Therapeutic potential of EVs loaded with CB2 receptor agonist in spinal cord injury via the Nrf2/HO-1 pathway

ABSTRACT

Background

Spinal cord injury (SCI) poses a challenge due to limited treatment options. Recently, the effect and mechanism of Exo-loaded cannabinoid receptor type 2 (CB2) agonist AM1241(Exo + AM1241) have been applied in other inflammatory diseases but not in SCI.

Methods

The SCI model was set up using C57BL/6 mice, followed by the treatment of Exo, AM1241, and Exo + AM1241. We assessed the effects of the following treatments on motor function recovery using BMS, and evaluated histological changes, apoptosis activity, inflammation, and oxidative stress in the SCI mice model. Additionally, the effect of following treatments on spinal cord neural stem cells (NSCs) was evaluated under lipopolysaccharides (LPS) induced inflammatory and oxidative models and, glutamate (Gluts) induced cell apoptosis models.

Result

Our results demonstrated that Exo + AM1241 treatment significantly improved motor function recovery, after SCI by decreasing proinflammatory cytokines, and suppressing astrocyte/microglia (GFAP/Iba1) activation in the injury zone. Additionally, this treatment reduces pro-apoptotic proteins (Bax and caspase 3), increases the levels of the anti-apoptotic protein Bcl-2, enhances antioxidant defenses by boosting SOD and GSH, and lowers oxidative stress markers such as MDA. It also activates the Nuclear factor erythroid-2 (Nrf2) related factor 2 signaling pathway, thereby enhancing tissue protection against damage and cell death.

1. Introduction

Spinal cord injury (SCI) is a profound and devastating clinical condition resulting from an external force, leading to severe neurological deficits and partial or total paralysis below the level of damage, with potential implications for mortality [1]. SCI initiates a cascade of pathological events that culminate in inflammation, oxidative stress, neural apoptosis, and ferroptosis, ultimately impairing both motor and sensory function below the site of injury [2,3]. The inflammatory response and oxidative stress are major factors that can have a profound impact on neural cells, potentially triggering neuronal cell apoptosis and significantly influencing nerve function recovery [4]. This condition not only drastically diminishes the quality of life and life expectancy of affected individuals but also contributes to psychological and emotional disturbances, social anxiety, and a substantial economic burden on both families and healthcare systems [5]. As such, there is an urgent need for effective treatments to restore function and improve the lives of those suffering from SCI.

Exosomes, nanoscale extracellular vesicles derived from various cell types, have emerged as promising vehicles for drug delivery and cellular communication. Exosomes facilitate the transfer of chemical and genetic material from parental to daughter cells, contributing to nerve repair and playing a critical role in diagnosing and treating several diseases in the central nervous system (CNS) [6,7]. Human umbilical cord mesenchymal stem cells(hUC-MSCs) derived exosomes have garnered significant attention in various medical fields and shown particular promise in enhancing cell survival, [8] modulating immune responses, [9] and promoting tissue regeneration [10–12]. As hUC-MSCs possess higher proliferation abilities than bone marrow MSCs, they are more readily available. They are natural bio-carriers composed of lipid-bilayer natural nanoparticles (NNPs) and can transport diverse genetic and biological materials to target cells and tissues. Exosomes, with enhanced safety, greater stability, and reduced immunogenicity compared to MSCs, exhibit a longer half-life in blood circulation and an ability to cross the blood–brain barrier. Notably, exosomes exhibit effects similar to their parental cells, devoid of mutation and tumorigenic potential, making them promising candidates for treating various neurological disorders, including SCI [13–15]. Particularly when compared to the characteristics of standard implanted stem cells and inorganic nanoparticles(iNPs). They can easily traverse the bio barriers with diameters ranging from 30–200 nm and excellent fat solubility [16]. Numerous studies have demonstrated exosomes’ pivotal role in promoting and regulating the neural stem cells (NSCs) microenvironment in the spinal cord and brain [17–21].

AM1241, a cannabinoid receptor-type 2 (CB2) agonist derived from Cannabis sativa, exhibits noteworthy anti-pain, anti-oxidative, and anti-inflammatory properties in various diseases, including brain and spine disorders [22,23]. Previous research highlights AM1241's potential to delay neurodegenerative disorders and improve inflammation in lateral sclerosis animal models [24]. AM1241 promoted the differentiation of human and murine neural progenitor cells (NPCs) into neuronal cells and improved neurogenesis in GFAP/Gp120 transgenic mice [25]. Our research team discovered that AM1241 facilitates the regenerating of dopamine neurons in a Parkinson's disease model by activating CB2 receptor through the Xist/miR, 133b-3p/Pitx3 axis [26]. Moreover, AM1241 has shown promising results in alleviating symptoms of neuroinflammatory conditions like Alzheimer's disease by modulating CB2 receptors [27]. Despite these findings, there is a notable gap in understanding the effective delivery of AM1241 via exosomes and its therapeutic potential in SCI, leaving the mechanisms behind motor function recovery unexplored.

In this study, we explore the therapeutic potential of combining hUC-MSC-derived exosomes with AM1241 (Exo + AM1241) to promote NSC differentiation, proliferation, and survival in the context of SCI. We hypothesize that Exo + AM1241 will synergistically enhance the regenerative capabilities of NSCs, leading to improved functional recovery following SCI. Through a series of in vitro and in vivo experiments, we aim to elucidate the mechanisms by which Exo + AM1241 influences NSC behavior and assess its efficacy as a novel treatment for SCI.

