Exosomal miR-223-3p from Mesenchymal Stem Cells Targets FBXW7 to Inhibit Intervertebral Disc Degeneration: Mechanism Insights

Exosomal miR-223-3p from Mesenchymal Stem Cells Targets FBXW7 to Inhibit Intervertebral Disc Degeneration: Mechanism Insights | 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 Research Article Exosomal miR-223-3p from Mesenchymal Stem Cells Targets FBXW7 to Inhibit Intervertebral Disc Degeneration: Mechanism Insights Rui Chen, Kaiyi Cao, Yuting Gong, Yuning Zhu, Yi Gao, Jing Yan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7737133/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted 7 You are reading this latest preprint version Abstract Background Intervertebral disc degeneration (IDD) represents a widespread musculoskeletal condition. Programmed cell death in nucleus pulposus cells (NPCs) significantly contributes to IDD pathogenesis. MicroRNAs (miRNAs) play critical roles in IDD development. Bone marrow mesenchymal stem cell (MSC)-derived exosomes can inhibit NPCs apoptosis and promote disc regeneration/repair by delivering miRNAs. Methods Rat bone marrow-derived mesenchymal stem cells (MSCs) were expanded in vitro , followed by exosome isolation via differential ultracentrifugation. Exosome characterization included assessment of size/concentration via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and nano-flow cytometry, alongside detection of exosomal markers (CD9, CD81, TSG101, Calnexin) by Western blotting. Exosome uptake by NPCs was confirmed using PKH26 labeling. MSCs were transfected with miR-223-3p mimic or inhibitor, followed by exosome isolation and co-culture with rat NPCs to assess miR-223-3p expression. The impact of miR-223-3p-overexpressing exosomes on TNF-α-induced NPCs injury was evaluated. An in vivo intervertebral disc degeneration (IDD) model was induced in rat caudal spines via percutaneous needle puncture. Therapeutic efficacy was assessed by intradiscal injection of MSC-derived exosomes loaded with miR-223-3p mimic. The regulatory role of exosomal miR-223-3p on FBXW7 in NPCs was determined using gain- and loss-of-function approaches. Rescue experiments investigated whether miR-223-3p attenuates NPCs injury by targeting FBXW7. Direct targeting of FBXW7 3'UTR by miR-223-3p was confirmed via dual-luciferase reporter assays using wild-type and mutant constructs. Results Isolated vesicles exhibited characteristic exosome morphology, size (~ 76.6 nm by nano-flow cytometry), and marker expression (CD9/CD81/TSG101-positive, Calnexin-negative). NPCs efficiently internalized PKH26-tagged exosomal vesicles. NPCs co-cultured with mimic-exosomes exhibited elevated miR-223-3p levels, while inhibitor-exosomes reduced them. In vitro , Exosomes loaded with miR-223-3p mimic markedly attenuated TNF-α-triggered programmed cell death in NPCs (flow cytometry: 17.64% vs. TNF-α group 26.58%), decreased pro-apoptotic protein expression (Bax, Caspase-3), and increased anti-apoptotic Bcl-2. In vivo , intradiscal delivery of miR-223-3p mimic-exosomes ameliorated IDD progression, evidenced by reduced Pfirrmann grades on MRI, higher disc height index (DHI%) on X-ray, decreased apoptosis-related protein expression in NPCs, and improved histology compared to the IDD group. Furthermore, miR-223-3p mimic-exosomes downregulated FBXW7 mRNA and protein in NPCs, while inhibitor-exosomes upregulated it. Modulating miR-223-3p inversely regulated apoptosis markers. Crucially, FBXW7 knockdown (siRNA) reversed the pro-apoptotic effects induced by miR-223-3p inhibition. Dual-luciferase reporter assays confirmed direct binding of miR-223-3p to the FBXW7 3'UTR, with significant activity reduction in wild-type versus mutant constructs. Conclusion MSC-derived exosomes deliver functional miR-223-3p to NPCs. Exosomal miR-223-3p suppresses NPCs apoptosis and attenuates IDD progression by directly targeting and downregulating FBXW7 expression. Intervertebral Disc Degeneration Nucleus Pulposus Cells Mesenchymal Stem Cells Exosomes miR-223-3p FBXW7 Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Intervertebral disc degeneration (IDD) represents a major orthopedic burden, constituting a primary cause of radiculopathy, spinal cord injury, and paralysis in individuals under 45 years, imposing significant socioeconomic costs [ 1 ] . The intervertebral disc (IVD), the largest avascular, immune-privileged, and nutrient-poor organ in the human body, relies on diffusion from peripheral vasculature to nourish the central nucleus pulposus cells (NPCs) [ 2 ] . Consequently, factors such as mechanical stress, trauma, smoking, and aging can compromise disc vascular supply, leading to nutrient deprivation and accelerating IDD [ 3 – 4 ] . Current clinical management, encompassing conservative approaches and surgical interventions (e.g., discectomy, spinal fusion), primarily alleviates symptoms without addressing the underlying degenerative pathology [ 5 ] . Therefore, elucidating novel molecular pathways and mechanisms driving IDD is paramount for advancing diagnostic and therapeutic strategies. Emerging evidence indicates that bone marrow-derived mesenchymal stem cells (MSCs) can adopt an NPCs-like phenotype following non-contact co-culture [ 6 ] . Moreover, MSCs transplantation effectively mitigates NPCs degeneration and apoptosis [ 7 , 8 ] . It is now established that MSCs exert reparative effects not only through direct differentiation but also via paracrine signaling, particularly through the release of exosomes—key mediators of microenvironmental regulation [ 9 ] . These nano-vesicles facilitate intercellular communication by transferring bioactive molecules (mRNA, miRNA, proteins) to recipient cells, modulating critical processes including proliferation, differentiation, and apoptosis [ 10 – 12 ] . Exosomes offer distinct advantages, including stability, ease of isolation and storage, low immunogenicity, and amenability to modification, positioning MSC-derived exosomes as promising candidates for IVD regeneration [ 10 ] . MicroRNAs (miRNAs), key functional cargos within MSCs exosomes, modulate the local microenvironment, inhibit NPCs apoptosis, and promote disc repair [ 13 ] . For instance, MSCs exosomal miR-532-5p was recently reported to attenuate TNF-α-induced NPCs apoptosis and enhance extracellular matrix (ECM) production [ 14 ] . Our group previously identified miR-223-3p as the most abundant miRNA within MSC-derived exosomes via miRNA microarray analysis, suggesting its potential role in regulating NPCs apoptosis and IDD progression. Concurrently, utilizing miRNA target prediction databases (miRDB, miRTarBase, TargetScan) and transcriptomic sequencing of NPCs co-cultured with miR-223-3p-overexpressing exosomes, we identified FBXW7 as a putative direct target of miR-223-3p in NPCs. FBXW7, an E3 ubiquitin ligase component, is critically involved in diverse cellular functions, including cell cycle regulation, differentiation, apoptosis, tumor suppression, and drug resistance [ 15 – 19 ] . However, its specific role in IDD pathogenesis remains unexplored. 2. Materials and Methods 2.1. Cell Culture Rat bone marrow MSCs (rBMSCs) and rat NPCs (rNPCs) were procured from Zhong Qiao Xin Zhou (Shanghai, China). Exosome-depleted fetal bovine serum (Exo-FBS, Vivacell, Shanghai, China) and standard FBS (KeyGEN BioTECH, Nanjing, China) were used. Cells were cultured in DMEM/F12 medium (KeyGEN BioTECH) supplemented with 10% FBS (NPCs) or 10% Exo-FBS (MSCs), 100 mg/mL streptomycin, 100 U/mL penicillin, and 1% L-glutamine at 37°C under 5% CO₂. Medium renewal occurred at 48-hour intervals. Cells at passage 2 were employed for subsequent experimentation. 2.2. Cell Transfection and Grouping miR-223-3p mimic, mimic control (miR-NC), miR-223-3p inhibitor, inhibitor control (inh-NC), FBXW7 siRNA (si-FBXW7), and siRNA control (si-NC) were purchased from Feng Hui (Xuzhou, China) and Sangon (Shanghai, China), respectively. Transfections were performed using RNAtransmate reagent (Sangon) per manufacturer’s protocol. Groups for specific experiments were defined as follows: Section 3.1 : NPCs: Normal (untreated), mimic control, miR-223-3p mimic. Section 3.2 : NPCs: mimic control, TNF-α, TNF-α + miR-223-3p mimic. Section 3.4 : NPCs: mimic control, miR-223-3p mimic, inhibitor control, miR-223-3p inhibitor. Section 3.3 : NPCs: inhibitor control, miR-223-3p inhibitor, miR-223-3p inhibitor + si-FBXW7. 2.3. Exosome Isolation and Characterization MSCs were cultured in exosome-depleted medium for 48 hours to collect conditioned medium. Approximately 300 mL of conditioned medium underwent differential centrifugation: 300 × g (10 min, 4°C), 2,000 × g (20 min, 4°C), and 10,000 × g (30 min, 4°C) with pellet removal at each step, followed by 0.22-µm filtration and final ultracentrifugation (100,000 × g, 70 min, 4°C; Beckman Coulter). The resultant pellet was washed with PBS and recentrifuged under identical ultracentrifugation conditions. The final exosome pellet was resuspended in PBS. Characterization included TEM (Hitachi, Japan), NTA (size/concentration), nano-flow cytometry (size), and Western blotting for exosomal markers (CD9, CD81, TSG101) and negative control (Calnexin). 2.4. Exosome Uptake Assay Isolated exosomes were labeled with PKH26 (MKbio, Shanghai, China) for 5 min, washed via ultracentrifugation (100,000 × g, 70 min, 4°C), and resuspended in PBS. Labeled exosomes were co-cultured with NPCs for 12 hours (37°C, 5% CO₂). Cells were then fixed, nuclei stained with DAPI (Beyotime, Shanghai, China), and visualized by fluorescence microscopy (Olympus, Japan). 