarding the neuroinflammation and macrophage polarization in SCI rats in vivo, the levels of related cytokines and markers were monitored. ROS clearance effects were evaluated by IHC staining detecting 8-OHdG levels. Figure 8A shows that 8-OHdG the levels of 8-OHdG were significantly higher in SCI model rats, but partially decreased in the ROS-hydrogel or Apelin-13@ROS-hydrogel treatment group, with Apelin-13@ROS-hydrogel decreasing 8-OHdG levels more. After ROS-hydrogel or Apelin-13@ROS-hydrogel treatment, in comparison with the SCI group, pro-inflammatory cytokine expression (TNF-α, IL-1β, IL-2, and iNOS) was significantly downregulated and anti-inflammatory cytokine (TGF-β1) was significantly upregulated (Fig. 8B), indicating that both hydrogels could decrease pro-inflammatory cytokines, thereby improving the tissue microenvironment post-injury. In the SCI group, MPO activity within the damaged spinal cord tissue was significantly increased, but decreased in the hydrogel treatment groups, with the Apelin-13@ROS-hydrogel intervention exerting a more pronounced effect (Fig. 8C). Regarding macrophage polarization, after ROS-hydrogel or Apelin-13@ROS-hydrogel treatment, the contents of M1 macrophage marker CD16/32 were notably decreased, and the contents of M2 marker Arg1 were significantly elevated (Fig. 8D-E), indicating that both hydrogels could regulate macrophage polarization, thereby improving the micro-inflammatory environment after injury. Apelin-13@ROS-hydrogel exhibits better improvement effects compared to those of the ROS-responsive hydrogel.

Fig. 8
figure 8

The anti-inflammatory effects of Apelin-13@ROS-hydrogel in SCI rats After 28 days of modeling and treatment, (A) the oxidative stress marker 8-OHdG expression in SCI rats was determined by IHC staining. (B) The mRNA expression levels of pro-inflammatory cytokines TNF-α, IL-1β, iNOS, IL-2, IL-4 and TGF-β1 were determined using qRT-PCR; (C) MPO activity at the injured site of SCI rats was detected using an MPO fluorescence assay kit. (D) On day 28 after SCI, the contents of microglia marker CD68, M1 macrophage marker CD16/32 and M2 macrophage marker Arg1 at the injured site of SCI rats were detected using IF staining. (E) On day 28 after SCI, the protein levels of M1 macrophage marker CD16 and M2 macrophage marker Arg1 at the injured site of SCI rats were detected using Immunoblotting. n = 6 or 3, ** p < 0.01, vs. sham group; ## p < 0.01, vs. SCI group; & Pp < 0.05, && p < 0.01, vs. SCI + ROS-hydrogel group

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Discussion

Apelin-13 has been reported to exhibit anti-inflammatory, antioxidant, and neuroprotective properties in various CNS damage models, including ischemic stroke, traumatic brain injury, and SCI [12,13,14]. Lin et al. [13] reported the downregulated expression of Apelin-13 in SCI tissues, which was further evidenced by our findings that the levels of Apelin-13 were significantly decreased at the injury sites of the SCI rats. Regarding the specific functions, Apelin-13 treatment improved the behavioral outcomes and motor ability of SCI rats since day 21 after modeling, suggesting the therapeutic potential of Apelin-13 in SCI wound healing. After silencing its receptor APJ [28, 29], Apelin-13-caused benefits were partially eliminated, indicating that the APJ receptor is crucial for Apelin-13-mediated improvements in SCI rats. Moreover, Apelin-13 administration improved neuroinflammation in the injured sites of SCI rats by decreasing pro-inflammatory TNF-α, IL-1β, IL-2 and iNOS but increasing anti-inflammatory TGF-β1; however, after silencing APJ, the effects of Apelin-13 were attenuated. These findings are consistent with previous studies showing that the Apelin-13/APJ system could attenuate neuroinflammation in different CNS diseases [30,31,32,33]. Furthermore, Apelin-13 facilitated the M2 polarization and suppressed the M1 polarization of macrophages in the injured sites, which is in line with previous studies indicating that Apelin-13 could regulate macrophage polarization in different contexts [34, 35]. Interestingly, it was also concluded that APJ silencing partially eliminated the effects of Apelin-13 on macrophage polarization, suggesting that the regulation of macrophage polarization by Apelin-13 is partly mediated by the APJ receptor. Considering that Apelin-13 functions on SCI rats were observed since day 21 after modeling, Apelin-13 is likely to improve SCI wound healing by attenuating neuroinflammation and correcting the disordered macrophage polarization sequence through the APJ receptor.

