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

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 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article 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Pathway Ke Huang, Jing Fang, Weiming Sun, Yujia Zeng, Bowen Shi, Bingkai Ren, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4699112/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction 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. 2. Chemicals and antibodies 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. Methods 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 6×10 5 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 q PCR 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. RESULTS 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 . 5. Discussion 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 . Declarations 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. References 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. 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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. Additional Declarations No competing interests reported. Supplementary Files file.png 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. Cite Share Download PDF Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 30 Oct, 2024 Reviews received at journal 29 Oct, 2024 Reviewers agreed at journal 14 Oct, 2024 Reviews received at journal 04 Aug, 2024 Reviewers agreed at journal 21 Jul, 2024 Reviewers invited by journal 19 Jul, 2024 Editor assigned by journal 19 Jul, 2024 Editor invited by journal 11 Jul, 2024 Submission checks completed at journal 09 Jul, 2024 First submitted to journal 07 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4699112","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":334464924,"identity":"526c81ce-08ca-4804-a1b4-4e0d56707e1f","order_by":0,"name":"Ke Huang","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Huang","suffix":""},{"id":334464925,"identity":"9b99a826-e281-4b02-9212-2df31fc56deb","order_by":1,"name":"Jing Fang","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Fang","suffix":""},{"id":334464926,"identity":"516a1d75-7d7e-498c-a66c-b9f2e0016dfd","order_by":2,"name":"Weiming Sun","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Weiming","middleName":"","lastName":"Sun","suffix":""},{"id":334464927,"identity":"4dd82942-d33d-4478-b21e-2d6ec42d3b49","order_by":3,"name":"Yujia Zeng","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Yujia","middleName":"","lastName":"Zeng","suffix":""},{"id":334464928,"identity":"242b408f-1357-4c46-883c-dc613a9b0ecd","order_by":4,"name":"Bowen Shi","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Bowen","middleName":"","lastName":"Shi","suffix":""},{"id":334464929,"identity":"9b4613b7-6fd6-44e3-a8fa-b3ee6d1c9a35","order_by":5,"name":"Bingkai Ren","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Bingkai","middleName":"","lastName":"Ren","suffix":""},{"id":334464930,"identity":"50cf9774-3b12-43bb-b2b4-9c242bfd7b52","order_by":6,"name":"Haidi Bi","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Haidi","middleName":"","lastName":"Bi","suffix":""},{"id":334464931,"identity":"feb48633-e6b0-4217-8661-0f7b7d842ed5","order_by":7,"name":"Lang Shuai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYFCCBIYPDAY2PPbNzAcgAgcIa2GcwVCRJmPAzpZAipYzh20M+HkMiNNizp5j2Mzbxsxjzszz7cHPNgY5vhsJjJ8L8Gix7HkD0sLGY9nMu92wt43BWPJGArP0DDxaDG7kmD/mbePhYTjMu02asY0hccONBDZmHvxaQLZIALXwPANpqSdOC88ZAx6DwzxsIC0JBgS1nHlW2DinIoFHspnNTLLnnIThzDMPm6XxajmevLHhjcF/e37+w88kfpTZyPMdTz74GZ8WBgYOAyYkBRJAzNiAVwMDA/sDxh8ElIyCUTAKRsEIBwBXnUoVEjFBeQAAAABJRU5ErkJggg==","orcid":"","institution":"Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Lang","middleName":"","lastName":"Shuai","suffix":""}],"badges":[],"createdAt":"2024-07-07 07:15:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4699112/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4699112/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-81332-y","type":"published","date":"2024-12-03T15:57:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61619986,"identity":"239dd34a-fc44-4737-afa3-cc6f341ee555","added_by":"auto","created_at":"2024-08-02 04:41:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":346174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation of spinal cord tissue and identification of BMMSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)A vascular clip was employed to apply pressure to the exposed spinal cord. (B) Twenty-eight days post-injury, spinal cord tissue specimens were harvested from each experimental group of rats for subsequent analysis. (C) Bone marrow mesenchymal stem cells (P3, 500 pixels). (D) Detection of surface antigens on P3 BMMSCs by flow cytometry.