2. Materials and methods

2.1. Materials

Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), and fetal bovine serum (FBS) purchased from Gibco (USA). Penicillin–streptomycin qualified for cell culture obtained from Sigma-Aldrich (USA). PBS (phosphate-buffered solution) was acquired by HyClone, (USA). TUNEL acquired from Beyotime Biotechnology (China). AM1241, obtained from Selleck (China). Lipopolysaccharides (LPS) and L-glutamate (Glu) from E. coli Sigma (USA). Antibodies such as anti-CD9 (#ab92726), anti-TSG101 (#ab193349), Anti-GFAP (#ab7260), Iba1 (#ab5076,) Anti-Bcl-2 (#ab182858), Anti-Bax (#ab182734), Anti-Caspase-3 (#ab184787), Anti-NeuN (# ABN90), DAPI (4, 6-diamidino-2-Phenylindole) and loading Control GAPDH (#ab9485) were purchased through Abcam (United Kindom), Sigma Aldrich (USA) and Invitrogen (USA). TRIzol was acquired from Invitrogen (USA). OCT-Freezing Medium purchased from Sakura (Japan). We obtained the Primer Script Reverse Transcriptase Kit and TB Green TM Premix Ex Taq Kit from Takara (Japan). Additionally, the Nucleoprotein and cytoplasmic protein extraction kit, along with the Bicinchoninic Acid Protein Assay Kit, were procured from Keygen (China). The detection of glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) levels was conducted using assay kits from Beyotime Institute of Biotechnology (Nantong, China), with procedures carried out under the manufacturer's protocol. Hematoxylin and eosin (HE) staining kits were purchased from elabscience, (USA).

2.2. Isolation, preparation, and identification of hUC-MSC-derived exosomes

Extraction of hUC-MSC cells was prepared under the supervision and ethical approval of the Stem-Cells Research Center of Tongji Hospital affiliated with Tongji University with informed and written consent from the donor. A fresh umbilical cord (UC) was prepared during cesarean section procedures using the described protocol [28]. Briefly, UC was rinsed with distilled H₂O/sodium solution three times till the cord blood was removed. UC was washed in 75% ethanol for 20 s before being disinfected with PBS and penicillin/streptomycin. After cleaning, the cord was cut into 1-1.5 mm² pieces and drifted in DMEM/F12 medium plus 10% FBS, 1% of penicillin/streptomycin (P/S) (V/V) and subsequently incubated at 37 °C under 5% CO ₂ – air atmosphere. After 45 min, a one-time wash with PBS was given to remove non-adhesive cells. hUC-MSC cells were passaged every 2 days ratio of 1:3. Cells from 9–10 passages of each hUC-MSC cell line were used to isolate exosomes for further experiments.

Exosomes were isolated from passage 9 hUC-MSCs cell culture media as previously described protocol [29]. Briefly, hUC-MSCs were washed thrice with PBS and incubated in FBS-free DMEM/F2 for 12 h. The supernatant was collected and differentially centrifuged at 300 × g for 15 min, 1000 × g for 15 min, and 10,000 × g for 30 min to separate cell debris, followed by ultracentrifugation at 100,000 × g for 70 min (optimaXPN-100, Beckman Coulter) and a second time after being washed with cold phosphate-buffered saline (PBS). All centrifugation procedures were maintained at 4°C. Finally, the supernatant was discarded, and the hUC-MSCs-exosome pellet was diluted with cold PBS. Exosomes were stored in aliquots at −80 ^oC until further use.

Exosomes are recognized for their vital roles in intercellular communication, characterized by their negative charges. In contrast, AM1241 bears a positive charge and is distinguished by its heightened lipophilicity and restricted water solubility, as evidenced by its log P value of 3.41. Consequently, AM1241 was dissolved in a 0.01% DMSO solution to create a concentrated stock solution. The exosome underwent ultrasonication with AM1241 on a shaker at 37 °C and 500 rpm for 2 h, facilitating the preparation of Exo + AM1241 through passive diffusion [27]. This process capitalized on the lipophilic nature of AM1241 and electrostatic adsorption. To restore the membrane integrity of exosomes, the mixture was incubated at 37 °C for 30 min in a hot water bath before being stored for further experiments.

The morphology of both collected hUC-MSC cells derived from blank exosomes and loaded with AM1241 was observed under Transmission Electron Microscopy (TEM, The Netherlands). The particle size distribution of blank exosomes and exosomes loaded with AM1241 were diluted to 1 mL/mL in distilled water and measured by the Nano Sight nanoparticle tracking analyzer at 25 ^oC (NTA, Malvern Instruments, UK). Western blotting analysis was utilized to identify surface markers of the extracted exosomes. The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto membranes. Then, specific marker proteins of exosomes like TSG 101, CD9, and Alix were detected using a western blot. GAPDH was included as a positive control.

2.3. Cell culture of NSCs

Embryonic RAT spinal cord NSCs were used in an in vitro study. In brief, Sprague–Dawley (SD) embryonic RAT (E15–E17) was immersed in 75% ethanol. Each embryonic RAT spinal cord was extracted under a stereoscopic microscope and cleaned using 1% PBS. NSCs were dissociated with 0.125% trypsin and 0.05% deoxyribonuclease and incubated at 37 °C with a 5% CO₂ humidified atmosphere. Later, to neutralize the trypsin effect, the suspension was dissolved with DMEM and FBS. Then, the cell suspension was filtered by BD Biosciences 70 μm mash cell strainers and centrifugated at 1000 rpm at 4 °C for 5 min. Cell suspensions were incubated with DMEM/F12 containing 2% B27 (Gibco), 1% N2 (Gibco), 20 ng/mL-1 bFGF (Peprotech), and 20 ng/mL−1 EGF (Peprotech). 0.025% trypsin was used to digest NSCs during passaging every 3 to 4 days with a passaging ratio of 1:4.

2.4. LPS-induced inflammation in NSCs

NSCs were pretreated with three different treatments: Exo + AM1241 (100 µg/mL Exosomes and 10 µM AM1241), Exosomes alone (100 µg/mL), and AM1241 alone (10 µM) for 24 h at 37 °C in a 5% CO₂ humiliator. After pretreatment, the cells were washed with the help of cold sterile PBS and all samples were subjected to LPS-induced inflammation (100 µg/mL) for 12 h under the same conditions. Post-treatment, the cells were washed with PBS, and the culture medium was refreshed. The effects on LPS-induced inflammation were evaluated using an ELISA method to measure the levels of pro-inflammatory cytokines in the cell culture supernatants.