2.5. Flow Cytometry (Apoptosis) NPCs were harvested using trypsin-EDTA, washed with PBS, and resuspended in Binding Buffer (1×10⁵ cells). Cells were stained with Annexin V-APC and propidium iodide (PI) (KeyGEN, Nanjing, China) for 5–15 min (RT, dark). Apoptosis was analyzed by flow cytometry (Annexin V-APC⁺/PI⁻ cells = apoptotic; Annexin V-APC⁺/PI⁺ cells = necrotic). 2.6. Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted from NPCs using Trizol (TianGen, Beijing, China). RNA concentration and purity were measured spectrophotometrically. Reverse transcription and qRT-PCR for miR-223-3p, FBXW7, Bax, Bcl-2, Caspase-3, U6 (internal control for miRNA), and β-actin (internal control for mRNA) were performed using a LightCycler 480 II system (Roche Diagnostics, USA) and Universal SYBR Green Fast qRT-PCR Mix. Reaction conditions: 95°C for 15 min; 40 cycles of 95°C for 10 s, 60°C for 20 s. Primer sequences: miR-223-3p: Fwd 5'-GCGCGTGTCAGTTTGTCAAAT-3', Rev 5'-AGTGCAGGGTCCCAGGTATT-3'. FBXW7: Fwd 5'-ACGGGTGAATTTATCCGAAAC-3', Rev 5'-ATTCACCCGTTTTCAAGTCCC-3'. Bax: Fwd 5'-CCCGAGAGGTCTTTTTCCGAG-3', Rev 5'-CCAGCCCATGATGGTTCTGAT-3'. Caspase-3: Fwd 5'-ATGGTTTGAGCCTGAGCAGA-3', Rev 5'-GGCAGCATCATCCACACATAC-3'. Bcl-2: Fwd 5'-CAGCTGCACCTGACGCCCTT-3', Rev 5'-GCCTCCGTTATCCTGGATCC-3'. U6: Fwd 5'-CTCGCTTCGGCAGCACA-3', Rev 5'-AACGCTTCACGAATTTGCGT-3'. β-actin: Fwd 5'-AGGGGCCGGACTCGTCATACT-3', Rev 5'-GGCGGCACCACCATGTACCCT-3'. Experiments were performed in triplicate. 2.7. Western Blotting Following co-culture, NPCs were lysed using RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitors (EDTA-free). Protein concentration was determined via BCA assay (Beyotime). Proteins were separated by SDS-PAGE (8% or 12.5%), transferred to NC membranes, blocked with 5% skim milk (Biosharp, Beijing, China), and incubated overnight (4°C) with primary antibodies against CD9(Proteintech, Wuhan, China), CD81(Proteintech, Wuhan, China), TSG101(Proteintech, Wuhan, China), Calnexin(Proteintech, Wuhan, China), FBXW7 (Abcam, UK), Bax(Proteintech, Wuhan, China), Bcl-2(Proteintech, Wuhan, China), Caspase-3(Proteintech, Wuhan, China), β-actin (Proteintech, Wuhan, China). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Proteintech; 1:15,000) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (Bio-Rad) and quantified by densitometry with β-actin as loading control. Three independent biological replicates were performed. 2.8. Rat IDD Model and Intervention Animal experiments were approved by the Animal Ethics Committee of Nanjing Lambda Pharmaceutical Co., Ltd. (No. 2024110106) and complied with the Guide for the Care and Use of Laboratory Animals (NRC). Thirty-six adult male Sprague-Dawley rats (3 months old) were randomly assigned to four groups. Four coccygeal discs (Co7/8, Co8/9, Co9/10, Co10/11) per rat were randomly allocated to: Control (no intervention), IDD (puncture), IDD + miR-223-3p mimic exosomes, or Sham. All rats were anesthetized with isoflurane gas (3% for induction, 2% for maintenance). A 22-gauge puncture needle was then inserted into the intervertebral space between two adjacent vertebral bodies to a depth of approximately ~ 5 mm. Following insertion, the needle was rotated 360 degrees and maintained in situ for 30 seconds before withdrawal to establish the IDD model [ 20 ] . In the treatment group, intradiscal injections of miR-223-3p mimic-loaded exosomes (concentration 2 µg/µL, total volume 20 µL) were administered to discs with confirmed successful modeling using a microsyringe, once weekly for 4 weeks. 2.9. Radiological Evaluation Six weeks post-intervention, rats were anesthetized. X-ray (Xinglian Experimental, Shenzhen, China) and T2-weighted MRI (Biospec 7T/20 USR, Bruker, Germany) of the coccyx were performed. Using a double-blind method, three experienced spine surgeons evaluated the imaging findings of the intervertebral discs. Disc height index (DHI%) was calculated from X-rays using ImageJ. Disc degeneration was graded on MRI according to the Pfirrmann classification. 2.10. Histological Analysis Disc specimens were fixed in 10% neutral buffered formalin for 24 hours, decalcified in 10% EDTA, dehydrated, paraffin-embedded, sectioned (4–5 µm), and stained with Hematoxylin and Eosin (H&E) for morphological assessment. 2.11. Isolation and Culture of NPCs from Rat Model NPC tissue was dissected from harvested discs, minced, and digested in 0.2% collagenase type II + 0.25% trypsin (Gibco, USA) for 3 hours. The cell suspension was filtered, washed, centrifuged, and cultured as described in Section 2.1 . 2.12. Dual-Luciferase Reporter Assay 293T cells (Jinke Biological, Tianjin, China) were seeded in 24-well plates (1×10⁵ cells/mL). Cells were co-transfected with: Group 1: hsa-miR-223-3p-NC + pmirGLO-FBXW7-3'UTR-WT Group 2: hsa-miR-223-3p-mimic + pmirGLO-FBXW7-3'UTR-WT Group 3: hsa-miR-223-3p-NC + pmirGLO-FBXW7-3'UTR-MUT Group 4: hsa-miR-223-3p-mimic + pmirGLO-FBXW7-3'UTR-MUT Transfection complexes were prepared using Lipofectamine 2000 (Invitrogen, USA) per manufacturer's protocol. After 24 hours, luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Beyotime) on a microplate reader (MD M5). Firefly luciferase activity was normalized to Renilla luciferase activity. 2.13. Statistical Analysis Data analysis and graph generation were performed using GraphPad Prism 9.0 (GraphPad Software, USA). ImageJ analyzed radiological images. Quantitative data are presented as mean ± standard deviation (X̄ ± SD) from ≥ 3 independent experiments. Comparisons between two groups used unpaired Student's t-tests. Multiple group comparisons employed one-way ANOVA. Statistical significance was defined as P < 0.05. 3. Results 3.1. Characterization of MSC Exosomes and miR-223-3p Delivery to NPCs rBMSCs exhibited a characteristic spindle-shaped morphology at ~ 40–50% confluence (Fig. 1 (a)). Exosomes isolated from transfected MSCs (miR-223-3p mimic/inhibitor) were characterized. NTA indicated a concentration of 3.3×10 10 particles/mL and an average size of 150.7 nm (Fig. 1 (b)). Nano-flow cytometry confirmed exosome concentration (8.64×10 9 particles/mL) and size (average 76.6 nm) (Fig. 1 (c)). TEM revealed characteristic cup-shaped vesicle morphology (Fig. 1 d). Western blot confirmed enrichment of exosomal markers (CD9, CD81, TSG101) and absence of the negative marker Calnexin (Fig. 1 (e)). PKH26-labeled exosomes were effectively internalized by NPCs (Fig. 1 (f)). qRT-PCR demonstrated that NPCs co-cultured with mimic-exosomes exhibited significantly elevated miR-223-3p levels, while inhibitor-exosomes reduced miR-223-3p expression compared to respective controls (Fig. 1 (g)), confirming functional miRNA transfer. 3.2. MSCs Exosomal miR-223-3p Attenuates TNF-α-Induced NPCs Apoptosis In Vitro qRT-PCR confirmed reduced miR-223-3p expression in TNF-α-treated NPCs compared to controls. Co-culture with miR-223-3p mimic-exosomes restored miR-223-3p levels (Fig. 2 (a)). Mimic-exosomes significantly counteracted TNF-α-induced apoptosis: qRT-PCR and Western blot showed decreased Bax and Caspase-3 expression and increased Bcl-2 expression in the TNF-α + mimic-exosome group versus TNF-α alone (Fig. 2 (b)-(c)). Flow cytometry corroborated these findings, revealing significantly reduced apoptosis rates in NPCs treated with mimic-exosomes (17.64%) versus TNF-α alone (26.58%) (Fig. 2 (d)). 3.3. MSCs Exosomal miR-223-3p Mitigates IDD Progression In Vivo MRI and X-ray assessment 6 weeks post-intervention demonstrated that intradiscal injection of miR-223-3p mimic-exosomes significantly ameliorated IDD severity. Pfirrmann grades were lower (Fig. 3 (a), (c)), and DHI% was higher (Fig. 3 (b), (d)) in the IDD + mimic-exosome group compared to the IDD group. H&E staining revealed less severe structural disruption in the treatment group compared to IDD controls, although both differed significantly from Control/Sham groups (Fig. 3 (e)). NPCs isolated from treated discs exhibited higher miR-223-3p levels (Fig. 3 (f)) and reduced expression of pro-apoptotic proteins (Bax, Caspase-3) alongside elevated Bcl-2 expression compared to NPCs from IDD discs (Fig. 3 (g)-(h)). 3.4. MSCs Exosomal miR-223-3p Suppresses FBXW7 Expression and Modulates Apoptosis in NPCs Co-culture with mimic-exosomes significantly downregulated FBXW7 mRNA and protein levels in NPCs, whereas inhibitor-exosomes upregulated FBXW7 expression compared to respective controls (Fig. 4 (a)-(c)). Concordantly, mimic-exosomes decreased Bax and Caspase-3 mRNA/protein and increased Bcl-2, while inhibitor-exosomes exerted opposing effects on these apoptosis markers (Fig. 4 (d)-(e)). This established an inverse correlation between exosomal miR-223-3p levels and FBXW7 expression, coupled with modulation of apoptosis pathways. 