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/2d537d19db6088354e496fe8.png"},{"id":61619989,"identity":"302cdfb1-326a-4949-a010-75281a0d18e2","added_by":"auto","created_at":"2024-08-02 04:41:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":282497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMMSCs improve both behavioral and histological recovery following SCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Assessment of motor function recovery over time in spinal cord injured rats using the BBB score. (B, C) From 14 to 28 days post-injury, the BBB scores of the BMMSCs group showed a significant increase compared to the SCI group, indicating that the BMMSCs treatment could facilitate the recovery of behavioral functions in rats with SCI. (D) Footprints left by groups of rats walking or running on surfaces are analysed by footprint testing to obtain valuable information on various aspects of motor function, including step length, stride length, gait and coordination. (E, F) Quantitative analysis of the base of support and stride length in rats. (G) HE staining for pathological analysis of sagittal and transverse sections. (H, I) The quantitative results of HE staining were employed to assess the size of tissue cavities in different groups of rats 28 days after SCI.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/75dc859cbdd0729cbcf9196b.png"},{"id":61619657,"identity":"488f7bf5-4e9d-4adc-a899-6f997ef1330e","added_by":"auto","created_at":"2024-08-02 04:33:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":470580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMMSCs enhance neuronal viability and promote the regeneration of axons after SCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) TUNEL staining was employed to assess the extent of apoptotic cell death in the injured spinal cord tissue (scale bar=50 µm). (B) Nissl staining of spinal cord tissue was conducted to evaluate the depletion of Nissl bodies within neurons. (C, D) Weperformed immunofluorescence staining for Bcl-xL and NeuN in the injured spinal cord tissue to assess the expression levels and localization of these proteins within neuronal populations (scale bar = 40 µm). (E, F) Western blotting was carried out to measure the expression of NeuN on day 28 following SCI. (G)Western blot analysis revealed that BMMSCs administration resulted in the downregulation of BAX and the upregulation of Bcl-xL expression within the spinal cord tissue. (H, I) BAX and Bcl-xL levels were relative to those of GAPDH in the spinal cord.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/d700b6b6762c8bcbf0be3d19.png"},{"id":61619988,"identity":"71caa030-d91b-4d40-8ba0-56ad17de4ef8","added_by":"auto","created_at":"2024-08-02 04:41:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":592756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMMSCs improve neuronal survival and axonal regeneration after SCI.\u003c/strong\u003e (A, B) At the 28-day time point following SCI, we employed double immunofluorescence imaging coupled with quantitative analysis to gauge the levels of MAP2 and GFAP expression within three distinct experimental groups: the Sham group, the SCI group, and the group treated with BMMSCs (scale bar =1 mm). The green fluorescence represents the expression of MAP2, predominantly localized in neuronal dendrites, while the red fluorescence indicates the expression of GFAP, primarily found in astrocytes. (C)The distance from the center of the injury to the closest neuronal cell body. (D, E) At the 28-day time point following SCI, we employed a merged immunofluorescence assay focused on GAP43 (labeled in green) and GFAP (labeled in red) to conduct an in-depth evaluation of cellular reactions following SCI, covering the spectrum of neuronal regeneration and glial activation (scale bar =1 mm). (F-H) The expression levels of MAP2 and GAP43 proteins were evaluated and quantified in each experimental group 28 days post-injury.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/a7ec5bec2d783fb019997952.png"},{"id":61619987,"identity":"72dff638-0f7f-47df-ae65-44f1af319cdf","added_by":"auto","created_at":"2024-08-02 04:41:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":431028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMMSCs enhance autophagy after SCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blottingwas employed to interrogate the involvement of p62, LC3II/I, Beclin1, VPS34, and CTSD in elucidating the nuanced intricacies governing autophagic processes. (B-F) The quantitative analysis of protein abundance was conducted for p62, LC3II/I ratio, Beclin1, VPS34, and CTSD across the experimental groups of rats. (G, J) Immunofluorescence staining was performed to investigate the localization and potential interaction between LC3 (a marker for autophagosomes) and NeuN (a neuronal marker) in the context of SCI (scale bar = 50 µm). The quantification of LC3 immunofluorescence staining is depicted alongside representative images. (H, I) Immunofluorescence staining was conducted to examine the expression and potential co-localization patterns of p62 (a selective autophagy substrate) and NeuN (a neuronal marker) in the context of SCI (scale bar=1 mm). Quantitative analysis of p62 immunofluorescence staining is provided adjacent to representative images.