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.