Despite these promising results, the continuous and stable release of Apelin-13 in the injury sites remains challenging due to the lengthy healing process. ROS contributes to impaired wound healing after SCI [36, 37]. ROS-responsive hydrogels, known for their biocompatibility and ability to reduce oxidative stress, have been reported to facilitate repair and regeneration of SCI [23, 38, 39]. Therefore, in this study, ROS-hydrogel has been employed as the delivery scaffold to achieve a continuous and stable release of Apelin-13 in the injury sites. As expected, ROS-hydrogel and Apelin-13@ROS-hydrogel caused faster degradation upon H2O2 stimulation, and the hydrogel acted as an in-situ gel system, releasing Apelin-13 continuously and in a stable manner. Unlike direct Apelin-13 administration, Apelin-13@ROS-hydrogel-caused improvements in the behavioral outcomes and motor ability of SCI rats were observed as early as 10 days after the modeling and continued throughout the whole monitoring process. As revealed by in vivo degradation analysis, the fluorescence of the FITC-Apelin-13@ROS-hydrogel gradually decreased over time, but even on day 21, strong fluorescence was still detected at the injury site, indicating sustained release of Apelin-13 at the delivery site. Considering that the improvement caused by Apelin-13 sustained significantly 28 days after modeling, the continuous and stable release of Apelin-13 was crucial in attenuating wound healing in SCI rats. Indeed, both hydrogels improved rats’ behavioral outcomes and motor ability, with Apelin-13@ROS-hydrogel exerting stronger ameliorative effects, confirming the improvement of SCI repair.

As aforementioned, the disrupted M1/M2 polarization sequence and the predominance of M1 macrophages following SCI lead to persistent inflammation and increased production of ROS, including hydrogen peroxide (H2O2), hydroxyl radicals (OH), and superoxide radicals (·O2−) [7, 8], ultimately delaying recovery [10, 11]. Since ROS-responsive hydrogels are known for their biocompatibility and ability to reduce oxidative stress, Apelin-13-loaded ROS-responsive hydrogels were speculated to exert better ROS clearance properties. As expected, the DPPH clearance, H2O2 clearance,·O2−, and ROS clearance capacities of Apelin-13@ROS-hydrogel outperformed the control hydrogel and ROS-responsive hydrogel, confirming the anti-oxidative functions of Apelin-13@ROS-hydrogel. Moreover, pro-inflammatory factor levels saw similar trends. In vitro, Apelin-13@ROS-hydrogel remarkably reduced pro-inflammatory mediators and M1 macrophage marker levels, but increased anti-inflammatory IL-10, IL-4, and TGF-β1 [40, 41] and M2 macrophage marker. In LPS-stimulated microglia BV2 cells, both hydrogels facilitate the M2 polarization of macrophages and exhibit anti-inflammatory effects, with Apelin-13@ROS-hydrogel showing stronger functions on LPS-stimulated BV2 cells. In SCI rats, both hydrogels reduced pro-inflammatory mediators and elevated anti-inflammatory mediators, suppressed macrophage M1 polarization, and promoted M2 polarization. Consistent with Apelin-13 administration, Apelin-13@ROS-hydrogel significantly improved neuroinflammation, facilitated the M2 polarization, and suppressed the M1 polarization of macrophages at the injury sites, which were partially eliminated by APJ silencing. These data confirm that Apelin-13@ROS-hydrogel facilitates the M2 polarization and suppresses the M1 polarization, therefore exerting anti-oxidative and anti-inflammatory functions.