3.5. FBXW7 is a Direct Target of miR-223-3p and Mediates its Anti-Apoptotic Effect Rescue experiments confirmed FBXW7's role downstream of miR-223-3p. Inhibitor-exosomes upregulated FBXW7, Bax, and Caspase-3 while downregulating Bcl-2 (Fig. 5 (a)-(b)). Co-transfection of FBXW7 siRNA with the miR-223-3p inhibitor significantly reversed these pro-apoptotic effects, normalizing FBXW7, Bax, Caspase-3, and Bcl-2 levels (Fig. 5 (c)-(d)). Flow cytometry confirmed reduced apoptosis in the inhibitor + si-FBXW7 group (16.05%) versus inhibitor alone (21.37%) (Fig. 5 (e)). The dual-luciferase reporter analysis conclusively demonstrated: miR-223-3p mimic significantly suppressed luciferase activity from the wild-type (WT) FBXW7 3'UTR reporter construct but not the mutant (MUT) construct (Fig. 5 (f)), confirming direct binding of miR-223-3p to the FBXW7 3'UTR. 4. Discussion Intervertebral disc degeneration (IDD) is a prevalent orthopedic disorder, representing the primary cause of nerve root and spinal cord injuries, and the most significant contributor to paralysis in individuals under 45 years of age, exerting considerable socioeconomic impacts [ 1 ] . The intervertebral disc (IVD), comprising the annulus fibrosus, cartilaginous end plates, and nucleus pulposus, functions as the largest immune-privileged, hypovascular, and avascular organ in the human body. Nutrient supply to the central nucleus pulposus cells (NPCs) relies primarily on diffusion from surrounding vasculature. NPCs orchestrate pivotal pathological cascades in IDD development, and factors such as mechanical stress, trauma, smoking, and aging can compromise disc vascularization, leading to nutrient deprivation and accelerating IDD progression [ 3 , 4 ] . Current therapeutic strategies for IDD fail to reverse the underlying pathological processes. Consequently, discovery of novel molecular regulators and delineation of their mechanistic roles in disc degeneration represent urgent research priorities to enable targeted therapeutic development. Stem cell-based regenerative strategies have advanced therapeutic development for IDD, particularly MSCs transplantation. Numerous studies indicate that MSCs implantation can delay NPCs pathology by modulating microenvironmental homeostasis [ 21 ] . However, the hostile disc milieu—marked by hypoxia, acidic pH, and nutrient deprivation—severely compromises engrafted MSC viability, constituting a major translational barrier [ 22 ] . Notably, MSC-derived exosomes function as critical intercellular signaling vectors, delivering bioactive cargo (mRNAs, miRNAs, proteins) that modulate recipient cell transcriptomes via paracrine mechanisms [ 9 ] . This paracrine paradigm establishes exosome-based nanotherapeutics as promising cell-free alternatives that circumvent microenvironmental limitations in IDD treatment. MicroRNAs (miRNAs) are endogenous non-coding RNAs (~ 19–25 nt) involved in regulating cell proliferation and apoptosis [ 23 – 26 ] . They primarily function by binding target mRNAs, leading to mRNA degradation or translational repression, resulting in differential expression of target genes and modulation of downstream signaling pathways [ 27 , 28 ] . Previous research highlights the significant role of miRNAs in IDD pathogenesis [ 29 ] . Our group's prior work, involving isolation of MSCs-derived exosomes and miRNA microarray profiling, identified miR-223-3p as one of the most highly enriched miRNAs within these vesicles. Studies suggest miR-223-3p may regulate autophagy by targeting ATG16L1 and is implicated in inflammation, potentially serving as a therapeutic target in keratitis [ 30 ] . Furthermore, Bao et al. demonstrated that exosomal miR-223-3p facilitates complex communication between colon cancer cells and macrophages, promoting cancer cell proliferation and migration [ 31 ] . In IDD, miR-223-3p expression is reduced by overexpression of MIR155HG, leading to upregulated NLRP3 expression and induction of apoptosis [ 32 ] . In the present study, exosomes were purified through differential ultracentrifugation and characterized. Recognizing that NTA sensitivity (~ 70 nm) and inclusion of surface hydration layers can overestimate size, we employed nano-flow cytometry for complementary validation, confirming exosome isolation (Fig. 1 (b)-(c)). Subsequent PKH26 labeling and DAPI staining confirmed NPCs uptake of exosomes (Fig. 1 (f)). Critically, qPCR analysis revealed significantly elevated miR-223-3p levels in NPCs co-cultured with exosomes derived from miR-223-3p mimic-transfected MSCs (Fig. 1 (g)), demonstrating efficient functional delivery. Exosomes derived from MSCs and carrying miRNAs hold significant therapeutic promise for IDD. For instance, MSCs-derived exosomal miR-532-5p was shown to attenuate TNF-α-induced NPCs apoptosis and promote extracellular matrix (ECM) production [ 14 ] . Similarly, Zhu et al. demonstrated that MSCs exosomes carrying miR-142-3p ameliorate NPCs apoptosis [ 33 ] . Our findings align with this paradigm: TNF-α-induced NPCs apoptosis was associated with upregulated Bax and Caspase-3, downregulated miR-223-3p and Bcl-2. Conversely, treatment with miR-223-3p mimic-loaded exosomes significantly suppressed Bax and Caspase-3 expression while elevating Bcl-2 levels (Fig. 2 ), indicating a protective role against NPCs apoptosis. Following the observation of miR-223-3p's effect on apoptosis in vitro , we investigated its role in vivo using a rat caudal IDD model. Consistent with in vitro results, in vivo studies confirmed that MSCs-exosomal miR-223-3p mitigates IDD progression. Radiological assessment (MRI and X-ray) revealed less severe disc degeneration in the IDD + miR-223-3p mimic group compared to the IDD group (Fig. 3 (a)-(d)), a finding corroborated by histopathological evaluation (Fig. 3 (e)). Furthermore, analysis of NPCs isolated from these discs showed decreased expression of pro-apoptotic proteins Bax and Caspase-3 and increased expression of anti-apoptotic Bcl-2 in the treatment group versus the IDD group (Fig. 3 (f)-(h)). Collectively, these data suggest MSCs-exosomal miR-223-3p delays IDD progression, at least partially, by attenuating NPCs apoptosis. Bioinformatic analysis from our prior work identified FBXW7 as a putative direct target of miR-223-3p in NPCs. The regulatory relationship between miR-223 and FBXW7 is well-documented across various tissues and diseases, including hematopoietic and digestive systems [ 34 – 40 ] , highlighting its broad regulatory capacity. In orthopedics, recent studies indicate that FBXW7 inhibition upregulates HIF1α and Runx2, promoting osteoblast differentiation [ 41 , 42 ] . Wang et al. reported that overexpression of miR-223-3p significantly reduced FBXW7 protein levels in mouse corneal epithelial cells, while miR-223-3p knockdown increased FBXW7 [ 43 ] . Our results are consistent: co-culture of NPCs with exosomes from miR-223-3p mimic-transfected MSCs significantly downregulated FBXW7 mRNA and protein expression, whereas exosomes from inhibitor-transfected MSCs reversed this effect (Fig. 4 (a)-(c)). Furthermore, Wang et al. showed adipose MSCs-derived exosomal miR-223-3p inhibits inflammation via FBXW7 suppression [ 43 ] . We extended this by examining apoptosis markers: miR-223-3p mimic-exosomes decreased FBXW7 expression, concurrently suppressing Bax and Caspase-3 while elevating Bcl-2 expression at both mRNA and protein levels (Fig. 4 (d)-(e)). The reversal of these effects by inhibitor-exosomes strongly suggests miR-223-3p participates in NPCs apoptosis regulation by inhibiting FBXW7. Functional rescue experiments further confirmed FBXW7 as a key mediator. Co-culture with inhibitor-exosomes increased FBXW7, Bax, and Caspase-3 expression while decreasing Bcl-2. Co-transfection of NPCs with FBXW7 siRNA alongside inhibitor-exosomes effectively reversed the upregulation of FBXW7, Bax, and Caspase-3 and the downregulation of Bcl-2 induced by miR-223-3p knockdown alone. This confirms that MSCs-exosomal miR-223-3p regulates NPCs apoptosis through FBXW7 suppression(Fig. 5 (a)-(d)). To definitively establish direct targeting, dual-luciferase reporter assays were performed. The results showed miR-223-3p mimic significantly reduced luciferase activity only in cells transfected with the reporter plasmid containing the wild-type (WT) FBXW7 3'UTR fragment. In contrast, no significant reduction was observed when the plasmid contained a mutated (MUT) binding site within the 3'UTR (Fig. 5 (f)). This series of evidence demonstrates that miR-223-3p specifically binds to the 3'UTR region of FBXW7 mRNA, implicating it in the post-transcriptional regulation of FBXW7 expression and confirming FBXW7 as a direct target gene of miR-223-3p. Capitalizing on prior mechanistic insights, this investigation targets IDD through engineered exosomal nanovectors—a promising therapeutic paradigm. Through integrative multi-level analysis (molecular to organismal), we delineate a previously unreported mechanism: MSC-derived exosomal miR-223-3p attenuates disc degeneration by targeting FBXW7 in NPCs, thereby promoting tissue repair. These findings bridge fundamental discovery and clinical translation, establishing a conceptual framework for MSC-exosome therapeutics in IDD management. However, limitations exist: The persistence, biodistribution, and targeting efficiency of MSCs exosomes in vivo across species remain unclear, and the precise efficiency of exosomal miR-223-3p delivery requires further elucidation. Furthermore, potential FBXW7 crosstalk with alternative miR-223-3p targets necessitates systems-level interrogation via proteomics/transcriptomics. Finally, clinical translation necessitates optimization of exosome isolation protocols and dosage regimens, consistent with challenges emphasized in exosome therapy trials. 