Inhibition of Ferroptosis by Mesenchymal Stem Cell-Derived Exosomes in Acute Spinal Cord Injury: Role of Nrf2/GCH1/BH4 Axis
Article information
Abstract
Objective
The therapeutic benefits of exosomes obtained from mesenchymal stem cells (MSCs) in acute spinal cord injury (SCI) have been demonstrated in recent years, but the precise mechanisms remain unknown. In this study, the efficacy and mechanisms of MSC-derived exosomes (MSC-Exo) in acute SCI were investigated.
Methods
By utilizing a BV2 ferroptosis cellular model and an SCI rat model, we investigated the effects of MSC-Exo on iron death related indicators and NF-E2 related factor 2 (Nrf2)/GTP cyclolase I (GCH1)/5,6,7,8-tetrahydrobiopterin (BH4) signaling axis, as well as their therapeutic effects on SCI rats.
Results
The results revealed that MSC-Exo effectively inhibited the production of ferrous iron, lipid peroxidation products malonaldehyde and reactive oxygen species, and ferroptosis-promoting factor prostaglandin-endoperoxide synthase 2. Concurrently, they upregulated ferroptosis suppressors FTH-1 (ferritin heavy chain 1), SLC7A11 (solute carrier family 7 member 11), FSP1 (ferroptosis suppressor protein 1), and GPX4 (glutathione peroxidase 4), contributing to enhanced neurological recovery in SCI rats. Further analysis showed the Nrf2/GTP/BH4 signaling pathway’s critical role in suppressing ferroptosis. Additionally, MSC-Exo was found to inhibit lipopolysaccharide-induced ferroptosis in BV2 cells and SCI rats by activating the Nrf2/GCH1/BH4 axis.
Conclusion
In summary, the study demonstrates that MSC-Exo mitigates microglial cell ferroptosis via the Nrf2/GCH1/BH4 axis, showing potential for preserving and restoring neurological function post-SCI.
INTRODUCTION
Spinal cord injury (SCI), characterized by high mortality and disability rates, commonly results in limb dysfunction, restricted mobility, and can be fatal. Traumatic events are the predominant cause of SCI, while non-traumatic instances arise from ailments like infections or tumors [1]. Acute SCI leads to the demise of neurons and glial cells, accompanied by oxidative stress, inflammation, and ischemia [2-4]. These conditions contribute to sensory loss, motor function impairment, and severe outcomes like paraplegia, tetraplegia, or even early mortality [5], with the extent of functional impairment varying based on the lesion’s severity and location. Despite notable progress in SCI management [6,7], it remains an incurable neurological disorder, necessitating lifelong patient care and treatment. Understanding the intricate regulatory mechanisms of SCI is crucial for developing innovative therapies to combat this debilitating condition.
Ferroptosis, a recently identified type of nonapoptotic cell demise, is distinguished by iron-dependent and accumulation of intracellular reactive oxygen species, and it had been proven to be implicated in various diseases such as ischemia-reperfusion injury, cancers, neurodegenerative disorders and SCI [8,9]. Ferroptosis has been found to be a major cause of secondary injury of SCI, and deferoxamine facilitates traumatic SCI recovery through suppression of ferroptosis [10]. Intraperitoneal administration of SRS16-86, a ferroptosis inhibitor, significantly ameliorates astrogliosis and promotes recovery of SCI in rats [11]. Building upon our prior investigations, we have established the involvement of ferroptosis in SCI. Notably, administration of sodium selenite has been observed to foster neurological recuperation in SCI-affected rat models, principally through the suppression of ferroptosis [12]. Despite these advancements, the comprehensive understanding of ferroptosis’s functionalities and regulatory pathways in SCI context remains an area of ongoing exploration.
In the realm of SCI therapeutics, mesenchymal stem cells (MSCs) emerge as a potent candidate. Their efficacy is attributed to their capacity to alleviate secondary injuries. This is achieved through a multi-pronged approach involving the suppression of inflammatory responses, secretion of paracrine factors, and their potential to differentiate into neural cell lineages, presenting a multifaceted therapeutic mechanism in SCI treatment [13,14].
Recent investigations have shed light on the therapeutic potential of MSC-derived exosomes (MSC-Exo) in addressing acute central nervous system injuries [15]. Focusing on SCI, studies have indicated that MSC-derived extracellular vesicles contribute to spinal cord functional recuperation. This is achieved by enhancing angiogenesis and axonal regeneration, diminishing cellular apoptosis, and attenuating inflammation and immune reactions [16]. Despite these advancements, the specific role of ferroptosis in these processes remains an under-explored domain.
Divalent metal transporter 1 (DMT1) mediates the translocation of free iron from the endosome to the cytoplasm, thereby creating an “unstable iron pool” that predisposes cells to ferroptosis. Recent research indicated that exosomes derived from human umbilical cord blood MSCs inhibited ferroptosis by downregulating DMT1, thereby attenuating myocardial injury [17]. Motivated by these findings, we hypothesize that the modulation of ferroptosis by MSC-Exo could be a critical factor in SCI recovery. This suggests a potential avenue for therapeutic intervention in SCI treatment.
