Introduction
Spinal cord injury (SCI) is an extremely serious nervous system disorder that permanently impairs motor and sensory abilities, along with various pathological alterations, including tissue hemorrhage, edema, and local inflammation [
1,
2]. Around 2.5 million people worldwide suffer from traumatic SCI, with over 130,000 new cases reported annually [
3]. SCI is divided into primary and secondary. Falls, traffic accidents, exercise, and violence can induce primary SCI [
4,
5]. Secondary SCI initiates shortly after the primary injury and can endure for several weeks or months, spreading the injury from the affected area to the surrounding tissue. Secondary SCI can also lead to inflammation, edema, necrotic cell death, and vascular damage. However, no effective treatments are available for SCI [
6,
7].
As a kind of stem cells, mesenchymal stem cells (MSCs) have self-renewal ability and multiple differentiation potentials. They come from various sources and have low immunogenicity, making them ideal grafts for tissue engineering repair. In recent years, the strong proliferative and differential capacities of mesenchymal stem cells (MSCs) have led to new progress in SCI treatment [
8]. As recognized anti-inflammatory response barriers, MSCs are currently the best SCI cell transplantation therapy choice. Due to the therapeutic effects of MSC transplantation for SCI, these cells have entered the clinical stage of SCI treatment [
9,
10].
Umbilical cord mesenchymal stem cells (UC-MSCs) exist in the umbilical cord tissue connected to the fetus after delivery. They are widely used as seed cells for tissue regeneration and repair due to their low ethical issues, multi-directional differentiation potential, immune regulation, and biological anti-inflammatory effects [
11,
12]. The differentiation potential of UC-MSCs into diverse cell types, such as bone, cartilage, and myocardium, has been evidenced by research under in vitro and in vivo induction conditions [
13,
14]. UC-MSCs rarely express human leucocyte antigen I (HLA-I), human leucocyte antigen DR (HLA-DR), and co-stimulatory molecules CD40 and CD80, thereby presenting low immunogenicity and good post-transplant receptor tolerance [
15,
16]. Therefore, UC-MSC transplantation might become the most promising method for treating SCI due to its advantages of convenient extraction of primary cells, less contamination, and low immunogenicity [
17,
18].
Exosomes originate from intracellular vesicles and are small membrane vesicles with a diameter ranging from approximately 30 to 200 nm. These vesicles have diverse proteins, lipids, and RNAs. Cells from all living systems can release these vesicles into the cerebrospinal fluid, blood, and extracellular fluid [
19]. After SCI occurrence, how to effectively improve its pathological and physiological changes has become a key point in the treatment and prognosis of rehabilitation effects. Compared with simple stem cell transplantation, the application of an exosome cell-free diagnosis and treatment scheme will not cause tumor risk, immune reaction, infection, and other complications caused by living cell transplantation [
20]. Moreover, due to the nano diameter of exosomes, they are not easily captured and degraded by tissues such as the lungs and liver after use, and can smoothly pass through the tissue barrier, thus concentrating in the lesion area [
21]. Therefore, exosomes have certain feasibility and theoretical basis for SCI treatment. Until now, many in vivo and in vitro studies have demonstrated that exosomes play a good role in SCI treatment [
22], promoting nerve regeneration and angiogenesis and reducing inflammatory reactions, cell apoptosis, and scar tissue [
23].
Herein, we explored the finctions of UC-MSC-derived exosomes in SCI. Combined with the transcriptome analysis, we found that these exomes might alleviate inflammatory responses in cells and tissues via the NF-κB/MAPK pathway. Overall, we provided new insights and assistance for future SCI treatments.
Materials and methods
Cell culture and treatment
BV2 microglia were purchased from Procell Biotechnology Co., Ltd. (Wuhan, Hubei, China). Cells were placed in α-MEM medium containing FBS (10%) and penicillin and streptomycin (1%), then cultured at 37 ℃ and 5% CO2. BV2 microglia were stimulated with LPS for 12 h, and ATP (5 mM Sigma, USA) was added for 30 min. The concentration of UC-MSCs added was 20 µ g/mL.
UC-MSCs were provided by Procell Biotechnology Co., Ltd. (Wuhan, Hubei, China). Cells were inoculated in a 1:2 ratio and subcultured in a T25 culture flask. Then, the fresh complete culture medium was added to 5 mL and placed in a cell culture incubator at 37 ℃, 5% CO2, and saturated humidity for static cultivation. When the cell fusion degree was about 60–70%, the original FBS-containing medium was removed and replaced by a fresh serum-free medium. Cell culture continued for about three days. The cell supernatant was collected when the cell fusion degree was about 80–95%.