5. Conclusions In conclusion, our findings demonstrate that MSC-derived exosomal miR-223-3p mitigates intervertebral disc degeneration (IDD) progression by targeting FBXW7 to regulate nucleus pulposus cell apoptosis. This work provides a mechanistic foundation for translating MSC-exosome therapeutics into clinical applications for IDD management. Declarations Data Availability Statement All data supporting the results have been displayed in the paperitself or uploaded as Supporting Information for OnlinePublication. Ethics Statement All animal experiments were approved by the Animal Ethics Committee of Nanjing Lambda Pharmaceutical Co., Ltd. (Approval No. 2024110106) and conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Conflicts of Interest The authors declare no conflicts of interest. Author Contributions Rui Chen, Kaiyi Cao, and Yuting Gong: Performed data collection and conducted experiments. Yuning Zhu and Yi Gao: Conducted data analysis and interpretation. Jing Yan and Yuning Zhu: Searched the literature. Rui Chen, Yi Gao, and Yuting Gong: Completed figure/image processing. Rui Chen: Drafted the manuscript. Quan Zhou: Reviewed and edited the manuscript. Quan Zhou and Wei Pan: Designed and funded the study. All authors read and approved the final manuscript. Rui Chen, Kaiyi Cao, and Yuting Gong contributed equally to this work. Quan Zhou and Wei Pan are the co-corresponding authors of this article. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 82372480), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (Grant No. SJCX24-1546), and the Huai'an Municipal Bureau of Science and Technology - Natural Science Research Plan (Grant No. HAB202320). References Yang S, Zhang F, Ma J, et al. Intervertebral disc ageing and degeneration: The antiapoptotic effect of oestrogen. Ageing Res Rev. 2020;57:100978. Humzah MD, Soames RW. Human intervertebral disc: structure and function. Anat Rec. 1988;220(4):337-356. Mohd II, Teoh SL, Mohd NN, et al. Discogenic Low Back Pain: Anatomy, Pathophysiology and Treatments of Intervertebral Disc Degeneration. Int J Mol Sci. 2022;24(1):208. Published 2022 Dec 22. 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Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted Editorial decision: Revision requested 10 Oct, 2025 Reviews received at journal 09 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers invited by journal 02 Oct, 2025 Editor assigned by journal 02 Oct, 2025 Submission checks completed at journal 01 Oct, 2025 First submitted to journal 28 Sep, 2025 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":159668,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of exosomal miR-223-3p derived from rat BM-MSCs and its internalization by NPCs. (a) Representative phase-contrast micrograph of rat BM-MSCs. Scale bar = 50 μm. (b-c) Nanoparticle tracking analysis (NTA) and nanoscale flow cytometry (nanoFCM) characterization of exosomes, with horizontal axis representing particle size (nm) and vertical axis indicating concentration (particles/mL). (d) Transmission electron microscopy (TEM) image revealing exosomal morphology. Scale bar = 100 nm. (e) Western blot (WB) analysis of exosomal marker proteins in both MSCs and their derived exosomes. (f) Fluorescent micrograph demonstrating internalization of PKH26-labeled exosomes (red) by NPCs (magnification 25.2×, scale bar = 20 μm). (g) qRT-PCR quantification of miR-223-3p expression levels. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737133/v1/0c9091188d78395e3b9803f0.jpeg"},{"id":93612522,"identity":"1609d487-5f30-4a2b-a506-8b8782b5ae16","added_by":"auto","created_at":"2025-10-15 16:20:14","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":254061,"visible":true,"origin":"","legend":"\u003cp\u003eMSC-derived exosomal miR-223-3p suppresses TNF-α-induced apoptosis in rat NPCs. (a-b) qRT-PCR analysis demonstrating mRNA expression levels of miR-223-3p and apoptosis-related markers (Bax, Bcl-2, and Caspase-3) in NPCs across experimental groups. (c) Western blot analysis of apoptosis-related protein expression patterns. (d) Flow cytometric quantification of apoptotic rates in NPCs. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737133/v1/10c99fe1aff80f301b6162e3.jpeg"},{"id":93611088,"identity":"65d090cf-97e5-417c-be0e-59ce8a8b5d6a","added_by":"auto","created_at":"2025-10-15 16:12:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":439220,"visible":true,"origin":"","legend":"\u003cp\u003eHistological and molecular evaluations. (a-b) Representative radiographic and MRI characteristics of four vertebral segments per mouse at 6 weeks post-intervention. (c-d) Pfirrmann grading classifications and %DHI indices across experimental groups. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (e) Hematoxylin-eosin (HE) staining of intervertebral disc tissues; scale bar = 500 μm. (f-h) mRNA expression levels of miR-223-3p and apoptosis-related proteins (Bax, Bcl-2, Caspase-3) in NPCs, as measured by qRT-PCR (panels f-g) and Western blot (panel h). ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737133/v1/583950739000ed2c7eb91b02.jpeg"},{"id":93611096,"identity":"e360abc8-4250-4606-b33a-553f07aaa672","added_by":"auto","created_at":"2025-10-15 16:12:14","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181034,"visible":true,"origin":"","legend":"\u003cp\u003eMSC-derived exosomal miR-223-3p suppresses apoptosis of NPCs via FBXW7. (a-c) Quantitative analysis of miR-223-3p expression levels (qRT-PCR) and FBXW7 mRNA/protein expression (qRT-PCR and Western blot, respectively) in NPCs across experimental groups. (d-e) mRNA and protein expression profiles of apoptosis-related markers (Bax, Bcl-2, Caspase-3) in NPCs were determined by qRT-PCR and Western blot. Statistical significance: ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737133/v1/4724620dcf7096de888f86fd.jpeg"},{"id":93611092,"identity":"cdceb449-1358-4a46-bbb9-32b421da283a","added_by":"auto","created_at":"2025-10-15 16:12:14","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":277357,"visible":true,"origin":"","legend":"\u003cp\u003eMSC-derived exosomal miR-223-3p targets FBXW7 to suppress nucleus pulposus cell apoptosis. (a-d) qRT-PCR and Western blot analyses demonstrate the mRNA and protein expression levels of FBXW7, along with apoptosis-related proteins Bax, Bcl-2, and Caspase-3, in NPCs across experimental groups. (e) Flow cytometry quantifies apoptosis rates in NPCs among groups. (f) Dual-luciferase reporter assay using WT or mutant (MU) FBXW7 3ʹ-UTR constructs. Co-transfection of WT FBXW7 3ʹ-UTR with miR-223-3p mimics significantly suppresses luciferase activity in 293T cells. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737133/v1/d75786ad7e462ce7d4dd58c3.jpeg"},{"id":98245093,"identity":"117293f4-5aa5-4372-b930-e9f218579eb5","added_by":"auto","created_at":"2025-12-15 16:16:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2139861,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7737133/v1/99544fe1-7da3-468a-bc34-ba2024a93547.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomal miR-223-3p from Mesenchymal Stem Cells Targets FBXW7 to Inhibit Intervertebral Disc Degeneration: Mechanism Insights","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIntervertebral disc degeneration (IDD) represents a major orthopedic burden, constituting a primary cause of radiculopathy, spinal cord injury, and paralysis in individuals under 45 years, imposing significant socioeconomic costs\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The intervertebral disc (IVD), the largest avascular, immune-privileged, and nutrient-poor organ in the human body, relies on diffusion from peripheral vasculature to nourish the central nucleus pulposus cells (NPCs)\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Consequently, factors such as mechanical stress, trauma, smoking, and aging can compromise disc vascular supply, leading to nutrient deprivation and accelerating IDD\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Current clinical management, encompassing conservative approaches and surgical interventions (e.g., discectomy, spinal fusion), primarily alleviates symptoms without addressing the underlying degenerative pathology\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, elucidating novel molecular pathways and mechanisms driving IDD is paramount for advancing diagnostic and therapeutic strategies.\u003c/p\u003e\u003cp\u003eEmerging evidence indicates that bone marrow-derived mesenchymal stem cells (MSCs) can adopt an NPCs-like phenotype following non-contact co-culture\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Moreover, MSCs transplantation effectively mitigates NPCs degeneration and apoptosis\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. It is now established that MSCs exert reparative effects not only through direct differentiation but also via paracrine signaling, particularly through the release of exosomes\u0026mdash;key mediators of microenvironmental regulation\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. These nano-vesicles facilitate intercellular communication by transferring bioactive molecules (mRNA, miRNA, proteins) to recipient cells, modulating critical processes including proliferation, differentiation, and apoptosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Exosomes offer distinct advantages, including stability, ease of isolation and storage, low immunogenicity, and amenability to modification, positioning MSC-derived exosomes as promising candidates for IVD regeneration\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMicroRNAs (miRNAs), key functional cargos within MSCs exosomes, modulate the local microenvironment, inhibit NPCs apoptosis, and promote disc repair\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. For instance, MSCs exosomal miR-532-5p was recently reported to attenuate TNF-α-induced NPCs apoptosis and enhance extracellular matrix (ECM) production\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Our group previously identified miR-223-3p as the most abundant miRNA within MSC-derived exosomes via miRNA microarray analysis, suggesting its potential role in regulating NPCs apoptosis and IDD progression.