Nicotine exacerbates intervertebral disc degeneration via NF-κB/MAPK-dependent inflammatory activation and cellular senescence

Nicotine exacerbates intervertebral disc degeneration via NF-κB/MAPK-dependent inflammatory activation and cellular senescence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nicotine exacerbates intervertebral disc degeneration via NF-κB/MAPK-dependent inflammatory activation and cellular senescence Desheng Wu, Hang Feng, Zhaoyu Ba, Ziqi Zhu, Tongde Wu, Kai Guo, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6586785/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Cigarette smoking constitutes a major modifiable risk factor for intervertebral disc degeneration (IVDD), yet the mechanistic underpinnings remain incompletely elucidated. This study aimed to elucidate the pathological effects of nicotine-the primary addictive component in tobacco-on IVDD, focusing on its role in inflammation, extracellular matrix (ECM) disruption, and cellular senescence. Utilizing in vivo models (smoke exposure, nicotine injection, and disc puncture) and in vitro human nucleus pulposus cell (NPC) cultures, we employed multi-omics approaches (metabolomics, transcriptomics, imaging, and functional assays) to investigate nicotine's impact on ECM metabolism, inflammatory signaling, and senescence. Nicotine and its primary metabolite, cotinine, accumulated in avascular disc tissue following inhalation or systemic administration. Nicotine dose- and time-dependently inhibited aggrecan and collagen II synthesis, concomitant with MMP-13 upregulation, indicating a catabolic shift in ECM homeostasis. In vivo, nicotine exacerbated puncture-induced IVDD, with synergistic ECM degradation and inflammation. RNA-seq revealed NF-κB and MAPK pathway activation, confirmed by rapid p65 and p38 phosphorylation and elevated IL-1β and IL-6 expression. Pharmacological inhibition of these pathways attenuated nicotine-induced ECM degradation and inflammation. Notably, nicotine triggered NPC senescence with a pro-inflammatory senescence-associated secretory phenotypes (SASP), synergizing with pre-existing disc injury to accelerate IVDD. Our study uncovers a dual-pathway mechanism in which nicotine activates NF-κB and MAPK signaling to amplify inflammatory cascades and SASP, synergistically accelerating IVDD progression. These insights underscore the urgency of smoking cessation, particularly in early-stage IVDD, and propose targeted inhibition of these pathways as a therapeutic strategy. Health sciences/Diseases/Rheumatic diseases/Osteoarthritis Health sciences/Medical research/Genetics research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 ​INTRODUCTION Low back pain (LBP) affects over 540 million people globally, representing a leading cause of disability and imposing significant socioeconomic burdens​ 1 . As the leading cause of disability worldwide, LBP imposes substantial socioeconomic burdens on individuals, families, and healthcare systems 2 . Intervertebral disc degeneration (IVDD) is a primary pathological contributor to LBP, often precipitating secondary spinal disorders such as disc herniation, spinal stenosis, and segmental instability 3 . The intervertebral disc (IVD) comprises three distinct structures: the gelatinous nucleus pulposus (NP), the concentric lamellae of the annulus fibrosus (AF), and the cartilaginous endplates. The NP, populated by nucleus pulposus cells (NPCs) embedded in an extracellular matrix (ECM) rich in type II collagen and aggrecan, maintains disc hydration, mechanical resilience, and nutrient exchange 4 . Notably, the IVD is an avascular tissue, relying on diffusion through the endplates and peripheral AF capillaries for metabolic sustenance 5 . A hallmark of IVDD is the dysregulation of ECM homeostasis, characterized by enhanced catabolism mediated by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family enzymes 6 . Concurrently, IVDD is associated with elevated pro-inflammatory cytokine expression, which exacerbates ECM degradation, oxidative stress, and