Corresponding Author Wenge Liu Department of Orthopedics, Fujian Medical University Union Hospital, No. 29, Xin Quan Road, Fuzhou 350001, China Email: wengeunion@fjmu.edu.cn

Co-corresponding Author Zhenyu Wang Department of Orthopedics, Fujian Medical University Union Hospital, No. 29, Xin Quan Road, Fuzhou 350001, China Email: zhenyu_wang@fjmu.edu.cn

*Hao Feng and Dehui Chen contributed equally to this study as co-first authors.

Received 2024 September 3; Revised 2024 November 11; Accepted 2024 November 15.

Abstract

Objective

Neuronal apoptosis is considered to be a critical process in spinal cord injury (SCI). Despite growing evidence of the antiapoptotic, anti-inflammatory, and modulation of ischemic injury tolerance effects of extracellular ubiquitin (eUb), existing studies have paid less attention to the impact of eUb in neurological injury disorders, particularly in SCI. This study aimed to investigate whether eUb can play a protective role in neurons, both in vitro and in vivo, and explores the underlying mechanisms.

Methods

By utilizing an oxygen glucose deprivation cellular model and a SCI rat model, we firstly investigated the therapeutic effects of eUb on SCI and further explored its effects on neuronal autophagy and mitochondria-dependent apoptosis-related indicators, as well as the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mechanical target of rapamycin (mTOR) signaling pathway.

Results

In the SCI models both in vivo and in vitro, early intervention with eUb enhanced neuronal autophagy and inhibited mitochondrial apoptotic pathways, significantly mitigating SCI. Further studies had shown that this protective effect of eUb was mediated through its receptor, CXC chemokine receptor type 4 (CXCR4). Additionally, eUb-enhanced autophagy and antiapoptotic effects were possibly associated with inhibiting the PI3K/Akt/mTOR pathway.

Conclusion

In summary, the study demonstrates that early eUb intervention can enhance autophagy and inhibit mitochondrial apoptotic pathways via CXCR4, protecting neurons and promoting SCI repair.

Keywords: Spinal cord injury; Extracellular ubiquitin; CXCR4; Autophagy; Mitochondrial apoptosis pathway

INTRODUCTION

Spinal cord injury (SCI) is a devastating central nervous system injury caused by primary mechanical force to the spinal cord, leading to severe motor, sensory, and autonomic nervous system dysfunction [1,2]. Currently, no effective clinical treatment exists to improve the poor prognosis following SCI, which imposes significant physical, psychological, and economic burdens on patients and society.

Primary SCI often results in ischemic injury, which compresses spinal cord tissues and triggers a series of secondary reactions, including ischemia, hypoxia, overproduction of free radicals, excitotoxicity, and inflammatory responses, which ultimately lead to apoptosis or necrosis of neurons, axonal demyelination, and neuroglial scar formation [3-5]. Secondary neuronal death is a major contributor to neurological dysfunction [6]. Therefore, reducing secondary neuronal death through early intervention is essential for improving outcomes post-SCI. The principal neuronal death pathways vary depending on the injury stage, with local ischemia, edema, and hypoxia caused by primary injury being the leading causes of neuronal apoptosis and irreversible dysfunction in the early stages of SCI [7].

Monomeric ubiquitin (Ub) is a 76-amino-acid highly conserved small-molecule protein found in eukaryotes (molecular weight approximately 8.6 kDa), playing a role in regulating various cellular processes [8,9]. Intracellular Ub functions include protein turnover regulation and the protection of cells from damaged or misfolded proteins via the Ub-proteasome pathway, affecting receptor internalization, apoptosis, and cellular tolerance to ischemic injury [10,11]. Meanwhile, Ub is also found in extracellular body fluids, which is designated as free Ub or extracellular Ub (eUb). Secretion into the extracellular space via an exocytic mechanism from normal cells, necrosis of damaged cells, and hemolysis have been suggested as potential mechanisms for the presence of Ub in body fluids [9]. eUb has been detected in the serum, cerebrospinal fluid, lungs, and urine of normal individuals, where it regulates various cellular processes [12]. Elevated levels of eUb have been reported in patients with sepsis, burns, ischemic heart disease, and central nervous system disorders such as Alzheimer’s disease and traumatic brain injury [12-15].

Ub is highly stable, with a thermal stability of 95°C at pH 7.0, which may be ideal for storing Ub as a lyophilized powder and/or solution at room temperature [8]. The systemic half-life of eUb has been determined to be 60 minutes after intravenous administration, and only 10% of intravenously administered Ub is recovered in the urine after 5 serum half-lives, suggesting that most Ub is cleared by extrarenal mechanisms [16,17]. Ub concentrations in cerebrospinal fluid after experimental brain injury peaked within 30–60 minutes after injury and then declined with a halflife of 1–2 hours. The similarity of Ub’s systemic and intrathecal half-lives also makes it unlikely that the lungs or liver determine its systemic clearance [17,18]. Meanwhile, extracellular Ub can also be taken up by a variety of eukaryotic cells, such as rat ventricular myocytes, human monocyte cell lines, and human T-cell lines [18]. Therefore, in addition to its possible extracellular degradation, cellular uptake and metabolism of Ub are attractive mechanisms for its removal from the systemic circulation. In addition, the pharmacokinetics of eUb injected into rats via intraperitoneal injection: half-life variability is within the range of 9–16 hours, t1/2< 20 hours [19].