Identification of exosomes
As previously described [
24], exosomes were extracted from the supernatant of UC-MSCs using ultracentrifugation. The morphology of extracted exosomes was analyzed using transmission electron microscopy (TEM), and the diameter and size were evaluated using nanoparticle tracking analysis (NTA). Then CD9, CD63, and CD81 expression were examined Western blot.
Reactive oxygen species (ROS)
BV2 cells were digested, counted, and placed in a 24-well plate, ensuring the number of cells was equal in each well. The probe was diluted with medium (1:1000) to 10 µM. After removing the old medium and washing the cells using PBS, 500 µL of the probe solution was added to each well and incubated for 20 min in an incubator at 37 ℃. The probe’s working solution was discarded, washed gently with PBS thrice, and 400 µL of the medium was added to each well. Finally, a fluorescence microscope was used for observation.
RNA sequencing (RNA-seq)
Total RNA extraction was conducted after cell digestion in QIAZOL (Qiagen). A TruSeq Stranded mRNA Sample Prep Kit (Illumina) was used to prepare the library. The 75-base single-end mode was applied for the sequencing on an Illumina HiSeq 2500 platform. Base calling was performed using Illumina Casava 1.8.2. The Integrated Differential Expression and Pathway (iDEP) analysis software (Version 94) was used for bioinformatics analysis.
Immunofluorescence
For BV2 microglia subcultures, cells were inoculated into the culture dish with the treated cover glass. After cells had grown into a monolayer, the cover glass was removed and soaked with PBS thrice (three min each). Then, the slide was fixed with paraformaldehyde for 15 min and soaked thrice with PBS. Next, the slices were permeated for 20 min and soaked thrice with PBS (three min each) at room temperature (RT). The slide was blocked for 30 min using normal goat serum at RT. Then, the slide was incubated with diluted primary antibodies at 4 ℃. On the second day, samples were incubated with the diluted fluorescent secondary antibody at 37 ℃ for 1 h. Finally, one drop of sealing agent was added, and samples were observed with a fluorescence microscope.
RT-PCR and Western blot
Quantitative RT-PCR and Western blot analyses were performed as previously described [
25,
26]. The primers for PCR are shown in Table
1.
Table 1
Primers for qRT-PCR analysis
INOS | CCCTTCAATGGTTGGTACATGG | ACATTGATCTCCGTGACAGCC |
TNF-α | CTCAAGCCCTGGTATGAGCC | GGCTGGGTAGAGAACGGATG |
IL-1β | AGCTTCAGGAAGGCAGTGTC | TCAGACAGCACGAGGCATTT |
IL-6 | AGAGACTTCCAGCCAGTTGC | AGTCTCCTCTCCGGACTTGT |
Animal experiments
Fifteen Sprague Dawley rats were divided into three groups: Sham, SCI, and SCI + exosomes. In the SCI group, 0.5 mL PBS was injected into the tail vein of rats, while the same volume of UC-MSCs (200 µg/mL) was injected into the tail vein of rats in the SCI + exosomes group.
Rats were anesthetized by intraperitoneal injection of 3.6% chloral hydrate at 1 mL/100 g. After 3–5 min, rats experienced unstable standing and weak or disappeared pain responses. Considering T8 and T9 as the center, a midline incision (1-1.5 cm length) was made on the back. The fascia and muscles were separated layer by layer, the spinal spinous processes of T8 to T10 segments were exposed, and the muscles on the spinal vertebrae of T8 and T10 segments were passively separated. The bone forceps were bitten to remove the spinous processes and lamina of T8 and T10 segments, and the T10 spinal cord was exposed. A 10 g metal rod was selected to fall freely at a distance of 30 mm and hit the T10 nerve segment. Tail swinging and hind limb retraction flutter marked the successful SCI. After the blow, the muscles, fascia, and skin were disinfected using sterile surgical suture needles and threads, followed by intraperitoneal injection of 2 mL glucose solution and intramuscular injection of 1 mL (8 U/mL) penicillin. Within one week after surgery, the wound was disinfected, and 1 mL (8 U/mL) of penicillin was injected intramuscularly. Experimental animals received help to urinate thrice a day until they could urinate autonomously. Two weeks after surgery, spinal cord tissue was taken from rats for subsequent experiments.