\u003c/p\u003e\u003cp\u003eConcurrently, utilizing miRNA target prediction databases (miRDB, miRTarBase, TargetScan) and transcriptomic sequencing of NPCs co-cultured with miR-223-3p-overexpressing exosomes, we identified FBXW7 as a putative direct target of miR-223-3p in NPCs. FBXW7, an E3 ubiquitin ligase component, is critically involved in diverse cellular functions, including cell cycle regulation, differentiation, apoptosis, tumor suppression, and drug resistance\u003csup\u003e[\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. However, its specific role in IDD pathogenesis remains unexplored.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cell Culture\u003c/h2\u003e\u003cp\u003eRat bone marrow MSCs (rBMSCs) and rat NPCs (rNPCs) were procured from Zhong Qiao Xin Zhou (Shanghai, China). Exosome-depleted fetal bovine serum (Exo-FBS, Vivacell, Shanghai, China) and standard FBS (KeyGEN BioTECH, Nanjing, China) were used. Cells were cultured in DMEM/F12 medium (KeyGEN BioTECH) supplemented with 10% FBS (NPCs) or 10% Exo-FBS (MSCs), 100 mg/mL streptomycin, 100 U/mL penicillin, and 1% L-glutamine at 37\u0026deg;C under 5% CO₂. Medium renewal occurred at 48-hour intervals. Cells at passage 2 were employed for subsequent experimentation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Cell Transfection and Grouping\u003c/h2\u003e\u003cp\u003emiR-223-3p mimic, mimic control (miR-NC), miR-223-3p inhibitor, inhibitor control (inh-NC), FBXW7 siRNA (si-FBXW7), and siRNA control (si-NC) were purchased from Feng Hui (Xuzhou, China) and Sangon (Shanghai, China), respectively. Transfections were performed using RNAtransmate reagent (Sangon) per manufacturer\u0026rsquo;s protocol. Groups for specific experiments were defined as follows: Section \u003cspan refid=\"Sec17\" class=\"InternalRef\"\u003e3.1\u003c/span\u003e: NPCs: Normal (untreated), mimic control, miR-223-3p mimic. Section \u003cspan refid=\"Sec18\" class=\"InternalRef\"\u003e3.2\u003c/span\u003e: NPCs: mimic control, TNF-α, TNF-α\u0026thinsp;+\u0026thinsp;miR-223-3p mimic. Section \u003cspan refid=\"Sec20\" class=\"InternalRef\"\u003e3.4\u003c/span\u003e: NPCs: mimic control, miR-223-3p mimic, inhibitor control, miR-223-3p inhibitor. Section \u003cspan refid=\"Sec19\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e: NPCs: inhibitor control, miR-223-3p inhibitor, miR-223-3p inhibitor\u0026thinsp;+\u0026thinsp;si-FBXW7.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Exosome Isolation and Characterization\u003c/h2\u003e\u003cp\u003eMSCs were cultured in exosome-depleted medium for 48 hours to collect conditioned medium. Approximately 300 mL of conditioned medium underwent differential centrifugation: 300 \u0026times; g (10 min, 4\u0026deg;C), 2,000 \u0026times; g (20 min, 4\u0026deg;C), and 10,000 \u0026times; g (30 min, 4\u0026deg;C) with pellet removal at each step, followed by 0.22-\u0026micro;m filtration and final ultracentrifugation (100,000 \u0026times; g, 70 min, 4\u0026deg;C; Beckman Coulter). The resultant pellet was washed with PBS and recentrifuged under identical ultracentrifugation conditions. The final exosome pellet was resuspended in PBS. Characterization included TEM (Hitachi, Japan), NTA (size/concentration), nano-flow cytometry (size), and Western blotting for exosomal markers (CD9, CD81, TSG101) and negative control (Calnexin).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Exosome Uptake Assay\u003c/h2\u003e\u003cp\u003eIsolated exosomes were labeled with PKH26 (MKbio, Shanghai, China) for 5 min, washed via ultracentrifugation (100,000 \u0026times; g, 70 min, 4\u0026deg;C), and resuspended in PBS. Labeled exosomes were co-cultured with NPCs for 12 hours (37\u0026deg;C, 5% CO₂). Cells were then fixed, nuclei stained with DAPI (Beyotime, Shanghai, China), and visualized by fluorescence microscopy (Olympus, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Flow Cytometry (Apoptosis)\u003c/h2\u003e\u003cp\u003eNPCs were harvested using trypsin-EDTA, washed with PBS, and resuspended in Binding Buffer (1\u0026times;10⁵ cells). Cells were stained with Annexin V-APC and propidium iodide (PI) (KeyGEN, Nanjing, China) for 5\u0026ndash;15 min (RT, dark). Apoptosis was analyzed by flow cytometry (Annexin V-APC⁺/PI⁻ cells\u0026thinsp;=\u0026thinsp;apoptotic; Annexin V-APC⁺/PI⁺ cells\u0026thinsp;=\u0026thinsp;necrotic).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Quantitative Real-Time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from NPCs using Trizol (TianGen, Beijing, China). RNA concentration and purity were measured spectrophotometrically. Reverse transcription and qRT-PCR for miR-223-3p, FBXW7, Bax, Bcl-2, Caspase-3, U6 (internal control for miRNA), and β-actin (internal control for mRNA) were performed using a LightCycler 480 II system (Roche Diagnostics, USA) and Universal SYBR Green Fast qRT-PCR Mix. Reaction conditions: 95\u0026deg;C for 15 min; 40 cycles of 95\u0026deg;C for 10 s, 60\u0026deg;C for 20 s. Primer sequences: miR-223-3p: Fwd 5'-GCGCGTGTCAGTTTGTCAAAT-3', Rev 5'-AGTGCAGGGTCCCAGGTATT-3'. FBXW7: Fwd 5'-ACGGGTGAATTTATCCGAAAC-3', Rev 5'-ATTCACCCGTTTTCAAGTCCC-3'. Bax: Fwd 5'-CCCGAGAGGTCTTTTTCCGAG-3', Rev 5'-CCAGCCCATGATGGTTCTGAT-3'. Caspase-3: Fwd 5'-ATGGTTTGAGCCTGAGCAGA-3', Rev 5'-GGCAGCATCATCCACACATAC-3'. Bcl-2: Fwd 5'-CAGCTGCACCTGACGCCCTT-3', Rev 5'-GCCTCCGTTATCCTGGATCC-3'. U6: Fwd 5'-CTCGCTTCGGCAGCACA-3', Rev 5'-AACGCTTCACGAATTTGCGT-3'. β-actin: Fwd 5'-AGGGGCCGGACTCGTCATACT-3', Rev 5'-GGCGGCACCACCATGTACCCT-3'. Experiments were performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Western Blotting\u003c/h2\u003e\u003cp\u003eFollowing co-culture, NPCs were lysed using RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitors (EDTA-free). Protein concentration was determined via BCA assay (Beyotime). Proteins were separated by SDS-PAGE (8% or 12.5%), transferred to NC membranes, blocked with 5% skim milk (Biosharp, Beijing, China), and incubated overnight (4\u0026deg;C) with primary antibodies against CD9(Proteintech, Wuhan, China), CD81(Proteintech, Wuhan, China), TSG101(Proteintech, Wuhan, China), Calnexin(Proteintech, Wuhan, China), FBXW7 (Abcam, UK), Bax(Proteintech, Wuhan, China), Bcl-2(Proteintech, Wuhan, China), Caspase-3(Proteintech, Wuhan, China), β-actin (Proteintech, Wuhan, China). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Proteintech; 1:15,000) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (Bio-Rad) and quantified by densitometry with β-actin as loading control. Three independent biological replicates were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Rat IDD Model and Intervention\u003c/h2\u003e\u003cp\u003e Animal experiments were approved by the Animal Ethics Committee of Nanjing Lambda Pharmaceutical Co., Ltd. (No. 2024110106) and complied with the Guide for the Care and Use of Laboratory Animals (NRC). Thirty-six adult male Sprague-Dawley rats (3 months old) were randomly assigned to four groups. Four coccygeal discs (Co7/8, Co8/9, Co9/10, Co10/11) per rat were randomly allocated to: Control (no intervention), IDD (puncture), IDD\u0026thinsp;+\u0026thinsp;miR-223-3p mimic exosomes, or Sham. All rats were anesthetized with isoflurane gas (3% for induction, 2% for maintenance). A 22-gauge puncture needle was then inserted into the intervertebral space between two adjacent vertebral bodies to a depth of approximately\u0026thinsp;~\u0026thinsp;5 mm. Following insertion, the needle was rotated 360 degrees and maintained in situ for 30 seconds before withdrawal to establish the IDD model\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In the treatment group, intradiscal injections of miR-223-3p mimic-loaded exosomes (concentration 2 \u0026micro;g/\u0026micro;L, total volume 20 \u0026micro;L) were administered to discs with confirmed successful modeling using a microsyringe, once weekly for 4 weeks.