In conclusion, our study demonstrated that Apelin-13@ROS-hydrogel can promote recovery in SCI rats by facilitating neuroinflammation, promoting M2 polarization, and suppressing M1 polarization of macrophages, as well as scavenging ROS through the continuous and stable release of Apelin-13 (Fig. 9). By utilizing a comprehensive approach that combines biochemical, cellular, and animal models to elucidate the effects of Apelin-13@ROS-hydrogel, this study laid a solid experimental basis for the clinical application of Apelin-13@ROS-hydrogel in SCI treatment. However, the study is limited by the lack of long-term follow-up to fully assess the chronic effects of the treatment. Future research should focus on optimizing the hydrogel formulation for clinical use and investigating the long-term safety and efficacy of Apelin-13@ROS-hydrogel in larger animal models. Additionally, exploring the molecular mechanisms underlying the observed effects will further enhance the therapeutic potential of this treatment.

Fig. 9
figure 9

The schematic diagram of the effect of Apelin-13@ROS-hydrogel on spinal cord injury recovery. Apelin-13@ROS-hydrogel promotes recovery in SCI rats by facilitating neuroinflammation, promoting M2 polarization, suppressing M1 polarization of macrophages, and scavenging ROS through the continuous and stable release of Apelin-13

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Data availability

All data and materials are available.

References

  1. Fehlings MG, Baptiste DC. Current status of clinical trials for acute spinal cord injury. Injury. 2005;36(Suppl 2):B113–22.

    Article  PubMed  Google Scholar 

  2. Hausmann ON. Post-traumatic inflammation following spinal cord injury. Spinal Cord. 2003;41(7):369–78.

    Article  CAS  PubMed  Google Scholar 

  3. Singh PL, et al. Current therapeutic strategies for inflammation following traumatic spinal cord injury. Neural Regen Res. 2012;7(23):1812–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Schwab JM, et al. Experimental strategies to promote spinal cord regeneration–an integrative perspective. Prog Neurobiol. 2006;78(2):91–116.

    Article  CAS  PubMed  Google Scholar 

  5. Sindrilaru A, et al. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J Clin Invest. 2011;121(3):985–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shechter R, London A, Schwartz M. Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates. Nat Rev Immunol. 2013;13(3):206–18.

    Article  CAS  PubMed  Google Scholar 

  7. Sorce S, Krause KH. NOX enzymes in the central nervous system: from signaling to disease. Antioxid Redox Signal. 2009;11(10):2481–504.

    Article  CAS  PubMed  Google Scholar 

  8. Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal cerebral ischemia. Surg Neurol. 2006;66(3):232–45.

    Article  PubMed  Google Scholar 

  9. Kigerl KA, et al. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci. 2009;29(43):13435–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Nathan C, Ding A. Nonresolving Inflamm Cell. 2010;140(6):871–82.

    CAS  Google Scholar 

  11. Werdin F, et al. Evidence-based management strategies for treatment of chronic wounds. Eplasty. 2009;9:e19.

    PubMed  PubMed Central  Google Scholar 

  12. Shao ZQ, et al. Apelin-13 inhibits apoptosis and excessive autophagy in cerebral ischemia/reperfusion injury. Neural Regen Res. 2021;16(6):1044–51.

    Article  CAS  PubMed  Google Scholar 

  13. Lin T et al. Apelin-13 protects neurons by attenuating early-stage Postspinal Cord Injury apoptosis in Vitro. Brain Sci, 2022. 12(11).

  14. Shen X, et al. Apelin-13 attenuates early brain injury through inhibiting inflammation and apoptosis in rats after experimental subarachnoid hemorrhage. Mol Biol Rep. 2022;49(3):2107–18.