Studies have demonstrated that eUb has pleiotropic effects, such as modulation of the immune response, anti-apoptosis, anti-inflammation, and neuroprotection [20-23], and may play essential biological roles in certain acute traumatic or ischemic conditions. For example, in mice with myocardial ischemia/reperfusion injury, Scofield et al. [24] found that eUb treatment reduced cardiac infarct size, improved cardiac function, and decreased the inflammatory response. Other studies have suggested that eUb significantly increases the survival of irradiated intestinal epithelial cells and exerts a protective effect against radiation-induced intestinal injury [25]. In traumatic brain injury, eUb treatment has been shown to have a direct neuroprotective effect, significantly reducing the volume of cortical contusion after closed traumatic brain injury [22]. However, little is known about the specific protective role and mechanism of eUb in traumatic diseases of the central nervous system, particularly in SCI, where it has not yet been reported. Therefore, we aimed to explore, both in vitro and in vivo, whether eUb intervention could exert a neuroprotective effect in the early stage of SCI and to investigate its possible specific mechanisms, thereby providing a promising candidate for protein therapy in early SCI intervention.

MATERIALS AND METHODS

1. Cell Culture and Treatment

Human neuroblastoma cells (SH-SY5Y, Cell Line Bank of the Chinese Academy of Sciences, China) were maintained in a medium containing 43% MEM (Minimum Essential Medium, Cytiva, Marlborough, MA, USA), 43% F-12 medium (Ham’s F-12 Nutrient Medium, Gibco, Grand Island, NY, USA), 10% Fetal Bovine Serum (Gibco), 1% Penicillin-Streptomycin (Gibco), 1% MEM Non-Essential Amino Acid Additive (NEAA, Coolaber, Beijing, China), 1% Pyruvate Solution (Coolaber), and 1% Glutamine (GlutaMAX-l, Coolaber). The cells were incubated in a humidified atmosphere at 37°C with 5% CO2 and 70%– 80% humidity.

To simulate ischemia and hypoxia conditions following SCI, an SH-SY5Y oxygen glucose deprivation (OGD) injury model was constructed. The cell culture medium was replaced with Hank’s balanced salt solution (Genview, Houston, TX, USA) and incubated in a low oxygen incubator containing 1% O2, 94% N2, and 5% CO2 at 70%-80% humidity and 37°C. Recombinant eUb (Biotechnology, China) was dissolved in a conditioned medium, and cells were pretreated for 15 minutes before constructing the OGD model. In the AMD+eUb group, cells were first pretreated with AMD3100 (Macklin, China) for 15 minutes and then treated with eUb (10 μg/mL).

2. Cell Counting Kit-8 (CCK-8) Assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cells were seeded in 96-well plates at a density of 5×10³ cells/well and treated once the cells reached 90% confluence. At the designated time points, 10 µL of CCK-8 solution (Lablead, Beijing, China) was added to each well. The CCK-8 solution was mixed with the conditioned medium and incubated at 37°C for approximately 90 minutes. Absorbance was measured at 450 nm using a multifunctional microplate reader (SpectraMax i3x, Molecular Devices, Sunnyvale, CA, Japan).

3. Live/Dead Cell Staining

Cell viability was also evaluated using a live/dead cell staining assay kit (Beyotime, Shanghai, China), with cells observed under a fluorescence microscope (Leica DMi8, Leica, Heidelberg, Germany). Live cells were stained with Calcein AM, emitting green fluorescence, while dead cells were stained with propidium iodide (PI), emitting red fluorescence.

4. Apoptosis Rate Detection Assay After OGD

Apoptotic SH-SY5Y cells were identified and quantified using an apoptosis detection kit (Yeasen, Shanghai, China). SH-SY5Y cells were seeded in 6-well plates at a density of 1×10⁵ cells/well and cultured until reaching 90% confluence. After 6 hours of OGD treatment, 50,000 cells were collected and resuspended in 100 μL of binding buffer. Five microliters of Annexin V-FITC and 10 μL of PI staining solution were added, mixed well, and incubated for 10–15 minutes in the dark at room temperature. After adding 400 μL of 1× binding buffer, the mixture was kept on ice. Samples were analyzed using a flow cytometer (BD Accuri C6 Plus, BD, Franklin Lakes, NJ, USA) within 1 hour. Early apoptotic cells (Annexin V-FITC-positive), late apoptotic cells (positive for both Annexin V-FITC and PI), and necrotic cells (PI-positive) were sorted from the general cell population, and apoptosis rates were calculated from 3 replicate experiments.

5. Western Blotting Assay

Total proteins were extracted from cells or spinal cord tissues using RIPA lysis buffer (Dingguo, Beijing, China) containing phenylmethylsulphonyl fluoride, phosphatase inhibitors, and protease inhibitors. Protein concentration was determined using the BCA protein assay kit (Boster, Zhongshan, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 6%, 12%, or 15% and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skimmed milk (BioFroxx, Hesse, Germany) for 3 hours at room temperature, followed by overnight incubation with the following antibodies at 4°C: anti-Ub (1:500; Proteintech, Shanghai, China), anti-Bax (1:1,000; Abcam, Cambridge, UK), anti-Bcl-2 (1:1,000; Abcam), anti-Cleaved casepase-3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-Caspase-3 (1:1,000; Cell Signaling Technology, USA), anti-Cleaved casepase-9 (1:1,000; Cell Signaling Technology), anti-Caspase-9 (1:1,000; Cell Signaling Technology), anti-Akt (1:1,000; Cell Signaling Technology), anti-p-Akt (1:500; Cell Signaling Technology), anti-PI3K (1:1,000; Abcam), anti-p-PI3K (1:1,000; Abcam), anti-mTOR (1:1,000; Abcam), anti-p-mTOR (1:1,000; Abcam), anti-CytC (1:1,000; Cell Signaling Technology), anti-p62 (1:1,000; Cell Signaling Technology), anti-Beclin1 (1:1,000; Cell Signaling Technology), anti-ATG7 (1:1,000, Abcam), anti-ATG5 (1:1,000; Abcam), anti-LC3B (1:1,000; Abcam) and antiα-Tubulin (1:1,000, Abcam).