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Radiological Evaluation\u003c/h2\u003e\u003cp\u003eSix weeks post-intervention, rats were anesthetized. X-ray (Xinglian Experimental, Shenzhen, China) and T2-weighted MRI (Biospec 7T/20 USR, Bruker, Germany) of the coccyx were performed. Using a double-blind method, three experienced spine surgeons evaluated the imaging findings of the intervertebral discs. Disc height index (DHI%) was calculated from X-rays using ImageJ. Disc degeneration was graded on MRI according to the Pfirrmann classification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Histological Analysis\u003c/h2\u003e\u003cp\u003eDisc specimens were fixed in 10% neutral buffered formalin for 24 hours, decalcified in 10% EDTA, dehydrated, paraffin-embedded, sectioned (4\u0026ndash;5 \u0026micro;m), and stained with Hematoxylin and Eosin (H\u0026amp;E) for morphological assessment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Isolation and Culture of NPCs from Rat Model\u003c/h2\u003e\u003cp\u003eNPC tissue was dissected from harvested discs, minced, and digested in 0.2% collagenase type II\u0026thinsp;+\u0026thinsp;0.25% trypsin (Gibco, USA) for 3 hours. The cell suspension was filtered, washed, centrifuged, and cultured as described in Section \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003e2.1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Dual-Luciferase Reporter Assay\u003c/h2\u003e\u003cp\u003e293T cells (Jinke Biological, Tianjin, China) were seeded in 24-well plates (1\u0026times;10⁵ cells/mL). Cells were co-transfected with:\u003c/p\u003e\u003cp\u003eGroup 1: hsa-miR-223-3p-NC\u0026thinsp;+\u0026thinsp;pmirGLO-FBXW7-3'UTR-WT\u003c/p\u003e\u003cp\u003eGroup 2: hsa-miR-223-3p-mimic\u0026thinsp;+\u0026thinsp;pmirGLO-FBXW7-3'UTR-WT\u003c/p\u003e\u003cp\u003eGroup 3: hsa-miR-223-3p-NC\u0026thinsp;+\u0026thinsp;pmirGLO-FBXW7-3'UTR-MUT\u003c/p\u003e\u003cp\u003eGroup 4: hsa-miR-223-3p-mimic\u0026thinsp;+\u0026thinsp;pmirGLO-FBXW7-3'UTR-MUT\u003c/p\u003e\u003cp\u003eTransfection complexes were prepared using Lipofectamine 2000 (Invitrogen, USA) per manufacturer's protocol. After 24 hours, luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Beyotime) on a microplate reader (MD M5). Firefly luciferase activity was normalized to Renilla luciferase activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Statistical Analysis\u003c/h2\u003e\u003cp\u003eData analysis and graph generation were performed using GraphPad Prism 9.0 (GraphPad Software, USA). ImageJ analyzed radiological images. Quantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (X̄ \u0026plusmn; SD) from \u0026ge;\u0026thinsp;3 independent experiments. Comparisons between two groups used unpaired Student's t-tests. Multiple group comparisons employed one-way ANOVA. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization of MSC Exosomes and miR-223-3p Delivery to NPCs\u003c/h2\u003e\u003cp\u003erBMSCs exhibited a characteristic spindle-shaped morphology at ~\u0026thinsp;40\u0026ndash;50% confluence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a)). Exosomes isolated from transfected MSCs (miR-223-3p mimic/inhibitor) were characterized. NTA indicated a concentration of 3.3\u0026times;10\u003csup\u003e10\u003c/sup\u003e particles/mL and an average size of 150.7 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)). Nano-flow cytometry confirmed exosome concentration (8.64\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL) and size (average 76.6 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c)). TEM revealed characteristic cup-shaped vesicle morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Western blot confirmed enrichment of exosomal markers (CD9, CD81, TSG101) and absence of the negative marker Calnexin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(e)). PKH26-labeled exosomes were effectively internalized by NPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(f)). qRT-PCR demonstrated that NPCs co-cultured with mimic-exosomes exhibited significantly elevated miR-223-3p levels, while inhibitor-exosomes reduced miR-223-3p expression compared to respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(g)), confirming functional miRNA transfer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2. MSCs Exosomal miR-223-3p Attenuates TNF-α-Induced NPCs Apoptosis In Vitro\u003c/h2\u003e\u003cp\u003eqRT-PCR confirmed reduced miR-223-3p expression in TNF-α-treated NPCs compared to controls. Co-culture with miR-223-3p mimic-exosomes restored miR-223-3p levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a)). Mimic-exosomes significantly counteracted TNF-α-induced apoptosis: qRT-PCR and Western blot showed decreased Bax and Caspase-3 expression and increased Bcl-2 expression in the TNF-α\u0026thinsp;+\u0026thinsp;mimic-exosome group versus TNF-α alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b)-(c)). Flow cytometry corroborated these findings, revealing significantly reduced apoptosis rates in NPCs treated with mimic-exosomes (17.64%) versus TNF-α alone (26.58%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(d)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3. MSCs Exosomal miR-223-3p Mitigates IDD Progression In Vivo\u003c/h2\u003e\u003cp\u003eMRI and X-ray assessment 6 weeks post-intervention demonstrated that intradiscal injection of miR-223-3p mimic-exosomes significantly ameliorated IDD severity. Pfirrmann grades were lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a), (c)), and DHI% was higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b), (d)) in the IDD\u0026thinsp;+\u0026thinsp;mimic-exosome group compared to the IDD group. H\u0026amp;E staining revealed less severe structural disruption in the treatment group compared to IDD controls, although both differed significantly from Control/Sham groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(e)). NPCs isolated from treated discs exhibited higher miR-223-3p levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(f)) and reduced expression of pro-apoptotic proteins (Bax, Caspase-3) alongside elevated Bcl-2 expression compared to NPCs from IDD discs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(g)-(h)).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4. MSCs Exosomal miR-223-3p Suppresses FBXW7 Expression and Modulates Apoptosis in NPCs\u003c/h2\u003e\u003cp\u003eCo-culture with mimic-exosomes significantly downregulated FBXW7 mRNA and protein levels in NPCs, whereas inhibitor-exosomes upregulated FBXW7 expression compared to respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a)-(c)). Concordantly, mimic-exosomes decreased Bax and Caspase-3 mRNA/protein and increased Bcl-2, while inhibitor-exosomes exerted opposing effects on these apoptosis markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d)-(e)). This established an inverse correlation between exosomal miR-223-3p levels and FBXW7 expression, coupled with modulation of apoptosis pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5. FBXW7 is a Direct Target of miR-223-3p and Mediates its Anti-Apoptotic Effect\u003c/h2\u003e\u003cp\u003eRescue experiments confirmed FBXW7's role downstream of miR-223-3p. Inhibitor-exosomes upregulated FBXW7, Bax, and Caspase-3 while downregulating Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a)-(b)). Co-transfection of FBXW7 siRNA with the miR-223-3p inhibitor significantly reversed these pro-apoptotic effects, normalizing FBXW7, Bax, Caspase-3, and Bcl-2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(c)-(d)). Flow cytometry confirmed reduced apoptosis in the inhibitor\u0026thinsp;+\u0026thinsp;si-FBXW7 group (16.05%) versus inhibitor alone (21.37%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(e)). The dual-luciferase reporter analysis conclusively demonstrated: miR-223-3p mimic significantly suppressed luciferase activity from the wild-type (WT) FBXW7 3'UTR reporter construct but not the mutant (MUT) construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(f)), confirming direct binding of miR-223-3p to the FBXW7 3'UTR.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIntervertebral disc degeneration (IDD) is a prevalent orthopedic disorder, representing the primary cause of nerve root and spinal cord injuries, and the most significant contributor to paralysis in individuals under 45 years of age, exerting considerable socioeconomic impacts\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The intervertebral disc (IVD), comprising the annulus fibrosus, cartilaginous end plates, and nucleus pulposus, functions as the largest immune-privileged, hypovascular, and avascular organ in the human body. Nutrient supply to the central nucleus pulposus cells (NPCs) relies primarily on diffusion from surrounding vasculature. NPCs orchestrate pivotal pathological cascades in IDD development, and factors such as mechanical stress, trauma, smoking, and aging can compromise disc vascularization, leading to nutrient deprivation and accelerating IDD progression\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Current therapeutic strategies for IDD fail to reverse the underlying pathological processes. Consequently, discovery of novel molecular regulators and delineation of their mechanistic roles in disc degeneration represent urgent research priorities to enable targeted therapeutic development.\u003c/p\u003e\u003cp\u003eStem cell-based regenerative strategies have advanced therapeutic development for IDD, particularly MSCs transplantation. Numerous studies indicate that MSCs implantation can delay NPCs pathology by modulating microenvironmental homeostasis\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. However, the hostile disc milieu\u0026mdash;marked by hypoxia, acidic pH, and nutrient deprivation\u0026mdash;severely compromises engrafted MSC viability, constituting a major translational barrier\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Notably, MSC-derived exosomes function as critical intercellular signaling vectors, delivering bioactive cargo (mRNAs, miRNAs, proteins) that modulate recipient cell transcriptomes via paracrine mechanisms\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. This paracrine paradigm establishes exosome-based nanotherapeutics as promising cell-free alternatives that circumvent microenvironmental limitations in IDD treatment.