    Article  CAS  PubMed  Google Scholar 

  15. Chen D et al. Intranasal delivery of Apelin-13 is neuroprotective and promotes Angiogenesis after ischemic stroke in mice. ASN Neuro, 2015. 7(5).

  16. Xin Q, et al. Neuroprotective effects of apelin-13 on experimental ischemic stroke through suppression of inflammation. Peptides. 2015;63:55–62.

    Article  CAS  PubMed  Google Scholar 

  17. Zhang D, et al. Injectable and reactive oxygen species-scavenging gelatin hydrogel promotes neural repair in experimental traumatic brain injury. Int J Biol Macromol. 2022;219:844–63.

    Article  CAS  PubMed  Google Scholar 

  18. Li X, et al. EGF and curcumin co-encapsulated nanoparticle/hydrogel system as potent skin regeneration agent. Int J Nanomed. 2016;11:3993–4009.

    Article  CAS  Google Scholar 

  19. Huang X, et al. Reactive oxygen species scavenging functional hydrogel delivers procyanidins for the treatment of traumatic brain Injury in mice. ACS Appl Mater Interfaces. 2022.

  20. Thi PL, et al. In situ forming and reactive oxygen species-scavenging gelatin hydrogels for enhancing wound healing efficacy. Acta Biomater. 2020;103:142–52.

    Article  CAS  PubMed  Google Scholar 

  21. Zhu C, et al. Multifunctional thermo-sensitive hydrogel for modulating the microenvironment in Osteoarthritis by polarizing macrophages and scavenging RONS. J Nanobiotechnol. 2022;20(1):221.

    Article  CAS  Google Scholar 

  22. Bai J, et al. Reactive oxygen species-scavenging Scaffold with Rapamycin for treatment of intervertebral disk degeneration. Adv Healthc Mater. 2020;9(3):e1901186.

    Article  PubMed  Google Scholar 

  23. Li Z, et al. A reactive oxygen species-responsive hydrogel encapsulated with bone marrow derived stem cells promotes repair and regeneration of spinal cord injury. Bioact Mater. 2023;19:550–68.

    PubMed  Google Scholar 

  24. Barros Filho TE, Molina AE. Analysis of the sensitivity and reproducibility of the Basso, Beattie, Bresnahan (BBB) scale in Wistar rats. Clin (Sao Paulo). 2008;63(1):103–8.

    Article  Google Scholar 

  25. Li Y, et al. Pericytes impair capillary blood flow and motor function after chronic spinal cord injury. Nat Med. 2017;23(6):733–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Choi YH, et al. Isoliquiritigenin ameliorates dextran sulfate sodium-induced colitis through the inhibition of MAPK pathway. Int Immunopharmacol. 2016;31:223–32.

    Article  CAS  PubMed  Google Scholar 

  27. Shin K, et al. Bioactivity of the putative apelin proprotein expands the repertoire of apelin receptor ligands. Biochim Biophys Acta Gen Subj. 2017;1861(8):1901–12.

    Article  CAS  PubMed  Google Scholar 

  28. Hosoya M, et al. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J Biol Chem. 2000;275(28):21061–7.

    Article  CAS  PubMed  Google Scholar 

  29. Masri B, et al. The apelin receptor is coupled to Gi1 or Gi2 protein and is differentially desensitized by apelin fragments. J Biol Chem. 2006;281(27):18317–26.

    Article  CAS  PubMed  Google Scholar 

  30. Luo H, et al. Apelin-13 suppresses Neuroinflammation Against Cognitive Deficit in a Streptozotocin-Induced Rat Model of Alzheimer’s Disease through activation of BDNF-TrkB signaling pathway. Front Pharmacol. 2019;10:395.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Duan J, et al. Neuroprotective effect of Apelin 13 on ischemic stroke by activating AMPK/GSK-3beta/Nrf2 signaling. J Neuroinflammation. 2019;16(1):24.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Xu W, et al. Apelin-13/APJ system attenuates early brain injury via suppression of endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation and oxidative stress in a AMPK-dependent manner after subarachnoid hemorrhage in rats. J Neuroinflammation. 2019;16(1):247.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang Y et al. Neuroprotective roles of Apelin-13 in neurological diseases. Neurochem Res. 2023.

