FBXW7 mediates intervertebral disc degeneration by regulating the JNK signaling pathway to induce nucleus pulposus cell apoptosis

FBXW7 mediates intervertebral disc degeneration by regulating the JNK signaling pathway to induce nucleus pulposus cell apoptosis | 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 FBXW7 mediates intervertebral disc degeneration by regulating the JNK signaling pathway to induce nucleus pulposus cell apoptosis Yi Gao, Yuning Zhu, Yuting Gong, Rui Chen, Jing Yan, Shuo Miao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7518753/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Elucidating the mechanistic role of FBXW7 in JNK pathway activation and subsequent nucleus pulposus apoptosis during disc degeneration. Background NPCs apoptosis constitutes a core pathological event during disc degeneration. Oxidative stress triggers JNK-mediated apoptotic signaling cascades in nucleus pulposus cells. As a core E3 ligase component, FBXW7 is involved in apoptosis regulation, but its role in NPCs apoptosis during IDD remains unexplored. Methods Bioinformatics analysis was performed on GEO datasets to identify differentially expressed genes in nucleus pulposus tissues of varying degeneration grades. Human NP samples with different degeneration levels were collected to assess FBXW7 expression. In vitro, NPCs were cultured, transfected with siRNA to silence FBXW7 or JNK, and subjected to apoptosis induction, followed by evaluation of apoptosis-related protein expression, cell apoptosis, and proliferation. In vivo IDD modeling employed rat disc puncture methodology, followed by local injection of FBXW7 shRNA to evaluate therapeutic effects on disc degeneration. Results FBXW7 and JNK expression were significantly upregulated in severely degenerated nucleus pulposus tissues. In vitro, FBXW7/JNK overexpression upregulated apoptotic effectors (Bax, Caspase-3) and downregulated Bcl-2, whereas FBXW7/JNK knockdown reduced apoptosis rates and mitigated TBHP-induced suppression of NPC proliferation. In vivo, FBXW7 shRNA injection delayed disc degeneration, attenuated T2-weighted MRI signal decline, and suppressed JNK expression. Conclusion FBXW7 and JNK are upregulated in advanced IDD. FBXW7 promotes NPCs apoptosis via the JNK pathway, and its knockdown alleviates disc degeneration progression in rats. Intervertebral disc degeneration FBXW7 JNK Apoptosis Nucleus pulposus cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction IDD: the foremost orthopedic disc pathology causing spinal dysfunction. IDD affects cervical, thoracic and lumbar regions, causing disc herniation, chronic back pain, spinal stenosis or spondylolisthesis. Discs between vertebrae comprise a gelatinous nucleus pulposus enclosed by fibrous annulus. The nucleus pulposus primarily contains proteoglycans and type II collagen, contrasting with the type I collagen-dominated annulus fibrosus [ 1 ] .The etiology of IDD is complex, with factors such as environment, genetics, aging, smoking, obesity, inflammation, occupation, and mechanical stress all contributing to its development [ 2 ] . Currently, conventional treatments for IDD include conservative therapy and surgical intervention. However, these approaches can only alleviate pain and reduce symptoms without addressing the root cause of the problem. Therefore, elucidating IDD etiology facilitates targeted intervention strategies [ 3 ] . Dysregulated apoptosis characterizes degenerative disorders including OA, IDD and malignancies. IDD's primary pathology centers on nucleus pulposus cells (NPCs) apoptosis. NPCs apoptosis triggers extracellular matrix breakdown, disrupting the structural balance of the intervertebral disc and thereby mediating disc degeneration. FBXW7 is an F-box-containing E3 ubiquitin ligase, with its gene located on chromosome 4 (4q31.3). The FBXW7 gene yields a canonical F-box protein, which can recognize and bind to phosphorylated sites in target proteins, mediating their ubiquitination and degradation. FBXW7 occupies a central position in the protein regulation network and exerts important functions in multiple aspects, including cell division, cell differentiation, apoptosis, cancer dissemination, and therapeutic unresponsiveness, primarily through the regulation of signaling pathways such as Wnt/β-catenin, JNK, RhoA, RPAP2, and ENO1 [ 4 – 6 ] . Most FBXW7 investigations concentrate on cancer biology, delineating its antiproliferative and proapoptotic functions [ 7 – 8 ] . Recent studies have revealed that FBXW7 can promote apoptosis of cardiomyocytes by degrading anti-apoptotic proteins (e.g., Mcl-1) [ 9 – 12 ] . Bioinformatics analysis of GEO datasets (GSE70362/150408/124272) revealed elevated FBXW7 expression in IDD disc tissues of IDD patients. No prior studies have investigated FBXW7's function in disc degeneration. Hoeck et al. demonstrated that FBXW7 can regulate apoptosis of neural progenitor cells via JNK-mediated signal transduction [ 13 ] . c-Jun N-terminal kinase (JNK), an essential mediator within the MAPK signaling cascade, mediates stress-responsive signaling. [ 14 – 15 ] . Liu et al. demonstrated JNK pathway activation drives disc degeneration via NPC apoptosis. [ 16 ] . To date, there have been no reports on whether FBXW7 affects intervertebral disc degeneration via stimulation of the JNK-mediated signaling axis. 2. Materials and methods 2.1 Bioinformatics Analysis This study integrated three datasets—GSE70362, GSE150408, and GSE124272—from the GEO database and analyzed them using Rstudio software. We performed differential gene expression analysis (volcano plot, P < 0.05) comparing normal and IDD groups. 2.2 Clinical Sample Collection A total of 20 human nucleus pulposus (NP) tissue samples (10 males and 10 females) were obtained from patients undergoing discectomy at Huai’an Hospital Affiliated to Xuzhou Medical University over a 24-month period from September 2022 through September 2024. The cohort comprised 14 degenerative and 6 traumatic disc specimens (Table 1 ). The inclusion criteria comprised patients who underwent surgery for vertebral fractures due to various etiologies and those with intervertebral disc degeneration (IDD) who failed conservative treatment. Patients with ankylosing spondylitis, lumbar spondylolisthesis, spinal stenosis, or spinal tumors were excluded. All specimens were surgically collected by orthopedic surgeons following informed consent. This study received hospital’s ethics committee authorization.Preoperative MRI(magnetic resonance imaging) scans preceded NP classification into mild (Pfirrmann I-III) and severe (IV-V) degenerative groups. (Table 2 ) [ 17 ] . Table 1 Clinical characteristics of intervertebral disc samples Group ID Sex Age (years) Etiology Spinal Level Pfirrmann Grade Low 1 M 20 Trauma L4/5 Ⅱ Low 2 M 25 Trauma L2/3 Ⅱ Low 3 M 34 IDD L4/5 Ⅱ Low 4 F 24 Trauma L3/4 Ⅱ Low 5 F 26 Trauma L2/3 Ⅲ Low 6 M 18 IDD L5/S1 Ⅱ Low 7 M 46 IDD L5/S1 Ⅲ Low 8 F 44 IDD L4/5 Ⅲ Low 9 F 43 IDD L3/4 Ⅲ Low 10 F 46 IDD L4/5 Ⅲ High 11 M 59 IDD L3/4 Ⅳ High 12 F 68 IDD L5/S1 Ⅳ High 13 F 59 IDD L2/3 Ⅴ High 14 M 23 Trauma L3/4 Ⅳ High 15 M 29 IDD L5/S1 Ⅴ High 16 F 22 IDD L4/5 Ⅴ High 17 F 27 Trauma L3/4 Ⅳ High 18 F 19 IDD L5/S1 Ⅳ High 19 M 41 IDD L4/5 Ⅴ High 20 M 52 IDD L5/S1 Ⅴ Table 2 Pfirrmann grading system for intervertebral disc degeneration Grade NP Structure NP Signal Intensity NP–AF demarcation Disc height Ⅰ Homogeneous, bright white High Clear Normal Ⅱ Inhomogeneous with horizontal bands High Distinct Normal Ⅲ Inhomogeneous, gray Intermediate Unclear Slightly decreased Ⅳ Inhomogeneous, gray to black Intermediate-low Lost Moderately decreased Ⅴ Inhomogeneous, black Low Lost Severely decreased 2.3 NPCs Isolation and Culture Human NP cells were derived from surgical disc samples. The tissue samples were washed in PBS to Clear residual blood and connective tissue, then minced into 1–2 mm³ fragments. These pieces were incubated at 37°C in DMEM culture medium (Kaiji Biotech, Nanjing, China) containing 0.25% collagenase II (Thermo Fisher Scientific, USA) for 5 hours. Cells were pelleted by centrifugation (1200 rpm, 5 min) with subsequent supernatant discard. Cells were resuspended in complete DMEM (10% FBS, penicillin/streptomycin) and cultured at 37°C/5% CO₂. Fresh complete medium replaced the existing culture solution at 48-hour intervals. Cells at passage 3 (P3) served for downstream assays. 