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/a4550dcbf44b83ceec97eae9.png"},{"id":61619659,"identity":"fb336f58-51f3-4432-ae02-f4f1245fb872","added_by":"auto","created_at":"2024-08-02 04:33:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":436051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMMSCs enhance mitophagy after SCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Transmission electron microscopy showing autophagy and mitochondrial autophagy. The autophagic vesicles are indicated by red arrows and the scale bars are set at lengths of 5.0 µm, 2.5 µm, and 2.0 µm, respectively. (B, C) Utilizing double immunofluorescence imaging and subsequent quantitative analysis, the expression levels of NIX (labeled in green) and NeuN (labeled in red) were assessed across the Sham, SCI, and BMMSC groups (scale bar =40 µm). (D) Western blotting was conducted to determine the levels of expression for Parkin, BNIP3, and NIX across the Sham, SCI and BMMSCs groups. (E-G) Quantification of Parkin, BNIP3 and NIX protein levels in each group of rats.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/1960be5da59628df9d82d399.png"},{"id":61619661,"identity":"ae64402e-b173-4e47-965c-049aa706a588","added_by":"auto","created_at":"2024-08-02 04:33:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":686744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMMSCs promote autophagy by regulating the AMPK, MAPK, and PI3K/AKT/mTOR/TFEB signaling pathway to improve functional recuperation following SCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Western blotting and quantitative analyses showed that BMMSCs promoted p-AMPK expression in tissue of the spinal cord. (C, D) Western blot analysis, coupled with quantitative assessment, demonstrated that BMMSCs induced increased expression of phosphorylated forms of JNK (p-JNK), Erk (p-Erk), and P38 (p-P38) in spinal cord tissue. (E, F) Western blotting and quantitative analyses showed that BMMSCs inhibited p-PI3K and p-AKT expression in tissue of the spinal cord. (G-J) Western blotting and quantitative analyses showed that BMMSCs inhibited p-mTOR expression and promoted TFEB expression in spinal cord tissue.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/60a521f84b983b95967842c9.png"},{"id":61619663,"identity":"775ffa94-153b-4529-8d0f-c179948b4910","added_by":"auto","created_at":"2024-08-02 04:33:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":182664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-202-3p mimics inhibit apoptosis and promote autophagy in vivo and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Q-PCR was employed to identify alterations in miR-202-3p expression in spinal cord tissues. The results indicated that BMMSCs can facilitate miR-202-3p expression. (B, C) PC-12 cells were subjected to transfection with miR-202-3p mimics to enhance miR-202-3p expression, miR-202-3p inhibitor to suppress miR-202-3p expression, and miR-202-3p NC to serve as a baseline control for comparison. QPCR analysis of the miR-202-3p level. (D-F) Western blotting and quantitative analyses showed that Beclin1 expression gradually increased and Bax expression gradually decreased with increasing miR-202-3p concentration (0, 10, 20, 40, 60, and 80 nM). (G)Western blotting images were provided as representatives, illustrating the protein levels of p62, LC3II/I, and BAX in each group of PC-12 cells. (H-I) Quantitative assessment of p62 and BAX protein levels within each cohort of PC-12 cells.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/a72b18b9f67a61555dd140e3.png"},{"id":61619660,"identity":"03d228a3-897f-42d6-bae5-75dbf7cb2551","added_by":"auto","created_at":"2024-08-02 04:33:52","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":258988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe therapeutic efficacy of BMMSCs in the context of SCI is illustrated in this schematic representation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMMSCs mediate miR-202-3p to enhance autophagic flux, attenuate neuronal apoptosis and ultimately promote restitution of function in rats with SCI by regulating the AMPK, MAPK and PI3K/AKT/mTOR/TFEB pathway.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/bf719e56c8cab3d201dc30bd.png"},{"id":70964780,"identity":"db4e268b-b281-4748-b6a6-6ad5a6119e9e","added_by":"auto","created_at":"2024-12-09 16:15:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4822105,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/06dbe2a6-9984-4286-94f1-1d04a56cd1da.pdf"},{"id":61619665,"identity":"858453b5-a574-41fd-b840-f51f8c9f30c7","added_by":"auto","created_at":"2024-08-02 04:33:52","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":99468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow chart of an experimental study in animal models and cell cultures:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo: \u003c/strong\u003e\u003c/em\u003eAnimal 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.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro:\u003c/strong\u003e\u003c/em\u003e 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.