  34. Izgut-Uysal VN, et al. The effect of apelin on the functions of peritoneal macrophages. Physiol Res. 2017;66(3):489–96.

    Article  CAS  PubMed  Google Scholar 

  35. Leeper NJ, et al. Apelin prevents aortic aneurysm formation by inhibiting macrophage inflammation. Am J Physiol Heart Circ Physiol. 2009;296(5):H1329–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sullivan PG, et al. Temporal characterization of mitochondrial bioenergetics after spinal cord injury. J Neurotrauma. 2007;24(6):991–9.

    Article  PubMed  Google Scholar 

  37. Qiu Z, et al. Lipopolysaccharide (LPS) aggravates high glucose- and Hypoxia/Reoxygenation-Induced Injury through activating ROS-Dependent NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. J Diabetes Res. 2019;2019:8151836.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Ying Y, et al. A shear-thinning, ROS-scavenging hydrogel combined with dental pulp stem cells promotes spinal cord repair by inhibiting ferroptosis. Bioact Mater. 2023;22:274–90.

    CAS  PubMed  Google Scholar 

  39. Liu D, et al. ROS-Scavenging hydrogels synergize with neural stem cells to enhance spinal cord Injury Repair via regulating Microenvironment and facilitating nerve regeneration. Adv Healthc Mater. 2023;12(18):e2300123.

    Article  PubMed  Google Scholar 

  40. Wei R, Jonakait GM. Neurotrophins and the anti-inflammatory agents interleukin-4 (IL-4), IL-10, IL-11 and transforming growth factor-beta1 (TGF-beta1) down-regulate T cell costimulatory molecules B7 and CD40 on cultured rat microglia. J Neuroimmunol. 1999;95(1–2):8–18.

    Article  CAS  PubMed  Google Scholar 

  41. Zhou WJ, et al. Anti-inflammatory cytokines in endometriosis. Cell Mol Life Sci. 2019;76(11):2111–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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Funding

This study was supported by Kewei Joint Project of Hunan Natural Science Foundation (2021JJ70070), Natural Science Foundation of Hunan Province (2021JJ31036, 2022JJ30912 and 2024JJ5539) and the Scientific Research Plan Project of Health Commission of Hunan Province (202205014861).

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Authors and Affiliations

  1. Department of Spine Surgery, The Third Xiangya Hospital, Central South University, Changsha, 410013, China

    Zhiyue Li, Jiahui Zhou & Yifan Zheng

  2. Health Management Medicine Center, The Third Xiangya Hospital, Central South University, Changsha, 410013, China

    Qun Zhao

  3. NanChang University Queen Mary School, Nanchang, 330038, China

    Yuyan Li

  4. School of Pharmaceutical Science, University of South China, Hengyang, 421001, China

    Linxi Chen

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Contributions

Zhiyue Li, Qun Zhao, and Jiahui Zhou designed the experiments. Zhiyue Li drafted the article. Zhiyue Li, Yuyan Li, and Yifan Zheng contributed to experiments, analysis and manuscript preparation. Linxi Chen revised the article critically for important intellectual content. Qun Zhao, Jiahui Zhou funded the research. All the authors read and approved the manuscript.

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Li, Z., Zhao, Q., Zhou, J. et al. A reactive oxygen species-responsive hydrogel loaded with Apelin-13 promotes the repair of spinal cord injury by regulating macrophage M1/M2 polarization and neuroinflammation. J Nanobiotechnol 23, 12 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-024-02978-4

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Journal of Nanobiotechnology

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