2.4 Cell Transfection Cells were cultured to 60–90% confluence. For transfection, Medium was exchanged for serum-free DMEM. In accordance with kit instructions, target siRNA was mixed with RNA TransMate transfection reagent (Sangon Biotech, Shanghai, China) in the appropriate ratio and incubated at room temperature yielding siRNA-carrier complexes after 10 min. The complex was then added to culture vessels, and vessels were gently agitated to ensure even distribution. After 6h, complete medium (DMEM + 10%FBS + antibiotics) was replenished, and cells were harvested at 48h post-culture for follow-up investigations. For overexpression of FBXW7, lentivirus was directly introduced into culture medium per manufacturer's protocol (Table 3 ), without the need for a transfection reagent, and following steps were performed as aforementioned. The FBXW7 overexpression lentivirus, FBXW7 siRNA, JNK siRNA, and RNA TransMate transfection reagent used in the experiments were all prepared by Sangon Biotech. Table 3 Recommended amounts of siRNA and RNA TransMate reagent for transfection in different culture plates Culture Plate Medium Volume (µL/well) siRNA (pmol/well) RNA TransMate(µL/well) Serum-Free Medium for Complexing 96-well plate 100 µL 3 pmol 0.2 µL 10 µL 48-well plate 200 µL 6 pmol 0.4 µL 20 µL 24-well plate 500 µL 15 pmol 1.0 µL 50 µL 12-well plate 1.0 ml 30 pmol 2.0 µL 100 µL 6-well plate 2.0 ml 60 pmol 4.0 µL 150 µL 35 mm 2.0 ml 60 pmol 4.0 µL 150 µL 60 mm 5.0 ml 150 pmol 10.0 µL 250 µL 2.5 Nucleus Pulposus Cell Apoptosis Model Tert-butyl hydroperoxide (TBHP) was applied to stimulate programmed cell death in NPCs. Confluent NPCs (> 80%) in 6-cm dishes underwent 2×PBS washes following medium aspiration. Following Wang et al.'s methodology, cells were treated with 100µM TBHP in complete medium (37°C, 5% CO₂, 6h) [ 18 ] . After medium replacement with complete culture medium, cells were cultured for 24h prior to analysis. Some groups required prior transfection with RNA or siRNA. 2.6 Western blot Total protein extraction was performed using RIPA lysis buffer (Beyotime, Shanghai), followed by concentration determination with a bicinchoninic acid (BCA) kit from the same manufacturer. Protein separation by electrophoresis was followed by PVDF membrane transfer (Millipore, USA). After 1h blocking in skim milk (Beyotime, Shanghai), primary antibodies were applied at 4°C overnight. Secondary antibody incubation was performed on the membranes for one hour under gentle agitation. Bands were detected with SuperSignal West Pico PLUS(Beyotime, Shanghai). ImageJ was employed for densitometric analysis. 2.7 qPCR Total RNA was isolated via Trizol (Takara, Japan), with concentration/purity (OD260/OD280 ratio, R) assessed by spectrophotometry.The reverse transcription reaction containing 1 µg total RNA was carried out with HiScript II Q RT SuperMix(Vazyme, Nanjing) under standardized conditions: 42°C (60 min) → 85°C (5 min). 20µL qPCR reactions used ChamQ SYBR mix (Vazyme, Nanjing) under cycling: 95°C 5min → 40 cycles (95°C 5s, 60°C 30s). Technical triplicates per sample and relative quantification was performed using GAPDH as the reference gene. The corresponding primer sequences can be found in Table 4 . The relative expression levels were calculated using the 2 −ΔΔCt method. Table 4 Primer Design and Synthesis Human Primers Primer Name Target Gene Sequence FBXW7 Forward 5'-GGCCAAAATGATTCCCAGCAA-3' Reverse 5'-ACTGGAGTTCGTGACACTGTTA-3' JNK Forward 5'-TGTGTGGAATCAAGCACCTTC-3' Reverse 5'-AGGCGTCATCATAAAACTCGTTC-3' GAPDH Forward 5'-GGAGCGAGATCCCTCCAAAAT-3' Reverse 5'-GGCTGTTGTCATACTTCTCATGG-3' Rat Primers Primer Name Target Gene Sequence FBXW7 Forward 5'-AAGGTCCCAGCAAGCATCAGA-3' Reverse 5'-ATTCACCCGTTTTCAAGTCCC-3' JNK Forward 5'-AGGAGCGAACTAAGAATGGCG-3' Reverse 5'-ACTGCTGTCTGTATCCGAGGC-3' GAPDH Forward 5'-CTACCCACGGCAAGTTCAACG-3' Reverse 5'-GCCAGTAGACTCCACGACATAC-3' 2.8 CCK8 NPCs were plated at 10⁴ cells/well in 96-well plates. According to the experimental design, overexpression vectors for FBXW7, FBXW7 siRNA, JNK siRNA, or corresponding controls were added, with three replicates for each group. Cells were maintained at 37°C with 5% CO₂ for 6h. Subsequently, Cells received TBHP treatment (6h) as designed. Complete medium replacement post-treatment sustained cell growth.The culture medium was refreshed at 24 hours, 48 hours, and 72 hours. Each well received 100µL CCK8/DMEM (1:9, TaoShu Bio) for 2h standard incubation. Optical density measurements were performed at 450 nm wavelength using a microplate spectrophotometer. 2.9 Apoptosis Analysis by Flow Cytometry NPCs apoptosis rates were determined through dual-labeling with Annexin V-FITC and PI (Kaiji Bio, Nanjing) according to established procedures. For adherent cells, they were collected after digestion with trypsin (without EDTA) (Beyotime, Shanghai, China). After two rounds of phosphate-buffered saline rinsing, cells were pelleted by low-speed centrifugation (5 minutes at 1000 rpm). Cell pellets were first resuspended in Binding Buffer, followed by 10-minute incubation at room temperature with Annexin V-FITC and PI in the dark. Finally, the stained cells were analyzed using a Beckman flow cytometer (Beckman, USA). 2.10 Rat IDD Model In this study, Percutaneous puncture established rat caudal disc degeneration [ 19 – 20 ] . Three-month-old male SD rats (n = 40, 500 ± 50 g) were studied. Four caudal discs (Co7/8-Co10/11) per rat were randomized into: control, IDD, IDD + FBXW7 shRNA (rescue), and sham groups. After fasting for 6 hours before surgery, the rats were anesthetized by gas anaesthesia using isoflurane (3% isoflurane for induction, 2% isoflurane for maintenance) [ 21 ] . Control group: no treatment. IDD group: 22G needle puncture (full disc penetration → 360° rotation → 30s retention). Rescue group: AAV9-FBXW7-shRNA was injected directly following needle puncture (2µL, 2.67×10¹³ VG/mL). Sham group:underwent paravertebral puncture avoiding disc injury. Postoperatively, rats were singly housed with ad libitum activity and daily monitoring. The rats were sacrificed at 6 weeks post-surgery for subsequent examinations and experiments. The AAV9-FBXW7-shRNA used in this study was prepared by Nanjing Zebrafish Biotech Co., Ltd. 2.11 X-ray/MRI Following 6-week induction, the rats were anesthetized by gas anaesthesia using isoflurane (3% isoflurane for induction, 2% isoflurane for maintenance). The anesthetized rats were secured on an X-ray examination table, and radiographs of the caudal vertebrae were taken using a medical diagnostic X-ray tube assembly (Xinglian Industry, Shenzhen, China). The images were obtained and analyzed via a PACS system.7T MRI (Bruker, Germany)acquired T2-weighted sagittal images at Jiangsu Medical Animal Facility. Disc degeneration severity was graded via Pfirrmann criteria 2.12 Paraffin Embedding and HE Staining Post-imaging, rats were sacrificed with caudal samples fixed in 4% PFA (Biosharp, Hefei) for 12h. Post-EDTA decalcification (10%), tissues were dehydrated and paraffin-processed. Histological examination was performed on 4–5 µm thick sections using HE staining to evaluate group-specific morphological variations in intervertebral discs. Microscopic imaging was performed with a PrimoStar system (Zeiss, Germany). 2.13 Ethical Statement The study protocol received formal ethical clearance (Approval No. XXX) from the Institutional Review Board of Huai'an Hospital affiliated with Xuzhou Medical University. The cohort comprised 20 NP tissues (50% male) collected during discectomies (2022–2024). All participants and family members provided written consent. Sprague-Dawley rats were obtained from an accredited vendor (Lambda, Nanjing). The animal use license and ethical review number are IACUC-2024110506. All in vivo studies complied with institutional animal care guidelines (Guide for the Care and Use of Laboratory Animals), with technical support provided by commercial partners. 3. Results 3.1 FBXW7/JNK Upregulation in Severely Degenerated NP Tissues FBXW7 was upregulated in IDD compared to normal discs (bioinformatics data). (Fig. 1 ). WB and qPCR quantified molecular changes across degeneration-grade NP samples. The results demonstrated that FBXW7 and JNK expression was markedly elevated in severe versus mild degeneration groups (Fig. 2 ). 3.2 Both si-FBXW7 and si-JNK Inhibit TBHP-Induced Apoptosis Flow cytometry revealed significantly higher NPCs apoptosis in TBHP-treated groups than controls. Knockdown groups (si-FBXW7/si-JNK) showed markedly lower apoptosis rates than TBHP-treated cells. CCK8 analysis revealed significant proliferation inhibition in TBHP-treated cells versus controls, while si-FBXW7 and si-JNK could partially reverse the inhibitory effect of TBHP (Fig. 3 ). Western blot quantified FBXW7, JNK, and apoptosis regulators (Caspase-3/Bax/Bcl-2). TBHP treatment elevated FBXW7/JNK/Caspase-3/Bax expression but suppressed Bcl-2 versus controls. Compared to TBHP alone, si-FBXW7 co-treatment downregulated FBXW7/JNK/Caspase-3/Bax while maintaining higher levels than controls; Bcl-2 remained suppressed(Fig. 4 ). Compared to TBHP group, si-JNK co-treatment downregulated JNK/Caspase-3/Bax (though still above controls) and further suppressed Bcl-2 (Fig. 5 ). qPCR revealed elevated FBXW7/JNK expression in TBHP-treated cells, which was attenuated by si-FBXW7 transfection (Fig. 4 c, Fig. 5 c). 