2.5. Glutamate-induced apoptosis model in NSCs

NSCs were cultured to a suitable density and transferred to a 6-well plate. Cultured Cells were divided into five groups: Sham, Glutamate, and pretreated with Exosome, AM1241, and Exo + AM1241. After pretreatment, cells were incubated at 37 °C with 5% CO₂ for 24 h. The culture media was then discarded, cells were washed by PBS, and all groups except Sham were treated with 100 μM Glutamate for another 24 h under the same conditions. The next day, cells were rinsed with PBS, fixed with 4% paraformaldehyde for 15-20 min at room temperature, and washed again with PBS.

2.6. In vivo experimental design and contusion injury model

A total of 75 healthy female C57BL/6 mice, weighing between 17 and 22 g and aged 8 to 10 weeks, were purchased from the Shanghai Experimental Animal Center, Shanghai, China. All mice were divided into five groups, each consisting of n = 15 mice. Four groups were subjected to the SCI contusion model using modified Allen's technique, while the sham group underwent laminoplasty without spinal cord damage. Following four groups with treatment as follow SCI group (SCI plus PBS), alone AM1241 group (SCI plus AM1241), alone Exosome group (SCI plus exosomes), and Exosome + AM1241 group (SCI plus Exo + AM1241). Each group of mice was separately housed in cages under suitable room temperature and a 12-hour/12-hour light cycle- regulated environment, through free access to food and drinking water. Present animal studies comply with the Animal Ethics Committee of Tongji University(T3-HB-LAL-2023-25).

Selection of female mice were decided due to shorter bladder which easy to empty after SCI. A contusive SCI mouse model was performed with a modified-Allen's weight drop impactor, as previously mentioned by Gruner [30]. In brief, mice were anesthetized with isoflurane (a self-inhalation instrument), and thoracic laminectomy was performed at the T10 level. The spine was stabilized by clamping the transverse processes at T9 and T11. A weight drop injury was performed on the exposed dorsal side of the cord using a 10 g rod dropped from a height of 12.5 mm. Following the impact injury, the muscles and skin were sutured closed in double layers. Then, the mice were placed inside a chamber with controlled temperature and humidity levels. We manually emptied the bladders of injured mice twice a day until they had fully regained the ability to void their bladders independently.

AM1241 (10 mg/kg) and Exo + AM1241 containing (10 mg/kg) AM1241 loaded on exosomes at 10 µL volume and uniformly mixed with the help of ultra-sonification. To restore the membrane integrity of exosomes, the mixture was incubated at 37 ^oC for half an hour in a hot water bath. On the 3rd day after SCI, all prepared treatment solutions (Exo, AM1241, and Exo + AM1241) were administered as a single intrathecal dose with a volume of 10 μL at the L5-L6 level. The control mice were administered regular PBS, whereas the sham group received nothing. Intrathecal injections were performed as per the protocol shown [23] with a Hamilton syringe (80301, USA) and a 26-gauge needle. After intrathecal injection, it was confirmed that mice showed no signs of neurological or motor alternations.

2.7. Assessment of Basso Mouse Scale (BMS) evaluation

All groups of mice (n = 12 /group) were assessed by Basso Mouse Scale (BMS) scoring system by two independent examiners blinded to the mice's conditions. Evaluations were conducted prior to surgery, 1-day post-surgery, and weekly after that until the completion of 8 weeks. In brief, the primary scoring system for the MBS was established on a scale of 0 to 9 points, where a score of 0 indicates total paralysis and a score of 9 indicates full mobility, as observed in healthy mice. Subjects were placed on a flat surface and monitored for 6 min. The single-blind approach was used to assess hind limb motor function by two independent blinded investigators. The subject's BMS score was calculated, and mean values were counted for both hind limbs. Body weight was measured after emptying the bladder to avoid an error.

2.8. Footprint analysis

Eight weeks post-treatment, gait, and motor coordination were assessed. Subjects had their front and rear paws coated with different-colored dyes, and they were placed on white absorbent paper surrounded by cages to promote straight-line walking. The footprint patterns were subsequently scanned, and coordination was measured using all captured images.

2.9. Immunofluorescences staining for spinal cord tissue sections

All subjects were sacrificed in the eighth-week post-treatment to obtain tissue samples. In brief, mice were anesthetized with sodium phenobarbital, and their hearts were perfused with PBS and 4% paraformaldehyde to rigidify the limbs. The vertebral column (C2 to L3) was fixed in 4% paraformaldehyde, followed by dehydration and immersion in 15% and 30% sucrose gradients for 48 h at 4°C. The spinal cord was exposed, cleaned, and then subjected to dehydration in a 30% sucrose gradient for 24 h at 4°C. Finally, the spinal cord was embedded in OCT. The spinal cord tissue was sliced longitudinally into 12 μm diameter-thick sections using a Leica-CM3050 S microtome from Leica, Germany, and stored at −80°C for analysis.

All group tissue sections were taken from −80 ^oC and kept for 15 min at room temperature. After fixing all samples in PFA for 5 min, washing with PBS three times for 15 min follows to remove the surrounding excess OCT. Permeabilization was achieved by incubating tissue slices into 0.4% Triton X-100 for 30 min and washing through PBS three times, staining blocking solution with 3% BSA in PBS for 1 h at room temperature. All samples were stained overnight at 4°C with primary antibodies against related proteins. Following antibodies were used (neuronal nuclei (NeuN (1:500, Pig, Sigma ABN90), GFAP (1:1000, Rabbit, Abcam, ab7260, USA), and Iba1 (1:500, Goat, Abcam, ab5076, USA)). Second-day samples were washed thrice in PBS and incubated with secondary antibodies for 2 h at room temperature in low light conditions. The tissue nuclei were then detected using the counter-stain DAPI. The sections were covered with coverslips and mounting medium (O8015, Sigma) and examined under a confocal microscope (LSM880, Zeiss, Germany) to conduct further analysis.