\u003c/p\u003e","description":"","filename":"file.png","url":"https://assets-eu.researchsquare.com/files/rs-4699112/v1/60f41c83458b220f7a591f6a.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"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","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpinal cord injury (SCI) ranks among the most prevalent types of\u0026nbsp;neurotrauma,\u0026nbsp;characterized by segmental paralysis, sensory deficits, and a spectrum of dysfunctions, imposing profound physical, socioeconomic, and psychosocial burdens on affected individuals\u003csup\u003e[1-3]\u003c/sup\u003e.\u0026nbsp;The development of SCI involves both initial and subsequent injuries.\u0026nbsp;\u003csup\u003e[4, 5]\u003c/sup\u003e.\u0026nbsp;Primary SCI pertains to the initial mechanical trauma inflicted upon the spinal cord resulting from\u0026nbsp;mechanical forces such\u0026nbsp;as squeezing, crushing, tearing or traction\u0026nbsp;\u003csup\u003e[6-8]\u003c/sup\u003e. Secondary injuries arise from a complex interplay of pathophysiological processes,\u0026nbsp;these factors include apoptosis, autophagy, inflammation, oxidative stress, and other related mechanisms, perturbing\u0026nbsp;the microenvironment and exacerbating neurological impairment\u003csup\u003e[9, 10]\u003c/sup\u003e. Hence, effective management of secondary damage assumes paramount importance in optimizing SCI recovery\u003csup\u003e[11-13]\u003c/sup\u003e. Notably, cellular autophagy and neuronal apoptosis stand out as pivotal targets for mitigating secondary injury within the pathophysiological cascade of SCI\u003csup\u003e[14, 15]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAutophagy, vital for clearing cytoplasmic waste and damaged organelles, is crucial in promoting neuroprotection and facilitating repair following SCI\u003csup\u003e[16]\u003c/sup\u003e. 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\u003csup\u003e[17]\u003c/sup\u003e. Studies have underscored the intricate interplay between cellular inflammation, oxidative stress, and apoptosis in SCI pathogenesis, highlighting the interconnectedness with autophagic processes\u003csup\u003e[18]\u003c/sup\u003e.\u0026nbsp;Enhancing autophagy has been identified\u0026nbsp;as a hopeful avenue for therapy to enhance the restoration of functionality following SCI.\u003c/p\u003e\n\u003cp\u003eThe 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\u003csup\u003e[19]\u003c/sup\u003e. BMMSCs have emerged as a valuable asset in the realm of spinal cord repair\u003csup\u003e[20]\u003c/sup\u003e, demonstrating notable neuroprotective effects\u003csup\u003e[21]\u003c/sup\u003e, facilitation of axonal regeneration\u003csup\u003e[22]\u003c/sup\u003e, and attenuation of astrocyte hyperactivation\u003csup\u003e[23]\u003c/sup\u003e. Moreover,\u0026nbsp;miRNAs, a class of eukaryotic bodily noncoding single-stranded RNAs,\u0026nbsp;are recognized for their regulatory roles in inflammation, apoptosis, autophagy, and other pathophysiological processes\u003csup\u003e[24]\u003c/sup\u003e. Various studies have implicated specific miRNAs in mediating the therapeutic benefits of BMMSCs in SCI. For instance,\u0026nbsp;BMMSC-derived exosomal miR-455-5p stimulates autophagy while suppressing neuronal apoptosis in rats with SCI\u0026nbsp;\u003csup\u003e[25]\u003c/sup\u003e. Additionally, research by Wei\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eet al\u003c/em\u003e\u003csup\u003e[26]\u003c/sup\u003e. 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 \u003cem\u003ein vitro\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003eDespite 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.\u003c/p\u003e"},{"header":"2. Chemicals and antibodies","content":"\u003cp\u003eMiRNA-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).\u003c/p\u003e"},{"header":"3. Methods","content":"\u003cp\u003e\u003cstrong\u003e3.1 Animal and SCI models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA\u0026nbsp;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\u0026nbsp;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\u0026nbsp;(Figure 1A).\u0026nbsp;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\u0026nbsp;three\u0026nbsp;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 \u0026times; 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Behavioural assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional 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\u0026nbsp;21 (representing full functional recovery)\u003csup\u003e[27]\u003c/sup\u003e,\u0026nbsp;was recorded\u0026nbsp;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\u0026apos; 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Tissue section preparation and HE and Nissl staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats 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\u0026nbsp;tissues underwent dehydration, embedding, and sectioning into longitudinal and transverse slices measuring 4-5 \u0026mu;m 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Tissue immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded spinal cord tissue sections, measuring 5 \u0026mu;m 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\u0026deg;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\u0026apos;,6-diamidino-2- phenylindole (DAPI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 TUNEL staining.