3.3 FBXW7 Regulates JNK to Promote Nucleus Pulposus Cell Apoptosis Western blot/flow cytometry elucidated FBXW7-JNK-apoptosis axis in NPCs. Evidence suggested overexpression of FBXW7 increased apoptosis in NPCs, while transfection with si-JNK partially attenuated this effect (Fig. 6 a, b). FBXW7 overexpression upregulated JNK/Bax/Caspase-3 but downregulated Bcl-2 versus controls. In FBXW7-overexpressing cells, JNK silencing partially normalized the expression of apoptotic regulators: decreasing JNK, Bax and Caspase-3, and increasing Bcl-2. (Fig. 6 c-e). 3.4 Animal Experiments Hematoxylin-eosin (HE) demonstrated that the NP tissues in control group exhibited large, intact ellipses with spindle-shaped nucleus pulposus cells, distinct nuclei and cytoplasm, and annulus fibrosus tissues arranged in a regular ring-like pattern. In contrast, the NP tissues in the IDD + FBXW7 shRNA group (rescue group) and the sham surgery group (Sham group) appeared flattened and elongated, with more pronounced changes in the annulus fibrosus structure and surrounding muscle. Additionally, in the Sham group, the muscles surrounding the intervertebral discs were visibly damaged. The IDD group showed overall fragmentation and disorganization, with NP tissues, annulus fibrosus, and surrounding muscles intermingled and losing their clear and distinct morphology (Fig. 7 ). Radiographs of the rat caudal vertebrae demonstrated that degenerated intervertebral discs exhibited characteristics such as narrowed intervertebral space, wedge-shaped changes in the intervertebral space, and osteophyte formation. MRI revealed significantly reduced T2-signal intensity in IDD rats versus controls, with rescue and Sham groups showing intermediate values (IDD < rescue/Sham < control) (Fig. 8 ). Finally, protein and RNA from disc tissues facilitated Western blot and qPCR assays. IDD tissues exhibited significantly elevated FBXW7/JNK expression relative to controls. However, injection of FBXW7 shRNA significantly attenuated the upregulation of FBXW7 and JNK in the IDD group (Fig. 9 ). 4. Discussion IDD drives chronic low back pain and functional impairment through ECM catabolism, inflammation, and NPCs apoptosis. As individuals age, the intervertebral discs experience water loss, reduced elasticity, annulus fibrosus rupture, and changes in the nucleus pulposus structure, leading to structural and functional degeneration of the discs. Studies have shown that apoptosis of NPCs, ECM degradation, and inflammatory responses are key pathological aspects of IDD [ 22 , 23 ] . In recent years, Mounting studies underscore JNK pathway involvement in disc degeneration via NPCs apoptosis. JNK, a crucial member of the MAPK family, modulates cell death, inflammatory activation, and ECM catabolism. Mechanical stress, oxidative stress, and inflammatory cytokines can all activate the JNK pathway, thereby promoting NPCs apoptosis and ECM destruction. However, The mechanistic interplay between JNK signaling and IDD progression remains incompletely characterized. [ 24 – 26 ] . This investigation elucidates FBXW7's mechanistic contribution to IDD through JNK pathway modulation, offering novel therapeutic insights. FBXW7's central role in the ubiquitin-proteasome system impacts cell cycle, proliferative capacity, and survival pathways [ 27 – 29 ] . FBXW7 has been implicated in diverse pathologies, spanning oncogenesis, neurodegeneration, cardiovascular dysfunction, and metabolic dysregulation [ 30 – 33 ] . Welcker et al. proposed that FBXW7 promotes the ubiquitination and proteasomal degradation of target proteins, thereby regulating cell differentiation, proliferation, and apoptosis [ 34 ] . Mao's research on breast cancer indicated that FBXW7 inhibits tumor cell proliferation and metastasis by degrading c-Myc and Notch1 [ 35 ] . Our analysis of nucleus pulposus tissues with different degrees of degeneration revealed high expression of FBXW7 in highly degenerated NP tissues. In cellular experiments, TBHP treatment upregulated FBXW7 expression and enhanced apoptosis versus controls. Knocking down FBXW7 partially rescued TBHP's pro-apoptotic impact. FBXW7 upregulation exerted anti-proliferative and pro-apoptotic effects, indicating that FBXW7 promotes NPCs apoptosis. FBXW7 was upregulated in IDD rats, whereas FBXW7 shRNA treatment attenuated degeneration.Therefore, FBXW7 may contribute to IDD by promoting NPCs apoptosis. JNK cascade serves as a master modulator of stress adaptation, closely associated with various apoptotic processes and involved in pathological conditions such as neurodegenerative diseases, tumors, fibrotic diseases, and chronic inflammation. The JNK cascade is upregulated in degenerative discs, modulating apoptosis, senescence, and ECM remodeling. JNK pathway activation is triggered by mechanical/oxidative stress and pro-inflammatory cytokines (TNF-α/IL-1β), leading to ECM degradation and NPCs apoptosis [ 36 – 39 ] . Additionally, JNK pathway stimulation accelerates IDD through ECM-degrading enzymes (MMPs/ADAMTs) [ 40 – 41 ] . In this study, elevated JNK levels characterized severely degenerated NP tissues and TBHP-treated apoptotic NPCs. Park's research suggested that JNK inhibition attenuates macrophage-derived inflammatory mediators (TNF-α, IL-6/8, CCL2/3), suppressing apoptosis and ECM degradation in chronic pain [ 42 ] . In cellular experiments, induction of NPCs apoptosis resulted in elevated levels of JNK/Bax/Caspase-3, while Bcl-2 expression was downregulated. However, knocking down JNK partially inhibited TBHP-induced apoptosis. Our experimental results are consistent with current studies, indicating that JNK promotes NPCs apoptosis. JNK pathway modulation by FBXW7 represents a novel therapeutic target for IDD. In highly degenerated NP tissues, the expression levels of FBXW7 and JNK are elevated. In vitro cellular experiments revealed that overexpression of FBXW7 promotes JNK expression and NPCs apoptosis. Knocking down JNK in this context partially inhibits NPCs apoptosis, suggesting that FBXW7 may induce NPCs apoptosis by regulating the JNK signaling pathway. Animal experiments showed that the IDD group exhibited elevated FBXW7/JNK expression relative to controls.Injection of FBXW7 shRNA significantly inhibited the upregulation of FBXW7 and JNK in degenerated NP tissues and IDD advancement. This evidence supports FBXW7 promotes NPCs apoptosis by regulating the JNK signaling pathway, thereby contributing to IDD development. Qiu et al. studied the role of FBXW7 in oral squamous cell carcinoma (OSCC) and found that transfection with FBXW7 promoted apoptosis in OSCC cells, with upregulated Bax levels and suppressed Bcl-2 expression in FBXW7-transfected OSCC cells [ 43 ] . We obtained similar results in vitro in NPCs transfected with FBXW7. Diverging from our observations, Nateri et al. reported that knocking down FBXW7 induces apoptosis in neurons and that FBXW7 antagonizes JNK signaling pathway-induced apoptosis [ 44 ] . Research from Qiu's and Nateri's groups reveals the intricate interplay of FBXW7 in apoptotic regulation. Notably, Zhang et al. reported FBXW7 downregulation in OA cartilage (human patients and murine models). In FBXW7 knockout mice, chondrocyte senescence was exacerbated. Mechanical overload reduced FBXW7 expression, decreased FBXW7-mediated degradation of MKK7, enhanced JNK-dependent signaling, and thereby promoted chondrocyte senescence. Additionally, Zhang et al. demonstrated in animal experiments that injection of AAV-FBXW7 alleviated OA symptoms in mice [ 45 ] . In the animal experiment part of our study, injection of AAV-FBXW7 shRNA alleviated IDD symptoms in rats. The results of our study contrast sharply with those of Nateri and Zhang. These findings indicate that FBXW7/JNK signaling interplay is not fixed but rather depends on the cell type and disease context. FBXW7's pro-apoptotic effects vary across cell lineages and pathological contexts. Regarding FBXW7's therapeutic potential, it is essential to fully consider its complex mechanisms under different conditions to achieve precise treatment. Moreover, FBXW7 may also indirectly affect JNK pathway activity by regulating other downstream targets, such as c-Myc and Notch. Future research can further explore the specific mechanisms of FBXW7 in the JNK pathway and its potential therapeutic value in IDD [ 46 – 49 ] . 