2.10. Histological study by H&E staining

Transverse sections, 10 μm in thickness, were prepared from a 1 cm length of the spinal cord at the injury epicenter. Additionally, sections from the liver, lung, brain, kidney, and heart were collected at a thickness of 16 μm. All sections were stained with Mayer's Hematoxylin and Eosin (H&E) staining based on the manufacturer's protocols. Tissue morphology was evaluated, and the injury site was captured at X4 and X20 magnification using a bright field Leica microscope.

2.11. TUNEL staining

In vitro, experiment NSCs initially plated at a density of 5 × 10⁵ cells/well in 6-well plates, and divided into five groups: sham, control (glutamate), and three pre-treatment groups with AM1241, Exosomes, and Exo + AM1241. After 24 h of incubation at 37 °C in a 5% CO₂ humidified atmosphere, the culture medium was replaced with PBS, and glutamate (100 μM/1 mL) was used to induce in vitro cell death in all groups except the sham. These cells were then further incubated for 24 h.

In vivo experiment, frozen spinal cord sections were fixed with 4% PFA and washed in PBS. Proteinase K treatment followed. Later, sections were equilibrated and subjected to the TUNEL reaction for 1 h at 37°C. After further PBS washing (3x, 10 min each), nuclei were stained with DAPI. TUNEL-positive neurons were counted under a fluorescence microscope to assess apoptosis levels in each group.

2.12. RT-qPCR

Total RNA was isolated from cells and spinal cord tissue through the basic TRIzol method (USA) per the manufacturer's protocols. Briefly, the integrity of RNA was determined using 1% agarose electrophoresis, and purity was determined photometrically using the Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA samples were transformed into cDNA using Takara’s Prime Script RT Reagent Kit. Gene expression quantification was conducted using RT-qPCR, with the primer details available in supplementary Table S1. The PCR process consisted of an initial step at 50 °C for 30 s, followed by 5 min at 95 °C, and then 40 cycles, each involving five seconds at 95 °C and 34 s at 60 °C. A QuantStudio3 Real-Time PCR System (Thermo Fisher, USA) and the TB Green TM Premix Ex Taq Kit were utilized to determine relative mRNA expression, with the ΔΔCt method employed. The housekeeping gene, GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), served as the internal control, and the mRNA levels of the target genes were normalized to the expression of GAPDH.

2.13. Assessment of oxidative stress levels

Spinal cord tissues and cell homogenates were prepared with cold phosphate-buffered saline. Oxidative stress markers kits included superoxide dismutase (SOD), reduced glutathione (MDA), and malondialdehyde (MDA) from Byotime Institute of Biotechnology Nanjing China were used. The final mixture for each marker was analyzed at 450 nm wavelength using a microplate reader from Molecular Devices. All procedures were directed following the manufacturer's protocols. The study included six replicates (n = 6).

2.14. Cell viability assay

We assess the AM1241 drug cytotoxicity in neural stem cells. Cells were seeded at a density of 6 × 10⁵ cells /well in a 96-well plate and incubated for 24 h at 37 ^oC. Then, cells were exposed to various concentrations of AM1241 ranging from 0 to 50 µg/mL (0, 5, 10, 20, and 50 µg/mL) for an additional 24 h. Subsequently, 20 µL of bromide (MTT,5 mg/mL, Solarbio, china) was added to each well, and the cells were incubated for an additional 4 h. cytotoxic effects were checked across different concentration groups.

2.15. Western Blot analysis

Proteins of interest from spinal tissue, cells, hUC-MSC cells, and exosomes were analyzed through Western Blot analysis. In brief, all samples were homogenized in a cold RIPA buffer, and protein concentrations were determined using the BCA assay kit from Beyotime (China). 15% SDS-PAGE was used to separate the proteins and transfer to PVDF membranes. Subsequently, the PVDF membranes were blocked for 2 h at room temperature with blocking buffer (5% skim milk in TBST) and incubated overnight with primary antibodies (1:1000) at 4 °C. After three washes with TBST (5 min each), the membranes were incubated for 60 min along with a secondary antibody (1:5000) at room temperature. Blottings were detected using an Image Quant LAS-4000-Mini instrument from GE Healthcare Life Sciences, USA, and enhanced chemiluminescence.

2.16. Statistical data analysis

The statistical data analysis was performed using Graph Prism 10.1 software, and results are presented as mean ± SD. Student t-pared tests were used for comparisons among two groups, while one-way or two-way ANOVA tests were employed for comparisons involving three or more groups. The significant levels are represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3. Result

3.1. Isolation and characterization of hUC-MSC-Exosomes

The exosome and Exo + AM1241 were obtained through cryogenic ultracentrifugation, and characterized through transmission electron microscopy (TEM). As shown in Figure 1A, exosomes have a completely thin membrane oval/round by complete envelope structure with an average size of approximately 125.4 nm. After loading AM1241, there were slight changes in morphology. To find out about conformation, the Malvern Zetasizer Nano ZS90 was used to check the particle size and number of exosomes, with and without AM1241. As a result, Figure 1B shows that the particle size distribution of Exosome and Exo + AM1241 was 125 ± 4 and 141 ± 8, respectively. The zeta potential of the exosomes was measured to be −12.48 mV, whereas the exosomes loaded with positively charged AM1241 had a zeta potential of −9.04 mV (Figure 1C). This indicates the effective loading of AM1241 into the exosomes. Western blot analysis of hUC-MSC and the exosome expressed the distinctive marker proteins CD9, TSG101, and Alix (Figure 1D). These results proved that exosomes were extracted successfully.

Figure 1.

Characterization of hUC-MSC cells derived Exosome (Exo) and Exosomes loaded AM1241(Exo + AM1241). (A) Representative TEM images of hUC-MSC cells derived Exosome and Exo + AM1241. (B) Particle size of Exosome and Exo + AM1241. (C) Zeta potential of Exosomes/ Loaded AM1241. (D) Representative Immunoblots of Protein Markers CD9, TSG101, and Alix in MSC Cells and Derived Exosomes.