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTUNEL stands for Terminal deoxynucleotidyl transferase dUTP Nick End Labelling, which was employed to detect apoptotic cells\u0026nbsp;in 5-\u0026mu;m-thick longitudinal paraffin sections following established protocols. The sections underwent deparaffinization and antigen retrieval procedures. Subsequently, cell permeabilization was achieved by treating the sections\u0026nbsp;with Triton X-100 for 12 minutes at 37\u0026deg;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Transmission electron microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAutophagic 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePC-12 cells and BMMSCs were procured from Shanghai Anwei Biotechnology Co\u0026nbsp;Ltd. Culturing of PC-12 cells involved maintenance\u0026nbsp;in high-glucose Dulbecco\u0026apos;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\u0026deg;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Transfection and grouping of cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells. When 50%-80% confluence is reached, the cells underwent transfection with Lipofectamine\u0026trade; 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 \u0026mu;mol/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 \u003cem\u003ein vivo and in vitro.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.9 Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRIPA 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\u0026deg;C,\u0026nbsp;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\u0026deg;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.10\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eq\u003c/strong\u003e\u003cstrong\u003ePCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor 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\u0026nbsp;(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.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.11 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical 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\u0026apos;s post hoc test for further analysis. In this context, we defined statistical significance as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 indicated significance, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 signified a higher level of significance, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 represented even greater significance, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 was considered highly significant.\u0026nbsp;\u003c/p\u003e"},{"header":"4. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e4.1 Morphology and antigen identification of BMMSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon 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\u0026nbsp;(Figure 1D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 BMMSCs facilitate the functional motor abilities recovery in rats\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;with SCI.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 (\u003cem\u003ep\u003c/em\u003e\u0026lt;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\u0026apos;s performance (\u003cem\u003ep\u003c/em\u003e\u0026lt;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 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001; Figure 2E, F). Altogether, the findings underscore the therapeutic potential of BMMSCs in enhancing motor function restoration in rats with SCI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 BMMSCs promote histological repair of the damaged spinal cord.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistological 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 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; \u003cem\u003ep\u003c/em\u003e\u0026lt;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 BMMSCs inhibit neuronal apoptosis after\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudies have revealed that attenuating neuronal apoptosis is crucial for improving functional recuperation following SCI\u003csup\u003e[28]\u003c/sup\u003e. 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 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; Figure 3C-D). Western blotting further substantiated the BMMSCs treatment partially reinstated the protein expression of NeuN (\u003cem\u003ep\u003c/em\u003e\u0026lt;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 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001; \u003cem\u003ep\u003c/em\u003e\u0026lt;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5 BMMSCs protect neurons and promote axonal regeneration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrotubule-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\u0026nbsp;Sham group, the SCI group showed a notable increase in the distance between the epicentre of the injury and the nearest neuron (\u003cem\u003ep\u003c/em\u003e \u0026lt;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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;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\u003csup\u003e[29]\u003c/sup\u003e.