5. Conclusion In summary, FBXW7 drives IDD pathogenesis via JNK-mediated NPCs apoptosis. This finding provides avenues for targeted therapy development. However, the complex regulatory mechanisms of FBXW7 in different cell types and pathological states suggest that its development as a therapeutic target requires careful consideration of tissue specificity and precise treatment strategies. Future research can further explore the specific mechanisms of FBXW7 in the JNK signaling pathway and investigate its clinical application potential from the perspectives of gene therapy or drug inhibitors. Declarations Author Contributions Yi Gao and Yuning Zhu: Performed data collection and conducted experiments.Yuting Gong and Rui Chen: Conducted data analysis and interpretation. Jing Yan and Shuo Miao: Searched the literature. Yi Gao, Yuning Zhu, and Yuting Gong: Completed figure/image processing. Yi Gao: Drafted the manuscript. Quan Zhou: Reviewed and edited the manuscript. Quan Zhou and Wei Pan: Designed and funded the study. Yi Gao and Yuning Zhu contributed equally to this work. Quan Zhou and Wei Pan are the co-corresponding authors of this article.Quan Zhou is the lead corresponding author.Wei Pan is the co-corresponding author. All authors read and approved the final manuscript. Funding This work was supported by General Program of the Health Commission of Jiangsu Province (Grant No.M2022125). Ethics Statement The acquisition of human intervertebral disc samples was approved by the Ethics Committee of the Affiliated Huai’an Hospital of Xuzhou Medical University, with informed consent obtained from the patients and their family members.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). Data Availability Statement The data that support the findings of this study are available on request from the corresponding author. 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1","display":"","copyAsset":false,"role":"figure","size":79867,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs: Normal vs IDD Comparison a. Volcano plot displays DEGs (red: upregulated; purple: downregulated). b.Violin plot analysis of FBXW7 expression in both normal and IDD groups (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/fd383643a01fb6e72be633e1.jpeg"},{"id":93945851,"identity":"85bc1efb-8148-4bba-ba11-438df4bdd41d","added_by":"auto","created_at":"2025-10-20 14:23:05","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50548,"visible":true,"origin":"","legend":"\u003cp\u003eFBXW7 and JNK (protein/mRNA) in degenerated NP tissues a、b: FBXW7/JNK expression by Western blot: representative images and quantitative analysis (low vs. high degeneration). c. Relative mRNA expression of FBXW7 and JNK in the two groups as determined by qPCR (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/b8d505995b208cbccd6a7f02.jpeg"},{"id":93945853,"identity":"57bc2062-5901-4703-8825-22a63be941a7","added_by":"auto","created_at":"2025-10-20 14:23:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64333,"visible":true,"origin":"","legend":"\u003cp\u003ea、b.Flow-cytometric analysis of NPCsapoptosis following TBHP exposure and transfection with FBXW7 siRNA or JNK siRNA. c.CCK-8 assay evaluating the effects of TBHP and FBXW7/JNK silencing on cell proliferation (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/16fe91456e3d6113872929c4.jpeg"},{"id":93945855,"identity":"b10544aa-4dc9-41ee-97f4-6651c99b97e1","added_by":"auto","created_at":"2025-10-20 14:23:05","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60160,"visible":true,"origin":"","legend":"\u003cp\u003ea. Effects of TBHP and FBXW7 siRNA on protein expression. b. Representative immunoblots with protein quantification. c. FBXW7/JNK mRNA expression (relative) (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/9bede77298ad5db8db8a9822.jpeg"},{"id":93947714,"identity":"40e5b4d1-b0d4-4c13-9ce4-db818324c386","added_by":"auto","created_at":"2025-10-20 14:31:05","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":70244,"visible":true,"origin":"","legend":"\u003cp\u003ea.Effects of TBHP and JNK siRNA on protein expression. b. Representative Western blot images and quantitative analysis of the indicated proteins. c. Relative mRNA expression of JNK (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/c73391bba327db6344ecadbc.jpeg"},{"id":93945854,"identity":"e199b994-319b-419d-8ddc-99bab022f2ff","added_by":"auto","created_at":"2025-10-20 14:23:05","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":93609,"visible":true,"origin":"","legend":"\u003cp\u003ea、b.Flow-cytometric evaluation of apoptosis in nucleus pulposus cells after FBXW7 overexpression and JNK knockdown. c、d. Representative Western blots and quantitative densitometry from the functional rescue experiments. e. Relative JNK mRNA levels in the rescue assay (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/2fcb7b8a120416232b7e0fa9.jpeg"},{"id":93945877,"identity":"25dd8ec0-7339-45e3-8830-0eb51d8ce0e8","added_by":"auto","created_at":"2025-10-20 14:23:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":12190207,"visible":true,"origin":"","legend":"\u003cp\u003eHE staining of rat caudal intervertebral disc tissues.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/fd7917e8d2b829b674afd6f6.png"},{"id":93947720,"identity":"2058f8eb-bfeb-408e-a65a-2af0e163acc4","added_by":"auto","created_at":"2025-10-20 14:31:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":110612,"visible":true,"origin":"","legend":"\u003cp\u003eRat caudal disc imaging: X-ray and MRI assessment. a. Rat caudal disc imaging (X-ray/MRI) pre-treatment b. X-ray/MRI of rat caudal discs at 6 weeks post-modeling.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/d7baa89ea3dd3bce30116893.png"},{"id":93945862,"identity":"712ea962-cbb3-4561-b04b-8af2bd677f81","added_by":"auto","created_at":"2025-10-20 14:23:05","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":62560,"visible":true,"origin":"","legend":"\u003cp\u003eFBXW7 and JNK expression (protein/mRNA) in rat NP samples. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/b32f49520c9d31779ed82d21.jpeg"},{"id":96363018,"identity":"8018d505-7d72-4ffc-97d1-d27be7078c07","added_by":"auto","created_at":"2025-11-20 10:03:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13850731,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/21302e46-1023-47cc-8246-008eb69cb4a8.pdf"},{"id":93945878,"identity":"b95a4bed-dca3-4f52-9f57-8f5a52aadeb3","added_by":"auto","created_at":"2025-10-20 14:23:06","extension":"ppt","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15981568,"visible":true,"origin":"","legend":"","description":"","filename":"WB.ppt","url":"https://assets-eu.researchsquare.com/files/rs-7518753/v1/0cce9c2ac91043e39a0bbb8b.ppt"}],"financialInterests":"No competing interests reported.","formattedTitle":"FBXW7 mediates intervertebral disc degeneration by regulating the JNK signaling pathway to induce nucleus pulposus cell apoptosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIDD: the foremost orthopedic disc pathology causing spinal dysfunction. IDD affects cervical, thoracic and lumbar regions, causing disc herniation, chronic back pain, spinal stenosis or spondylolisthesis. Discs between vertebrae comprise a gelatinous nucleus pulposus enclosed by fibrous annulus. The nucleus pulposus primarily contains proteoglycans and type II collagen, contrasting with the type I collagen-dominated annulus fibrosus \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.The etiology of IDD is complex, with factors such as environment, genetics, aging, smoking, obesity, inflammation, occupation, and mechanical stress all contributing to its development \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Currently, conventional treatments for IDD include conservative therapy and surgical intervention. However, these approaches can only alleviate pain and reduce symptoms without addressing the root cause of the problem. Therefore, elucidating IDD etiology facilitates targeted intervention strategies \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Dysregulated apoptosis characterizes degenerative disorders including OA, IDD and malignancies. IDD's primary pathology centers on nucleus pulposus cells (NPCs) apoptosis. NPCs apoptosis triggers extracellular matrix breakdown, disrupting the structural balance of the intervertebral disc and thereby mediating disc degeneration.\u003c/p\u003e\u003cp\u003eFBXW7 is an F-box-containing E3 ubiquitin ligase, with its gene located on chromosome 4 (4q31.3). The FBXW7 gene yields a canonical F-box protein, which can recognize and bind to phosphorylated sites in target proteins, mediating their ubiquitination and degradation. FBXW7 occupies a central position in the protein regulation network and exerts important functions in multiple aspects, including cell division, cell differentiation, apoptosis, cancer dissemination, and therapeutic unresponsiveness, primarily through the regulation of signaling pathways such as Wnt/β-catenin, JNK, RhoA, RPAP2, and ENO1\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Most FBXW7 investigations concentrate on cancer biology, delineating its antiproliferative and proapoptotic functions \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Recent studies have revealed that FBXW7 can promote apoptosis of cardiomyocytes by degrading anti-apoptotic proteins (e.g., Mcl-1)\u003csup\u003e[\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Bioinformatics analysis of GEO datasets (GSE70362/150408/124272) revealed elevated FBXW7 expression in IDD disc tissues of IDD patients. No prior studies have investigated FBXW7's function in disc degeneration.