6. Real-Time Fluorescent Quantitative Polymerase Chain Reaction Assay

Total RNA was extracted and purified using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China), and RNA concentration was determined using an ultra-microUV spectrophotometer (Thermo, Nanodrop One, Wilmington, DE, USA). RNA was reverse transcribed into cDNA using a 5X All-In-One RT MasterMix kit (ABM, Vancouver, Canada). Ub mRNA levels were assessed using a real-time fluorescence quantitative polymerase chain reaction (qPCR) instrument (Life, ABI 7500 Real-Time PCR system, Singapore) and PerfectStart Green qPCR SuperMix (ABM). The primers were synthesized by Sangya Bio (Harbin, China), and the relative gene expression was calculated by the 2-ΔΔCt method, with β-actin as an internally standardized reference gene. The primers used were as follows:

Ubiquitin, forward: 5´-GGTCCTGCGTCTGAGAGGT-3´;

Ubiquitin, reverse: 5´-GCCTTCACATTTTCGATGGTGT-3´;

β-actin, forward: 5´-GAGAAAATCTGGCACCACACC-3´;

β-actin, reverse: 5´-GGATAGCACAGCCTGGATAGCAA-3´.

7. Animals and Experimental Groups

Adult male Sprague-Dawley rats (clean grade), weighing 220–250 g, were obtained from Shanghai SLAC Laboratory Animal Co. (Shanghai, China). All rats were housed under standard conditions with normal access to food and water, with 4–5 rats per cage, in a room maintained at a constant temperature of 22°C–24°C, a humidity of 45%–55%, and a 12-hour light/dark cycle. Thirty rats were randomly assigned to 3 groups: sham surgery group, SCI group, and SCI+eUb group (n = 10). Any death or surgical failure during the experiment were compensated by using spare animals. All experiments were approved by the Animal Ethics Committee of Fujian Medical University (approval number: IACUC FJMU 2024-0018) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

8. Construction of SCI Model and Administration of AMD3100 in Rats

A rat SCI model was established using Allen’s modified method. Rats were anesthetized with 1% pentobarbital (50 mg/kg, intraperitoneally) prior to the procedure. After successful anesthesia, a posterior median longitudinal incision, approximately 2 cm in length, was made centered on the T10 spinous process, sequentially incising the skin, subcutaneous tissue and paravertebral muscles to reveal the vertebral plate. The T10 spinous process and part of the vertebral plate were removed to expose the spinal cord. The rats were then immobilized on a modified Allen’s impactor (RWD Life Science, San Diego, CA, USA), with the T10 spinal cord segment accurately positioned for impact. The impingement head had a diameter of 2.5 mm, a weight of 15 g, and a drop height of 10 cm. Immediately after impact, edema developed in and around the impact area, and the posterior median vein of the spinal cord ruptured, resulting in a hematoma formation. The rats exhibited tails spasms, and after the trunk and hind limbs retracted and fluttered, muscle tone in both hind limbs disappeared, leading to paralysis.

In animal experiments, scholars treated partially hepatectomized rats by multiple intraperitoneal injections of eUb (200 μg/dose) and achieved better intervention results [19,26]. Similar to the methods of these studies, we also used multiple intraperitoneal injections of eUb (10 mg/kg) to intervene in rats (220–250 g). In addition, we performed early serial interventions with eUb immediately after surgery, which is consistent with the practice of many eUb-related studies [16,27]. The SCI+eUb group received intraperitoneal injections of eUb (10 mg/kg) immediately after modeling, as well as 24-hour, 3-day, and 5-day postinjury. In contrast, the remaining 2 groups received intraperitoneal injections of 1× phosphate-buffered saline, the vehicle used to dissolve eUb, at the same time points as a control. To prevent wound and urinary tract infections, rats were given daily intraperitoneal injections of cefuroxime sodium (100 mg/kg) for 3-day postsurgery. Postoperatively, the rats’ bladders were manually massaged and gently squeezed twice daily to assist with urination until urinary retention resolved.

9. Assessment of Motor Ability

Motor ability was assessed using a 21-point (0–21) Basso-Beatie-Bresnahan (BBB) scale. Rats were scored immediately after they awoke from anesthesia, and rats with scores higher than 5 were excluded as modeling failures and were replaced by re-modeling with spare rats. All rats were observed individually in double-blind pairs, and their behaviors were assessed before surgery and at 8-hour, 1 day, 3-day, 5-day, and 7-day postsurgery. The rats were placed in the observation area before assessment and allowed to walk freely in a designated area for 5 minutes during the assessment period, once they had adapted to the environment. Gait and motor coordination were evaluated 7 days after surgery. The forelimbs and hindlimbs of the rats were marked with green and red colors, respectively. Engineering drawings were placed flat on a tabletop, and a 3-sided slot was constructed to form a simple “tunnel.” The rat was placed at the entrance of the tunnel and encouraged to walk forward, leaving tracks on the drawing. The overall footprint image was used to assess gait and motor coordination.