cellular senescence, collectively accelerating IVDD 7 . Large-scale epidemiological studies unequivocally identify smoking as a modifiable risk factor for IVDD 8 , 9 . A longitudinal study of 5,180 forestry workers demonstrated that smoking significantly correlated with radicular neck and sciatic pain 10 , while another large-scale investigation (n = 25,610) confirmed its association with chronic LBP 11 . Notably, MRI-based analyses in monozygotic twins revealed an 18% higher IVDD severity in smokers compared to non-smokers 12 . Meta-analyses further indicate that smokers exhibit a 27% increased risk of IVDD, with disease severity correlating positively with smoking intensity and duration 13 . Additionally, smoking adversely impacts spinal surgical outcomes, increasing non-union rates in anterior cervical discectomy/fusion and postoperative airway complications 14 , 15 . Previous mechanistic studies highlight nicotine-the primary addictive component of tobacco-as a key mediator of smoking-induced IVDD. Nicotine compromises disc microcirculation, impairing nutrient supply and metabolic waste clearance 16 . Furthermore, tobacco smoke or nicotine extracts upregulate IL-1β and ADAMTS5 expression while suppressing aggrecan and collagen synthesis, thereby disrupting ECM equilibrium 17 – 19 . Emerging evidence implicates cellular senescence as a pivotal driver​of IVDD 20 . Senescent NPCs acquire a senescence-associated secretory phenotype (SASP), releasing pro-inflammatory cytokines, chemokines, and proteases that amplify ECM catabolism, propagate senescence bystander effects, and sustain a degenerative microenvironment 21 , 22 . Despite epidemiological associations, the direct pathogenic role​of nicotine in IVDD remains poorly define. With the global smoking population projected to reach 1.6 billion by 2025 (WHO), elucidating nicotine’s pathological role in IVDD is clinically imperative 23 . Here, this study systematically delineates nicotine’s penetration into avascular disc tissue, its activation of NF-κB and MAPK-mediated inflammation, and induction of senescence-mechanisms that collectively exacerbate IVDD. MATERIALS AND METHODS Ethics statement​ ​ All protocols were approved by the Institutional Ethics Committee of Henan Provincial People’s Hospital, Zhengzhou University, in compliance with the Declaration of Helsinki. Animal procedures strictly followed the guidelines of Zhengzhou University’s Animal Care and Use Committee (Ethical Application Ref: No. 1-233). Human NP tissues were obtained from non-smoking patients undergoing discectomy, with informed consent. IVDD status was classified using preoperative MRI (Pfirrmann grade I-III: control; IV-V: IVDD) 24 . Reagents and antibodies ​​ Nicotine was purchased from Sigma-Aldrich. Cigarettes (Double Happiness; Shanghai Tobacco Group) contained 15 mg tar, 1.3 mg nicotine, and 14 mg CO per unit. Fetal bovine serum (FBS), trypsin and penicillin-streptomycin were purchased from Gibco (Grand Island, NY, USA). Phosphate buffered solution (PBS) and Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) were purchased from Hyclone (South Logan, UT, USA). The type II collagenase were purchased from (St. Louis, MO, USA). The primary antibody of Collagen II, MMP-13, IL-1β and P16INK4a were acquired from Abcam (Cambridge, MA, USA). The antibodies against GAPDH, P65, P38 and P-p38 were purchased from CST (Danvers, MA, USA). The antibodies for P-p65 and IL-6 were purchased from SANTA (Dallas, TX, USA). The antibody against Aggrecan was purchased from Thermo Fisher (Waltham, MA, USA). NF-κB inhibitor JSH-23 25 and MAPK inhibitor BIRB796 26 were purchased from APExBIO (Houston, TX, USA). Experimental design ​​ For in vitro studies, human NPCs were serum-starved overnight in DMESM/F12 containing 2% FBS prior to experimentation. Nicotine solutions (25, 100, and 200 µg ml⁻¹) were prepared in complete medium (10% FBS, 1.5% penicillin/streptomycin). To investigate nicotine's temporal effects on ECM metabolism, NPCs were treated with 100 µg ml⁻¹ nicotine for 0, 3, 5, and 