Mechanistic study of Lip@ZIF-8(Mfn2/SMART1)-mediated ubiquitination regulation in restoring mitochondrial homeostasis and inhibiting cartilage endplate calcification

This study aimed to elucidate the molecular mechanisms by which SMAD ubiquitin regulatory factor 1 (Smurf1)-mediated ubiquitination and degradation of mitofusin 2 (Mfn2) lead to mitochondrial homeostasis imbalance and progressive cartilage endplate (CEP) calcification in intervertebral disc degeneration (IVDD), and to evaluate the therapeutic potential of the constructed pH-responsive PEGylated lipid-coated zeolitic imidazolate framework-8 (ZIF-8) nanoparticles co-delivering Mfn2 mRNA and Smurf1-targeting PROTAC (designated as SMART1) (Lip@ZIF-8(Mfn2/SMART1)) for reconstructing the Smurf1-Mfn2-AMP-activated protein kinase (AMPK)-autophagy axis and suppressing CEP calcification. In this study, a rat IVDD model was first established via intervertebral disc puncture, and CEP tissues were collected from the Sham and IVDD groups for proteomic and metabolomic analyses. Differential analyses were performed in combination with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, metabolic pathway analysis, and the construction of an integrated proteomic-metabolomic network to delineate the global dysregulation of the Smurf1-Mfn2-AMPK-mitochondrial metabolic regulatory network. On this basis, single-cell RNA sequencing (scRNA-seq) was conducted on CEP tissues from IVDD and Lip@ZIF-8(Mfn2/SMART1)-treated groups to assess the systemic remodeling effects of the treatment on CEP cellular states. For mechanistic investigations, primary CEP cells and 293T cells were isolated and cultured, and a β-glycerophosphate (β-GP)-induced osteogenic-like calcification model of CEP cells was established. Lentivirus-mediated Mfn2 overexpression, SMART1 treatment, and AMPK inhibitor intervention were applied to systematically evaluate the functional role of the Smurf1-Mfn2-AMPK-autophagy axis in calcification regulation. Co-immunoprecipitation (Co-IP), Glutathione S-transferase (GST) pull-down assays, and in vitro reconstituted ubiquitination reactions were further performed to validate the molecular mechanism underlying Smurf1-mediated ubiquitination and degradation of Mfn2. In functional experiments, alkaline phosphatase (ALP) and Alizarin Red staining, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot (WB), JC-1 mitochondrial membrane potential assays, transmission electron microscopy (TEM), and mCherry-GFP-LC3 dual-fluorescence tracing were comprehensively employed to assess mitochondrial homeostasis, autophagic flux, and osteogenic-like transdifferentiation. Subsequently, a Lip@ZIF-8(Mfn2/SMART1) was constructed, and its physicochemical properties were characterized. Cellular uptake, lysosomal colocalization, and CCK-8 assays were conducted to evaluate its biocompatibility and functional release efficiency. In vivo, the nanosystem was locally injected into intervertebral discs, and in vivo imaging system (IVIS), micro-computed tomography (micro-CT), histological staining, immunohistochemistry (IHC), and molecular analyses were integrated to systematically evaluate the in vivo retention and distribution characteristics of Lip@ZIF-8(Mfn2/SMART1), as well as its regulatory effects on CEP calcification, mitochondrial homeostasis, and the Smurf1-Mfn2-AMPK-autophagy axis. Integrated proteomic and metabolomic analyses revealed that Smurf1 was significantly upregulated, whereas Mfn2 was markedly downregulated in CEP tissues from IVDD, accompanied by impaired AMPK signaling and disrupted mitochondrial oxidative metabolism. Animal experiments demonstrated that, in a rat CEP calcification model, aberrant activation of Smurf1, suppression of Mfn2 and AMPK signaling, and blockade of autophagic flux occurred concurrently, leading to mitochondrial structural damage, enhanced osteogenic-like transdifferentiation, and aggravated pathological calcification. Mechanistic investigations confirmed that Smurf1 directly interacted with Mfn2 and functioned as its upstream E3 ubiquitin ligase, mediating Mfn2 ubiquitination and degradation. In vitro functional assays showed that combined Smurf1 inhibition and Mfn2 supplementation synergistically enhanced AMPK activity and restored autophagic flux and mitochondrial homeostasis. The constructed Lip@ZIF-8(Mfn2/SMART1) exhibited favorable physicochemical stability, acid-triggered release properties, and cellular compatibility, enabling efficient cellular uptake and lysosomal escape in CEP cells. Both in vitro and in vivo experiments demonstrated that Lip@ZIF-8(Mfn2/SMART1) simultaneously downregulated Smurf1, upregulated Mfn2, and activated the AMPK-autophagy axis, thereby markedly improving mitochondrial structure and function and suppressing osteogenic-like differentiation and calcification of the CEP. Lip@ZIF-8(Mfn2/SMART1) enabled the synergistic delivery of Mfn2 mRNA and SMART1 to reconstruct the Smurf1-Mfn2-AMPK-autophagy regulatory axis, restore mitochondrial function, and effectively inhibit CEP calcification, thereby providing a mechanistic basis and candidate nanotherapeutic strategy for future IVDD intervention.