Ferroptosis, fundamentally driven by iron-dependent lipid peroxidation [18], implicates oxidative stress regulatory pathways as pivotal players. Within this context, the GTP cyclolase I (GCH1)/tetrahydrobiopterin (BH4) signaling axis, linked to oxidative stress, emerges as notable [19]. BH4, essential for nitric oxide synthases, when deficient, leads to superoxide generation [20]. GCH1, as the rate-limiting enzyme in BH4 synthesis, governs the GTP to BH4 conversion, with its suppression amplifying oxidative stress in vivo [21]. Intriguingly, recent discoveries position GCH1/BH4 as an independent ferroptosis regulatory system, separate from the GSH/glutathione peroxidase 4 (GPX4) axis [22]. Under the oxidative stress induced by the accumulation of reactive oxygen species (ROS), overexpression of NF-E2 related factor 2 (Nrf2) transcriptionally activated the GCH1/BH4 pathway. This upregulation of GCH1 expression facilitated the restoration of intracellular BH4 levels, effectively mitigating ROS generation triggered by radiation exposure and thereby ameliorating oxidative stress. Consequently, the activation of Nrf2 leads to an antioxidant defense mechanism via the GCH1/BH4 axis [23]. Enhanced Nrf2 expression correlates with inflammatory response moderation and ferroptosis resistance post-RSL3 stimulation in microglia and macrophages [24]. Additionally, emerging evidence indicates that the overexpression of heme oxygenase 1 (HO-1) increases intracellular iron, disrupting redox balance and leading to ferroptosis, while Nrf2 mitigates oxidative stress and ferroptosis in SCI through the downregulation of HO-1 [25,26]. However, the exact role of the Nrf2/GCH1/BH4 pathway in ferroptosis, and its potential interplay with MSC-derived exosomes, remains an area ripe for investigation.
This study is designed to delve into the role of MSC-derived exosomes in ferroptosis within the context of SCI, specifically probing the mechanisms of the Nrf2/GCH1/BH4 signaling pathway.
MATERIALS AND METHODS
1. Cell Culture, Transfection, and Treatment
The isolation and cultivation of bone marrow MSCs were conducted following the methodologies outlined in earlier research [27]. In brief, C57BL/6 mice (male, 4-week-old) were euthanized, and the hind limbs were dissected and placed on ice in DMEM (ThermoFisher Scientific, Waltham, MA, USA). Connective and muscle tissues were meticulously excised from the femur and tibia. Subsequently, bone marrow cells were extracted from these bones using a 27-gauge needle affixed to a 10-mL syringe, ensuring a thorough and efficient retrieval process. Bone marrow cells, at a concentration of 2.5× 107 cells/mL, were subjected to a cultivation process in 10-cm dishes at a temperature of 37°C for a duration of 3 hours. During this period, nonadherent cells were systematically removed through medium replacement. The culture was maintained for an additional 72 hours, with the medium being refreshed at 8-hour intervals. The adherent cells underwent washing and received fresh culture medium every 3 days. Following a 2-week period, these cells were washed and treated with 0.25% trypsin/1 mM ethylenediaminetetraacetic acid for 2 minutes. The dissociated cells were then harvested and transferred to a 25 cm2 flask for further culture. After 2–3 passages, the MSCs harvested exhibited typical spindle shape morphology with a diameter of 25–30 μm (Supplementary Fig. 1A). The features of MSCs surface markers include positive CD44 and negative CD11b (Supplementary Fig. 1B). After induction, the cells differentiated into adipocytes, osteoblasts, and cartilage (Supplementary Fig. 1C–E).
This study was conducted with the approval of the Experimental Animal Ethics Committee of Xiangya Hospital Central South University and approved (approval number: 202109058), adhering to all relevant ethical guidelines.
As the primary supportive cells for neurons, microglia play a crucial role in the nutrition and regeneration of spinal cord neuronal cells. Microglia rapidly adapt to changes in the microenvironment following SCI, thereby also playing a vital role in secondary damage and subsequent recovery of SCI. Therefore, we chose the most common microglial cell, the BV2 cells, for the cellular experiments. The advantages of BV2 cells include characteristics of primary microglia, immortality, and the ability for sustained growth, while limitations include their derivation from mouse microglia, which may not fully replicate the features and complexity of spinal cord microglia in vivo. Previous studies have shown that lipopolysaccharide (LPS) induced ferroptosis in various tissue and cell models, as well as BV2 cells. In addition to ferroptosis, LPS also significantly induce cellular inflammatory responses and other forms of cell death, which is similar to the pathological mechanisms of SCI. Therefore, we chose LPS-induced ferroptosis in BV2 cells for the mechanism study related to SCI.