3.2. In Vitro, Exo+AM1241 drives NSC differentiation and promotes proliferation independent of growth factors

To examine hUC-MSC-derived exosomes loaded with AM1241 on NSC differentiation, we performed immunostaining for neurons and astrocytes using NeuN and GFAP markers. The control group received a regular medium, while the AM1241, Exosome, and Exo + AM1241 groups received their respective treatments, and incubated for 24 h. Immunostaining showed fewer NeuN-positive neurons in the control group compared to AM1241. The Exo + AM1241 group had the highest neural cell count among all groups (Figure 2A). GFAP staining was higher in the control group than in Exo + AM1241, with Exo + AM1241 being more effective than AM1241 or Exosome alone.

Figure 2.

hUC-MSC derived exosomes loaded AM1241 promote NSCs proliferation and differentiation absence of growth factor (A) Differentiation of NSCs at 24 h: Immunostaining by NeuN and GFAP. (B) NSCs Morphology structure by normal microscopy (C) Exo + AM1241 helps the proliferation of NSCs without EGF/bFGF. (D) different concentrations of AM1241toxicity effect on NSC viability.

Previous studies have used high concentrations of EGF and bFGF to promote NSC differentiation [31]. Our investigation focuses on Exo + AM1241's potential to enhance NSC proliferation without EGF/bFGF. We prepared NSC cultures with and without EGF/bFGF as control and negative control, respectively, and administered AM1241, exosomes, and Exo + AM1241 treatments for 24 h. The Exo + AM1241 group had higher cell counts than the negative control, indicating its proliferative efficacy (Figure 2B & 2C). A toxicity assessment of AM1241 at varying dosages (5, 10, 20, and 50 µg/mL) showed consistent cell viability, with 20 µg treated cells comparable to normal cells (Figure 2D).

3.3. Exo + AM1241 treatment decreases inflammation, oxidative stress, and apoptosis in neuronal cells

We demonstrated glutamate-induced apoptosis in NSC cells, with the NSCs + Glu group showing increased apoptosis compared to the sham group. The Exo + AM1241 group significantly reduced apoptotic cells compared to NSCs + Glu, while AM1241 and exosome groups had a lesser effect (Figure 3A and 3B). Pro-apoptotic proteins Bax and Caspase-3 were upregulated in NSCs + Glu, but Exo + AM1241 treatment reduced their expression (Figure 3C-3F). Exo + AM1241 also increased the anti-apoptotic protein Bcl-2 compared to NSCs + Glu, whereas AM1241 alone had no significant effect on Bax expression.

Figure 3.

In Vitro Pretreated Exo + AM1241 Protects NSCs by Inhibiting LPS-Induced Inflammation, Oxidative Stress, and Glutamate-Induced Neural Loss. (A) Apoptosis in primary rat spinal cord neurons was visualized using TUNEL (green) and DAPI (blue) staining, with white arrows indicating apoptotic cells in the control group. (B) Histogram: TUNEL+/DAPI + area, showing Exo + AM1241 pretreatment significantly reduced glutamate-induced apoptic cells (n = 3, Scale bar = 50 µm). (C) Immunoblotting of apoptosis-related proteins. (D-F) Relative expression of apoptosis proteins normalized to GAPDH: Exo + AM1241 increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bax and cleaved caspase-3 (n = 3). (G) GSH activity. (H) MDA content. (I) SOD activity (n = 3). (J) ELISA of IL-1β, IL-6, and TNF-α in LPS-induced NSC (activated) microglial cells: Exo + AM1241 significantly suppressed cytokine production compared to single treatments (n = 6, mean ± SD, p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001).

Our study assessed Exo + AM1241's impact on oxidative stress in LPS-induced neural cells. LPS treatment significantly increased intracellular malondialdehyde (MDA) and reduced glutathione (GSH) and superoxide dismutase (SOD). AM1241 alone had no significant effect on MDA, GSH, and SOD levels (Figure 3G and 3I). However, Exo + AM1241 pre-treatment significantly reduced oxidative stress compared to exosomes alone, mitigating LPS-induced oxidative stress. An ELISA assay showed increased IL-1β, TNF-α, and IL-6 levels in the LPS-induced NSCs group, whereas Exo + AM1241 pre-treatment significantly decreased these cytokines (Figure 3J).

3.4. Exo + AM1241 treatment enhances locomotor improvement by protecting neurons

In vivo, we comprehensively assessed the neuroprotective efficacy of exosomes loaded with AM1241 in SCI model experimental timeline as shown in Figure 4A. A standardized BMS test evaluated a mouse model after a T10 spinal contusion injury. SCI group BMS scores were zero until the first week, but motor function improved in the AM1241, Exosome, and Exo + AM1241 groups, surpassing the SCI group at 3, 4, 5, 6, and 8 weeks. From week 3, Exo + AM1241 group BMS scores were consistently higher than those in the AM1241, Exosome, and SCI groups (Figure 4B). The SCI group's body weight increased faster than the other groups, with decreasing weight observed in the AM1241 group compared to Exo + AM1241 (Figure 4C).

Figure 4.