7 days. Concentration-dependent responses were assessed using 0-200 µg ml⁻¹ nicotine over 5 days. mRNA expression of ECM components (Aggrecan, Collagen II) and catabolic enzymes (MMP-13, ADAMTS-4) was quantified by RT-qPCR, with protein levels analyzed via western blot. NF-κB and MAPK pathway activation was evaluated by immunofluorescence and phosphorylation status of p65 and p38 at 15 min, 30 min, and 2 h post-treatment (100 µg ml⁻¹ nicotine). Pharmacological inhibition studies employed JSH-23 (30 µM, NF-κB inhibitor) and BIRB796 (400 nM, p38 inhibitor) pre-treated 1 h before nicotine exposure. Cellular senescence was assessed by β-galactosidase (β-gal) staining and p16 INK 4 a expression in nicotine-treated NPCs and surgical specimens from heavy smokers (> 2 packs/day, > 10 years). RNA sequencing of nicotine-treated NPCs (100 µg ml⁻¹, 48 h) was performed to identify global transcriptomic changes. For in vivo studies, 8-week-old SD rats (Shanghai SLAC Laboratory Animal Co.) were randomized into five experimental groups: Control group, Smoking group, Nicotine group (Intraperitoneal nicotine injection), Puncture group, Puncture + Nicotine group. Metabolic profiling of serum and disc tissues was conducted at week 1 post-intervention. Intervertebral disc degeneration grading was evaluated using X-ray and MRI. Histological assessment of intervertebral disc tissues was performed through Hematoxylin & Eosin (H&E) staining and Safranine O-Fast Green staining. Immunohistochemical analysis was employed to examine the expression of extracellular matrix proteins and evaluate inflammatory status in the disc tissues (Fig. 1 a). Human nucleus pulposus cells culture ​​ Microscopically, NP tissues were meticulously dissected and minced into 1–2 mm³ fragments. Tissue fragments underwent sequential enzymatic digestion: first with 0.25% trypsin-EDTA for 30 min at 37°C, followed by 0.1% type II collagenase for 4 h at 37°C with gentle agitation. The resulting cell suspension was centrifuged at 300 × g for 5 min, and pelleted NPCs were resuspended in complete growth medium composed of DMEM/F12 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine. Primary cells were maintained under standard hypoxic conditions (2% O₂, 5% CO₂, 37°C) to mimic the native intervertebral disc microenvironment. The culture medium was refreshed every 72 h until 80–90% confluence, at which point cells were passaged using 0.25% trypsin-EDTA at a 1:2 split ratio. All experiments utilized passage 2–3 cells to preserve phenotypic stability and minimize dedifferentiation artifacts. Cell viability assay ​​ The viability of NPCs was assayed with the cell counting kit-8 (CCK-8; Dojindo Co, Kumamoto, Japan) according to the manufacturer’s protocol. NPCs were uniformly seeded in 96-well plates at a density of 1.5 × 10⁴ cells/well. The cells were then treated with nicotine at concentrations of 0, 0.2, 0.5, 1, 10, 50, 100, 200, and 300 µg ml⁻¹ for 24 h. Following treatment, 100 µL of DMEM containing CCK-8 reagent was added to each well, followed by incubation at 37°C for 2 h. Optical density values were measured at 450 nm using a microplate reader, and cell viability was calculated accordingly. Western blot The total protein of NPCs was extracted using the total protein extraction kit (Beyotime Biotechnology, Shanghai, China). The determination of the protein concentration was conducted using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal aliquots of protein from each sample were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to 22 µm polyvinylidene fluoride membranes (Millipore, USA) using a wet-blotting method. Then, the membranes were blocked by 5% nonfat milk in TBST buffer and incubated overnight at 4°C with primary antibodies, including GAPDH (1:5,000), Collagen II (1:5,000), Aggrecan (1:500), MMP-13 (1:3,000), IL-1β (1:1,000), P65 (1:1,000y), P-p65 (1:200, SANTA), P38 (1:1,000), P-p38 (1:1,000), p16 INK 4 a (1:1,000). Afterwards, the membranes were incubated with secondary antibodies for 1 h at 37°C. After washing with PBS three