10. Spinal Cord Tissue Processing, Hematoxylin-eosin Staining, and Nissl Staining

One week after surgery, rats were anesthetized and perfused with 0.9% NaCl via the heart, followed by 4% paraformaldehyde (PFA). Immediately after perfusion, the target segment of the spinal cord, approximately 1 cm in length (0.5 cm above and below the injury site), was removed and fixed in 4% PFA for 24 hours. The tissue was then paraffin-embedded and sectioned into 4-μm-thick slices mounted on poly-L-lysine-coated slides. Hematoxylin-eosin (H&E) and Nissl staining were performed on the deparaffinization section for histopathological examination. Images were captured using a light microscope (Leica), and the number of surviving neurons was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

11. Statistical Analysis

All data are expressed as mean ± standard deviation. Data from at least 3 independent replicates were analyzed using IBM SPSS Statistics ver. 25.0 (IBM Co., Armonk, NY, USA) and GraphPad Prism v8.0 (GraphPad Software Inc., La Jolla, CA, USA). A t-test was used for comparison between the 2 groups, while a 1-way analysis of variance (ANOVA) was used for comparisons among multiple groups, followed by Tukey multiple comparison test. A p-value < 0.05 was considered statistically significant.

RESULTS

1. OGD Model Induces Ischemic Injury in SH-SY5Y Cells

Reduced blood flow to the spinal cord due to primary SCI can cause ischemic hypoxia and accelerate tissue damage, which constitutes the primary pathological process in the early stages of SCI [28]. Consequently, the OGD model is widely recognized and commonly used to simulate the extracellular environment following early SCI [29-31]. Based on this, we utilized the OGD model to simulate early SCI in our study. Using the no OGD injury (standard culture) group as a control, SH-SY5Y cells were subjected to OGD injury for 1, 2, 4, 6, and 8 hours. The CCK-8 results indicated that the cell viability of SH-SY5Y decreased in a time-dependent manner. At 6 hours of OGD, a significant reduction (approximately 50%) in cell viability was observed (Fig. 1A). Furthermore, after 6 hours of OGD injury, substantial morphological changes in SH-SY5Y cells were observed under the microscope, including disorganized cellular arrangements and cell rupture or lysis (Fig. 1B).

Fig. 1.

Oxygen glucose deprivation (OGD)-induced SH-SY5Y cell injury. (A) Relative survival of SH-SY5Y cells was assessed by the CCK-8 assay at 1, 2, 4, 6, and 8 hours after OGD injury. (B) Morphological changes of SH-SY5Y cells at different time points after OGD injury were observed under a light microscope. All images were captured at ×100 magnification. (C) Cells were exposed to OGD for 6 hours and evaluated for viability using Calcein/propidium iodide (PI) staining. Calcein AM stained live cells green, while PI marked dead cells with red fluorescence. (D, E) After 6 hours of OGD treatment, the apoptosis rate was measured by Annexin V-FITC/PI double staining and flow cytometry. One-way analysis of variance was used to calculate the p-value for the mean±standard deviation of 3 independent experiments. ***p<0.001. ****p<0.0001.

Live-dead staining results showed a significant increase in the number of dead cells after SH-SY5Y cells were treated with OGD for 6 hours compared to the control group (Fig. 1C). Annexin V-FITC/PI apoptosis double staining and flow cytometry assay also revealed that 6 hours of OGD significantly increased the apoptosis rate of SH-SY5Y cells (Fig. 1D and E). In addition, Western blotting results demonstrated that 6 hours of OGD significantly upregulated the ratios of apoptosis-related proteins Bax/Bcl-2, Cleaved-caspase-3/Caspase-3, Cleaved-caspase-9/Caspase-9, and p-Akt/Akt, as well as cytochrome C (CytC) expression (Fig. 2A and B). These findings indicate that the OGD injury model of SH-SY5Y cells was successfully constructed and that 6 hours of OGD could significantly induce mitochondria-dependent apoptosis. Therefore, we selected 6 hours of OGD as the time point of OGD injury in subsequent in vitro studies.

Fig. 2.

Oxygen glucose deprivation (OGD) 6-hour treatment significantly induced mitochondria-dependent apoptosis in vitro. (A) Western blot analysis was performed to examine the expression of apoptosis-related proteins Bax/Bcl-2, Cleaved-caspase-3/ Caspase-3, Cleaved-caspase-9/Caspase-9, Cyt-C, and phosphor-Akt (p-Akt)/Akt following OGD exposure. (B) Semiquantitative analysis of apoptosis-related protein expression levels. One-way analysis of variance was used to calculate the p-value for the mean±standard deviation of 3 independent experiments. **p<0.01. ***p<0.001. ****p<0.0001.

