Bone Marrow Mesenchymal Stem Cells Modulate miR-202-3p to Suppress Neuronal Apoptosis Following Spinal Cord Injury Through Autophagy Activation via the AMPK, MAPK, and PI3K/AKT/mTOR Signaling Pathway

doi:10.21203/rs.3.rs-4699112/v1

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Bone Marrow Mesenchymal Stem Cells Modulate miR-202-3p to Suppress Neuronal Apoptosis Following Spinal Cord Injury Through Autophagy Activation via the AMPK, MAPK, and PI3K/AKT/mTOR Signaling Pathway

https://doi.org/10.21203/rs.3.rs-4699112/v1

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Background: Bone marrow mesenchymal stem cells (BMMSCs) have garnered attention as promising therapeutic modalities for spinal cord injury (SCI) due to their neuroregenerative, anti-apoptotic, and functional recovery-enhancing properties. The central role of microRNAs (miRNAs) in mediating the beneficial outcomes resulting from BMMSCs in SCI has been highlighted in recent studies, suggesting that targeted modulation of specific miRNAs holds potential for augmenting SCI recovery. Our previous investigation implicated miR-202-3p in the reparative processes of injured spinal cords, although the precise mechanistic underpinnings remain elusive.

Methods: In vivo, BMMSCs were administered to SCI rats, while in vitro, miR-202-3p was transfected into PC-12 cells. Motor capabilities recovery was assessed via Basso-Beattie-Bresnahan (BBB) scores and footprinting tests; the evaluation of neuronal and spinal cord tissue repair was conducted using Nissl staining, TUNEL staining, hematoxylin and eosin (HE) staining, and immunofluorescence; and the impacts of miR-202-3p on cellular autophagy, neuronal apoptosis, and relevant pathways were evaluated using Western blotting, quantitative polymerase chain reaction (qPCR), and transmission electron microscopy (TEM).

Results: Functionally, BMMSCs utilized miR-202-3p to improve motor recovery in SCI rats. Histopathologically, they contributed to the repair of damaged spinal cords and the regeneration of nerve axons. At the molecular level, BMMSCs stimulated autophagy and suppressed neuronal apoptosis by regulating the AMPK, MAPK, and PI3K/AKT/mTOR pathway.

Conclusions: Collectively, our findings demonstrate that BMMSCs coordinate miR-202-3p to inhibit mTOR activation via the AMPK, MAPK, and PI3K/AKT pathways, thereby promoting TFEB dephosphorylation, modulating autophagy and neuronal apoptosis, and ultimately fostering functional recovery post-SCI.

Biological sciences/Neuroscience

Biological sciences/Stem cells

BMMSCs

SCI

miR-202-3p

autophagy

neuronal apoptosis

Spinal cord injury (SCI) ranks among the most prevalent types of neurotrauma, characterized by segmental paralysis, sensory deficits, and a spectrum of dysfunctions, imposing profound physical, socioeconomic, and psychosocial burdens on affected individuals^[1-3]. The development of SCI involves both initial and subsequent injuries. ^{[4, 5]}. Primary SCI pertains to the initial mechanical trauma inflicted upon the spinal cord resulting from mechanical forces such as squeezing, crushing, tearing or traction ^[6-8]. Secondary injuries arise from a complex interplay of pathophysiological processes, these factors include apoptosis, autophagy, inflammation, oxidative stress, and other related mechanisms, perturbing the microenvironment and exacerbating neurological impairment^{[9, 10]}. Hence, effective management of secondary damage assumes paramount importance in optimizing SCI recovery^[11-13]. Notably, cellular autophagy and neuronal apoptosis stand out as pivotal targets for mitigating secondary injury within the pathophysiological cascade of SCI^{[14, 15]}.

Autophagy, vital for clearing cytoplasmic waste and damaged organelles, is crucial in promoting neuroprotection and facilitating repair following SCI^[16]. Neuronal apoptosis, characterized by programmed cell death of neurons, represents a substantial contributor to neurological deficits and is linked to unfavorable prognostic outcomes in SCI cases^[17]. Studies have underscored the intricate interplay between cellular inflammation, oxidative stress, and apoptosis in SCI pathogenesis, highlighting the interconnectedness with autophagic processes^[18]. Enhancing autophagy has been identified as a hopeful avenue for therapy to enhance the restoration of functionality following SCI.

The inherent limited regenerative potential of neurons poses a formidable obstacle in the development of efficacious clinical interventions for SCI. Recent investigations have highlighted cell transplantation as a promising avenue for enhancing the recuperation of motor, sensory, and autonomic functions postoperative^[19]. BMMSCs have emerged as a valuable asset in the realm of spinal cord repair^[20], demonstrating notable neuroprotective effects^[21], facilitation of axonal regeneration^[22], and attenuation of astrocyte hyperactivation^[23]. Moreover, miRNAs, a class of eukaryotic bodily noncoding single-stranded RNAs, are recognized for their regulatory roles in inflammation, apoptosis, autophagy, and other pathophysiological processes^[24]. Various studies have implicated specific miRNAs in mediating the therapeutic benefits of BMMSCs in SCI. For instance, BMMSC-derived exosomal miR-455-5p stimulates autophagy while suppressing neuronal apoptosis in rats with SCI ^[25]. Additionally, research by Wei et al^[26]. demonstrated that miR-383 inhibition elevated GDNF protein levels, boosting the therapeutic efficacy of human BMMSCs in managing SCI. Furthermore, our prior investigations unveiled that miR-202-3p overexpression in vitro diminished the expression of inflammation markers, while its inhibition led to their upregulation, implicating miR-202-3p in the reparative processes of injured spinal cords.

Despite extensive exploration of miRNAs and apoptosis in SCI utilizing diverse animal models, including rodents, the specific impact of BMMSC transplantation on miRNA expression profiles and cellular autophagy post-SCI remains uncharted territory. Furthermore, the potential of BMMSCs to mitigate neuronal apoptosis through autophagy activation remains elusive. Hence, this study was designed to elucidate the impacts and associated biochemical pathways of miR-202-3p-mediated cellular autophagy in the context of BMMSC transplantation for spinal cord repair. These findings are poised to offer novel insights and theoretical foundations for advancing the clinical management of SCI.