In this study, BV2 cells, sourced from the Cell Bank of the Chinese Academy of Science in Shanghai, China, were subjected to a 24-hour treatment with LPS at a concentration of 100 ng/mL. For Nrf2 knockdown, Nrf2 siRNA (si-Nrf2) was obtained from RiboBio (Guangzhou, Guangdong, China), and BV2 cells were transfected with si-Nrf2 using Lipofectamine RNAiMAX (ThermoFisher Scientific). After 72 hours, cells were collected for subsequent assays. In some assays, BV2 cells were cocultured with MSC-derived exosomes at 1 µg/mL, tert-butylhydroquinone (TBHQ, Selleck Chemicals, Houston, TX, USA) at 25 µM and FIN56 (Selleck Chemicals, Houston, TX, USA) at 5 µM, respectively.
2. Exosome Isolation and Characterization
MSCs were cultured in a serum-depleted environment, following which exosomes were isolated utilizing the Total Exosome Isolation reagent from ThermoFisher Scientific. The characterization of these exosomes was meticulously performed using nanoparticle tracking analysis (NTA) conducted by Malvern Instruments (Westborough, MA, USA) and transmission electron microscopy (TEM) provided by ThermoFisher Scientific. To further validate the exosome identity, the presence of markers such as CD63, CD81, and TSG101 was confirmed through Western blot analysis.
3. Transmission Electron Microscopy
The examination of exosomes via TEM was carried out in accordance with previous study [28]. In brief, exosomes were concentrated by centrifuging at 100,000 g for a period of 90 minutes. After the removal of the supernatant, the exosome pellets were fixed using a 2.5% glutaraldehyde solution, maintained at 4°C for an hour. Subsequently, the glutaraldehyde solution was discarded, and the pellets were subjected to a tripartite wash using a 0.1-M sodium cacodylate solution. Pellets were post-fixed in 2% Osmium tetroxide at 4°C for 1 hour and washed 3 times in 0.1-M sodium cacodylate solution. A series of gradient acetone solution (50%, 60%, 70%, 80%, 90%, 95%, and 100%) were prepared, and pellets were incubated in each acetone solution for 10 minutes. The mixture of acetone and low viscosity (3:1, 1:1, and 1:3) was prepared, and pellets were consecutively incubated in the mixture of acetone and low viscosity (3:1, 1:1, and 1:3) for 30 minutes. Finally, pellets were incubated in pure low viscosity embedding mixture overnight, embed and baked at 65°C for 24 hours. Sections (60 nm) were sliced and stained with 2% uranyl acetate for 20 minutes and lead citrate for 10 minutes followed by observation under TEM.
4. Immunofluorescence Staining
BV2 cells underwent fixation in a 4% paraformaldehyde solution and were permeabilized using 0.2% Triton X-100. Following washing and blocking, the cells were incubated overnight with rabbit anti-GPX4 antibodies (2 μg/mL) and anti-FSP1 antibodies (1 μg/mL), both sourced from Abcam (Cambridge, UK). The cells were then thoroughly rinsed and treated with an Alexa Fluor 488-conjugated secondary antibody for one hour. Poststaining with 4′,6-diamidino-2-phenylindole (DAPI) provided by Beyotime (Shanghai, China), the cells were prepared for imaging. Observations were made using a confocal microscope from Nikon (Tokyo, Japan). The results of immunofluorescence staining were analyze using Image J software (National Institutes of Health, Bethesda, MD, USA) and semiquantitative analysis was performed based on the average optical density.
5. Methylthiazolyldiphenyl-Tetrazolium Bromide Assay
In the prescribed treatment protocol for BV2 cells, the culture medium was initially discarded. Thereafter, a mixture of 100 µL of fresh medium and 10 µL of methylthiazolyldiphenyl-tetrazolium bromide (MTT) reagent was added to each culture. The cells were then incubated for a duration of 4 hours. Following this incubation period, dimethyl sulfoxide was introduced into the cultures. The absorbance at 540 nm was subsequently measured to assess cell viability. The MTT reagent used in this procedure was procured from Sigma (St. Louis, MO, USA).
6. Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from BV2 cells employing the Trizol reagent, sourced from Beyotime. This RNA was then subjected to reverse transcription into cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN, Germantown, MD, USA). Ferritin heavy chain 1 (FTH1), prostaglandin-endoperoxide synthase 2 (PTGS2), solute carrier family 7 member 11 (SLC7A11), Nrf2, and GCH1 were examined by quantitative polymerase chain reaction with SYBR Green (ThermoFisher Scientific) and normalized to GAPDH. Primers were shown in Table 1. The 2−∆∆Ct method was used for calculation.
Quantitative real-time polymerase chain reaction primers
7. Experimental Rat SCI Model
As the most commonly used animal model for SCI currently, Sprague-Dawley (SD) rats offer the benefits of low cost, easy availability, and similarities in electrophysiology and morphology to the human spinal cord. Therefore, this study selected SD rats for the animal experiments. However, considering the complexity of human SCI, the SD rat SCI model cannot fully simulate the pathological process of human SCI. SD rats (male, 8-week-old) were purchased from SJA Laboratory Animal (Changsha, Hunan, China) and blindly divided into control, SCI, SCI+ normal saline and SCI+MSC-Exo groups. SCI rats were anesthetized, and the spinal cord was processed for laminectomy at the T9/10 position. Then, a contusion injury was performed via dropping a mass of 10 g at a vertical height of 40 mm on the spinal cord. Control rats received same procedures without the contusion injury. Rats in SCI+MSC-Exo were intrathecally injected with MSC-Exo (100 µg). After 14 days, spinal cord tissues (T10) were collected for pathological examination, Western blotting and oxidative stress analysis. In addition, we conducted Basso, Beattie, and Bresnahan (BBB) score for all the rats at 1, 3, 7, 14 days after SCI. All experimental procedures involving rats received approval from the Animal Care and Use Committee at Xiangya Hospital, Central South University, ensuring compliance with ethical standards.