times, the bands were observed in a darkroom and quantified using the ImageJ software (National Institutes of Health USA). Immunofluorescence NPCs were cultured in 24-well plates (4x104 cells/well), treated as mentioned above and fixed with 4% paraformaldehyde for 30 min. Following washing with PBS that contained 0.1% Tween-20 (PBST), the cells were incubated with 0.2% Triton X-100 for 20 min. Then, the samples were blocked for 30 min in 5% goat serum albumin. Subsequently, the samples were incubated with primary antibodies against Aggrecan (1:50), Collagen II (1:100), MMP-13 (1:500), IL-1β (1:1,00), P65 (1:400), P38 (1:50) and p16 INK 4 a (1:200) overnight at 4°C. The samples were then were washed and incubated with FITC-conjugated secondary antibodies for 1 h in the dark and labeled with DAPI for 5 min. Finally, the fluorescent images were obtained using a fluorescence microscope. Quantitative real-time PCR The RNA/DNA/Protein Isolation Kit was used to extract total RNA from NPCs, and RNA and qRT SuperMix were then used to synthesize cDNA, following to the manufacturer’s instructions (Vazyme-innovation in enzyme technology, China). GAPDH was used to standardize the cycle thresholds (Ct) of the transcripts that were obtained. The △△Ct method was used to calculate the relative mRNA levels of each target gene. Primer sequences are presented in Supplementary Table S1 . mRNA sequencing and data analysis Total RNA was extracted from rat NP cells using TRIzol reagent, and the BGI (Shenzhen, China) was commissioned to perform mRNA transcriptome sequencing. Differential expression analyses were conducted using the DESeq2 package, with p < 0.05, to identify differentially expressed genes. Gene ontology (GO) terms for enriched genes were determined using GOSeq, and in-house scripts were used to analyze significantly differentially expressed genes in KEGG pathways. The threshold was set as follows: | log2 (fold change) | >1 and p < 0.05. SA-β-gal staining The level of senescence was measured by SA-β-gal staining kit (Beyotime, Shanghai, China) according to the instruction. Cells were seeded in a six-well plate and then washed twice with PBS. Cells were fixed with 0.2% glutaraldehyde for 15 min at room, washed three times with PBS and then stained with X-gal staining solution at pH 6.0 overnight. SA-β-gal-positive cells were counted in six randomly selected images under a microscope, and the percentages of SA-β-gal-positive cells were averaged and quantified. Passive smoking model in rats ​ We established a chronic smoke exposure system using a custom-designed chamber equipped with real-time CO/O₂/PM₂.₅ monitoring and automated ventilation control (Supplementary Fig. 1). Rats were exposed to mainstream smoke from two combusted cigarettes (1.3 mg nicotine each) per session, with CO concentrations maintained at 180–208 ppm through feedback-regulated airflow. Exposure cycles consisted of 1 h smoke inhalation followed by 20 min clearance, repeated twice daily (6 total hours/day). The protocol ran 5 days/week for 12 weeks, as previously optimized in smoke-induced COPD models 27 , 28 . Food and water were available ad libitum during exposures. Nicotine intraperitoneal injection model Nicotine solution was prepared in normal saline at a concentration of 1 mg ml⁻¹, protected from light and stored at 4°C. Prior to injection, solutions were equilibrated to room temperature for 30 minutes. Daily intraperitoneal injections (2 mg kg⁻¹ body weight) were administered in the lower abdominal quadrant, with a regimen of 5 consecutive days per week over 8 weeks, as previously validated 29 . Dose selection and administration protocols were based on pharmacokinetic studies demonstrating sustained nicotine exposure mimicking chronic smoking patterns in human 30 . The annulus fibrosus needle puncture model The puncture procedure was performed as previously described 31 . Briefly, rats were anesthetized via intraperitoneal injection of 2% (w/v) pentobarbital sodium (40 mg kg⁻¹) 32 . The target coccygeal intervertebral discs (Co7/8) were identified by manual palpation and confirmed radiographically through vertebral counting from the sacral region. A 26-gauge needle was percutaneously inserted through the full thickness of the AF under aseptic conditions. The needle was rotated 360° and maintained within the disc space for 1 minute to induce controlled annular injury. Puncture depth (about 5 mm) was standardized based on preliminary radiographic measurements of AF and NP dimensions in coccygeal discs. Metabolomic profiling and analysis ​​ Upon completion of modeling, blood and intervertebral disc specimens were collected from experimental rats. Metabolomic analysis was conducted by Shanghai Biotree Biotech Co., Ltd. (Shanghai, China) using ultra-performance liquid chromatography (UPLC, Shimadzu Nexera X2) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS, Sciex X500R). Raw data processing included peak detection, extraction, alignment, and integration using MetaboAnalyst 5.0. Compound annotation was performed against the BiotreeDB (v2.1) in-house library with a matching score threshold of 0.3. Multivariate statistical analysis (PCA and OPLS-DA) was conducted to identify differential metabolites ( p < 0.05 by Student's t-test). X-ray and MRI assays The X-ray films of the rat tails were captured using an X-ray system (uDR 588i, United Imaging, Shanghai, China) to evaluate disc gross appearance and disc height status. The Disc Height Index (DHI) was adopted to assess disc height loss using the method as previously described 33 . Magnetic resonance imaging was performed on all rats to evaluate the signal and structural changes in sagittal T2-weighted images using a 3.0 T MRI system (uMR 770, United Imaging, Shanghai, China). IVDD grade was assessed using the Pfirrmann system. Histopathologic analysis The isolated rat spines and adjacent vertebral bodies were first preserved for 24 h in 4% paraformaldehyde before being decalcified in a 10% EDTA solution. Following decalcification, the tissues were immersed in paraffin and the blocks of paraffin were sectioned into homogeneous slices with a thickness of 5 µm. Histopathological characteristics were assessed using hematoxylin and eosin (H&E) staining and Safranin O-fast green staining. Histological grading of the intervertebral discs was performed as previously described 34 . Immunohistochemistry The sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity for 10 min and 5% bovine serum albumin was used to block nonspecific binding sites for 30 min at 37°C. Subsequently, sections were incubated overnight at 4°C with primary antibodies Aggrecan (1:100), Collagen II (1:100), MMP-13 (1:100), IL-1β (1:1,00), IL-6 (1:50) and p16 INK 4 a (1:1,00). Following this, sections were incubated with appropriate secondary antibodies for 1 h at ambient temperature. The DAB detection system (Sangon Biotech, Shanghai, China) was employed to visualize the immunoreactivity. Finally, sections were dehydrated, sealed, and digitally scanned using a slide scanner. Statistical analysis Statistical analysis was conducted utilizing SPSS 22.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 9 (GraphPad Software, USA). The collected data were tested for normality by Shapiro-Wilk normality test. The independent samples t-test assessed the statistical difference between two groups, while one-way analysis of variance (ANOVA) was employed for multiple data group comparison. Data were expressed as mean ± standard deviation (SD). Statistical significance was set at p < 0.05. RESULTS Nicotine and its metabolites accumulate in IVD ​​ While nicotine distribution has been documented in vascularized organs, its penetration into avascular IVD tissues remained unclear. Our multi-modal metabolomic investigation using liquid chromatography-tandem mass spectrometry provides first evidence of nicotine accumulation in disc tissues through diverse exposure routes (Fig. 1 a). Plasma analysis revealed significantly elevated nicotine and cotinine (nicotine's primary metabolite) levels in smoke-exposed rats compared to