MiRNA-202-3p mimics, miR-202-3p NC and miRNA-202-3p inhibitor were acquired through ChemShine Biotechnology, Inc. Primary antibodies targeting Erk/P38/JNK, p-Erk/P38/JNK, mTOR, p-mTOR, GFAP, Neun, GAP43, MAP2, AKT, p-AKT, Bcl-xL, LC3II/I, and Histone3 were sourced from Cell Signaling Technology. The p62 antibody was obtained from ABMART. Antibodies against p-AMPK were acquired from Bioswamp, while those against PI3K were obtained from Abcam and p-PI3K from HUABIO. Beclin1 antibodies were purchased from BOSTER, and antibodies against GAPDH, TFEB, NIX, and BAX were sourced from Proteintech. Antibodies targeting AMPK, VPS34, CTSD, Parkin, and BNIP3 were procured from Immunoway. Rapamycin was obtained from MCE. Zolgene custom-designed the polymerase chain reaction (PCR) primers. and reagents for reverse transcription and Vazyme provided the kits for reverse transcription and quantitative polymerase chain reaction (qPCR).

3.1 Animal and SCI models

A cohort of 54 adult female Sprague-Dawley (SD) rats weighing around 220 g was acquired via Zhejiang Viton Lever Co. All procedures involving the animals adhered to the regulatory framework outlined in the Standards for National Management and Utilisation of Experimental Rats in China. Subsequently, the animals underwent random allocation to three distinct experimental cohorts: Sham operation (Sham group), SCI induced by extrusion (SCI group), and SCI intervention with BMMSCs (BMMSCs group). Prior to the surgical intervention, anaesthesia was induced through intraperitoneal administration of a 1% (w/v) solution of pentobarbital sodium. The dose was 40 mg/kg. Following successful anesthesia and positioning in the prone orientation, the dorsal region was shaved to expose the spinous processes and vertebral segments within the thoracic spine, specifically at the levels of T9 through T11, and the surgical site was aseptically prepared (Figure 1A). The spinal cord was exposed by removing the vertebral plate centered on T10, followed by a 10-second compression using vascular clamps to establish the SCI model. Postoperatively, upon the rats regaining consciousness, intraperitoneal administration of 50,000 U/kg penicillin was carried out to prevent infections, fluid loss was replenished with 5 ml saline over three days. Twice daily, Expressing the bladder manually was conducted until bladder voiding occurring naturally occurred. In the BMMSCs group, 0.5 ml of third-generation (P3) BMMSCs (1 × 107/ml) was intravenously injected via the caudal vein 3 days post-injury, while in the SCI group, injection was made with an equal volume of PBS.

3.2 Behavioural assessment

Functional recovery following SCI was evaluated utilizing the Basso, Beattie, and Bresnahan (BBB) assessment and Footprinting. The BBB scale evaluations took place in an unconfined setting at postoperative days 1, 3, 7, 14, 21, and 28, with sensory and motor functions evaluated by three blinded investigators. The assessment scale, spanning from 0 (representing paralysis of the whole body) to 21 (representing full functional recovery)^[27], was recorded independently by each investigator, and the composite score was derived by computing the mean of the evaluations. Following this, at the 28-day juncture, rats' forelimbs and hindlimbs were delineated with red and blue ink correspondingly, before allowing the animals to traverse a white paper-padded pathway to document their footprints. Two impartial observers independently examined the footprints for the experimental parameters without prior knowledge.

3.3 Tissue section preparation and HE and Nissl staining

Rats were sedated with 1% (w/v) solution of pentobarbital sodium and then perfused via the left ventricle with 0.9% sodium chloride solution. Following perfusion, the affected spinal cord tissues (as depicted in Figure 1B) were extracted and immersed in 4% paraformaldehyde (w/v) solution for fixation over a period of 24 hours. Subsequently, the tissues underwent dehydration, embedding, and sectioning into longitudinal and transverse slices measuring 4-5 μm in thickness. Nissl staining and haematoxylin and eosin (HE) staining were performed on these sections. Pathological alterations, including changes in the spinal cord tissue cavity area, inflammatory cell infiltration, and neuronal apoptosis, were evaluated through image acquisition using a pathology section scanner for further detailed analysis.

3.4 Tissue immunofluorescence

Paraffin-embedded spinal cord tissue sections, measuring 5 μm in thickness, were prepared in both longitudinal and transverse orientations. After antigen retrieval, the sections were treated with a 5% bovine serum albumin (BSA) solution for 2 hours at room temperature to block non-specific binding. Subsequently, they were incubated overnight at 4°C with the primary antibody to facilitate specific binding to the target antigens, including anti-GFAP (1:300; CST), anti-Neun (1:500; CST), anti-LC3II/I (1:300; CST), anti-p62 (1:400; ABMART), anti-MAP2 (1:100; CST), anti-Bcl-xL (1:300; CST) and anti-GAP43 (1:200; CST). Following this, the sections underwent incubation with a secondary antibody for 1 hour at room temperature. To visualize the nuclei, counterstaining was conducted using a solution containing 4',6-diamidino-2- phenylindole (DAPI).

3.5 TUNEL staining.

TUNEL stands for Terminal deoxynucleotidyl transferase dUTP Nick End Labelling, which was employed to detect apoptotic cells in 5-μm-thick longitudinal paraffin sections following established protocols. The sections underwent deparaffinization and antigen retrieval procedures. Subsequently, cell permeabilization was achieved by treating the sections with Triton X-100 for 12 minutes at 37°C. The sections were exposed to the experimental reagent mixtures for 0.9 hours at room temperature. Following the incubation period, DAPI was used to stain nuclei for 5 minutes. Before coverslipping, an anti-fade mounting medium was applied dropwise. Upon UV excitation, DAPI-labeled nuclei emitted blue fluorescence, while TUNEL-positive apoptotic nuclei exhibited red fluorescence.

3.6 Transmission electron microscopy

Autophagic processes within spinal cord tissues were visualized through transmission electron microscopy (TEM) analysis. The spinal cord tissues were immersed at room temperature to achieve fixation. Subsequently, the sections underwent dehydration, embedding, fixation, and staining with conventional peroxynitrite acetate. TEM imaging was conducted utilizing an HT 7700 transmission electron microscope manufactured by HITACHI.

3.7 Cell culture

PC-12 cells and BMMSCs were procured from Shanghai Anwei Biotechnology Co Ltd. Culturing of PC-12 cells involved maintenance in high-glucose Dulbecco's Modified Eagle Medium (DMEM), enriched with 10% fetal bovine serum (FBS), and supplemented with 100 units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin. This was performed in a 37°C humidified incubator with 5% CO2, and passage occurred once cells reached approximately 80% confluence. Regular medium changes every 2 days were performed, and cell morphology and growth were monitored using an inverted microscope in preparation for subsequent transfection experiments. Primary BMMSCs were cultured in specialized stem cell medium with medium changes every 2-3 days. Following passage, cells were expanded to the third cell culture passage (P3) was used for further experiments, and flow cytometry was utilized for the characterization of the cellular phenotype of P3 BMMSCs.