Exo + AM1241 Treatment Enhances Locomotor Improvement in SCI Mouse Model. (A) Experimental timeline showing Exo + AM1241 administration and locomotor function assessment post-SCI. (B) BMS scores over 8 weeks post-SCI showed significant locomotor improvement in mice treated with Exo + AM1241 compared to control groups (SCI, Exo alone, and AM1241 alone). (n = 12, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001). (C) Weight of post-SCI mice showed no significant differences among the treatment groups (SCI, Exosome, AM1241 alone, and Exo + AM1241). (D) Improved coordination and movement in the Exo + AM1241-treated mice during stepping tasks post-SCI compared to SCI group. (E) Footprint analysis shows improved gait coordination in mice post-SCI, with blue footprints for upper limbs and red for lower limbs in the Exo + AM1241-treated group. (F) H&E-stained spinal cord sections showed preserved tissue and reduced lesions in the Exo + AM1241 group. (G-H) Immunofluorescence of spinal cord sections shows white arrows indicating neural cells and histogram shows increased neural cells (NeuN = red color) in Exo + AM1241-treated group (n = 3, Scale bar = 50 µm). (I) RT-qPCR analysis result of NeuN expression level in Spinal cord sections of SCI / treated Exo + AM1241 mice. (J-K) Western blot analysis result of NeuN protein expression in SCI mice model. (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001)

Gait analysis after eight weeks showed that the SCI group had lower limb instability, marked by red coloration, and reduced forepaw-hind paw coordination. Mice treated with Exo + AM1241 demonstrated significantly faster gait recovery and improved motor coordination compared to the AM1241, Exosome, and SCI groups (Figure 4D and 4E). The Exo + AM1241 group also showed improved stability and consistent lower limb steps, while the AM1241 group exhibited reduced lower limb movement and limb dragging.

Histological examination using hematoxylin and eosin (HE) staining revealed a larger injury site diameter (blue circle) in SCI group. In contrast, Exo + AM1241 treatment reduced injury site diameter compared to the SCI and single treatment groups (Figure 4F). To further explore the neuroprotective potential of Exo + AM1241, we conducted immunostaining analyses. The SCI group showed reduced NeuN expression compared to the Sham group. Conversely, Exo + AM1241 treatment significantly increased neuron numbers, indicating a neuroprotective effect, with higher neuron survival rates than exosomes alone (Figure 4G and 4H). To validate the immunostaining results for neural survival, our TR-qPCR and western blot analyses demonstrated an increase in neural protein levels in the Exo + AM1241 treatment group compared to the SCI group. However, the single treatment with AM1241 did not result in significant changes in NeuN protein levels compared to the SCI and Exo + AM1241 groups (Figure 4I and 4K).

3.5. Early Exo + AM1241 treatment reduces chronic inflammation in SCI models

In SCI, inflammation hinders recovery by activating proinflammatory cytokines and glial cells, making modulation crucial for neural repair. Our immunohistochemistry analysis showed a significant increase in intensity of activated astrocytes (GFAP = red) and microglial cells (Iba-1 = green) in the SCI group compared to Sham (Figure 5A). Exo + AM1241 treatment remarkably reduced activated astrocytes and microglial cells by 53% and 75%, respectively, compared to SCI. Single AM1241 treatment had more Iba-1 and GFAP-positive cells than Exo + AM1241 (Figure 5B and 5C). Gene expression of GFAP and Iba-1 was higher in the SCI group than in Sham, with Exo + AM1241 significantly decreasing their levels. AM1241 alone and Exosomes alone showed the least reduction compared to Exo + AM1241 (Figure 5D and 5E).

Figure 5.

Exo + AM1241 Treatment decreased Inflammation at the injured site in SCI mice model. (A) Spinal cord sections of SCI / treated AM1241, Exosome, and Exo + AM1241 mice were immunostained for activated-astrocytes (GFAP = red) and microglial cells (Iba1 = green) expression. (B-C) Quantify the intensity of immunostained images of GFAP (red) and Iba1(green) (n = 3, Scale bar = 50 µm). (D-E) RT-qPCR analysis of GFAP and Iba1 gene expression normalized to GAPDH (n = 3). (F) RT-qPCR analysis result of IL-1β, IL-6, and TNF-α expression level spinal cord tissue in SCI / treated AM1241, Exosome, and Exo + AM1241 mice (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001) (G) ELISA assay analysis of IL-1β, IL-6, and TNF-α in SCI / treated Exo + AM1241 mice spinal cord tissue (n = 6, mean ± SD p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001)

Exo + AM1241 treatment effectively reduced inflammation and modulated gene expression in SCI, as shown by ELISA (Figure 5F) and mRNA analysis (Figure 5G). The SCI group exhibited increased IL-6, TNF-α, and IL-1β secretion compared to the Sham group. Exo + AM1241 inhibited proinflammatory cytokines, unlike AM1241 alone. The Exosome group had the least inflammatory effects. Exo + AM1241 shows promise for reducing inflammation and supporting neural recovery in SCI.

3.6. Exo + AM1241 Inhibits ROS and attenuates apoptosis through Nrf-2/HO-1 pathway activated after SCI

SCI group exhibited a significant increase in oxidative stress, characterized by elevated levels of MDA (malondialdehyde), decreased SOD (superoxide dismutase), and GSH (glutathione). However, Exo + AM1241 administration substantially attenuated oxidative stress markers compared to the untreated SCI group. Exo + AM1241 treatment led to a marked upregulation in SOD, and GSH levels, indicating a robust anti-oxidative effect. Additionally, MDA levels were restored to a more balanced state, highlighting the efficacy of Exo + AM1241 in mitigating oxidative stress associated with SCI. In contrast, treatment with AM1241 alone did not significantly impact oxidative stress, underscoring the specific benefits achieved through Exo + AM1241 administration (Figure 6A).

Figure 6.

Exo + AM1241 treatment reduces oxidative stress via Nrf2/HO-1 signaling pathways and inhabits neural death in SCI mice model. (A) Histogram analysis shows anti-oxidants marker GSH activity, malondialdehyde (MDA) contents, and anti-oxidants marker SOD activity in spinal cord tissue (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001). (B) TUNEL (green) and DAPI (blue) staining show apoptosis in spinal tissue of the SCI model, with white arrows indicating apoptotic cells. The histogram indicates that Exo + AM1241 treatment significantly reduced neural apoptosis (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001). (C) Immunoblotting of spinal cord tissue showed the relative expression of apoptosis-related proteins normalized to GAPDH. Exo + AM1241 treatment increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bax and cleaved caspase-3. (n = 3, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001). (D) Immunoblotting analysis shows Nrf2 and HO-1 in spinal cord tissue SCI mice model. The histogram shows the relative expression levels of Nrf2 and HO-1 and normalized to GAPDH from three independent experiments. (n = 3, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001)

Neural death following SCI causes severe damage and impedes neural tissue recovery. Our study revealed a significant increase in apoptotic-positive cells in the SCI group compared to the sham group. Exo + AM1241 treatment significantly reduced apoptotic cells compared to the SCI and single-treatment groups (AM1241, Exosome) (Figure 6B). Western blotting results showed that Exo + AM1241 treatment reversed the elevated Bax and caspase-3 proteins and restored Bcl-2 protein levels compared to the SCI group (Figure 6C). The single AM1241 treatment showed no significant effects. These findings indicate that Exo + AM1241 can alleviate spinal cord neural injury and help restore motor functions.