\u003c/p\u003e\u003cp\u003eMicroRNAs (miRNAs) are endogenous non-coding RNAs (~\u0026thinsp;19\u0026ndash;25 nt) involved in regulating cell proliferation and apoptosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. They primarily function by binding target mRNAs, leading to mRNA degradation or translational repression, resulting in differential expression of target genes and modulation of downstream signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Previous research highlights the significant role of miRNAs in IDD pathogenesis\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Our group's prior work, involving isolation of MSCs-derived exosomes and miRNA microarray profiling, identified miR-223-3p as one of the most highly enriched miRNAs within these vesicles. Studies suggest miR-223-3p may regulate autophagy by targeting ATG16L1 and is implicated in inflammation, potentially serving as a therapeutic target in keratitis\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Furthermore, Bao et al. demonstrated that exosomal miR-223-3p facilitates complex communication between colon cancer cells and macrophages, promoting cancer cell proliferation and migration\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In IDD, miR-223-3p expression is reduced by overexpression of MIR155HG, leading to upregulated NLRP3 expression and induction of apoptosis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In the present study, exosomes were purified through differential ultracentrifugation and characterized. Recognizing that NTA sensitivity (~\u0026thinsp;70 nm) and inclusion of surface hydration layers can overestimate size, we employed nano-flow cytometry for complementary validation, confirming exosome isolation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)-(c)). Subsequent PKH26 labeling and DAPI staining confirmed NPCs uptake of exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(f)). Critically, qPCR analysis revealed significantly elevated miR-223-3p levels in NPCs co-cultured with exosomes derived from miR-223-3p mimic-transfected MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(g)), demonstrating efficient functional delivery.\u003c/p\u003e\u003cp\u003eExosomes derived from MSCs and carrying miRNAs hold significant therapeutic promise for IDD. For instance, MSCs-derived exosomal miR-532-5p was shown to attenuate TNF-α-induced NPCs apoptosis and promote extracellular matrix (ECM) production\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Similarly, Zhu et al. demonstrated that MSCs exosomes carrying miR-142-3p ameliorate NPCs apoptosis\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Our findings align with this paradigm: TNF-α-induced NPCs apoptosis was associated with upregulated Bax and Caspase-3, downregulated miR-223-3p and Bcl-2. Conversely, treatment with miR-223-3p mimic-loaded exosomes significantly suppressed Bax and Caspase-3 expression while elevating Bcl-2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating a protective role against NPCs apoptosis.\u003c/p\u003e\u003cp\u003eFollowing the observation of miR-223-3p's effect on apoptosis \u003cem\u003ein vitro\u003c/em\u003e, we investigated its role \u003cem\u003ein vivo\u003c/em\u003e using a rat caudal IDD model. Consistent with \u003cem\u003ein vitro\u003c/em\u003e results, \u003cem\u003ein vivo\u003c/em\u003e studies confirmed that MSCs-exosomal miR-223-3p mitigates IDD progression. Radiological assessment (MRI and X-ray) revealed less severe disc degeneration in the IDD\u0026thinsp;+\u0026thinsp;miR-223-3p mimic group compared to the IDD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)-(d)), a finding corroborated by histopathological evaluation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(e)). Furthermore, analysis of NPCs isolated from these discs showed decreased expression of pro-apoptotic proteins Bax and Caspase-3 and increased expression of anti-apoptotic Bcl-2 in the treatment group versus the IDD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(f)-(h)). Collectively, these data suggest MSCs-exosomal miR-223-3p delays IDD progression, at least partially, by attenuating NPCs apoptosis.\u003c/p\u003e\u003cp\u003eBioinformatic analysis from our prior work identified FBXW7 as a putative direct target of miR-223-3p in NPCs. The regulatory relationship between miR-223 and FBXW7 is well-documented across various tissues and diseases, including hematopoietic and digestive systems\u003csup\u003e[\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38 CR39\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, highlighting its broad regulatory capacity. In orthopedics, recent studies indicate that FBXW7 inhibition upregulates HIF1α and Runx2, promoting osteoblast differentiation\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Wang et al. reported that overexpression of miR-223-3p significantly reduced FBXW7 protein levels in mouse corneal epithelial cells, while miR-223-3p knockdown increased FBXW7\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Our results are consistent: co-culture of NPCs with exosomes from miR-223-3p mimic-transfected MSCs significantly downregulated FBXW7 mRNA and protein expression, whereas exosomes from inhibitor-transfected MSCs reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a)-(c)). Furthermore, Wang et al. showed adipose MSCs-derived exosomal miR-223-3p inhibits inflammation via FBXW7 suppression\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. We extended this by examining apoptosis markers: miR-223-3p mimic-exosomes decreased FBXW7 expression, concurrently suppressing Bax and Caspase-3 while elevating Bcl-2 expression at both mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d)-(e)). The reversal of these effects by inhibitor-exosomes strongly suggests miR-223-3p participates in NPCs apoptosis regulation by inhibiting FBXW7.\u003c/p\u003e\u003cp\u003eFunctional rescue experiments further confirmed FBXW7 as a key mediator. Co-culture with inhibitor-exosomes increased FBXW7, Bax, and Caspase-3 expression while decreasing Bcl-2. Co-transfection of NPCs with FBXW7 siRNA alongside inhibitor-exosomes effectively reversed the upregulation of FBXW7, Bax, and Caspase-3 and the downregulation of Bcl-2 induced by miR-223-3p knockdown alone. This confirms that MSCs-exosomal miR-223-3p regulates NPCs apoptosis through FBXW7 suppression(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a)-(d)). To definitively establish direct targeting, dual-luciferase reporter assays were performed. The results showed miR-223-3p mimic significantly reduced luciferase activity only in cells transfected with the reporter plasmid containing the wild-type (WT) FBXW7 3'UTR fragment. In contrast, no significant reduction was observed when the plasmid contained a mutated (MUT) binding site within the 3'UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(f)). This series of evidence demonstrates that miR-223-3p specifically binds to the 3'UTR region of FBXW7 mRNA, implicating it in the post-transcriptional regulation of FBXW7 expression and confirming FBXW7 as a direct target gene of miR-223-3p.\u003c/p\u003e\u003cp\u003eCapitalizing on prior mechanistic insights, this investigation targets IDD through engineered exosomal nanovectors\u0026mdash;a promising therapeutic paradigm. Through integrative multi-level analysis (molecular to organismal), we delineate a previously unreported mechanism: MSC-derived exosomal miR-223-3p attenuates disc degeneration by targeting FBXW7 in NPCs, thereby promoting tissue repair. These findings bridge fundamental discovery and clinical translation, establishing a conceptual framework for MSC-exosome therapeutics in IDD management. However, limitations exist: The persistence, biodistribution, and targeting efficiency of MSCs exosomes \u003cem\u003ein vivo\u003c/em\u003e across species remain unclear, and the precise efficiency of exosomal miR-223-3p delivery requires further elucidation. Furthermore, potential FBXW7 crosstalk with alternative miR-223-3p targets necessitates systems-level interrogation via proteomics/transcriptomics. Finally, clinical translation necessitates optimization of exosome isolation protocols and dosage regimens, consistent with challenges emphasized in exosome therapy trials.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, our findings demonstrate that MSC-derived exosomal miR-223-3p mitigates intervertebral disc degeneration (IDD) progression by targeting FBXW7 to regulate nucleus pulposus cell apoptosis. This work provides a mechanistic foundation for translating MSC-exosome therapeutics into clinical applications for IDD management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the results have been displayed in the paperitself or uploaded as Supporting Information for OnlinePublication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Ethics Committee of Nanjing Lambda Pharmaceutical Co., Ltd. (Approval No. 2024110106) and conducted in strict accordance with the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e (National Research Council, 2011).