2. Expression of Ub in SH-SY5Y Cells After OGD Injury

To investigate the expression of intracellular Ub molecules in damaged neurons during spinal cord ischemic injury, we first examined the changes in mRNA levels of Ub in SH-SY5Y cells at different OGD injury time points. The results showed that the mRNA level of Ub was significantly upregulated at 6 hours of OGD compared to the cells incubated under normoxic conditions, with this upregulation showing a gradual increase as OGD time was prolonged (Fig. 3A). Consistent with these findings, Western blotting results demonstrated that the protein expression of Ub also increased with longer OGD exposure (Fig. 3B and C). We further analyzed the changes in ubiquitinated protein levels in SH-SY5Y cells using Western blotting, which showed that OGD injury significantly increased the levels of ubiquitinated proteins in SH-SY5Y cells (Fig. 3D and E). In summary, these results suggest that Ub may play a crucial role in the response of SH-SY5Y cells to OGD injury.

Fig. 3.

Extracellular ubiquitin (eUb) was protective in SH-SY5Y cells subjected to oxygen glucose deprivation (OGD) injury. (A) The mRNA levels of Ub in SH-SY5Y cells at different OGD injury time points were quantified by quantitative reverse transcription polymerase chain reaction analysis. (B, C) Western blot analysis was performed to assess Ub protein expression levels in SH-SY5Y cells after various durations of OGD exposure. (D, E) Western blot analysis of ubiquitinated protein levels in SH-SY5Y cells. (F) The CCK-8 assay was used to assess the relative survival of SH-SY5Y cells after treatment with different concentrations of eUb. (G, H) Cells were pretreated with eUb (0–1,000 μg/mL) for 15 minutes and then subjected to OGD treatment for 6 hours. (G) Cell viability was then measured by CCK-8 assay, and (H) cell morphology was photographed using a light microscope. All images were captured under a ×100 objective lens. One-way analysis of variance was used to calculate the p-value for the mean± standard deviation of 3 independent experiments. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001.

3. eUb Enhances the Viability of OGD-Injured Neurons In Vitro

To investigate whether Ub can play a protective role in neurons subjected to OGD injury, we pretreated SH-SY5Y neurons with different concentrations of purified eUb before OGD injury. It has been reported that many authors, in order to investigate the protective effect of eUb on injured cells, used eUb for pretreatment 15–30 minutes before cell injury treatment [11,23]. Among them, in order to verify the protective effect of eUb on cardiomyocytes during ischemia/hypoxia, Ji et al. [23] applied eUb for pretreatment 15 minutes before I/H injury and achieved better intervention results. Therefore, we also chose to pretreat cells with eUb 15 minutes before OGD model construction.

CCK-8 experiments demonstrated that none of the eUb concentrations affected the viability of SH-SY5Y cells cultured under normoxic conditions (Fig. 3F). Subsequentially, we found that higher concentrations (10, 50, 100, 500, and 1,000 μg/mL) of eUb significantly and dose-dependently reversed the decrease in cellular viability induced by OGD damage (Fig. 3G). Microscope observations revealed that these eUb treatment concentrations helped protect the cells from OGD damage and better maintained normal cell morphology (Fig. 3H). These results suggest that eUb intervention can significantly reduce OGD-induced cellular damage.

4. eUb Protects Neurons From OGD Damage In Vitro by Blocking the Mitochondrial Apoptotic Pathway

To further validate the protective effect of eUb on neurons, we pretreat SH-SY5Y cells with a 10 μg/mL concentration of eUb for 15 minutes in subsequent in vitro studies before OGD injury based on the above results and economic factors. We then measured the expression of proteins related to mitochondria-dependent apoptosis using Western blotting. We found that OGD injury upregulated the expression of Bax/Bcl-2, Cleaved-caspase-3/Caspase-3, Cleaved-caspase-9/Caspase-9 ratios, and Cyt-C compared with the control group. These changes were significantly reduced by eUb intervention (Fig. 4A and B). These results indicate that eUb significantly inhibits mitochondria-dependent apoptosis induced by OGD injury.

Fig. 4.

Extracellular ubiquitin (eUb) inhibited mitochondria-dependent apoptosis induced by oxygen glucose deprivation (OGD) in vitro. (A) Western blot analysis was performed to examine the expression of mitochondria-dependent apoptosis-related proteins and proteins associated with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mechanical target of rapamycin (mTOR) signaling pathway after OGD treatment. (B) Semiquantitative detection of the expression levels of mitochondria- dependent apoptosis-related proteins and PI3K/Akt/mTOR signaling pathway-related proteins. One-way analysis of variance was used to calculate the p-value for the mean±standard deviation of 3 independent experiments. p-PI3K, phospho- PI3K; p-Akt, phosphor-Akt; p-mTOR, phosphor-mTOR. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001.

It is well known that the PI3K/Akt/mTOR signaling pathway plays an essential role in the physiology and pathology of SCI, regulating autophagy and apoptosis in spinal cord-injured neurons [32]. Therefore, we also examined the changes in the expression of proteins related to the PI3K/Akt/mTOR pathway. The results showed that the ratios of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR were significantly higher in the OGD group compared to the control group. However, eUb treatment significantly reversed these changes, suggesting that eUb inhibits the activation of the PI3K/Akt/mTOR signaling pathway induced by OGD injury (Fig. 4A and B). Taken together, it is reasonable to speculate that the inhibition of the OGD-induced mitochondrial apoptotic pathway by eUb may be associated with the inhibition of the PI3K/Akt/mTOR pathway.