\u0026nbsp;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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; Figure 3F-H). Collectively, these outcomes underscore the neuroprotective effects of BMMSCs in safeguarding neurons and fostering axonal regeneration post-SCI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6 BMMSCs enhance autophagy after\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAutophagy has garnered attention as an exciting way to treat central nervous system (CNS) diseases,\u0026nbsp;exerting a pivotal role in modulating programmed cell death triggered by neuroinflammation\u003csup\u003e[30]\u003c/sup\u003e. 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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05;\u003cem\u003e\u0026nbsp;p\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001; Figure 5A-F). Furthermore, immunofluorescence demonstrated diminished p62 density and heightened LC3II signal within the BMMSCs group relative to the SCI group (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.7 BMMSCs enhance mitophagy after\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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\u003csup\u003e[31]\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ehave 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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001; \u003cem\u003ep\u003c/em\u003e \u0026lt;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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001, Figure 6 D-G). Notably, the BMMSCs group exhibited enhanced protein expression levels of the markers in comparison to the SCI group (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; Figure 6 D-G). Collectively, these findings suggest that BMMSCs augment mitochondrial autophagy, potentially through their pro-autophagic properties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.8 BMMSCs promote autophagy through the AMPK, MAPK and PI3K/AKT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e/mTOR/TFEB signaling cascade to promote functional recuperation following SCI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur 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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; \u003cem\u003ep\u003c/em\u003e \u0026lt;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 (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001; Figure 7 C-D). Furthermore, BMMSCs were observed to attenuate the levels of phosphorylated PI3K and AKT (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; \u003cem\u003ep\u003c/em\u003e \u0026lt;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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.9 MiR-202-3p mimics inhibit apoptosis and promote autophagy \u003cem\u003ein vivo and\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein vitro.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur previous study illustrated that \u003cem\u003ein vitro\u003c/em\u003e overexpression of miR-202-3p\u0026nbsp;led to\u0026nbsp;a notable\u0026nbsp;reduction of\u0026nbsp;pro-inflammatory cytokines levels such as TNF-\u0026alpha;, IL-6, and IL-1\u0026beta;, whereas downregulation of miR-202-3p expression resulted in an elevation in these cytokines. This observation provides evidence that miR-202-3p may be\u0026nbsp;implicated in the reparative processes associated with SCI. Consequently, we hypothesized that BMMSCs may exert their effects through miR-202-3p-mediated autophagy. \u003cem\u003eIn vivo\u003c/em\u003e, our examination of spinal cord tissue disclosed markedly elevated levels of miR-202-3p in the BMMSCs treatment\u0026nbsp;group\u0026nbsp;relative to the SCI group\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e \u0026lt;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\u0026nbsp;miR-202-3p\u0026nbsp;NC.\u0026nbsp;The transfection experiments yielded compelling evidence indicating a substantial upregulation of miR-202-3p expression within the group transfected with miR-202-3p mimics.\u0026nbsp;Notably, the inhibitor\u0026apos;s mechanism of action involves sequestering endogenous free miRNA rather than degrading it, resulting in an\u0026nbsp;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\u0026nbsp;(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 \u003cem\u003ein vivo and in vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. Discussion","content":"\u003cp\u003eSCI can lead to substantial and enduring functional deficits, imposing significant burdens on individuals, families, and society\u003csup\u003e[32-34]\u003c/sup\u003e.\u0026nbsp;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\u003csup\u003e[35]\u003c/sup\u003e. 