\u003c/p\u003e\u003cp\u003eHoeck et al. demonstrated that FBXW7 can regulate apoptosis of neural progenitor cells via JNK-mediated signal transduction \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. c-Jun N-terminal kinase (JNK), an essential mediator within the MAPK signaling cascade, mediates stress-responsive signaling. \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Liu et al. demonstrated JNK pathway activation drives disc degeneration via NPC apoptosis. \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. To date, there have been no reports on whether FBXW7 affects intervertebral disc degeneration via stimulation of the JNK-mediated signaling axis.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Bioinformatics Analysis\u003c/h2\u003e\u003cp\u003eThis study integrated three datasets\u0026mdash;GSE70362, GSE150408, and GSE124272\u0026mdash;from the GEO database and analyzed them using Rstudio software. We performed differential gene expression analysis (volcano plot, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) comparing normal and IDD groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Clinical Sample Collection\u003c/h2\u003e\u003cp\u003eA total of 20 human nucleus pulposus (NP) tissue samples (10 males and 10 females) were obtained from patients undergoing discectomy at Huai\u0026rsquo;an Hospital Affiliated to Xuzhou Medical University over a 24-month period from September 2022 through September 2024. The cohort comprised 14 degenerative and 6 traumatic disc specimens (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe inclusion criteria comprised patients who underwent surgery for vertebral fractures due to various etiologies and those with intervertebral disc degeneration (IDD) who failed conservative treatment. Patients with ankylosing spondylitis, lumbar spondylolisthesis, spinal stenosis, or spinal tumors were excluded. All specimens were surgically collected by orthopedic surgeons following informed consent. This study received hospital\u0026rsquo;s ethics committee authorization.Preoperative MRI(magnetic resonance imaging) scans preceded NP classification into mild (Pfirrmann I-III) and severe (IV-V) degenerative groups. (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e .\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical characteristics of intervertebral disc samples\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEtiology\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSpinal Level\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePfirrmann 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colname=\"c6\"\u003e\u003cp\u003eL4/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅡ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTrauma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL3/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅡ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" 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colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL5/S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅡ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL5/S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅢ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL4/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅢ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL3/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅢ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL4/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅢ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL3/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅣ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL5/S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅣ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL2/3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅤ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTrauma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL3/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅣ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL5/S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅤ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL4/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅤ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTrauma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL3/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅣ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL5/S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅣ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL4/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅤ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIDD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eL5/S1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eⅤ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePfirrmann grading system for intervertebral disc degeneration\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrade\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNP Structure\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNP Signal Intensity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNP\u0026ndash;AF demarcation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDisc height\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅠ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHomogeneous, bright white\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eClear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅡ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInhomogeneous with horizontal bands\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDistinct\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅢ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInhomogeneous, gray\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIntermediate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUnclear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSlightly decreased\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅣ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInhomogeneous, gray to black\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIntermediate-low\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLost\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eModerately decreased\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅤ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInhomogeneous, black\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLost\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSeverely decreased\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 NPCs Isolation and Culture\u003c/h2\u003e\u003cp\u003eHuman NP cells were derived from surgical disc samples. The tissue samples were washed in PBS to Clear residual blood and connective tissue, then minced into 1\u0026ndash;2 mm\u0026sup3; fragments. These pieces were incubated at 37\u0026deg;C in DMEM culture medium (Kaiji Biotech, Nanjing, China) containing 0.25% collagenase II (Thermo Fisher Scientific, USA) for 5 hours. Cells were pelleted by centrifugation (1200 rpm, 5 min) with subsequent supernatant discard. Cells were resuspended in complete DMEM (10% FBS, penicillin/streptomycin) and cultured at 37\u0026deg;C/5% CO₂. Fresh complete medium replaced the existing culture solution at 48-hour intervals. Cells at passage 3 (P3) served for downstream assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell Transfection\u003c/h2\u003e\u003cp\u003eCells were cultured to 60\u0026ndash;90% confluence. For transfection, Medium was exchanged for serum-free DMEM. In accordance with kit instructions, target siRNA was mixed with RNA TransMate transfection reagent (Sangon Biotech, Shanghai, China) in the appropriate ratio and incubated at room temperature yielding siRNA-carrier complexes after 10 min. The complex was then added to culture vessels, and vessels were gently agitated to ensure even distribution. After 6h, complete medium (DMEM\u0026thinsp;+\u0026thinsp;10%FBS\u0026thinsp;+\u0026thinsp;antibiotics) was replenished, and cells were harvested at 48h post-culture for follow-up investigations. For overexpression of FBXW7, lentivirus was directly introduced into culture medium per manufacturer's protocol (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), without the need for a transfection reagent, and following steps were performed as aforementioned. The FBXW7 overexpression lentivirus, FBXW7 siRNA, JNK siRNA, and RNA TransMate transfection reagent used in the experiments were all prepared by Sangon Biotech.