8. Western Blotting
Protein (20 µg) extracted from BV2 cells and spinal cord tissues was electrophoresed and transferred to PVDF membranes from Bio-Rad (Hercules, CA, USA). Subsequently, membranes were incubated with rabbit antibodies against CD63 (0.5 μg/mL, Abcam), CD81 (0.1 μg/mL, Abcam), TSG101 (0.02 μg/mL, Abcam), GPX4 (2 μg/mL, Abcam), and FSP1 (1 μg/mL, Abcam) overnight. Membranes were rinsed and incubated with an horseradish peroxidase-conjugated secondary antibody (ThermoFisher Scientific). Enhanced chemiluminescence substrate (Abcam) was added to visualize bands. The grayscale values of each band were analyze using Image J software, then the relative quantitative analysis was performed by calculating the ratio of each group to the internal reference.
9. Enzyme-Linked Immunosorbent Assay
BH4 were determined with enzyme-linked immunosorbent assay (ELISA) kits following manuals. Tetrahydrobiopterin (BH4) ELISA kit was bought from EIAab (Wuhan, Hubei, China).
10. Iron Assay
To determine Fe2+ level, BV2 cells were lysed on ice, and supernatants were harvested after centrifugation. The level of Fe2+ in BV2 cells was examined with the Iron Assay Kit (Sigma) following the manual.
11. Oxidative Stress Examination
Superoxide dismutase (SOD) and malonaldehyde (MDA) were examined with SOD (Sigma) and MDA (BioVision, Milpitas, CA, USA) assay kits, respectively. For ROS staining, cells and spinal cord tissues were stained with CellRox green (ThermoFisher Scientific) at 1 µM for 30 minutes, washed and stained with DAPI (Beyotime).
12. Hematoxylin and Eosin Staining
The spinal cord samples were embedded in paraffin and cut into 5-μm thick sections. The sections were stained with hematoxylin-eosin reagents (Servicebio, Wuhan, China), and the hematoxylin and eosin (H&E) staining process included 5 steps: dewaxing, staining, dehydration, transparency, and sealing. When finished, the number of neural cells, arrangement regularity, and cavity sizes were observed using an optical microscope (Eclipse E100; Nikon).
13. Statistical Analysis
The data presented herein are expressed as the mean±standard deviation. For comparing 2 groups, the Student t-test was utilized. In cases involving multiple groups, the variance was analyzed using 1-way analysis of variance. A p-value of less than 0.05 was considered to denote statistical significance.
RESULTS
1. LPS-Induced Ferroptosis in BV2 Cells
Upon treatment with LPS, BV2 cells demonstrated notable alterations in cell morphology, indicative of damage, along with a discernible reduction in cell count (Fig. 1A). Moreover, LPS obviously impaired BV2 cell proliferation (Fig. 1B). To demonstrate that the form of cell death induced by LPS was ferroptosis [29,30], we examined ferroptosis-related factors GPX4, PTGS2, FTH1, and SLC7A11. LPS-treated BV2 cells showed significantly increased expression of PTGS2 and decreased expression of GPX4, FTH1, and SLC7A11 (Fig. 1C). In addition, LPS-treated BV2 cells showed markedly elevated Fe2+, ROS and MDA (Fig. 1D–F). These observations implied that LPS could induce ferroptosis in BV2 cells.
Lipopolysaccharide (LPS) induced ferroptosis in BV2 cells. BV2 cells were treated with LPS at 100 ng/mL for 24 hours. (A) Morphological observation of BV2 cells (scale bar, 100 μm). (B) Cell proliferation was evaluated with methylthiazolyldiphenyl- tetrazolium bromide (n=3). (C) Quantitative real-time polymerase chain reaction analysis of GPX4, PTGS2, FTH1, and SLC7A11 (n=3). (D) The relative Fe2+ level (n=3). (E) Level of reactive oxygen species (ROS) (n=3). (F) Level of malonaldehyde (MDA) (n=3). GPX4, glutathione peroxidase 4; PTGS2, prostaglandin-endoperoxide synthase 2; FTH1, ferritin heavy chain 1; SLC7A11, solute carrier family 7 member 11. *p<0.05. **p<0.01.
2. Characterization of MSC-Derived Exosomes
Bone marrow MSCs were isolated from the femurs and tibias and cultured for exosome isolation. The morphology of passage 1, 2, and 3 MSCs was examined, and the classical spindle-shaped morphology was observed (Fig. 2A). Subsequently, exosomes were isolated from bone marrow MSCs followed by exosome characterization. NTA and TEM validated that MSC-derived exosomes (MSC-Exo) showed typical round or round-like vesicle structure with a diameter of 40 to 200 nm (Fig. 2B and C). Moreover, high expression of exosome markers CD63, CD81, and TSG101 was observed in MSC-derived exosomes, but not in MSCs (Fig. 2D). These results confirmed typical exosome characterization of MSC-derived exosomes.