5. Early Intervention with eUb In Vivo Protects Against SCI and Promotes Functional Recovery from SCI

To investigate whether eUb has a neuroprotective effect in vivo, we constructed a rat spinal cord contusion model using the modified Allen’s method (see schematic in Fig. 5A) and performed a series of experiments. First, we performed BBB scoring before and at 8 hours, and 1, 3, 5, and 7 days after surgery to assess the therapeutic effect of early eUb intervention on motor function recovery after SCI. The results demonstrated that the BBB scores in the SCI+eUb group were significantly higher than those in the SCI group starting from day 5 postinjury (Fig. 5B). Secondly, we assessed gait by manually analyzing footprints 7 days after surgery. All animals in the SCI and SCI+eUb groups showed a significant decrease in hind paw motor coordination compared to the sham-operated group. However, animals in the SCI+eUb group exhibited substantial recovery in gait and motor coordination compared to the SCI group (Fig. 5C).

Fig. 5.

Early treatment with extracellular ubiquitin (eUb) protected spinal cord tissues and promoted the recovery of motor function after spinal cord injury (SCI) in rats. (A) Schematic diagram of the rat spinal cord contusion model constructed according to the modified Allen method. (B) Basso-Beatie-Bresnahan (BBB) scores of rats in each group at different time points after spinal cord contusion. (C) Schematic diagram of the walking footprints of rats 1 week after SCI. Green: paw prints - front paw; red: paw prints - hind paw. One-way analysis of variance was used to calculate the p-value for the mean±standard deviation of 3 independent experiments. (D) Hematoxylin-eosin staining of representative spinal cord cross-sections from each group. Scale bar= 1,000 μm or 250 μm. (E, F) The number of surviving neurons in each group was detected by Nissl staining. Scale bar=250 μm. One-way analysis of variance was used to calculate the p-value for the mean±standard deviation of 3 independent experiments. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001.

Furthermore, we used H&E staining to identify and analyze the histological structure of the rat spinal cord 1 week after injury. The results showed that the SCI group had significant and extensive destruction and loss of spinal cord tissue compared to the sham-operated group. However, the SCI+eUb group displayed significantly less spinal cord tissue destruction and loss than the SCI group (Fig. 5D).

To further investigate whether early intervention with eUb could protect SCI neurons, we performed Nissl staining on anterior horn motor neurons 7 days after injury to analyze neurons survival. The results indicated that the SCI group had significantly fewer spinal cord anterior horn neurons compared to the sham-operated group. In contrast, early intervention with eUb significantly increased the number of spinal cord anterior horn neurons (Fig. 5E and F).

In summary, early intervention with eUb protects spinal cord neurons from SCI, promotes the repair of spinal cord tissue damage, and enhances functional recovery after SCI.

6. In Vivo eUb Increases Autophagy and Inhibits Apoptosis in Spinal Cord Neurons After SCI in Rats

Autophagy is a cellular self-protection mechanism and moderate activation of autophagy early after SCI is considered neuroprotective and involved in regulating neuronal apoptosis [33,34]. Additionally, various signaling pathways, including the PI3K/Akt/mTOR pathway, regulate autophagy after SCI. Therefore, based on the above results, we explored whether the neuroprotective effect of eUb is also related to autophagy. We examined the expression of autophagy-related proteins by Western blotting. The results revealed that the ratio of LC3B II/I and the expression of Beclin1, ATG5, and ATG7 proteins were significantly upregulated after SCI, accompanied by a significant downregulation of the autophagy flow-related protein P62. eUb treatment further amplified these changes. These results indicate that SCI induces cellular autophagy, and eUb intervention enhances this autophagic activation (Fig. 6A and B).

Fig. 6.

Extracellular ubiquitin (eUb) enhanced autophagy and inhibited mitochondria-mediated apoptosis in rat spinal cord injury (SCI) neurons. (A) Western blot analysis was performed to examine the expression of proteins related to cellular autophagy and mitochondria-dependent apoptosis after SCI and/or eUb treatment. (B) Semiquantitative detection of the expression levels of proteins related to cellular autophagy and mitochondria-dependent apoptosis after SCI and/or eUb treatment. One-way analysis of variance was used to calculate the p-value for the mean±standard deviation of 3 independent experiments. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001.

We also examined the expression levels of mitochondrial apoptosis pathway-related proteins, including Bax, Bcl-2, Caspase-3, Cleaved-caspase-3, Caspase-9, and Cleaved-caspase-9. The results showed that the ratios of Bax/Bcl-2, Cleaved-caspase-3/Caspase-3, and Cleaved-caspase-9/Caspase-9 were significantly upregulated in the SCI group compared to the sham-operated group. However, eUb treatment significantly reversed these changes (Fig. 6A and B). These findings further confirm that early intervention with eUb can protect spinal cord-injured neurons by enhancing cellular autophagy and inhibiting the mitochondrial apoptotic pathway.

7. eUb Protects Injured Neurons via CXCR4 Receptors

To further investigate the role of the Ub cell surface receptor CXCR4 in eUb-mediated neuroprotection, we used the AMD3100 which is a well-recognized and highly specific antagonist of the CXCR4 in our study. Western blotting results showed that eUb treatment further upregulated the ratio of autophagy-related proteins LC3B II/ I as well as the expression of Beclin1, ATG5, and ATG7, while inhibiting the expression of P62 after OGD injury. Simultaneously, eUb intervention decreased the ratios of mitochondrial apoptosis pathway-related proteins Bax/Bcl-2, Cleaved-caspase-3/Caspase-3, and Cleaved-caspase-9/Caspase-9. However, these protective effects were markedly reversed by AMD3100 (Fig. 7A and B). Interestingly, we also found that after OGD injury, eUb intervention significantly downregulated the ratios of PI3K/Akt/mTOR pathway-associated proteins pPI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR, and this inhibitory effect was also counteracted by AMD3100 (Fig. 8A and B). These results suggest that eUb enhances autophagy and exerts anti-apoptosis effects by interacting with the CXCR4 receptor.