3.8 Transfection and grouping of cells.

The experimental setup included multiple groups: the control group, the miR-202-3p mimics group, the miR-202-3p inhibitor group, and the miR-202-3p negative control (NC) group. PC-12 cells were distributed into 6-well plates with each well harboring a population of 6×10⁵ cells. When 50%-80% confluence is reached, the cells underwent transfection with Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) along with the respective miRNAs. Following a 6-hour transfection period, the medium was replaced, and incubation with Rapamycin (0.5 μmol/L) was sustained for 48 hours. Subsequently, samples were collected for Western blot analysis to assess miR-202-3p influence on proteins associated with cellular autophagy and apoptosis. Additionally, miR-202-3p expression levels were assessed through qPCR both in vivo and in vitro.

3.9 Western blotting

RIPA lysis buffer was utilized for extract total protein from both spinal cord tissue samples and cellular specimens. Protein quantification was carried out employing a BCA protein kit (Solarbio). Subsequently, protein samples of equal quantity underwent SDS‒PAGE separation, transfer to nitrocellulose (NC) membranes, and incubation with a 5% skim milk powder solution for a duration of 2 hours. Following blocking, primary antibodies were applied to the membranes and then incubated at 4°C, targeting Beclin1 (1:1000), p62 (1:3000), Erk (1:1000), p-Erk (1:1000), mTOR (1:1000), p-mTOR (1:1000), CTSD (1:1000), VPS34 (1:1000), NeuN (1:1000), Bcl-xL (1:1000), GAP43 (1:1000), MAP2 (1:1000), and GAPDH (1:5000). Subsequent to primary antibody incubation, the membranes underwent a 2-hour incubation period at 37°C with secondary antibodies. Following washing steps, protein bands on the membrane were visualized using fluorescent reagents and captured with Image Lab software (Bio-Rad). ImageJ software was employed for quantitative analysis of protein expression levels.

3.10 qPCR

For the evaluation of miRNA-202-3p expression levels, total RNA extraction was performed from PC-12 cells and spinal cord tissues employing a specialized miRNA extraction kit (UElandy). The utilization of a NanoDrop 2000 (Thermo Scientific, USA) spectrophotometer enabled the determination of both the quality and quantity of miRNAs, followed by cDNA synthesis through reverse transcription. Subsequently, U6 was employed as an internal reference for qPCR analysis utilizing SYBR Premix Ex Taq (Bio-Rad Laboratories, Inc.).

3.11 Statistical analysis

Statistical analyses were performed using GraphPad Prism 10.0 software. Data distribution normality was evaluated utilizing the Shapiro-Wilk test. For normally distributed data, one-way analysis of variance (ANOVA) was followed by Tukey's post hoc test for further analysis. In this context, we defined statistical significance as follows: *p < 0.05 indicated significance, **p < 0.01 signified a higher level of significance, ***p < 0.001 represented even greater significance, and ****p < 0.0001 was considered highly significant.

4.1 Morphology and antigen identification of BMMSCs

Upon examination under an inverted microscope, P3 BMMSCs exhibited robust adhesion to the substrate, displaying a consistent fibroblast-like morphology characterized by uniform distribution and swirling growth patterns (Figure 1C). Subsequently, the flow cytometry analysis revealed that P3 BMMSCs exhibited positive expression of CD44 and CD90, indicating the presence of these markers on the cell surface. Conversely, the cells showed negative expression of CD45 and CD34, suggesting the absence of these markers. This surface antigen profile aligns with the characteristic phenotype of BMMSCs (Figure 1D).

4.2 BMMSCs facilitate the functional motor abilities recovery in rats with SCI.

The evaluation of mobility abilities utilizing the BBB score revealed that on the initial postoperative day, rats with SCI displayed total paralysis with a score of 0, indicating the absence of hindlimb activity (Figure 2A). By the third day following the injury, a gradual restoration of hindlimb functionality became evident, with no notable distinction observed between the group treated with BMMSCs and the SCI group. Nevertheless, by day 14 after the injury, a notable enhancement in hindlimb function became evident in the BMMSCs group compared to the SCI group (p<0.0001; Figure 2B). Remarkably, 28 days following the occurrence of the injury, the BMMSCs group demonstrated a markedly higher mean BBB score of 10, significantly exceeding the SCI group's performance (p<0.0001; Figure 2C). During the footprint analysis, SCI rats exhibited noticeable dragging in comparison to the distinct footprints observed in the Sham group. Conversely, rats treated with BMMSCs showed an increase in hind paw prints and overlapping fore and hind paw prints compared to the SCI group (Figure 2D). Quantitative assessments further revealed that the BMMSCs group exhibited a notable decrease in bases of support and stride lengths, both of which were statistically significant (p<0.05; p<0.0001; Figure 2E, F). Altogether, the findings underscore the therapeutic potential of BMMSCs in enhancing motor function restoration in rats with SCI.

4.3 BMMSCs promote histological repair of the damaged spinal cord.

Histological alterations in spinal cord morphology were assessed via HE staining. Comparative analysis unveiled conspicuous deformities and cavitations accompanied by substantial infiltration of inflammatory cells within the spinal cords of the group with SCI compared to the Sham group, suggesting extensive tissue damage. Following BMMSCs treatment, spinal cord integrity was notably restored, with attenuated infiltration of inflammatory cells and reduced cystic cavity size in contrast to the SCI group (p<0.01; p<0.01; Figure 2G-I). These observations underscore the potential of BMMSCs therapy in mitigating spinal cord tissue injury and facilitating repair processes within the damaged spinal cord.

4.4 BMMSCs inhibit neuronal apoptosis after SCI.

Studies have revealed that attenuating neuronal apoptosis is crucial for improving functional recuperation following SCI^[28]. TUNEL staining unveiled a heightened incidence of neuronal loss in the SCI group contrasted with the Sham group, yet therapy utilizing BMMSCs mitigated this phenomenon (Figure 3A). Furthermore, assessment of neuronal preservation via Nissl staining revealed a greater density of intact neurons in the Sham group, in contrast to the presence of atrophied neuronal cell bodies and Nysted granule lysis in the SCI group. Notably, BMMSCs treatment significantly augmented neuronal numbers and restored neuronal morphology (Figure 3B), a finding corroborated by immunofluorescence staining (p<0.05; Figure 3C-D). Western blotting further substantiated the BMMSCs treatment partially reinstated the protein expression of NeuN (p<0.001, Figure 3E-F). Additionally, the BMMSCs group demonstrated decreased levels of the proapoptotic protein BAX and heightened expression of the antiapoptotic protein Bcl-xL in contrast to the SCI group (p<0.001; p<0.01; Figure 3G-I). To summarize, our results indicate that BMMSCs possess the capacity to effectively suppress neuronal apoptosis within the injured spinal cord.