Nuclear factor erythroid 2 (Nrf2) related factor 2, a key transcription factor in the cellular response to oxidative stress, is a potential protective mechanism in spinal cord injury through its pathway. In our study, we investigated whether Exo + AM1241 treatment could attenuate oxidative stress and facilitate the recovery of neural cells by activating the Nrf-2/HO-1 signaling pathway. Western blot analysis results depicted in the figure revealed a decrease in Nrf2 and HO-1 levels in the SCI group compared to the sham mice model, indicative of oxidative stress in response to injury (Figure 6D). However, following Exo + AM1241 treatment, there was a significant upregulation of Nrf2 and HO-1 levels, suggesting a potential therapeutic effect in mitigating oxidative stress and promoting neural cell recovery.

Additionally, HE analysis for vital organs (liver, lung, brain, kidney, and heart) showed no histopathological changes in all treatment groups, demonstrating no side effects of Exo + AM1241 inoculation (Supplementary Fig. S1).

4. Discussion

The present study explores the potential of exosomes isolated from hUC-MSC cells loaded with the cannabinoid receptor type 2 (CB2) agonist AM1241 (Exo + AM1241) in enhancing the differentiation, proliferation, and neuroprotective functions of neural stem cells (NSCs) in vitro and promoting functional recovery following spinal cord injury (SCI) in vivo. Our findings indicate that Exo + AM1241 exhibits significant neuroprotective and anti-inflammatory properties, underscoring its therapeutic potential for treating traumatic SCI.

Exosomes are characterized by their bilayer, cup-shaped morphology, typically range in size from 30 to 150 nm and exhibit a negative zeta potential. They are also positively identified by specific protein markers such as CD9, CD63, CD81, and TSG101 [32,33]. In this study, exosomes were successfully isolated from human umbilical cord mesenchymal stem cells (hUC-MSCs). The extracted exosomes displayed the expected size and morphology, with an average diameter of approximately 125.4 nm, and expressed key exosomes markers, including CD9, TSG101, and Alix. The measured zeta potential was −12.48 mV, consistent with the typical negative surface charge observed in exosomes. The zeta potential of nanoparticles (NPs) may also act as a parameter of their colloidal stability, where values below −10 mV suggest a high level of stability [27]. Importantly, the encapsulation of AM1241 into these exosomes did not result in any significant changes to their morphology or size, indicating that the drug loading process did not compromise the structural integrity or essential characteristics of the exosomes. This finding supports the notion that hUC-MSC-derived exosomes serve as highly efficient and stable carriers for AM1241, maintaining their intrinsic properties while potentially enhancing the therapeutic efficacy of the encapsulated compound. This structural stability, along with their favorable surface charge, highlights the suitability of hUC-MSC-derived exosomes as efficient carriers for AM1241, making them a robust delivery system for targeted therapeutic applications.

Several studies have investigated the effects of bFGF, EGF and NGF enhance neural survival in vitro, [34] promote neuron proliferation in vivo, [35] and support progenitor cell growth, [36] while other researchers have explored effect of various other compounds’ on NSC differentiation and proliferation [37]. Present study demonstrated that combination of Exo + AM1241 significantly enhances the differentiation of NSCs into neuronal cells, as indicated by the increased expression of neuronal marker NeuN and reduced expression of astrocytic marker GFAP. This suggests that Exo + AM1241 promotes neuronal lineage commitment over glial differentiation, a process that is advantageous for neural repair and functional recovery. Furthermore, Exo + AM1241 markedly improved NSC proliferation independent of exogenous growth factors like EGF and bFGF highlighting its potential to enhance neurogenesis without the need for external growth factors. This is a crucial finding, as it highlights the potential of Exo + AM1241 to support NSC expansion in conditions where growth factors are limited or absent, thereby simplifying the therapeutic application and reducing the risk of potential side effects associated with growth factor supplementation.

Some studies have pointed out that the imbalance between ROS and antioxidant functions in the body plays a crucial role in CNS functioning. Excessive oxidative stress possesses neurotoxic effects and is a critical factor contributing to secondary damage in SCI [38,39]. Our results showed that Exo + AM1241 significantly attenuates oxidative stress in neuronal cells exposed to lipopolysaccharide (LPS). Exo + AM1241 treatment led to a marked reduction in intracellular malondialdehyde (MDA) levels, (an indicator of lipid peroxidation), while enhancing the activity of antioxidant markers such as glutathione (GSH) and superoxide dismutase (SOD) in LPS-stimulated neuronal cells. Consistently our animal study showed that Exo + AM1241 significantly mitigates oxidative stress markers, including MDA, and enhances the levels of antioxidants such as SOD and GSH. These findings highlight the potent antioxidative properties of Exo + AM1241. Traumatic SCI has been shown to trigger a complex neuroinflammatory response, involving microglial activation and the increased expression of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 [40]. Numerous studies suggested that SCI is associated with inflammatory response and highlighted the importance of controlling the overexpression and proinflammatory factors in managing secondary injuries after SCI [41–47]. In our study Exo + AM1241 significantly lowered the levels of pro-inflammatory cytokines in LPS-induced neural cells, including IL-1β, TNF-α, and IL-6, demonstrating its robust anti-inflammatory effects. aligned with these results our animal experiments demonstrate decreased expression of pro-inflammatory cytokines in the Exo + AM1241-treated group. Additionally, immunostaining results demonstrated reduced activated microglial cells and astrocytes (Iba-1 and GFAP). The combined antioxidative and anti-inflammatory actions of Exo + AM1241 are crucial for minimizing secondary injury and creating a more favorable microenvironment for neural repair and regeneration.