Behavioral evaluation
Experimental rats were evaluated with Basso Beattie Bresnahan (BBB) 3, 7, 14, 21, and 28 days after surgery. The bladder was evacuated before observation to avoid affecting the rats’ activity. Then, animals were placed on a flat and unsmooth experimental platform and observed for four min. Behavioral scores were conducted on the animal’s body positioning and hind limb function. The raters are non-experimental personnel familiar with the scoring criteria, and the average value was taken based on three scores.
Enzyme-linked immunosorbent assay (ELISA)
Rat spinal cord samples were collected, placed in 0.25 mL of extraction buffer, and ground. The homogenate was centrifuged at 20,000 g at 4 ℃ for 0.5 h. The supernatant was equally divided and stored at -80 ℃ for cytokine detection. INOS, IL-6, IL-1β, TNF-α and were quantitatively analyzed using ELISA kits.
Hematoxylin and Eosin (HE) and Nissl stainings
We take pathological sections from two weeks after SCI for experimentation. For HE staining, sections were placed into a hematoxylin aqueous solution for staining for a few minutes, followed by acid and ammonia water for color separation for a few seconds. After one hour of rinsing using running water and 10 min of dehydration using 70 and 90% alcohol solutions, sections were stained for three min using eosin. After dehydration using 100% alcohol and transparentizing using xylene, stained sections were sealed and observed under a microscope.
For Nissl staining, sections were stained with Nissl staining solution at 56 ℃ for 10–15 min, directly differentiated with 95% alcohol differentiation solution for a few seconds, and quickly washed with water. Finally, samples were dehydrated with anhydrous alcohol twice for two minutes, transparentized with xylene twice for five minutes, and sealed with neutral resin.
Immunohistochemistry
Two weeks after establishing the SCI rat model, fresh spinal cord tissues were collected and fixed with 4% paraformaldehyde.Then, tissues were dehydrated, embedded in paraffin, and sliced to a thickness of 3 μm using a paraffin slicer. After dewaxing and antigen repair, the primary antibody was added to the slices overnight, and the second antibody was applied for one hour the next day. Finally, the staining was observed under a microscope.
Statistical analysis
Data were analyzed using SPSS statistical software. Results are presented as means ± standard deviations. One-way analysis of variance (ANOVA) was conducted to analyze the data. p < 0.05 was considered statistically significant.
Discussion
SCI causes changes in the function and structure of the spinal cord for various reasons, often resulting in complete or partial mobility loss [
27]. Falls, car accidents, falls from heights, fights, and sports injuries can lead to SCI. Moreover, SCI exerts a dual toll on patients, severely impacting their mental and physical health and imposing significant economic stress [
28]. The number of new SCI patients worldwide is about 250,000 to 500,000 annually, with more than one million SCI patients in China [
2]. The patients themselves have a serious psychological burden but also burden the family and society. One of the most formidable medical challenges is regenerating and repairing SCI. However, there is a lack of effective methods to promote neurological recovery, which can only provide supportive relief for patients with lifelong disability [
29].
Herein, we extracted exosomes derived from UC-MSCs and successfully identified them using Western blot, NTA, and other methods. Then, we differently treated BV2 microglia: one group was treated with LPS + ATP and the other with LPS + ATP + exosomes. Microglial cells are the main immune cell type in the parenchyma of the central nervous system (CNS), accounting for 5–10% of the total number of cells [
30]. Microglia can guide endothelial cells to influence the formation of blood vessels in the parenchyma [
31]. In a healthy CNS, microglia have branched protrusions through which they can dynamically monitor parenchyma to detect infections or injuries rapidly [
32]. Microglia mainly have two phenotypes, M1 and M2. Microglia in the M1-polarized state have phagocytic functions and produce pro-inflammatory cytokines and bactericidal molecules [
7]. Alternately activated M2 phenotypes are involved in the repair of damaged cells and inflammatory responses in resistant tissues [
33]. The microenvironment of tissues is the main factor influencing the polarization state of tissues [
34].