controls (Fig. 1 b), validating our exposure model. Strikingly, mass spectrometry analysis of disc tissues demonstrated marked nicotine accumulation in the nicotine injection group (Fig. 1 c) and elevated cotinine levels in smoke-exposed animals (Fig. 1 d). These findings establish that nicotine penetrates the avascular disc microenvironment and accumulates at biologically relevant concentrations, regardless of administration route. Nicotine/tobacco exposure induces IVDD To investigate tobacco-related IVDD pathogenesis, we established three experimental models: passive smoke exposure simulating conventional cigarette use, intraperitoneal nicotine injection mimicking novel delivery systems (e.g., vaping and nicotine pouches), and AF puncture with nicotine administration to assess combined mechanical-chemical injury. X-ray imaging revealed the Smoking and Nicotine groups exhibited slight increases in vertebral translucency but no significant structural alterations. In contrast, the Puncture group displayed marked disc height reduction, blurred bony endplates, and localized defects. The Puncture + Nicotine group demonstrated exacerbated degeneration, with further disc height loss, severe endplate erosion resembling moth-eaten defects, and peri-endplate osteophyte formation (Fig. 2 a, b). MRI analysis of smoking and nicotine groups exhibited no overt degeneration. In contrast, the Puncture group displayed reduced disc height, structural disorganization, and diminished T2 signal. while, the Puncture + Nicotine group exhibited near-complete structural collapse, extreme disc space narrowing, and Pfirrmann grade 5 degeneration (Fig. 2 c, d). Histological evaluation revealed distinct degeneration patterns: Smoking and Nicotine groups showed early matrix disorganization with reduced NP volume, partial annular disarray, and fibrotic folding, while Puncture + Nicotine specimens displayed complete architectural collapse featuring fibrotic nuclear replacement and annular lamellar disintegration (Fig. 2 e, f). These findings establish that nicotine exposure induces progressive IVDD through both direct biochemical effects and mechanical vulnerability potentiation. The accelerated degeneration in combined mechanical-chemical injury models underscores nicotine's synergistic toxicity in compromised discs, suggesting heightened clinical risk for smokers with pre-existing disc pathology. Tobacco products exposure disrupt intervertebral discs matrix homeostasis Our investigations revealed substantial nicotine-induced disruption of ECM homeostasis in both in vivo and in vitro models. Immunohistochemical analysis demonstrated marked reductions in aggrecan and collagen II expression within tobacco or nicotine-exposed discs compared to controls. The most severe depletion was observed in the Puncture + Nicotine group, highlighting nicotine’s synergistic exacerbation of ECM degradation in pre-injured discs (Fig. 3 a-d). Cellular viability assays revealed no significant cytotoxicity across a broad nicotine concentration range (0.2–300 µg mL⁻¹) (Fig. 3 e), suggesting nicotine's degenerative effects operate through mechanisms beyond direct cellular toxicity. Subsequent experiments employing pathologically relevant concentrations (25–200 µg mL⁻¹) demonstrated dose- and time-dependent suppression of ECM synthesis. RT-qPCR analysis showed progressive downregulation of aggrecan and collagen II mRNA level (Fig. 3 f-i). Western blot analysis confirmed dose-dependent suppression of aggrecan and collagen II protein expression, with progressive reduction over extended exposure periods (Fig. 3 j-l). Immunofluorescence further confirmed diminished aggrecan and collagen II immunoreactivity in nicotine-treated NPCs (Fig. 3 m, n). These findings establish that nicotine penetrates avascular disc tissues and exerts suppressive effects on critical ECM components through non-cytotoxic mechanisms. Notably, while imaging studies showed subtle changes in Smoking and Nicotine groups, histological and molecular analyses revealed early ECM