Characterization of mesenchymal stem cell (MSC)-derived exosomes. (A) Primary MSCs were isolated and cultured. The morphology of passage 1 (P1), 2 (P2), and 3 (P3) MSCs was examined and imaged. (B) Nanoparticle tracking analysis of exosome. (C) Transmission electron microscopy examination. (D) Protein levels of CD63, CD81, and TSG101 in MSC-derived exosomes and MSCs (n=3).
3. MSC-Exo Suppressed Ferroptosis in LPS-Treated BV2 Cells
To investigate the activity of MSC-Exo in ferroptosis, BV2 cells were treated with LPS, LPS+MSC-Exo, or LPS+exosomedepleted supernatant from MSCs. Control cells were treated with saline.
BV2 cells were treated with LPS at 0, 25, 50, or 100 ng/mL for 48 hours, and cell proliferation was analyzed with MTT (n= 3). LPS-induced BV2 cells were cocultured with MSC-Exo at 0.5, 1, or 1.5 μg/mL for 48 hours, and cell proliferation was analyzed with MTT. As shown in Fig. 3A and B, BV2 cell proliferation was inhibited by LPS but promoted by MSC-Exo in a dose-dependent manner. Therefore, we chose the concentration of 100 ng/mL and 1.5 μg/mL for subsequent experiments.
Mesenchymal stem cell-derived exosomes (MSC-Exo) suppressed ferroptosis in BV2 cells. (A) BV2 cells were treated with lipopolysaccharide (LPS) at 0, 25, 50, or 100 ng/mL for 48 hours. Cell proliferation was analyzed with methylthiazolyldiphenyltetrazolium bromide (MTT) (n=3). (B) LPS-induced BV2 cells were cocultured with MSC-Exo at 0.5, 1, or 1.5 μg/mL for 24 hours. Cell proliferation was analyzed with MTT (n=3). BV2 cells were treated with LPS at 100 ng/mL, LPS+MSC-Exo at 1.5 μg/mL or LPS+exosome-depleted supernatant. (C) Quantitative real-time polymerase chain reaction analysis of GPX4, PTGS2, FTH1, and SLC7A11 (n=3). (D) The relative ratio of reactive oxygen species (ROS)-positive BV2 cells (n=3). (E) Levels of malonaldehyde (MDA) (n=3). (F) The relative Fe2+ level (n=3). (G) Cell proliferation was analyzed with MTT (n=3). GPX4, glutathione peroxidase 4; PTGS2, prostaglandin-endoperoxide synthase 2; FTH1, ferritin heavy chain 1; SLC7A11, solute carrier family 7 member 11. *p<0.05. **p<0.01. ***p<0.001.
LPS downregulated FTH1, SLC7A11, and GPX4, but upregulated PTGS2 (Fig. 3C). MSC-Exo enhanced the expression of FTH1, SLC7A11, and GPX4, and inhibited the expression of PTGS2, while exosome-depleted supernatant showed no effect (Fig. 3C). Furthermore, LPS-induced ROS was attenuated by MSC-Exo in BV2 cells (Fig. 3D), and the elevated level of MDA were reversed by MSC-Exo (Fig. 3E). No significant change was observed in BV2 cells treated with exosome-depleted supernatant (Fig. 3D and E). Furthermore, we found that LPS raised Fe2+ level in BV2 cells, but the effect was reversed by MSC-Exo. LPS-induced Fe2+ upregulation was not affected by exosomedepleted supernatant (Fig. 3F). As shown in Fig. 3G, BV2 cell proliferation was obviously inhibited by LPS but MSC-Exo partially alleviated this inhibition, while no similar change was observed in cells treated with exosome-depleted supernatant.
As GPX4 and FSP1 are key regulators in ferroptosis, we additionally examined their expression by IF staining. Compared to untreated control cells, LPS-treated BV2 cells exhibited reduced expression of GPX4 and FSP1, but simultaneous MSC-Exo treatment increased their expression (Fig. 4).
Mesenchymal stem cell-derived exosomes (MSC-Exo) suppressed ferroptosis in lipopolysaccharide (LPS)-treated BV2 cells. BV2 cells were treated with LPS at 100 ng/mL or LPS in combination with MSC-Exo at 1.5 μg/mL for 24 hours. (A–C) IF staining of GPX4 (red) and FSP1 (green). GPX4, glutathione peroxidase 4; FSP1, ferroptosis suppressor protein 1; DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 25 μm. *p<0.05. **p<0.01.
Collectively, these findings demonstrated that MSC-Exo attenuated LPS-induced ferroptosis in BV2 cells.