Apoptosis of neural cells significantly contributes to functional deficits following SCI [48,49] and is tightly regulated by genes such as Bcl-2 and caspases [50]. Following SCI, apoptosis can persist for an extended period after injury, leading to significant neuronal loss [51]. The delicate balance between Bcl-2, which promotes cell survival, and Bax, which induces apoptosis, is pivotal in determining cell fate. Activation of Bax triggers the release of cytochrome c from the mitochondria, which subsequently activates Apaf-1 and caspase-9, initiating a downstream apoptotic cascade involving caspase-3 and caspase-7, ultimately leading to programed cell death [52]. Our findings indicate that Exo + AM1241 exerts a protective effect against glutamate-induced apoptosis in neural stem cells (NSCs), as demonstrated by the reduced expression of pro-apoptotic proteins Bax and Caspase-3, along with an increase in the anti-apoptotic protein Bcl-2. Additionally, TUNEL staining and western blot analysis of SCI tissue revealed a marked reduction in apoptotic cells and the expression of pro-apoptotic proteins (Bax and caspase-3) along with an increase in the anti-apoptotic protein Bcl-2 in the Exo + AM1241 treated group. This indicates that Exo + AM1241 promotes cell survival by modulating apoptotic pathways and contributes to the preservation of neural tissue and functional recovery in SCI.

We comprehensively assessed the neuroprotective efficacy of exosomes loaded with the agonist AM1241 in vivo. Our study revealed that Exo + AM1241 treatment significantly improved locomotor function and motor coordination compared to control and single-treatment groups. Behavioral assessments, such as the BMS scores and gait analysis, revealed that Exo + AM1241 consistently outperformed other treatments in restoring motor functions over the study period. These findings were further supported by histological analyses that show reduced injury site diameter and increased neuronal survival in the Exo + AM1241 group. The immunostaining results confirmed enhanced neural repair, as indicated by the increased number of neural cells. consistent with result of increased mRNA and protein of neural cells in Exo + AM1241 treatment group compared to control group.

The beneficial effects of Exo + AM1241 appear to be mediated through multiple mechanisms, including enhanced neuronal differentiation and survival, reduction of inflammation and oxidative stress, and inhibition of apoptosis. Our investigation into the underlying mechanisms revealed that Exo + AM1241 activates the Nrf2/HO-1 signaling pathway, which plays a critical role in cellular defense against oxidative stress. Previous studies have shown that this pathway exerts beneficial effects through the protection against oxidative injury, regulation of apoptosis, modulation of inflammation as well as contribution to angiogenesis [53,54]. Moreover, the disturbances in this pathway are associated with the pathogenesis of numerous disorders, including neurodegenerative diseases and stimulation of this pathway plays a neuroprotective role [55]. The upregulation of Nrf2 and HO-1 in the Exo + AM1241-treated group suggests that this pathway may mediate the observed antioxidative and neuroprotective effects, promoting neural cell recovery and resilience to injury-induced oxidative damage shown in Figure 7.

Figure 7.

Graphical Scheme. 1. Scheme of Exosome-Loaded AM1241 2. A diagram illustrating, Exosome-Loaded AM1241 enhances cognitive function in SCI mice by reducing inflammation, oxidative stress, and neural apoptosis while promoting neuronal regeneration, primarily through the activation of the Nrf2/Ho-1 signaling pathway.

Although our results demonstrated the potent in vitro and in vivo therapeutic potential of Exo + AM1241 against SCI, future studies should focus on optimizing the dosage, and administration protocols and exploring the long-term efficacy and safety of Exo + AM1241 in clinical settings. Additionally, addressing the variability in individual responses to the treatment to ensure broader applicability, and the scalability of this treatment for widespread clinical use needs further evaluation.

5. Conclusion

In summary, our study demonstrates that Exo + AM1241 enhances NSC differentiation, proliferation, and survival while mitigating oxidative stress and inflammation. These effects contribute to improved functional recovery following SCI, positioning Exo + AM1241 as a promising therapeutic strategy for neurodegenerative diseases and neural injuries.

Glossary

Abbreviation: SCI: Spinal cord injury; hUC-MSCs: Human umbilical cord mesenchymal stem cells; NSCs: Neural stem cells; CB2: Cannabinoid receptor type 2; N NPs: Natural nanoparticles; INPs: Inorganic nanoparticles; PCs: Neural progenitor cells; EGF: Epidermal growth factor; bFGF: Basic fibroblast growth factor; GFAP: Glial fibrillary acidic protein; NeuN: Neuronal nuclei; GSH: Glutathione; SOD: Superoxide dismutase; MDA: Malondialdehyde; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; LPS: Lipopolysaccharides; IL-1β: Interleukin-1 beta; TNF-α: Tumor necrosis factor-alpha; IL-6: Interleukin-6; BMS: Basso Mouse Scale; Iba-1: Ionized calcium-binding adaptor molecule 1; Nrf2: Nuclear erythroid 2 related factor 2; HO-1: Heme oxygenase-1

Funding Statement

Present research funded by the Postdoctoral Research start-up fund of Lishui Peoples Hospital funding no. 2024bsh001 and the municipal public welfare self-financing technology application research project of Lishui.no. 2022SJZC074&2022SJZC079.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

II Shaikh, R Bhandari, LM Cheng, X Jian: Conceptualization. II Shaikh, R Bhandari, Shekhar S, Xu Z: Animal experiments, Data collection and analysis, Drawing figures, Writing- Original draft preparation. KA Shahzad, C Shao, LM Cheng, X Jian: Supervision, Writing- Reviewing and Editing.

Data availability statement

The data supporting this study's findings are available from the corresponding authors, Shaikh, LM Cheng, and X Jian, upon reasonable request.