4.5 BMMSCs protect neurons and promote axonal regeneration.

Microtubule-associated protein 2 (MAP2) serves as a mature neuronal marker crucial for regulating microtubule architecture, stability, and organelle transport within axonal and dendritic compartments. Immunofluorescence staining, coupled with quantitative analysis, revealed a reduced population of MAP2-positive axons within the SCI group in comparison to the Sham group. Additionally, compared to the Sham group, the SCI group showed a notable increase in the distance between the epicentre of the injury and the nearest neuron (p <0.01; Figure 4A-C). Conversely, the BMMSCs group exhibited heightened density and intensity of MAP2-positive axons, facilitating axonal growth towards the injury site (p <0.05; p <0.05; Figure 4A-C). Growth-associated protein 43 (GAP43), pivotal in nerve regeneration and axon guidance, plays a crucial role in fostering new axonal connections^[29]. Notably, the SCI group displayed a dearth of significant GAP43-positive axons within the fibrotic scar encircled by activated astrocytes, whereas BMMSCs treatment engendered a notable increase in GAP43-positive axons compared to the SCI group (p <0.05; Figure 4D, E). In line with the results observed in immunofluorescence analysis, Western blotting corroborated the upregulation of MAP2 and GAP43 expression following BMMSCs intervention (p <0.05; p <0.05; Figure 3F-H). Collectively, these outcomes underscore the neuroprotective effects of BMMSCs in safeguarding neurons and fostering axonal regeneration post-SCI.

4.6 BMMSCs enhance autophagy after SCI.

Autophagy has garnered attention as an exciting way to treat central nervous system (CNS) diseases, exerting a pivotal role in modulating programmed cell death triggered by neuroinflammation^[30]. To evaluate autophagic responses in spinal cord lesions post-SCI, we scrutinized the levels of protein markers associated with autophagosomes LC3II/I, Beclin1, VPS34, CTSD, and the autophagic cargo receptor protein p62 using immunofluorescence and Western blotting. Western blot analysis demonstrated a reduction in p62 protein levels concurrent with LC3II/I, Beclin1, VPS34 and CTSD upregulated after treatment with BMMSCs (p <0.01; p <0.05; p <0.05; p <0.05; p <0.001; Figure 5A-F). Furthermore, immunofluorescence demonstrated diminished p62 density and heightened LC3II signal within the BMMSCs group relative to the SCI group (p <0.05; p <0.01; Figure 5 G-J). BMMSCs not only augmented levels of autophagosome and autophagolysosome-associated markers but also alleviated the accumulation of autophagic substrates, potentially attributable to the overall enhancement of autophagic activity instigated by BMMSCs post-SCI.

4.7 BMMSCs enhance mitophagy after SCI.

To further substantiate the influence of BMMSCs on autophagic processes in SCI rats, we performed TEM analysis on sections of spinal cord tissue. TEM assessment unveiled a notable escalation with an abundance of autophagosomes within the BMMSCs group relative to the SCI group, as denoted by the red arrows, indicative of BMMSCs-induced autophagy activation (Figure 6A). Additionally, we observed alterations in mitochondrial morphology in the BMMSCs group. Contrasting with the oval-shaped mitochondria with internal cristae observed in the Sham and SCI groups, the mitochondria exhibited a contracted and rounded appearance post-BMMSCs treatment, suggestive of mitochondrial stress. Prior investigations^[31]have underscored the intricate interplay between autophagy and mitochondrial autophagy. Hence, we postulated that BMMSCs could potentiate mitochondrial autophagy, potentially through autophagy activation. To validate this conjecture, Parkin, BNIP3 and Nix expression was assessed in all groups via Western blot analysis and immunofluorescence. Immunofluorescence analyses revealed a higher abundance of Nix-positive neurons in the Sham group relative to the SCI group, with a significant augmentation in Nix-positive neurons following BMMSCs treatment (p <0.001; p <0.05; Figure 6 B-C). The Western blotting unveiled revealed heightened expressions of Parkin, BNIP3, and Nix proteins within the SCI group as opposed to the Sham group (p <0.001; p <0.05; p <0.0001, Figure 6 D-G). Notably, the BMMSCs group exhibited enhanced protein expression levels of the markers in comparison to the SCI group (p <0.01; p <0.01; p <0.01; Figure 6 D-G). Collectively, these findings suggest that BMMSCs augment mitochondrial autophagy, potentially through their pro-autophagic properties.

4.8 BMMSCs promote autophagy through the AMPK, MAPK and PI3K/AKT

/mTOR/TFEB signaling cascade to promote functional recuperation following SCI

Our study aimed to clarify the fundamental mechanisms through which BMMSCs modulate autophagy and mitochondrial autophagy. TFEB serves as a pivotal regulator within the autophagy-lysosomal signaling cascade. Its potential involvement in autophagy regulation was investigated. In the SCI group, Western blot analysis unveiled heightened expression of p-AMPK and augmented the relocation of TFEB to the nucleus, concomitant with reduced levels of p-mTOR compared to the Sham group (p <0.05; p <0.05; p <0.0001; Figure 7 A-B, G-J). Remarkably, BMMSCs treatment further augmented p-AMPK levels and nuclear translocation of TFEB while diminishing p-mTOR levels (p <0.01; p <0.01; p <0.01; Figure 7 A-B, G-J). Moreover, the investigation revealed that BMMSCs facilitated the stimulation of Erk, JNK, and p38 MAPK signaling cascade, as evidenced through elevated levels of p-Erk, p-JNK, and p-P38 relative to the SCI group (p <0.05; p <0.01; p <0.001; Figure 7 C-D). Furthermore, BMMSCs were observed to attenuate the levels of phosphorylated PI3K and AKT (p <0.01; p <0.01; Figure 7 E-F). Taken together, these findings suggest that the activation of the AMPK, Erk/JNK/P38, and PI3K/AKT pathways inhibits mTOR activation and facilitates TFEB dephosphorylation, thereby enhancing mitochondrial autophagy in the context of SCI (Figure 7 G-J).