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRecommended amounts of siRNA and RNA TransMate reagent for transfection in different culture plates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCulture Plate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedium Volume (\u0026micro;L/well)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003esiRNA (pmol/well)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRNA TransMate(\u0026micro;L/well)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSerum-Free Medium for Complexing\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e96-well plate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 pmol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.2 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e48-well plate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e200 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 pmol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.4 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e24-well plate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e500 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15 pmol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.0 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12-well plate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0 ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30 pmol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.0 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6-well plate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0 ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60 pmol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.0 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e150 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e35 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0 ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60 pmol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.0 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e150 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e60 mm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.0 ml\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e150 pmol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.0 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e250 \u0026micro;L\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Nucleus Pulposus Cell Apoptosis Model\u003c/h2\u003e\u003cp\u003eTert-butyl hydroperoxide (TBHP) was applied to stimulate programmed cell death in NPCs. Confluent NPCs (\u0026gt;\u0026thinsp;80%) in 6-cm dishes underwent 2\u0026times;PBS washes following medium aspiration. Following Wang et al.'s methodology, cells were treated with 100\u0026micro;M TBHP in complete medium (37\u0026deg;C, 5% CO₂, 6h) \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. After medium replacement with complete culture medium, cells were cultured for 24h prior to analysis. Some groups required prior transfection with RNA or siRNA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Western blot\u003c/h2\u003e\u003cp\u003e Total protein extraction was performed using RIPA lysis buffer (Beyotime, Shanghai), followed by concentration determination with a bicinchoninic acid (BCA) kit from the same manufacturer. Protein separation by electrophoresis was followed by PVDF membrane transfer (Millipore, USA). After 1h blocking in skim milk (Beyotime, Shanghai), primary antibodies were applied at 4\u0026deg;C overnight. Secondary antibody incubation was performed on the membranes for one hour under gentle agitation. Bands were detected with SuperSignal West Pico PLUS(Beyotime, Shanghai). ImageJ was employed for densitometric analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 qPCR\u003c/h2\u003e\u003cp\u003eTotal RNA was isolated via Trizol (Takara, Japan), with concentration/purity (OD260/OD280 ratio, R) assessed by spectrophotometry.The reverse transcription reaction containing 1 \u0026micro;g total RNA was carried out with HiScript II Q RT SuperMix(Vazyme, Nanjing) under standardized conditions: 42\u0026deg;C (60 min) \u0026rarr; 85\u0026deg;C (5 min). 20\u0026micro;L qPCR reactions used ChamQ SYBR mix (Vazyme, Nanjing) under cycling: 95\u0026deg;C 5min \u0026rarr; 40 cycles (95\u0026deg;C 5s, 60\u0026deg;C 30s). Technical triplicates per sample and relative quantification was performed using GAPDH as the reference gene. The corresponding primer sequences can be found in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer Design and Synthesis Human Primers\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTarget Gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFBXW7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-GGCCAAAATGATTCCCAGCAA-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ACTGGAGTTCGTGACACTGTTA-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eJNK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-TGTGTGGAATCAAGCACCTTC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-AGGCGTCATCATAAAACTCGTTC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-GGAGCGAGATCCCTCCAAAAT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-GGCTGTTGTCATACTTCTCATGG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eRat Primers\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTarget Gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSequence\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFBXW7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-AAGGTCCCAGCAAGCATCAGA-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ATTCACCCGTTTTCAAGTCCC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eJNK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-AGGAGCGAACTAAGAATGGCG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ACTGCTGTCTGTATCCGAGGC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-CTACCCACGGCAAGTTCAACG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-GCCAGTAGACTCCACGACATAC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 CCK8\u003c/h2\u003e\u003cp\u003eNPCs were plated at 10⁴ cells/well in 96-well plates. According to the experimental design, overexpression vectors for FBXW7, FBXW7 siRNA, JNK siRNA, or corresponding controls were added, with three replicates for each group. Cells were maintained at 37\u0026deg;C with 5% CO₂ for 6h. Subsequently, Cells received TBHP treatment (6h) as designed. Complete medium replacement post-treatment sustained cell growth.The culture medium was refreshed at 24 hours, 48 hours, and 72 hours. Each well received 100\u0026micro;L CCK8/DMEM (1:9, TaoShu Bio) for 2h standard incubation. Optical density measurements were performed at 450 nm wavelength using a microplate spectrophotometer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Apoptosis Analysis by Flow Cytometry\u003c/h2\u003e\u003cp\u003eNPCs apoptosis rates were determined through dual-labeling with Annexin V-FITC and PI (Kaiji Bio, Nanjing) according to established procedures. For adherent cells, they were collected after digestion with trypsin (without EDTA) (Beyotime, Shanghai, China). After two rounds of phosphate-buffered saline rinsing, cells were pelleted by low-speed centrifugation (5 minutes at 1000 rpm). Cell pellets were first resuspended in Binding Buffer, followed by 10-minute incubation at room temperature with Annexin V-FITC and PI in the dark. Finally, the stained cells were analyzed using a Beckman flow cytometer (Beckman, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Rat IDD Model\u003c/h2\u003e\u003cp\u003eIn this study, Percutaneous puncture established rat caudal disc degeneration \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Three-month-old male SD rats (n\u0026thinsp;=\u0026thinsp;40, 500\u0026thinsp;\u0026plusmn;\u0026thinsp;50 g) were studied. Four caudal discs (Co7/8-Co10/11) per rat were randomized into: control, IDD, IDD\u0026thinsp;+\u0026thinsp;FBXW7 shRNA (rescue), and sham groups. After fasting for 6 hours before surgery, the rats were anesthetized by gas anaesthesia using isoflurane (3% isoflurane for induction, 2% isoflurane for maintenance)\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eControl group: no treatment.\u003c/p\u003e\u003cp\u003eIDD group: 22G needle puncture (full disc penetration \u0026rarr; 360\u0026deg; rotation \u0026rarr; 30s retention).\u003c/p\u003e\u003cp\u003eRescue group: AAV9-FBXW7-shRNA was injected directly following needle puncture (2\u0026micro;L, 2.67\u0026times;10\u0026sup1;\u0026sup3; VG/mL).\u003c/p\u003e\u003cp\u003eSham group:underwent paravertebral puncture avoiding disc injury.\u003c/p\u003e\u003cp\u003ePostoperatively, rats were singly housed with ad libitum activity and daily monitoring. The rats were sacrificed at 6 weeks post-surgery for subsequent examinations and experiments. The AAV9-FBXW7-shRNA used in this study was prepared by Nanjing Zebrafish Biotech Co., Ltd.