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRui Chen, Kaiyi Cao, and Yuting Gong: Performed data collection and conducted experiments. Yuning Zhu and Yi Gao: Conducted data analysis and interpretation. Jing Yan and Yuning Zhu: Searched the literature. Rui Chen, Yi Gao, and Yuting Gong: Completed figure/image processing. Rui Chen: Drafted the manuscript. Quan Zhou: Reviewed and edited the manuscript. Quan Zhou and Wei Pan: Designed and funded the study. All authors read and approved the final manuscript. Rui Chen, Kaiyi Cao, and Yuting Gong contributed equally to this work. Quan Zhou and Wei Pan are the co-corresponding authors of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 82372480), the Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (Grant No. SJCX24-1546), and the Huai\u0026apos;an Municipal Bureau of Science and Technology - Natural Science Research Plan (Grant No. HAB202320).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYang S, Zhang F, Ma J, et al. Intervertebral disc ageing and degeneration: The antiapoptotic effect of oestrogen. Ageing Res Rev. 2020;57:100978.\u003c/li\u003e\n \u003cli\u003eHumzah MD, Soames RW. Human intervertebral disc: structure and function. 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Circular RNA circ-BNC2 (hsa_circ_0008732) inhibits the progression of ovarian cancer through microRNA-223-3p/ FBXW7 axis. J Ovarian Res. 2022 Aug 14;15(1):95.\u003c/li\u003e\n \u003cli\u003eKumar V, Palermo R, Talora C, et al. Notch and NF-kB signaling pathways regulate miR-223/FBXW7 axis in T-cell acute lymphoblastic leukemia. Leukemia. 2014;28:2324\u0026ndash;35.\u003c/li\u003e\n \u003cli\u003eWang H, Chen J, Zhang S, et al. MiR-223 regulates autophagy associated with cisplatin resistance by targeting FBXW7 in human non-small cell lung cancer. Cancer Cell Int. 2020;20:258\u0026ndash;71.\u003c/li\u003e\n \u003cli\u003eLiu Z, Ma T, Duan J, et al. MicroRNA223induced inhibition of the FBXW7 gene affects the proliferation and apoptosis of colorectal cancer cells via the Notch and Akt/mTOR pathways. Mol Med Rep. 2021;23(2):154\u0026ndash;62.\u003c/li\u003e\n \u003cli\u003eZhou X, Jin W, Jia H, et al. MiR-223 promotes the cisplatin resistance of human gastric cancer cells via regulating cell cycle by targeting FBXW7. J Exp Clin Cancer Res. 2015;34:28\u0026ndash;41.\u003c/li\u003e\n \u003cli\u003eKurashige J, Watanabe M, Iwatsuki M, et al. Overexpression of microRNA-223 regulates the ubiquitin ligase FBXW7 in oesophageal squamous cell carcinoma. Br J Cancer. 2012;106:182\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eZhang W, Duan W, Mo Z, et al. Upregulation of SNHG14 suppresses cell proliferation and metastasis of colorectal cancer by targeting miR-92b-3p. J Cell Biochem. 2020;121:1998\u0026ndash;2008.\u003c/li\u003e\n \u003cli\u003eLi Y, Wang J, Ma Y, et al. miR-101-loaded exosomes secreted by bone marrow mesenchymal stem cells requires the FBXW7/HIF1\u0026alpha;/FOXP3 axis, facilitating osteogenic differentiation. J Cell Physiol. 2021 Jan 12.\u003c/li\u003e\n \u003cli\u003eKumar Y, Kapoor I, Khan K, et al. E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation. J Biol Chem. 2015;290(52):30975-87.\u003c/li\u003e\n \u003cli\u003eWang G, Zhu Y, Liu Y, et al. Mesenchymal Stem Cells-Derived Exosomal miR-223-3p Alleviates Ocular Surface Damage and Inflammation by Downregulating Fbxw7 in Dry Eye Models. Invest Ophthalmol Vis Sci. 2024 Oct 1;65(12):1.\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":"journal-of-orthopaedic-surgery-and-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"josr","sideBox":"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)","snPcode":"13018","submissionUrl":"https://submission.nature.com/new-submission/13018/3","title":"Journal of Orthopaedic Surgery and Research","twitterHandle":"@MSKmedBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intervertebral Disc Degeneration, Nucleus Pulposus Cells, Mesenchymal Stem Cells, Exosomes, miR-223-3p, FBXW7, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7737133/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7737133/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eIntervertebral disc degeneration (IDD) represents a widespread musculoskeletal condition. Programmed cell death in nucleus pulposus cells (NPCs) significantly contributes to IDD pathogenesis. MicroRNAs (miRNAs) play critical roles in IDD development. Bone marrow mesenchymal stem cell (MSC)-derived exosomes can inhibit NPCs apoptosis and promote disc regeneration/repair by delivering miRNAs.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eRat bone marrow-derived mesenchymal stem cells (MSCs) were expanded \u003cem\u003ein vitro\u003c/em\u003e, followed by exosome isolation via differential ultracentrifugation. Exosome characterization included assessment of size/concentration via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and nano-flow cytometry, alongside detection of exosomal markers (CD9, CD81, TSG101, Calnexin) by Western blotting. Exosome uptake by NPCs was confirmed using PKH26 labeling. MSCs were transfected with miR-223-3p mimic or inhibitor, followed by exosome isolation and co-culture with rat NPCs to assess miR-223-3p expression. The impact of miR-223-3p-overexpressing exosomes on TNF-α-induced NPCs injury was evaluated. An \u003cem\u003ein vivo\u003c/em\u003e intervertebral disc degeneration (IDD) model was induced in rat caudal spines via percutaneous needle puncture. Therapeutic efficacy was assessed by intradiscal injection of MSC-derived exosomes loaded with miR-223-3p mimic. The regulatory role of exosomal miR-223-3p on FBXW7 in NPCs was determined using gain- and loss-of-function approaches. Rescue experiments investigated whether miR-223-3p attenuates NPCs injury by targeting FBXW7. Direct targeting of FBXW7 3'UTR by miR-223-3p was confirmed via dual-luciferase reporter assays using wild-type and mutant constructs.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIsolated vesicles exhibited characteristic exosome morphology, size (~\u0026thinsp;76.6 nm by nano-flow cytometry), and marker expression (CD9/CD81/TSG101-positive, Calnexin-negative). NPCs efficiently internalized PKH26-tagged exosomal vesicles. NPCs co-cultured with mimic-exosomes exhibited elevated miR-223-3p levels, while inhibitor-exosomes reduced them. \u003cem\u003eIn vitro\u003c/em\u003e, Exosomes loaded with miR-223-3p mimic markedly attenuated TNF-α-triggered programmed cell death in NPCs (flow cytometry: 17.64% vs. TNF-α group 26.58%), decreased pro-apoptotic protein expression (Bax, Caspase-3), and increased anti-apoptotic Bcl-2. \u003cem\u003eIn vivo\u003c/em\u003e, intradiscal delivery of miR-223-3p mimic-exosomes ameliorated IDD progression, evidenced by reduced Pfirrmann grades on MRI, higher disc height index (DHI%) on X-ray, decreased apoptosis-related protein expression in NPCs, and improved histology compared to the IDD group. Furthermore, miR-223-3p mimic-exosomes downregulated FBXW7 mRNA and protein in NPCs, while inhibitor-exosomes upregulated it. Modulating miR-223-3p inversely regulated apoptosis markers. Crucially, FBXW7 knockdown (siRNA) reversed the pro-apoptotic effects induced by miR-223-3p inhibition. Dual-luciferase reporter assays confirmed direct binding of miR-223-3p to the FBXW7 3'UTR, with significant activity reduction in wild-type versus mutant constructs.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eMSC-derived exosomes deliver functional miR-223-3p to NPCs. Exosomal miR-223-3p suppresses NPCs apoptosis and attenuates IDD progression by directly targeting and downregulating FBXW7 expression.\u003c/p\u003e","manuscriptTitle":"Exosomal miR-223-3p from Mesenchymal Stem Cells Targets FBXW7 to Inhibit Intervertebral Disc Degeneration: Mechanism Insights","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 16:12:09","doi":"10.21203/rs.3.rs-7737133/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-10T06:33:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-10T01:32:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253793321560458708752517590325440355567","date":"2025-10-02T10:05:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-02T06:16:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-02T06:14:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-01T05:19:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Orthopaedic Surgery and Research","date":"2025-09-29T02:17:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-orthopaedic-surgery-and-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"josr","sideBox":"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)","snPcode":"13018","submissionUrl":"https://submission.nature.com/new-submission/13018/3","title":"Journal of Orthopaedic Surgery and Research","twitterHandle":"@MSKmedBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"34ea35f4-df51-48b1-8731-1339bb06b493","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:12:07+00:00","versionOfRecord":{"articleIdentity":"rs-7737133","link":"https://doi.org/10.1186/s13018-025-06468-7","journal":{"identity":"journal-of-orthopaedic-surgery-and-research","isVorOnly":false,"title":"Journal of Orthopaedic Surgery and Research"},"publishedOn":"2025-12-10 15:58:48","publishedOnDateReadable":"December 10th, 2025"},"versionCreatedAt":"2025-10-15 16:12:09","video":"","vorDoi":"10.1186/s13018-025-06468-7","vorDoiUrl":"https://doi.org/10.1186/s13018-025-06468-7","workflowStages":[]},"version":"v1","identity":"rs-7737133","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7737133","identity":"rs-7737133","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}