Apoptosis is a prominent SCI feature. Due to mechanical trauma and other reasons, some cells in the lesion site become necrotic when SCI occurs, while others undergo apoptosis. Also, evident apoptosis of neurons and oligodendrocytes can be observed in the white matter [
35]. By analyzing the apoptosis time of SCI rats, it was found that neuron apoptosis occurred as early as four hours after injury and peaked eight hours later. Apoptosis of glial cells was detected four hours after injury and peaked 24 h later, while apoptosis of oligodendrocytes was detected 24 h after injury and peaked eight days later. A reduction in cell count was also observed up to three weeks after SCI [
36]. Therefore, apoptosis of neurons, glial cells, and oligodendrocytes might play an adverse role in SCI. On the one hand, apoptosis during SCI reduces the number of neurons and oligodendrocytes, which cannot meet the needs of nerve regeneration. On the other hand, the apoptotic signals secreted by apoptotic cells, while further inducing the apoptosis of other cells, can cause serious inflammatory reactions, aggravate the pathophysiological SCI microenvironment, and are not conducive to the differentiation of nerve cells and injury repair. Additionally, the inhibition of BV2 microglia apoptosis and SCI tissue by the UC-MSC-derived exosomes was observed by Western blot, immunofluorescence, and immunohistochemistry experiments in vivo and in vitro. In the acute phase of traumatic SCI, a series of physiological changes occur at the injury site, generating a large number of ROS [
37]. Due to its multi-origin, persistence, and chain reactions, ROS generated will further enhance inflammatory response at the SCI site, extend the damage to proteins, lipids, and nucleic acids, and induce neuronal necrosis or apoptosis [
38]. Here, we found that LPS + ATP promoted cell production of ROS, while UC-MSC-derived exosomes inhibited it, which might be an important reason exosomes promote SCI recovery.
Inflammation plays an important role in SCI. Failure of the blood-spinal barrier and blood vessel rupture caused by SCI leads to spinal tissue bleeding, followed by the invasion of various immunoinflammatory cells in the blood, such as macrophages, T and B lymphocytes, neutrophils, and monocytes into the spinal tissue. The inflammatory cells mentioned above release numerous pro-inflammatory factors in the SCI microenvironment, including TNF-α and IL-6. The serum content of TNF-a in SCI patients increases immediately after injury and with increased injury time, and many TNF-positive cells are detected in the injured spinal cord [
39]. The concentrations of IL-6 and IL-1β increase significantly at and around the injury site 3 to 24 h after injury [
39]. TNF-α can promote macrophage migration to the injured area and accelerate neuronal death. The increase of IL-1β and IL-6 concentration can cause activation and proliferation of astrocytes and macrophages/microglia, accelerate the formation of connective tissue scar, and aggravate injury severity. The infiltration of immune cells and inflammatory factors further aggravate the inflammatory response of the spinal cord [
40]. We used Western blot, immunofluorescence, and immunohistochemistry analyses to demonstrate that UC-MSC-derived exosomes can inhibit the inflammatory response of BV2 microglia and SCI tissue in vivo and in vitro.
The activation of macrophages often accompanies the occurrence of inflammation. LPS can bind to the TLR4 receptor on the macrophage surface to activate downstream Akt and MAPK/NF-κB signaling pathways mediated by this receptor [
41]. Hence, inflammatory genes and protein levels are significantly connected to the transcription factor NF-κB and MAPK in cells. Our RNA-seq of BV2 microglia suggested that UC-MSC-derived exosomes might act on the MAPK/NF-κB signaling pathway. Studies have shown that LPS-induced inflammatory responses are mainly mediated by MAPK/NF-κB signaling pathways [
42], of which NF-κB is the central link. Through inducing IKK kinase activation, LPS can cause the phosphorylation of IκBα protein and the ubiquitination degradation of the phosphorylated IκBα protein, resulting in the release of NF-κB dimer into the nucleus. MAPK and NF-kB signaling pathways are closely related to the occurrence of inflammation, and they have many overlaps in signal transduction. The MAPK signaling pathway is believed to be the main upstream site for NF-kB activation because 1) MAPK is activated by multi-effect regulatory factors of multiple damage response genes, and the continuous activation of MAPK leads to the continuous production of inflammatory cytokines, activating MAPK and NF-kB signaling pathways, and leading to the cascade “waterfall” effect; (2) after MAPK activation, IkB can be phosphorylated, resulting in IkB degradation and release of p50-p65/RelA heterodimers, which directly lead to NF-kB activation and translocation, driving NF-kB target genes transcription in the nucleus [
43,
44]. Therefore, regulation of the NF-κB/MAPK signal transduction pathway plays an important role in controlling the occurrence and development of inflammation. We demonstrated that UC-MSC-derived exosome could inhibit the NF-κB/MAPK signaling pathway in BV2 microglia and SCI rat tissues in vitro and in vivo.
In summary, we found that extracellular vesicles derived from UC-MSCs can alleviate inflammatory responses and promote SCI recovery by inhibiting the NF-κB/MAPK signaling pathway. These findings could offer novel perspectives on future SCI treatments.
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