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.\u003c/p\u003e\n\u003cp\u003eAutophagy, 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\u003csup\u003e[36, 37]\u003c/sup\u003e. Despite ongoing debates, it is postulated that the activation of autophagy following SCI confers the revitalization of neurons. These effects are\u0026nbsp;characterized by microtubule stabilization, facilitation of axonal regeneration, and a decreased incidence of apoptosis during the recovery phase\u003csup\u003e[38-40]\u003c/sup\u003e.\u0026nbsp;Our\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003eSCI pathogenesis is complex, involving diverse pathological cascades, with neuronal apoptosis playing a pivotal role in secondary injury progression\u003csup\u003e[41]\u003c/sup\u003e. 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\u003csup\u003e[42]\u003c/sup\u003e. Our prior work demonstrated that the induction of miR-202-3p expression attenuated the inflammation following SCI\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e. 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.\u003c/p\u003e\n\u003cp\u003eTo 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.\u003c/p\u003e\n\u003cp\u003eTo 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.\u0026nbsp;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\u003csup\u003e[43, 44]\u003c/sup\u003e. 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\u0026nbsp;\u003csup\u003e[45]\u003c/sup\u003e.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\u003csup\u003e[46, 47]\u003c/sup\u003e.\u0026nbsp;When mTOR activation is inhibited,\u0026nbsp;TFEB undergoes dephosphorylation, subsequently translocating to the\u0026nbsp;cytoblast\u003csup\u003e[48]\u003c/sup\u003e.\u0026nbsp;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\u003csup\u003e[49]\u003c/sup\u003e. 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\u003csup\u003e[50]\u003c/sup\u003e. 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 .\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e 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)\u003c/p\u003e\n\u003cp\u003eThis 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026apos;s\u0026rsquo; Contributions:\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was financially supported by grants from the National Natural Science Foundation of China（No. 82160441）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data presented in this study are available on reasonable request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e 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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiang Z, Yang Z, Xie H, Rao J, Xu X, Lin Y, Wang C, Chen C. 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Int J Mol Med. 2021;47(1):256-266.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"BMMSCs, SCI, miR-202-3p, autophagy, neuronal apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4699112/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4699112/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e, BMMSCs were administered to SCI rats, while\u003cem\u003e in vitro\u003c/em\u003e, 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e 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.\u003c/p\u003e","manuscriptTitle":"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","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-02 04:33:47","doi":"10.21203/rs.3.rs-4699112/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-30T05:25:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-29T08:21:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174633510861899530969797923000422610724","date":"2024-10-14T06:19:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-05T01:34:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313857182799455588135205414757991438880","date":"2024-07-21T23:59:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-19T16:14:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-19T16:07:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-11T15:44:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-09T13:40:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-07T07:14:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96636eaf-ae84-4bc1-b2ba-60fdc0ec2394","owner":[],"postedDate":"August 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35419919,"name":"Biological sciences/Neuroscience"},{"id":35419920,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2024-12-09T16:03:58+00:00","versionOfRecord":{"articleIdentity":"rs-4699112","link":"https://doi.org/10.1038/s41598-024-81332-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-12-03 15:57:44","publishedOnDateReadable":"December 3rd, 2024"},"versionCreatedAt":"2024-08-02 04:33:47","video":"","vorDoi":"10.1038/s41598-024-81332-y","vorDoiUrl":"https://doi.org/10.1038/s41598-024-81332-y","workflowStages":[]},"version":"v1","identity":"rs-4699112","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4699112","identity":"rs-4699112","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}