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 X-ray/MRI\u003c/h2\u003e\u003cp\u003eFollowing 6-week induction, the rats were anesthetized by gas anaesthesia using isoflurane (3% isoflurane for induction, 2% isoflurane for maintenance). The anesthetized rats were secured on an X-ray examination table, and radiographs of the caudal vertebrae were taken using a medical diagnostic X-ray tube assembly (Xinglian Industry, Shenzhen, China). The images were obtained and analyzed via a PACS system.7T MRI (Bruker, Germany)acquired T2-weighted sagittal images at Jiangsu Medical Animal Facility. Disc degeneration severity was graded via Pfirrmann criteria\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Paraffin Embedding and HE Staining\u003c/h2\u003e\u003cp\u003ePost-imaging, rats were sacrificed with caudal samples fixed in 4% PFA (Biosharp, Hefei) for 12h. Post-EDTA decalcification (10%), tissues were dehydrated and paraffin-processed. Histological examination was performed on 4\u0026ndash;5 \u0026micro;m thick sections using HE staining to evaluate group-specific morphological variations in intervertebral discs. Microscopic imaging was performed with a PrimoStar system (Zeiss, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Ethical Statement\u003c/h2\u003e\u003cp\u003eThe study protocol received formal ethical clearance (Approval No. XXX) from the Institutional Review Board of Huai'an Hospital affiliated with Xuzhou Medical University. The cohort comprised 20 NP tissues (50% male) collected during discectomies (2022\u0026ndash;2024). All participants and family members provided written consent.\u003c/p\u003e\u003cp\u003eSprague-Dawley rats were obtained from an accredited vendor (Lambda, Nanjing). The animal use license and ethical review number are IACUC-2024110506. All in vivo studies complied with institutional animal care guidelines (Guide for the Care and Use of Laboratory Animals), with technical support provided by commercial partners.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1 FBXW7/JNK Upregulation in Severely Degenerated NP Tissues\u003c/h2\u003e\u003cp\u003eFBXW7 was upregulated in IDD compared to normal discs (bioinformatics data). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). WB and qPCR quantified molecular changes across degeneration-grade NP samples. The results demonstrated that FBXW7 and JNK expression was markedly elevated in severe versus mild degeneration groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Both si-FBXW7 and si-JNK Inhibit TBHP-Induced Apoptosis\u003c/h2\u003e\u003cp\u003eFlow cytometry revealed significantly higher NPCs apoptosis in TBHP-treated groups than controls. Knockdown groups (si-FBXW7/si-JNK) showed markedly lower apoptosis rates than TBHP-treated cells. CCK8 analysis revealed significant proliferation inhibition in TBHP-treated cells versus controls, while si-FBXW7 and si-JNK could partially reverse the inhibitory effect of TBHP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWestern blot quantified FBXW7, JNK, and apoptosis regulators (Caspase-3/Bax/Bcl-2). TBHP treatment elevated FBXW7/JNK/Caspase-3/Bax expression but suppressed Bcl-2 versus controls. Compared to TBHP alone, si-FBXW7 co-treatment downregulated FBXW7/JNK/Caspase-3/Bax while maintaining higher levels than controls; Bcl-2 remained suppressed(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared to TBHP group, si-JNK co-treatment downregulated JNK/Caspase-3/Bax (though still above controls) and further suppressed Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). qPCR revealed elevated FBXW7/JNK expression in TBHP-treated cells, which was attenuated by si-FBXW7 transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 FBXW7 Regulates JNK to Promote Nucleus Pulposus Cell Apoptosis\u003c/h2\u003e\u003cp\u003eWestern blot/flow cytometry elucidated FBXW7-JNK-apoptosis axis in NPCs. Evidence suggested overexpression of FBXW7 increased apoptosis in NPCs, while transfection with si-JNK partially attenuated this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b).\u003c/p\u003e\u003cp\u003eFBXW7 overexpression upregulated JNK/Bax/Caspase-3 but downregulated Bcl-2 versus controls. In FBXW7-overexpressing cells, JNK silencing partially normalized the expression of apoptotic regulators: decreasing JNK, Bax and Caspase-3, and increasing Bcl-2. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Animal Experiments\u003c/h2\u003e\u003cp\u003eHematoxylin-eosin (HE) demonstrated that the NP tissues in control group exhibited large, intact ellipses with spindle-shaped nucleus pulposus cells, distinct nuclei and cytoplasm, and annulus fibrosus tissues arranged in a regular ring-like pattern. In contrast, the NP tissues in the IDD\u0026thinsp;+\u0026thinsp;FBXW7 shRNA group (rescue group) and the sham surgery group (Sham group) appeared flattened and elongated, with more pronounced changes in the annulus fibrosus structure and surrounding muscle. Additionally, in the Sham group, the muscles surrounding the intervertebral discs were visibly damaged. The IDD group showed overall fragmentation and disorganization, with NP tissues, annulus fibrosus, and surrounding muscles intermingled and losing their clear and distinct morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRadiographs of the rat caudal vertebrae demonstrated that degenerated intervertebral discs exhibited characteristics such as narrowed intervertebral space, wedge-shaped changes in the intervertebral space, and osteophyte formation. MRI revealed significantly reduced T2-signal intensity in IDD rats versus controls, with rescue and Sham groups showing intermediate values (IDD\u0026thinsp;\u0026lt;\u0026thinsp;rescue/Sham\u0026thinsp;\u0026lt;\u0026thinsp;control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFinally, protein and RNA from disc tissues facilitated Western blot and qPCR assays. IDD tissues exhibited significantly elevated FBXW7/JNK expression relative to controls. However, injection of FBXW7 shRNA significantly attenuated the upregulation of FBXW7 and JNK in the IDD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIDD drives chronic low back pain and functional impairment through ECM catabolism, inflammation, and NPCs apoptosis. As individuals age, the intervertebral discs experience water loss, reduced elasticity, annulus fibrosus rupture, and changes in the nucleus pulposus structure, leading to structural and functional degeneration of the discs. Studies have shown that apoptosis of NPCs, ECM degradation, and inflammatory responses are key pathological aspects of IDD\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In recent years, Mounting studies underscore JNK pathway involvement in disc degeneration via NPCs apoptosis. JNK, a crucial member of the MAPK family, modulates cell death, inflammatory activation, and ECM catabolism. Mechanical stress, oxidative stress, and inflammatory cytokines can all activate the JNK pathway, thereby promoting NPCs apoptosis and ECM destruction. However, The mechanistic interplay between JNK signaling and IDD progression remains incompletely characterized. \u003csup\u003e[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis investigation elucidates FBXW7's mechanistic contribution to IDD through JNK pathway modulation, offering novel therapeutic insights. FBXW7's central role in the ubiquitin-proteasome system impacts cell cycle, proliferative capacity, and survival pathways \u003csup\u003e[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. FBXW7 has been implicated in diverse pathologies, spanning oncogenesis, neurodegeneration, cardiovascular dysfunction, and metabolic dysregulation \u003csup\u003e[\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Welcker et al. proposed that FBXW7 promotes the ubiquitination and proteasomal degradation of target proteins, thereby regulating cell differentiation, proliferation, and apoptosis \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Mao's research on breast cancer indicated that FBXW7 inhibits tumor cell proliferation and metastasis by degrading c-Myc and Notch1\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Our analysis of nucleus pulposus tissues with different degrees of degeneration revealed high expression of FBXW7 in highly degenerated NP tissues. In cellular experiments, TBHP treatment upregulated FBXW7 expression and enhanced apoptosis versus controls. Knocking down FBXW7 partially rescued TBHP's pro-apoptotic impact. FBXW7 upregulation exerted anti-proliferative and pro-apoptotic effects, indicating that FBXW7 promotes NPCs apoptosis. FBXW7 was upregulated in IDD rats, whereas FBXW7 shRNA treatment attenuated degeneration.Therefore, FBXW7 may contribute to IDD by promoting NPCs apoptosis.