4. MSC-Exo Restrained Ferroptosis Through Activation of the Nrf2/GCH1/BH4 Signaling in BV2 Cells
As the Nrf2/GCH1/BH4 signaling plays key roles in the regulation of ferroptosis, we examined the expression of Nrf2 and GCH1 and BH4 generation in BV2 cells. We found that Nrf2 and GCH1 were downregulated and BH4 generation was reduced in LPS-treated BV2 cells when compared with the control group (Fig. 5A and B). TBHQ, an activator of Nrf2, significantly elevated the expression of Nrf2 and GCH1 and BH4 generation (Fig. 5A and B). In contrast, knockdown of Nrf2 further enhanced LPS-mediated downregulation of Nrf2, GCH1, and BH4 (Fig. 5A and B). Moreover, elevated ROS-positive cells were reduced by simultaneous TBHQ treatment but further upregulated by Nrf2 knockdown (Fig. 5C). LPS-mediated upregulation of PTGS2 and downregulation of FTH1, SLC7A11, and GPX4 in BV2 cells were partly abrogated by TBHQ treatment but reinforced by Nrf2 knockdown (Fig. 5D). Our data suggested that the Nrf2/GCH1/BH4 signaling suppressed ferroptosis in BV2 cells.
The Nrf2/GCH1/BH4 signaling suppressed ferroptosis in BV2 cells. BV2 cells were treated with lipopolysaccharide (LPS), LPS in combination with tert-butylhydroquinone (TBHQ) at 25 μM for 24 hours. Nrf2-knockdwon cells were treated with LPS. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Nrf2 and GCH1 (n=3). (B) BH4 generation was examined using enzyme-linked immunosorbent assay (n=3). (C) Reactive oxygen species (ROS) (green) staining in BV2 cells. Scale bar, 200 μm. (D) qRT-PCR analysis of GPX4, PTGS2, FTH1, and SLC7A11 (n=3). Nrf2, NF-E2 related factor 2; GCH1, GTP cyclolase I; BH4, tetrahydrobiopterin; GPX4, glutathione peroxidase 4; PTGS2, prostaglandin-endoperoxide synthase 2; FTH1, ferritin heavy chain 1; SLC7A11, solute carrier family 7 member 11. *p<0.05. **p<0.01. ***p<0.001.
Furthermore, we examined whether MSC-Exo regulated the Nrf2/GCH1/BH4 signaling to modulate ferroptosis. Reduced levels of Nrf2, GCH1, and BH4 in LPS-treated BV2 cells were partially enhanced by MSC-Exo treatment (Fig. 6A and B). In addition, MSC-Exo inhibited LPS-induced ROS, and the inducer of ferroptosis FIN56 markedly promoted ROS in LPS-treated BV2 cells (Fig. 6C). LPS-mediated suppression of GPX4, FSP1, FTH1, and SLC7A11 was reversed by MSC-Exo, and FIN56-treated cells showed lowest expression (Fig. 6D and E). LPS-induced expression of PTGS2 and Fe2+ was partially suppressed by MSC-Exo, and FIN56 treatment dramatically enhanced the expression of PTGS2 and Fe2+ (Fig. 6E). Taken together, our findings indicated that MSC-Exo may suppressed ferroptosis via activating the Nrf2/GCH1/BH4 signaling.
Mesenchymal stem cell-derived exosomes (MSC-Exo) restrained ferroptosis through activation of the Nrf2/GCH1/BH4 signaling. BV2 cells were treated with LPS, LPS+MSC-Exo, or LPS+FIN56 (5 μM) for 24 hours. (A) Quantitative real-time polymerase chain reaction analysis of Nrf2 and GCH1 (n=3). (B) BH4 generation was examined using enzyme-linked immunosorbent assay (n=3). (C) Reactive oxygen species (ROS) (green) staining in BV2 cells. Scale bar, 200 μm. (D) Protein levels of GPX4 and FSP1 (n=3). (E) Expression analysis of Fe2+, PTGS2, FTH1, and SLC7A11 (n=3). Nrf2, NF-E2 related factor 2; GCH1, GTP cyclolase I; BH4, tetrahydrobiopterin; GPX4, glutathione peroxidase 4; FSP1, ferroptosis suppressor protein 1; PTGS2, prostaglandin- endoperoxide synthase 2; FTH1, ferritin heavy chain 1; SLC7A11, solute carrier family 7 member 11. *p<0.05. **p<0.01. ***p<0.001.