4.9 MiR-202-3p mimics inhibit apoptosis and promote autophagy in vivo and in vitro.

Our previous study illustrated that in vitro overexpression of miR-202-3p led to a notable reduction of pro-inflammatory cytokines levels such as TNF-α, IL-6, and IL-1β, whereas downregulation of miR-202-3p expression resulted in an elevation in these cytokines. This observation provides evidence that miR-202-3p may be implicated in the reparative processes associated with SCI. Consequently, we hypothesized that BMMSCs may exert their effects through miR-202-3p-mediated autophagy. In vivo, our examination of spinal cord tissue disclosed markedly elevated levels of miR-202-3p in the BMMSCs treatment group relative to the SCI group (p <0.0001; Figure 8A). To better elucidate the impact of miR-202-3p on neuronal cells, PC-12 cells underwent transfection with miR-202-3p mimics, miR-202-3p inhibitor, and miR-202-3p NC. The transfection experiments yielded compelling evidence indicating a substantial upregulation of miR-202-3p expression within the group transfected with miR-202-3p mimics. Notably, the inhibitor's mechanism of action involves sequestering endogenous free miRNA rather than degrading it, resulting in an insignificant decrease in miR-202-3p levels in the inhibitor group (Figure 8 B-C). Subsequently, we explored how miR-202-3p may modulate autophagy and apoptosis after SCI, given its crucial function in preserving cellular balance and enhancing cell survival under challenging circumstances. Western blot analysis revealed that transfection with miR-202-3p mimics upregulated LC3-II/I protein expression and downregulated p62 and BAX protein expression. Conversely, transfection with the miR-202-3p inhibitor led to the downregulation of LC3-II/I protein expression and the upregulation of p62 and BAX protein expression (Figure 8 D-F). Notably, the pro-autophagic and anti-apoptotic effects of miR-202-3p were more pronounced with increasing concentrations of miR-202-3p mimics (Figure 8 G-I). In summary, our findings indicate that miR-202-3p can suppress apoptosis and enhance autophagy both in vivo and in vitro.

SCI can lead to substantial and enduring functional deficits, imposing significant burdens on individuals, families, and society^[32-34]. BMMSCs have emerged as a prominent focus of research due to their strong capability for self-renewal and their potential to differentiate into multiple lineages^[35]. Multiple studies have underscored the crucial function of BMMSCs in the treatment of diverse conditions, encompassing SCI, as well as the regeneration of bone and cartilage, and management of autoimmune disorders. Our investigation demonstrates the favorable impact of BMMSCs on functional recuperation post-SCI. Mechanistically, our findings propose that the therapeutic efficacy of BMMSCs may be attributed to miR-202-3p modulation of the AMPK, MAPK, and PI3K/AKT/mTOR/TFEB pathways, resulting in the stimulation of mitochondrial autophagy and suppression of neuronal apoptosis.

Autophagy, an intracellular degradation process facilitated by lysosomes, serves as a fundamental mechanism for eliminating misfolded albuminoid or dysfunctional cellular components, thus exerting a critical influence on human health and disease^{[36, 37]}. Despite ongoing debates, it is postulated that the activation of autophagy following SCI confers the revitalization of neurons. These effects are characterized by microtubule stabilization, facilitation of axonal regeneration, and a decreased incidence of apoptosis during the recovery phase^[38-40]. Our study not only showed the advantageous impact of BMMSCs on SCI outcomes but also revealed through Western blotting, TEM, and immunofluorescence analyses that these favorable effects were predominantly attributed to the enhancement of autophagic processes.

SCI pathogenesis is complex, involving diverse pathological cascades, with neuronal apoptosis playing a pivotal role in secondary injury progression^[41]. In this investigation, we postulated that BMMSCs could mitigate neuronal apoptosis while enhancing autophagy in SCI. To substantiate the neuroprotective effects of BMMSCs post-SCI, we evaluated neuronal apoptosis levels through Western blotting and TUNEL staining. Consistent with our hypothesis, Bax expression markedly decreased in SCI rats following BMMSC treatment, accompanied by an elevation in Bcl-xL levels. TUNEL assay results further confirmed the efficacy of BMMSCs in preventing neuronal apoptosis post-SCI. MiRNAs represent a subclass of abbreviated endogenous non-coding RNA molecules, typically spanning 21-25 nucleotides, pivotal in orchestrating the pathophysiological mechanisms underlying SCI. Significantly, the transfer of miRNAs via exosomes to neuronal, neuroglial, and endotheliocyte following SCI has demonstrated the capacity to modulate cellular processes, thereby either facilitating or impeding reparative mechanisms^[42]. Our prior work demonstrated that the induction of miR-202-3p expression attenuated the inflammation following SCI in vitro. Given the reparative potential of BMMSCs in SCI and the significant interplay between miRNAs and BMMSCs, we postulated that miR-202-3p may represent a mechanistic link in BMMSC transplantation for SCI treatment.

To interrogate this hypothesis, we initially scrutinized the alterations in miR-202-3p levels post-SCI. In our study, an increase in miR-202-3p expression was noted in the SCI group in comparison with the Sham group, potentially attributed to autophagy induction. Notably, miR-202-3p expression levels were further augmented following treatment with BMMSCs. To unravel the impact of miR-202-3p on neuronal cells, we transfected PC-12 cells with miR-202-3p mimics, miR-202-3p inhibitor, and miR-202-3p NC. The transfection outcomes illustrated a significant increase in miR-202-3p levels in the mimics group, while the inhibitor group did not exhibit a notable decrease in miR-202-3p levels. This phenomenon may be attributed to the mode of action of the miR-202-3p inhibitor, which sequesters endogenous free miRNA rather than degrading it. Furthermore, Western blotting analyses revealed that transfecting PC-12 cells with miR-202-3p mimics resulted in an upregulation of LC3-II/I and Beclin1 protein expression, concomitant with a downregulation of p62 and BAX protein levels. Notably, the impact was more pronounced with escalating concentrations of miR-202-3p mimics. Collectively, the results of this study indicate that BMMSCs leverage miR-202-3p to ameliorate the prognosis of SCI by fostering autophagy and suppressing apoptosis.