\u003c/p\u003e\u003cp\u003eJNK cascade serves as a master modulator of stress adaptation, closely associated with various apoptotic processes and involved in pathological conditions such as neurodegenerative diseases, tumors, fibrotic diseases, and chronic inflammation. The JNK cascade is upregulated in degenerative discs, modulating apoptosis, senescence, and ECM remodeling. JNK pathway activation is triggered by mechanical/oxidative stress and pro-inflammatory cytokines (TNF-α/IL-1β), leading to ECM degradation and NPCs apoptosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Additionally, JNK pathway stimulation accelerates IDD through ECM-degrading enzymes (MMPs/ADAMTs) \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. In this study, elevated JNK levels characterized severely degenerated NP tissues and TBHP-treated apoptotic NPCs. Park's research suggested that JNK inhibition attenuates macrophage-derived inflammatory mediators (TNF-α, IL-6/8, CCL2/3), suppressing apoptosis and ECM degradation in chronic pain \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. In cellular experiments, induction of NPCs apoptosis resulted in elevated levels of JNK/Bax/Caspase-3, while Bcl-2 expression was downregulated. However, knocking down JNK partially inhibited TBHP-induced apoptosis. Our experimental results are consistent with current studies, indicating that JNK promotes NPCs apoptosis.\u003c/p\u003e\u003cp\u003eJNK pathway modulation by FBXW7 represents a novel therapeutic target for IDD. In highly degenerated NP tissues, the expression levels of FBXW7 and JNK are elevated. In vitro cellular experiments revealed that overexpression of FBXW7 promotes JNK expression and NPCs apoptosis. Knocking down JNK in this context partially inhibits NPCs apoptosis, suggesting that FBXW7 may induce NPCs apoptosis by regulating the JNK signaling pathway. Animal experiments showed that the IDD group exhibited elevated FBXW7/JNK expression relative to controls.Injection of FBXW7 shRNA significantly inhibited the upregulation of FBXW7 and JNK in degenerated NP tissues and IDD advancement. This evidence supports FBXW7 promotes NPCs apoptosis by regulating the JNK signaling pathway, thereby contributing to IDD development.\u003c/p\u003e\u003cp\u003eQiu et al. studied the role of FBXW7 in oral squamous cell carcinoma (OSCC) and found that transfection with FBXW7 promoted apoptosis in OSCC cells, with upregulated Bax levels and suppressed Bcl-2 expression in FBXW7-transfected OSCC cells\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. We obtained similar results in vitro in NPCs transfected with FBXW7. Diverging from our observations, Nateri et al. reported that knocking down FBXW7 induces apoptosis in neurons and that FBXW7 antagonizes JNK signaling pathway-induced apoptosis \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Research from Qiu's and Nateri's groups reveals the intricate interplay of FBXW7 in apoptotic regulation. Notably, Zhang et al. reported FBXW7 downregulation in OA cartilage (human patients and murine models). In FBXW7 knockout mice, chondrocyte senescence was exacerbated. Mechanical overload reduced FBXW7 expression, decreased FBXW7-mediated degradation of MKK7, enhanced JNK-dependent signaling, and thereby promoted chondrocyte senescence. Additionally, Zhang et al. demonstrated in animal experiments that injection of AAV-FBXW7 alleviated OA symptoms in mice\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. In the animal experiment part of our study, injection of AAV-FBXW7 shRNA alleviated IDD symptoms in rats. The results of our study contrast sharply with those of Nateri and Zhang. These findings indicate that FBXW7/JNK signaling interplay is not fixed but rather depends on the cell type and disease context. FBXW7's pro-apoptotic effects vary across cell lineages and pathological contexts. Regarding FBXW7's therapeutic potential, it is essential to fully consider its complex mechanisms under different conditions to achieve precise treatment. Moreover, FBXW7 may also indirectly affect JNK pathway activity by regulating other downstream targets, such as c-Myc and Notch. Future research can further explore the specific mechanisms of FBXW7 in the JNK pathway and its potential therapeutic value in IDD \u003csup\u003e[\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, FBXW7 drives IDD pathogenesis via JNK-mediated NPCs apoptosis. This finding provides avenues for targeted therapy development. However, the complex regulatory mechanisms of FBXW7 in different cell types and pathological states suggest that its development as a therapeutic target requires careful consideration of tissue specificity and precise treatment strategies. Future research can further explore the specific mechanisms of FBXW7 in the JNK signaling pathway and investigate its clinical application potential from the perspectives of gene therapy or drug inhibitors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYi Gao and Yuning Zhu: Performed data collection and conducted experiments.Yuting Gong and Rui Chen: Conducted data analysis and interpretation. Jing Yan and Shuo Miao: Searched the literature. Yi Gao, Yuning Zhu, and Yuting Gong: Completed figure/image processing. \u0026nbsp;Yi Gao: Drafted the manuscript. Quan Zhou: Reviewed and edited the manuscript. \u0026nbsp;Quan Zhou and Wei Pan: Designed and funded the study. Yi Gao and Yuning Zhu contributed equally to this work. Quan Zhou and Wei Pan are the co-corresponding authors of this article.Quan Zhou is the lead corresponding author.Wei Pan is the co-corresponding author.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by General Program of the Health Commission of Jiangsu Province (Grant No.M2022125).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe acquisition of human intervertebral disc samples was approved by the Ethics Committee of the Affiliated Huai’an Hospital of Xuzhou Medical University, with informed consent obtained from the patients and their family members.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 \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e (National Research Council, 2011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author.\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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun Y, Li Z, Duan J, et al. From structure to therapy: the critical influence of cartilaginous endplates and microvascular network on intervertebral disc degeneration [J]. Front Bioeng Biotechnol, 2024, 12: 1489420.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOichi T, Taniguchi Y, Oshima Y, et al. 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Cancer Rep (Hoboken), 2021, 4(4): e1369.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intervertebral disc degeneration, FBXW7, JNK, Apoptosis, Nucleus pulposus cells","lastPublishedDoi":"10.21203/rs.3.rs-7518753/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7518753/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eElucidating the mechanistic role of FBXW7 in JNK pathway activation and subsequent nucleus pulposus apoptosis during disc degeneration.\u003c/p\u003e\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNPCs apoptosis constitutes a core pathological event during disc degeneration. Oxidative stress triggers JNK-mediated apoptotic signaling cascades in nucleus pulposus cells. As a core E3 ligase component, FBXW7 is involved in apoptosis regulation, but its role in NPCs apoptosis during IDD remains unexplored.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eBioinformatics analysis was performed on GEO datasets to identify differentially expressed genes in nucleus pulposus tissues of varying degeneration grades. Human NP samples with different degeneration levels were collected to assess FBXW7 expression. In vitro, NPCs were cultured, transfected with siRNA to silence FBXW7 or JNK, and subjected to apoptosis induction, followed by evaluation of apoptosis-related protein expression, cell apoptosis, and proliferation. In vivo IDD modeling employed rat disc puncture methodology, followed by local injection of FBXW7 shRNA to evaluate therapeutic effects on disc degeneration.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eFBXW7 and JNK expression were significantly upregulated in severely degenerated nucleus pulposus tissues. In vitro, FBXW7/JNK overexpression upregulated apoptotic effectors (Bax, Caspase-3) and downregulated Bcl-2, whereas FBXW7/JNK knockdown reduced apoptosis rates and mitigated TBHP-induced suppression of NPC proliferation. In vivo, FBXW7 shRNA injection delayed disc degeneration, attenuated T2-weighted MRI signal decline, and suppressed JNK expression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eFBXW7 and JNK are upregulated in advanced IDD. FBXW7 promotes NPCs apoptosis via the JNK pathway, and its knockdown alleviates disc degeneration progression in rats.\u003c/p\u003e","manuscriptTitle":"FBXW7 mediates intervertebral disc degeneration by regulating the JNK signaling pathway to induce nucleus pulposus cell apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 14:23:00","doi":"10.21203/rs.3.rs-7518753/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"34ea35f4-df51-48b1-8731-1339bb06b493","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-14T20:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-20 14:23:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7518753","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7518753","identity":"rs-7518753","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}