5. Administration of MSC-Exo Significantly Improved Neurological Rehabilitation After SCI in Rats
Following the establishment of a rat model of SCI via laminectomy and contusion injury, MSC-Exo were administered intrathecally. A 2-week observation period revealed a significant enhancement in the BBB locomotor rating scale in the hind limbs of the SCI rats, as depicted in Fig. 7A. Immediately following SCI, rats in the SCI, SCI+normal saline, and SCI+MSC-Exo groups exhibited severe hind limb paralysis. Hind limb motor function began to recover at 3 days after SCI and showed a gradual increase during the 14-day experimental period. The BBB scores of the SCI+MSC-Exo group were higher relative to those of the SCI group and SCI+normal saline group at 14 days after SCI. TEM analyses of the injured spinal cord tissues from both SCI and MSC-Exo groups revealed varying degrees of ferroptosis, characterized by phenomena such as hemolysis, mitochondrial shrinkage, and increased membrane density, as illustrated in Fig. 7B. Comparative analysis with control rats indicated pronounced neural cell damage and heightened inflammatory cell infiltration in the SCI group, as observed through H&E staining. However, the administration of MSCExo markedly mitigated these damages and reduced inflammatory cell infiltration (Fig. 7C). Similarly, SCI obviously increased the cavity area of spinal cord tissue, while MSC-Exo administration effectively reduced the cavity area (Fig. 7D). Additionally, the expression levels of GPX4, Nrf2, and GCH1 were found to be downregulated in SCI rats, with an upregulation of PTGS2, which was reversed upon MSC-Exo treatment, as shown in Fig. 7E. Furthermore, elevated levels of ROS, MDA, and Fe2+ in the SCI group were normalized post MSC-Exo treatment (Fig. 7F–H). These findings suggest that MSC-Exo significantly enhance neurological rehabilitation following SCI in rats, potentially through the inhibition of ferroptosis via the Nrf2/GCH1 signaling pathway.
Mesenchymal stem cell-derived exosomes (MSC-Exo) administration improved neurological rehabilitation after spinal cord injury (SCI) in rats. Rats were divided into control, SCI, SCI+normal saline, and SCI+MSC-Exo groups (n=3). (A) Basso, Beattie, and Bresnahan (BBB) scores of the hind limbs of SCI rats. (B) Varying degrees of ferroptosis such as hemolysis, mitochondrial shrinkage, and increased membrane density were observed in the injured spinal cord tissues. Scale bar, 2 μm. (C) Hematoxyling and eosinstaining of spinal cord tissues. Scale bars: upper, 100 μm; lower, 25 μm. (D) Cavity area of spinal cord tissues (n=3). (E) Protein levels of GPX4, PTGS2, Nrf2, and GCH1 in spinal cord tissues (n=3). (F) The reactive oxygen species (ROS)-positive rate in spinal cord tissues (n=3). (G) Levels of malonaldehyde (MDA) in spinal cord tissues (n=3). (H) The relative Fe2+ level in spinal cord tissues (n=3). GPX4, glutathione peroxidase 4; PTGS2, prostaglandin-endoperoxide synthase 2; Nrf2, NF-E2 related factor 2; GCH1, GTP cyclolase I. *p<0.05. **p<0.01. ***p<0.001.
DISCUSSION
SCI detrimentally impacts spinal cord functionality and contributes to a heightened mortality rate among affected individuals [31]. In the realm of SCI management, therapies based on MSCs have emerged as a promising avenue [32]. Recent experimental investigations in SCI have underscored the neuroprotective potential of MSC-Exo, positioning them as an innovative therapeutic strategy [33]. Notably, exosomes derived from bone marrow MSCs have been documented to repair traumatic SCI by curbing the activation of A1 neurotoxic reactive astrocytes [34]. In our current investigation, we discerned that MSC-Exo safeguard LPS-treated BV2 cells by impeding ferroptosis. Furthermore, the in vivo administration of these MSC-Exo was observed to effectively alleviate SCI symptoms.
Owing to their capacity for multilineage differentiation, MSCs can evolve into nerve cells, aiding in the repair of damaged neural tissues [35]. However, recent research increasingly attributes the therapeutic efficacy of MSCs to their paracrine functions [36]. MSCs discharge paracrine factors such as growth and inflammatory mediators in the form of exosomes, which play a pivotal role in modulating oxidative stress, inflammation, and apoptosis, thereby exhibiting neuroprotective activities [37]. The remarkable biocompatibility and low immunogenicity of exosomes enhance their potential for clinical applications across various diseases. In the context of cancers and neurodegenerative disorders, MSC-Exo have been identified as key players [38,39]. The use of exosomes from human Wharton’s jelly MSCs has emerged as a promising approach in the prevention and treatment of perinatal brain injury [40]. A recent study revealed that MSC-Exo combat ferroptosis in acute liver injury by preserving SLC7A11 function, thus promoting liver repair [41]. Shao et al. [42] have documented that MSCExo impedes ferroptosis in neuronal cells via the lncGm36569/ miR-5627-5p/FSP1 axis in acute SCI. In our study, we observed that MSC-Exo hindered LPS-induced ferroptosis in BV2 cells and enhanced neurorecovery post-SCI in rats, reinforcing the view that MSC-Exo hold neuroprotective capabilities.
Accumulating evidence suggests that targeting ferroptosis suppression could evolve into an efficacious strategy for enhancing SCI treatment [43]. Consequently, a deep understanding of the regulatory mechanisms governing ferroptosis is vital for its strategic manipulation. Several studies have identified key regulators within ferroptosis metabolic pathways, such as GPX4, FSP1, PTGS2, FTH1, and SLC7A11 [44]. Our findings indicate that LPS diminishes the expression of GPX4, FTH1, and SLC7A11, while augmenting PTGS2 expression in BV2 cells. Notably, these alterations were partially reversed by the application of MSC-Exo.