Fig. 7.

The effects of extracellular ubiquitin (eUb) on promoting autophagy and reducing apoptosis are closely related to its interaction with the CXCR4 receptor. (A) Cells were pretreated with AMD3100 (100 μm) for 15 minutes, followed by eUb and oxygen glucose deprivation (OGD) treatment. Western blot analysis was performed to examine the mitochondrial apoptotic pathway and the expression of autophagy-related proteins. (B) Semiquantitative detection of the expression levels of mitochondrial apoptotic pathways and autophagy-related proteins. One-way analysis of variance was used to calculate the p-value for the mean± standard deviation of 3 independent experiments. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001.

Fig. 8.

AMD3100 limited the inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mechanical target of rapamycin (mTOR) signaling pathway by eUb via blocking CXCR4. (A) Western blot analysis was performed to examine the expression of proteins related to the PI3K/Akt/mTOR signaling pathway after AMD3100 treatment. (B) Semiquantitative detection of the expression levels of PI3K/Akt/mTOR signaling pathway-related proteins. One-way analysis of variance was used to calculate the p-value for the mean±standard deviation of 3 independent experiments. p-PI3K, phospho-PI3K; p-Akt, phosphor-Akt; p-mTOR, phosphor-mTOR. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001.

DISCUSSION

SCI is a complex and devastating condition that can severely disrupt life and poses a significant public health challenge worldwide. Neurons are the most critical component of the spinal cord, and neuronal loss is the leading cause of poor functional recovery after SCI [35]. Reducing secondary neuronal loss or protecting neurons from cell death is a primary goal in SCI treatment. Consequently, this study focused on whether eUb can exert neuroprotection effects in the early stage of SCI and explored its potential mechanisms. We found that eUb protects injured spinal cord neurons and promotes the repair of spinal cord tissue damage. Early intervention with eUb upregulates autophagy levels in spinal cord neurons via CXCR4 and reduces the overactivation of the mitochondria-dependent apoptotic pathway. In addition, this protective effect of eUb appears closely related to the inhibition of the PI3K/Akt/mTOR pathway.

Apoptosis is a programmed, energy-dependent form of cell death that begins within hours following the primary injury to the spinal cord [3]. Among the apoptosis pathways, the mitochondria-mediated endogenous apoptotic pathway (also referred to as the mitochondrial apoptotic pathway) is a major signaling route for apoptosis and plays a crucial role in secondary SCI. The early stage of SCI is characterized by ischemic damage [36], where stimuli such as ischemia and hypoxia disrupt the intracellular balance between pro-apoptotic and antiapoptotic proteins (Bcl-2 family proteins). This disruption leads to increased mitochondrial membrane permeability and the release of apoptotic factors, such as Cyt-C, which activate the caspase family (e.g., Caspase-9, Caspase-3) and trigger apoptosis [37,38]. In the present study, the cell survival and apoptosis rates of SH-SY5Y cells were significantly altered postinjury, indicating the successful establishment of an OGD injury model to mimic ischemic injury in spinal cord neurons. Furthermore, the ratio of Bax/Bcl-2 and the expression of activated Caspase-3, Caspase-9, and Cyt-C were significantly upregulated in the in vivo rat SCI model and in the in vitro OGD model, suggesting that mitochondria-dependent apoptosis occurs in spinal cord neurons during the early phase of SCI.

In recent years, the role of apoptosis in SCI has received increasing attention. Several studies have shown that eUb regulates apoptosis and cellular tolerance to ischemic injury. Zhang et al. [39] suggested that eUb infusion reduces apoptosis in melanoma cells, which promotes lung metastasis and decreases long-term survival in melanoma mice. Another study found that Ub treatment postmyocardial ischemic injury improved cardiac function and was associated with reduced cardiomyocyte apoptosis, hypertrophy, and serum cytokine/chemokine levels [27]. In brain injury, eUb treatment also decreases the volume of brain contusion in rats and promotes microglia/macrophage activation after brain injury, thereby accelerating the healing process [22,40]. To investigate the effect of eUb on neuronal apoptosis in the early stage of SCI, this study employed an in vivo rat spinal cord contusion model and an in vitro OGD injury model. The results showed that early intervention with eUb significantly reversed the injury-induced Bax/Bcl-2 ratio and upregulated the expression of Cleaved-caspase-3, Cleaved-caspase-9, and Cyt-C, indicating that eUb significantly inhibited injury-induced mitochondria-dependent apoptosis.