To clarify the mechanisms behind the promotion of autophagy by BMMSCs in SCI, we delved into the upstream regulatory pathways of autophagy. Our investigation unveiled that the therapeutic effects of BMMSCs in SCI are intricately linked to the modulation of three pivotal signaling cascades: the PI3K/AKT, AMPK, and MAPK pathways. TFEB, a nuclear transcription factor, has been associated with a spectrum of human pathologies and physiological mechanisms, encompassing autophagy, apoptosis, inflammatory responses, and cellular homeostatic mechanisms^{[43, 44]}. The dephosphorylation and subsequent relocation to the nucleus of TFEB are essential for robust autophagy induction and the reestablishment of cellular energy homeostasis via the synthesis of adenosine triphosphate (ATP) from intrinsic metabolic substrates ^[45].AMPK acts as a pivotal controller of autophagy, triggered in scenarios of nutrient deprivation, inadequate energy levels, or oxidative stress.AMPK activation can directly or indirectly suppress mTOR activity, thereby enhancing autophagy^{[46, 47]}. When mTOR activation is inhibited, TFEB undergoes dephosphorylation, subsequently translocating to the cytoblast^[48]. In our current study, we observed that BMMSCs elevate AMPK phosphorylation while inhibiting mTOR phosphorylation. The MAPK pathway, encompassing Erk, JNK, and P38 MAPK cascades, is implicated in cellular processes such as autophagy and apoptosis^[49]. Our findings suggest that BMMSCs activate Erk/JNK/P38 signaling and downregulate mTOR/TFEB, thereby promoting autophagy. However, the MAPK pathway, particularly the P38 MAPK pathway, may exhibit a dual role in autophagy regulation, influenced by the stimulus nature and the duration and intensity of pathway activation^[50]. Given the intricate interplay among Erk, JNK, and P38 MAPK pathways and the complex regulation of autophagy, additional research into the molecular intricacies of MAPK signaling and its impact on autophagy modulation is warranted. Moreover, BMMSCs can enhance autophagy by suppressing the PI3K/AKT pathway. Across the PI3K/AKT, AMPK, and Erk/JNK/P38 pathways, mTOR activation was ultimately inhibited, promoting TFEB dephosphorylation to augment autophagy in SCI. In summary, our results underscore that BMMSCs harness miR-202-3p to enhance autophagic flux, mitigate neuronal apoptosis, and thereby promote functional recuperation in SCI rats by modulating the AMPK, MAPK, and PI3K/AKT/mTOR/TFEB pathway. While preclinical studies exhibit promise, critical considerations such as ethical concerns, therapeutic efficacy, complications, immune responses, and tumorigenic potential must be addressed before clinical translation of BMMSC therapy (Figure 9). Future investigations should prioritize elucidating the enduring effects of BMMSCs therapy, refining delivery methodologies, and executing rigorous clinical trials .

Ethics approval and consent to participate: All procedures in this study were performed in accordance with the international ethical statutes and law for the protection of animals and were approved by the Animal Research Committee of the First Affiliated Hospital of Nanchang University (Approval No. CDYFY-IACUC 202211 QR 007; Approval Date: 14 November 2022)

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the First Affiliated Hospital of Nanchang University. All methods are reported following ARRIVE guidelines(https://arriftguidelines.org) for the reporting of animal experiments.

Competing interests: The authors declare that they have no competing interests.

Author's’ Contributions: Ke Huang and Jing Fang authored the manuscript and were responsible for data collection and analysis. Ke Huang, Jing Fang and Yujia Zeng established the rat SCI model. Weiming Sun, Bowen Shi and Bingkai Ren conducted the cell experiments. Lang Shuai and Haidi Bi designed and supervised the study.

Funding: This research was financially supported by grants from the National Natural Science Foundation of China（No. 82160441）.

Data Availability Statement: The data presented in this study are available on reasonable request from the corresponding author.

Acknowledgments: The authors thank the Public Platform Laboratory of the First Affiliated Hospital of Nanchang University for providing the experimental conditions. Also, thanks to Home for Researchers for providing the online drawing platform.