Antioxidant synthesis stands as a primary defense against ferroptosis [45]. BH4, a critical antioxidant, mitigates lipid peroxidation and confers cellular protection against ferroptosis [46]. The synthesis of BH4 relies on the activity of GCH1, which catalyzes the transformation of guanosine triphosphate into dihydroneopterin triphosphate, eventually leading to BH4 production [47]. The GCH1/BH4 signaling pathway has been recognized for its role in ferroptosis suppression [22]. Nrf2, a pivotal transcription factor in antioxidant defense [48], stimulates GCH1 transcription and boosts BH4 synthesis [23], suggesting the implication of the Nrf2/GCH1/BH4 axis in ferroptosis regulation. In our study, we observed that the Nrf2/GCH1/BH4 signaling pathway could counteract ferroptosis in LPS-treated BV2 cells. Moreover, we report that MSC-Exo could reduce LPS-induced ferroptosis potentially through activating the Nrf2/GCH1/BH4 pathway. This observation was corroborated by similar findings obtained in our SCI rat model experiments, further validating the neuroprotective role of MSC-Exo in SCI contexts through modulation of ferroptosis.
There is also a close association between Nrf2/GCH1/BH4 signaling axis and key ferroptosis regulators, which are mainly mediated by Nrf2. That is to say, Nrf2 regulate ferroptosis through multiple signal axes. For instant, in liver injury, Nrf2 inhibit ROS production by activating the HO-1/GPX4 axis, thereby inhibiting ferroptosis [49]. Nrf2/GPX4 axis effectively inhibit ferroptosis in DOX induced cardiopathy [50]. In the PTGS2/COX-2/Nrf2 signaling, there is an interaction between PTGS2/COX-2 and Nrf2 through electrophilic oxo-derivative (EFOX). PTGS2/COX-2 regulates EFOX synthesis, while EFOX induce Nrf2 dissociation, nuclear translocation, and other functions, thereby regulating oxidative stress.
However, current methods for the isolation and identification of exosomes vary widely, with no standardized approach. There is also considerable debate regarding how to define the dosage for clinical applications of exosomes. The prevalent storage methods commonly utilize isotonic buffers to prevent pH changes during storage, in order to maintain the integrity of exosomes. Due to the lack of data on the impact of processing, storage duration, and preservatives on the structural stability and functional efficacy of exosomes, further research is necessary to establish a gold standard for exosome storage. It should also be recognized that the reproducibility of MSC-Exo-based therapies for SCI in humans may be poor. This is attributed to the differences between animal and human spinal cords, which may lead to less than optimal outcomes when exosome therapies effective in animals are applied to human SCI.
To validate the clinical translatability of MSC-Exo in SCI treatment, further research should progress in the following directions: continue studies on the mechanisms and potential side effects of MSC-Exo in the treatment of SCI, providing more theoretical basis for clinical use; further standardize the acquisition, storage, and utilization methods of MSC-Exo, laying the foundation for future clinical treatments of SCI; prior to clinical application for SCI treatment, rigorous, large-scale animal experiments and preclinical studies are required to ensure the safety and clinical efficacy of MSC-Exo therapy.
CONCLUSION
The findings of this research elucidate that exosomes derived from MSCs play a pivotal role in inhibiting ferroptosis, thereby facilitating functional recovery following SCI. It appears that the regulatory influence of MSC-Exo on ferroptosis might be contingent upon the Nrf2/GCH1/BH4 signaling axis. This study not only provides an initial insight into the modulation of ferroptosis in BV2 cells and SCI rat models but also paves the way for potential exosome-based therapeutic approaches for the treatment of SCI.
Supplementary Materials
Supplementary Fig. 1 can be found via https://doi.org/10.14245/ns.2448038.019.
Characterization of the mesenchymal stem cells (MSCs). (A) Photos of MSCs cells on day 7 of the firstgeneration culture, day 3 of the second-generation culture, and day 3 of the third-generation culture, respectively. Scale bar, 100 μm. (B) Flow cytometry was used to detect the number of positive cells for CD44 and CD11b in MSCs, and control serves as a negative control for flow cytometry testing. (C) Oil red O staining was used to detect the adipogenic differentiation ability of MSCs. Scale bar, 25 μm. (D) Alkaline phosphatase staining was used to detect the osteogenic differentiation ability of MSCs. Scale bar, 100 μm. (E) Alcian blue staining to detect the chondrogenic differentiation ability of MSCs. Scale bar, 100 μm.
Notes
Conflict of Interest
The authors have nothing to disclose.
Funding/Support
This study was supported by Hunan Provincial Natural Science Foundation Project (No. 2022JJ40772) and Changsha Municipal Natural Science Foundation (No. kq2014285).
Author Contribution
Conceptualization: YC, BL, JQ, YL, YT; Data curation: YC, JQ, YL; Formal analysis: YC, BL, JQ, ZL; Funding acquisition: YC; Methodology: YC, BL, YT; Project administration: BL; Visualization: YC, ZL, YL, YT; Writing – original draft: YC, JQ, ZL, YL, YT; Writing – review & editing: YC, JQ, YT.