Interestingly, we also observed that eUb intervention upregulated the ratio of LC3B II/ I and the expression levels of autophagy-related genes Beclin1, Atg5, and Atg7, while decreasing the expression level of P62. It is well established that autophagy is an essential mechanism for maintaining intracellular homeostasis and can be activated by stimuli such as hypoxia, oxidative stress, or nutrient deprivation. Several studies have confirmed that moderately enhanced autophagy can protect spinal cord neurons from SCI-induced apoptosis and promote functional recovery after SCI [41-43]. Furthermore, recent studies have shown that Ub-mediated ubiquitination is closely linked to autophagy, influencing autophagic flux and regulating the expression of various autophagy-related proteins [44-47]. The deubiquitinating enzyme USP14 can negatively regulate autophagy by controlling K63 ubiquitination of Beclin1, and inhibition of USP14 may provide a strategy to promote autophagy [48]. In myocardial ischemic injury, eUb protects cardiomyocytes from ischemic/hypoxic injury by upregulating autophagy levels and inhibiting mitochondrial apoptotic pathways [23]. These findings align with our results, leading us to hypothesize that eUb may also exert neuroprotective effects by promoting autophagy to inhibit neuronal death, including apoptosis, following SCI. However, this hypothesis requires further experimental validation.

It has been reported that eUb can be internalized into cells to exert biological effects through various mechanisms, with the CXCR4-dependent mechanism being one of them [49-51]. The chemokine receptor CXCR4 is a G protein-coupled receptor with a seven-transmembrane structure consisting of 352 amino acids [52]. In 2010, Saini et al. [49] provided the first evidence that CXCR4 can function as a Ub cell surface receptor and that pretreatment with the CXCR4 inhibitor AMD3100 prevented the cellular internalization of eUb. Other studies have shown that eUb can stimulate angiogenesis in cardiac microvascular endothelial cells and regulate cardiac fibroblast function and phenotype through interaction with CXCR4 [21,53]. Additionally, Ji et al. [23] found that eUb protects cardiomyocytes from ischemia/hypoxia-induced injury by inhibiting the mitochondrial apoptotic pathway via CXCR4. Inhibition of the CXCR4 receptor using AMD3100 abolished the cardioprotective effects of eUb.

In conclusion, these studies provide evidence that eUb acts through CXCR4. Similarly, in this part of the study, we observed that eUb enhances cellular autophagy and inhibits mitochondrial apoptotic pathways, thereby protecting spinal cord neurons from ischemic injury. In contrast, the antiapoptotic and autophagy-enhancing effects of eUb were limited under the intervention of the CXCR4 inhibitor AMD3100. This confirms that the protective effects of eUb on apoptosis and autophagy are closely related to the involvement of CXCR4 signaling.

Our study also found that neurons exposed to OGD injury in vitro exhibited activation of the PI3K/Akt/mTOR signaling pathway. Several studies have reported that the PI3K/Akt/mTOR pathway is essential in SCI pathological processes and neuronal recovery mechanisms. For example, it has been shown that fibroblast growth factor 18 and melatonin can promote autophagy and inhibit cell death by modulating the Akt/mTOR signaling pathway, thereby promoting motor recovery after SCI [54,55]. Inhibiting mTOR or using the mTOR inhibitor rapamycin can enhance autophagy and reduce apoptosis, thereby promoting neurological function recovery after SCI [56,57]. In addition, Daniels et al. [58] found that eUb plays a protective role in β-AR-stimulated cardiomyocyte apoptosis, and activation of the PI3K/Akt signaling pathway may be the mechanism involved in the antiapoptotic effects of eUb. It has been suggested that eUb inhibits apoptosis in damaged cardiomyocytes and that this effect is closely associated with the PI3K/Akt pathway. Our study found that eUb modulated the activation of the PI3K/Akt pathway while enhancing neuronal autophagy and inhibiting mitochondria-dependent apoptosis, consistent with the results of Daniels’ study. However, unlike Daniels’ study, our experiments revealed that eUb intervention inhibited the PI3K/Akt/mTOR pathway in damaged neurons, and this effect was blocked by AMD3100. The differences in these studies may be due to different cell types and injury models, as well as the presence of cellular metabolic disturbances, suggesting that eUb-regulated signaling pathways can vary under different conditions.

This is the first SCI-related study to provide evidence that eUb enhances neuronal autophagy and reduces early neuronal apoptosis. However, there are some limitations to this study. Firstly, how eUb inhibits the PI3K/Akt/mTOR pathway and the relationship between eUb’s antiapoptotic effects, its role in enhancing autophagy, and its interaction with the PI3K/Akt/mTOR pathway were not clarified, which will be the focus of our subsequent study. Secondly, instead of primary spinal cord neurons, we used SH-SY5Y cells for our in vitro study.

CONCLUSION

In summary, the results of the present study suggest that early administration of eUb in SCI can protect injured spinal cord neurons and facilitate the repair of spinal cord tissue by enhancing autophagy and inhibiting mitochondrial apoptotic pathways via CXCR4. These findings provide a new avenue for therapeutic strategy in SCI, indicating that extracellular Ub may emerge as a promising treatment for the early management of SCI.

Notes

Conflict of Interest

The authors have nothing to disclose.

Funding/Support

This study was supported by National Natural Science Foundation of China (Grant number: 82072407) and Fujian provincial health technology project (Grant number: 2023CXA013).

Author Contribution

Conceptualization: HF, DC, HC, ZW, WL; Data curation: HF, DC, HC, DWu, DWang; Formal analysis: HF, DC, HC, DWu, DWang; Funding acquisition: ZW, WL; Methodology: HF, DC, HC, ZY, LZ; Project administration: HF, ZW, WL; Visualization: HF, DC, HC, DWu, ZY; Writing – original draft: HF, DC, HC, DWu, DWang, ZY; Writing – review & editing: HF, DC, LZ, ZW, WL.

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