Liang Z, Yang Z, Xie H, Rao J, Xu X, Lin Y, Wang C, Chen C. Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization. Neural Regen Res. 2024;19(10):2259-2269.
Liu J, Cao J, Yu X, Chang J, Sui T, Cao X. Necroptosis pathway emerged as potential diagnosis markers in spinal cord injury. J Cell Mol Med. 2024;28(7):e18219.
Xu J, Ren Z, Niu T, Li S. Mechanism of Fat Mass and Obesity-Related Gene-Mediated Heme Oxygenase-1 m6A Modification in the Recovery of Neurological Function in Mice with Spinal Cord Injury. Orthop Surg. 2024;16(5):1175-1186.
Liu Y, Luo Z, Xie Y, Sun Y, Yuan F, Jiang L, Lu H, Hu J. Extracellular vesicles from UTX-knockout endothelial cells boost neural stem cell differentiation in spinal cord injury. Cell Commun Signal. 2024;22(1):155.
Zhang Y, Deng Q, Hong H, Qian Z, Wan B, Xia M. Caffeic acid phenethyl ester inhibits neuro-inflammation and oxidative stress following spinal cord injury by mitigating mitochondrial dysfunction via the SIRT1/PGC1α/DRP1 signaling pathway. J Transl Med. 2024;22(1):304.
Li F, Sun X, Sun K, Kong F, Jiang X, Kong Q. Lupenone improves motor dysfunction in spinal cord injury mice through inhibiting the inflammasome activation and pyroptosis in microglia via the nuclear factor kappa B pathway. Neural Regen Res. 2024;19(8):1802-1811.
Miao X, Lin J, Li A, Gao T, Liu T, Shen J, Sun Y, Wei J, Bao B, Zheng X. AAV-mediated VEGFA overexpression promotes angiogenesis and recovery of locomotor function following spinal cord injury via PI3K/Akt signaling. Exp Neurol. 2024;375:114739.
Wang J, Ji C, Ye W, Rong Y, Ge X, Wang Z, Tang P, Zhou Z, Luo Y, Cai W. Deubiquitinase UCHL1 promotes angiogenesis and blood-spinal cord barrier function recovery after spinal cord injury by stabilizing Sox17. Cell Mol Life Sci. 2024;81(1):137.
Xu Y, Hu X, Li F, Zhang H, Lou J, Wang X, Wang H, Yin L, Ni W, Kong J, Wang X, Li Y, Zhou K, Xu H. GDF-11 Protects the Traumatically Injured Spinal Cord by Suppressing Pyroptosis and Necroptosis via TFE3-Mediated Autophagy Augmentation. Oxid Med Cell Longev. 2021;2021:8186877.
Yan L, Han X, Zhang M, Kou H, Liu H, Cheng T. Melatonin exerts neuroprotective effects in mice with spinal cord injury by activating the Nrf2/Keap1 signaling pathway via the MT2 receptor. Exp Ther Med. 2023;27(1):37.
Akbari-Gharalari N, Ghahremani-Nasab M, Naderi R, Aliyari-Serej Z, Karimipour M, Shahabi P, Ebrahimi-Kalan A. Improvement of spinal cord injury symptoms by targeting the Bax/Bcl2 pathway and modulating TNF-α/IL-10 using Platelet-Rich Plasma exosomes loaded with dexamethasone. AIMS Neurosci. 2023;10(4):332-353.
Gu Q, Sha W, Huang Q, Wang J, Zhu Y, Xu T, Xu Z, Zhu Q, Ge J, Tian S, Lin X. Fibroblast growth factor 21 inhibits ferroptosis following spinal cord injury by regulating heme oxygenase-1. Neural Regen Res. 2024;19(7):1568-1574.
Wei FL, Wang TF, Wang CL, Zhang ZP, Zhao JW, Heng W, Tang Z, Du MR, Yan XD, Li XX, Guo Z, Qian JX, Zhou CP. Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas-Sting Axis in Spinal Cord Injury. Adv Sci (Weinh). 2024;11(4):e2305442.
Hu J, Huang K, Bao F, Zhong S, Fan Q, Li W. Low-dose lipopolysaccharide inhibits spinal cord injury-induced neuronal apoptosis by regulating autophagy through the lncRNA MALAT1/Nrf2 axis. PeerJ. 2023;11:e15919.
Gao K, Niu J, Dang X. Neuroprotection of melatonin on spinal cord injury by activating autophagy and inhibiting apoptosis via SIRT1/AMPK signaling pathway. Biotechnol Lett. 2020;42(10):2059-2069.
Zhang L, Han P. Neural stem cell-derived exosomes suppress neuronal cell apoptosis by activating autophagy via miR-374-5p/STK-4 axis in spinal cord injury. J Musculoskelet Neuronal Interact. 2022;22(3):411-421.
Xiao S, Zhong N, Yang Q, Li A, Tong W, Zhang Y, Yao G, Wang S, Liu J, Liu Z. Aucubin promoted neuron functional recovery by suppressing inflammation and neuronal apoptosis in a spinal cord injury model. Int Immunopharmacol. 2022;111:109163.
Li X, Xu J, Su W, Su L, Chen X, Yang J, Lin X, Yang L. GPNMB Modulates Autophagy to Enhance Functional Recovery After Spinal Cord Injury in Rats. Cell Transplant. 2024;33:9636897241233040.
Rong Y, Liu W, Wang J, Fan J, Luo Y, Li L, Kong F, Chen J, Tang P, Cai W. Neural stem cell-derived small extracellular vesicles attenuate apoptosis and neuroinflammation after traumatic spinal cord injury by activating autophagy. Cell Death Dis. 2019;10(5):340.
Tian D, You X, Ye J, Chen G, Yu H, Lv J, Shan F, Liang C, Bi Y, Jing J, Zheng M. hBcl2 overexpression in BMSCs enhances resistance to myelin debris-induced apoptosis and facilitates neuroprotection after spinal cord injury in rats. Sci Rep. 2024;14(1):1830.
Liu S, Zhang H, Wang H, Huang J, Yang Y, Li G, Yu K, Yang L. A Comparative Study of Different Stem Cell Transplantation for Spinal Cord Injury: A Systematic Review and Network Meta-Analysis. World Neurosurg. 2022;159:e232-e243.
Huang LY, Sun X, Pan HX, Wang L, He CQ, Wei Q. Cell transplantation therapies for spinal cord injury focusing on bone marrow mesenchymal stem cells: Advances and challenges. World J Stem Cells. 2023;15(5):385-399.
Jia Y, Lu T, Chen Q, Pu X, Ji L, Yang J, Luo C. Exosomes secreted from sonic hedgehog-modified bone mesenchymal stem cells facilitate the repair of rat spinal cord injuries. Acta Neurochir (Wien). 2021;163(8):2297-2306.
Li C, Li X, Zhao B, Wang C. Exosomes derived from miR-544-modified mesenchymal stem cells promote recovery after spinal cord injury. Arch Physiol Biochem. 2020;126(4):369-375.
Liu B, Zheng W, Dai L, Fu S, Shi E. Bone marrow mesenchymal stem cell derived exosomal miR-455-5p protects against spinal cord ischemia reperfusion injury. Tissue Cell. 2022;74:101678.
Wei GJ, An G, Shi ZW, Wang KF, Guan Y, Wang YS, Han B, Yu EM, Li PF, Dong DM, Wang LP, Teng ZW, Zhao DL. Suppression of MicroRNA-383 Enhances Therapeutic Potential of Human Bone-Marrow-Derived Mesenchymal Stem Cells in Treating Spinal Cord Injury via GDNF. Cell Physiol Biochem. 2017;41(4):1435-1444.
Yang Z, Liang Z, Rao J, Xie H, Zhou M, Xu X, Lin Y, Lin F, Wang C, Chen C. Hypoxic-preconditioned mesenchymal stem cell-derived small extracellular vesicles promote the recovery of spinal cord injury by affecting the phenotype of astrocytes through the miR-21/JAK2/STAT3 pathway. CNS Neurosci Ther. 2024;30(3):e14428.
Huang Y, Ren H, Gao X, Cai D, Shan H, Bai J, Sheng L, Jin Y, Zhou X. Amlodipine Improves Spinal Cord Injury Repair by Inhibiting Motoneuronal Apoptosis Through Autophagy Upregulation. Spine (Phila Pa 1976). 2022;47(17):E570-E578.
Hou Y, Luan J, Huang T, Deng T, Li X, Xiao Z, Zhan J, Luo D, Hou Y, Xu L, Lin D. Tauroursodeoxycholic acid alleviates secondary injury in spinal cord injury mice by reducing oxidative stress, apoptosis, and inflammatory response. J Neuroinflammation. 2021;18(1):216.
Zhang H, Ni W, Yu G, Geng Y, Lou J, Qi J, Chen Y, Li F, Ye H, Ma H, Xu H, Zhao L, Cai Y, Wang X, Xu H, Xiao J, Zhou K. 3,4-Dimethoxychalcone, a caloric restriction mimetic, enhances TFEB-mediated autophagy and alleviates pyroptosis and necroptosis after spinal cord injury. Theranostics. 2023;13(2):810-832.
Wu C, Chen H, Zhuang R, Zhang H, Wang Y, Hu X, Xu Y, Li J, Li Y, Wang X, Xu H, Ni W, Zhou K. Betulinic acid inhibits pyroptosis in spinal cord injury by augmenting autophagy via the AMPK-mTOR-TFEB signaling pathway. Int J Biol Sci. 2021;17(4):1138-1152.
Gao L, Zhang C, Zhu Y, Zhang N, Zhang C, Zhou S, Feng G, Huang F, Zhang L. Serum response factor promoting axonal regeneration by activating the Ras-Raf-Cofilin signaling pathway after the spinal cord injury. CNS Neurosci Ther. 2024;30(2):e14585.
Zhang B, Lin F, Dong J, Liu J, Ding Z, Xu J. Peripheral Macrophage-derived Exosomes promote repair after Spinal Cord Injury by inducing Local Anti-inflammatory type Microglial Polarization via Increasing Autophagy. Int J Biol Sci. 2021;17(5):1339-1352.
Zhao C, Zhou T, Li M, Liu J, Zhao X, Pang Y, Liu X, Zhang J, Ma L, Li W, Yao X, Feng S. Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway. Neural Regen Res. 2024;19(2):434-439.
Wang X, Ye L, Zhang K, Gao L, Xiao J, Zhang Y. Small Extracellular Vesicles Released from miR-211-5p-Overexpressed Bone Marrow Mesenchymal Stem Cells Ameliorate Spinal Cord Injuries in Rats. eNeuro. 2024;11(2).
He Z, Du J, Zhang Y, Xu Y, Huang Q, Zhou Q, Wu M, Li Y, Zhang X, Zhang H, Cai Y, Ye K, Wang X, Zhang Y, Han Q, Xiao J. Kruppel-like factor 2 contributes to blood-spinal cord barrier integrity and functional recovery from spinal cord injury by augmenting autophagic flux. Theranostics. 2023;13(2):849-866.
Li RY, Hu Q, Shi X, Luo ZY, Shao DH. Crosstalk between exosomes and autophagy in spinal cord injury: fresh positive target for therapeutic application. Cell Tissue Res. 2023;391(1):1-17.
Li N, Chen L, Zhao X, Gu C, Chang Y, Feng S. Targeting ANXA7/LAMP5-mTOR axis attenuates spinal cord injury by inhibiting neuronal apoptosis via enhancing autophagy in mice. Cell Death Discov. 2023;9(1):309.
Zhang X, Jing Y, Qin C, Liu C, Yang D, Gao F, Yang M, Du L, Li J. Mechanical stress regulates autophagic flux to affect apoptosis after spinal cord injury. J Cell Mol Med. 2020;24(21):12765-12776.
Zhou K, Chen H, Xu H, Jia X. Trehalose Augments Neuron Survival and Improves Recovery from Spinal Cord Injury via mTOR-Independent Activation of Autophagy. Oxid Med Cell Longev. 2021;2021:8898996.
Xu Y, Geng Z, Sun Y, Zhu G, Xiao L, Wang Z, Li B, Liu X, Shi J, Song X, Hu J, Qi Q. Complanatuside A improves functional recovery after spinal cord injury through inhibiting JNK signaling-mediated microglial activation. Eur J Pharmacol. 2024;965:176287.
Gu J, Wu J, Wang C, Xu Z, Jin Z, Yan D, Chen S. BMSCs-derived exosomes inhibit macrophage/microglia pyroptosis by increasing autophagy through the miR-21a-5p/PELI1 axis in spinal cord injury. Aging (Albany NY). 2024;16(6):5184-5206.
Zhou XY, Lai YH, Zhang J, Li Y, Wu XM, Yang YZ, Zhang XF, Ma LZ, Zheng KM, Liu YD, Wang Z, Chen SL. Advanced oxidation protein products attenuate the autophagy-lysosome pathway in ovarian granulosa cells by modulating the ROS-dependent mTOR-TFEB pathway. Cell Death Dis. 2024;15(2):161.
Zhou K, Zheng Z, Li Y, Han W, Zhang J, Mao Y, Chen H, Zhang W, Liu M, Xie L, Zhang H, Xu H, Xiao J. TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress. Theranostics. 2020;10(20):9280-9302.
Biswas VK, Sen K, Ahad A, Ghosh A, Verma S, Pati R, Prusty S, Nayak SP, Podder S, Kumar D, Gupta B, Raghav SK. NCoR1 controls Mycobacterium tuberculosis growth in myeloid cells by regulating the AMPK-mTOR-TFEB axis. PLoS Biol. 2023;21(8):e3002231.
Wu H, Ding J, Li S, Lin J, Jiang R, Lin C, Dai L, Xie C, Lin D, Xu H, Gao W, Zhou K. Metformin Promotes the Survival of Random-Pattern Skin Flaps by Inducing Autophagy via the AMPK-mTOR-TFEB signaling pathway. Int J Biol Sci. 2019;15(2):325-340.
Tang YJ, Li K, Yang CL, Huang K, Zhou J, Shi Y, Xie KG, Liu J. Bisperoxovanadium protects against spinal cord injury by regulating autophagy via activation of ERK1/2 signaling. Drug Des Devel Ther. 2019;13:513-521.
Young NP, Kamireddy A, Van Nostrand JL, Eichner LJ, Shokhirev MN, Dayn Y, Shaw RJ. AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes. Genes Dev. 2016;30(5):535-52.
Fang RY, Liu YW, Goan YG, Lin JJ, Su JH, Wu WT, Wu YJ. Suppression of Migration and Invasion by 4-Carbomethoxyl-10-Epigyrosanoldie E from the Cultured Soft Coral Sinularia sandensis through the MAPKs Pathway on Oral Cancer Cells. Adv Pharmacol Pharm Sci. 2024;2024:6695837.
Zhang J, Jiang N, Ping J, Xu L. TGF‑β1‑induced autophagy activates hepatic stellate cells via the ERK and JNK signaling pathways. Int J Mol Med. 2021;47(1):256-266.

No competing interests reported.

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Flow chart of an experimental study in animal models and cell cultures: In vivo: Animal subjects were evaluated for autophagy, mitochondrial autophagy, and apoptosis on day 7 post-injury, and for behavioral and pathological recovery on day 28 post-injury. In vitro: PC-12 cells were transfected with miR-202-3p mimics, miR-202-3p inhibitor, and miR-202-3p negative control (NC) using Lipofectamine™ RNAiMAX for 6 hours. Subsequently, the cells were stimulated with Rapamycin for 48 hours to assess levels of autophagy and apoptosis.

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Bone Marrow Mesenchymal Stem Cells Modulate miR-202-3p to Suppress Neuronal Apoptosis Following Spinal Cord Injury Through Autophagy Activation via the AMPK, MAPK, and PI3K/AKT/mTOR Signaling Pathway

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