destabilization, confirming ECM disorder prior to structural failure. Nicotine Disrupts ECM Homeostasis via Catabolic Enzyme Activation​ Enhanced ECM catabolism is a hallmark of IVDD 35 . Histopathological evaluation revealed distinct ECM degradation patterns across experimental groups. Immunohistochemical analysis revealed elevated MMP-13 expression in smoking and nicotine groups compared to controls, with the most pronounced upregulation observed in the Puncture + Nicotine group (Fig. 4 a, b). RT-qPCR demonstrated that nicotine exposure upregulated MMP-13 mRNA levels in human NPCs in a concentration and time-dependent manner (Fig. 4 c, d). Immunofluorescence confirmed increased MMP-13 protein expression in nicotine-treated NPCs (Fig. 4 e). Western blot analysis further validated these findings, showing progressive MMP-13 protein accumulation over 3–7 days of nicotine exposure (Fig. 4 f-g). RNA sequencing of nicotine-exposed NPCs revealed broad activation of matrix-degrading enzymes, including MMP family (e.g., MP -3, -9, -13) and ADAMTS family (e.g., ADAMTS-4, -5) (Fig. 4 h). This catabolic signature was accompanied by downregulation of ECM maintenance genes (Collagen II, Aggrecan), establishing nicotine's dual disruption of matrix homeostasis. Nicotine induces inflammatory responses in IVD Chronic low-grade inflammation mediated by proinflammatory cytokines represents a core pathological mechanism of IVDD 36 . RNA sequencing revealed significant enrichment of inflammation-related pathways, with multiple pro-inflammatory cytokines implicated in nicotine-induced functional impairment (Fig. 5 a). RT-qPCR demonstrated dose- and time-dependent upregulation of IL-1β mRNA expression in nicotine-exposed NPCs (Fig. 5 b, c). Immunofluorescence confirmed elevated IL-1β protein levels following nicotine treatment (Fig. 5 d), with Western blot analysis showing progressive accumulation of IL-1β protein over time (Fig. 5 e, f). In vivo validation through immunohistochemical analysis revealed striking nicotine-mediated inflammatory amplification. While control discs exhibited minimal IL-1β and IL-6 expression, Smoking and Nicotine groups showed marked increases in cytokine-positive cells (Fig. 5 g-i). Notably, punctured discs receiving nicotine injections demonstrated exacerbated IL-1β and IL-6 expression compared to puncture-only controls, establishing nicotine's synergistic inflammatory effects in degenerated discs. Nicotine activates NF-κB and MAPK signaling pathways to drive IVDD To elucidate the molecular mechanisms underlying nicotine-induced ECM dysregulation and inflammation, we performed transcriptomic analysis of nicotine-exposed NPCs. KEGG pathway enrichment revealed significant upregulation of genes associated with NF-κB and MAPK signaling (Fig. 6 a), both pivotal regulators of inflammatory cascades in IVDD. Immunofluorescence assays demonstrated rapid nuclear translocation of the NF-κB subunit p65 and MAPK component p38 within 20 minutes of nicotine exposure, with p65 activation being more pronounced (Fig. 6 b, c). Western blot analysis confirmed time-dependent phosphorylation of p65 and p38, peaking at 15–30 minutes post-exposure and declining by 2 h (Fig. 6 d, e). This transient activation pattern suggests nicotine initiates acute signaling events that trigger downstream degenerative processes. To functionally validate these pathways, we employed specific inhibitors of NF-κB (JSH-23) and p38 MAPK (BIRB796). Pharmacological blockade significantly attenuated nicotine-induced upregulation of IL-1β and MMP-13 (Fig. 6 f-h). The greater efficacy of NF-κB inhibition aligns with its predominant activation pattern observed in our earlier experiments. Nicotine Disrupts ECM Homeostasis via Catabolic Enzyme Activation Cellular senescence is increasingly recognized as a pivotal mechanism in IVDD pathogenesis. Transcriptomic KEGG pathway analysis revealed significant enrichment of senescence-associated signaling pathways in nicotine-treated NPCs (Fig. 6 a). To validate this finding, we performed SA-β-gal staining and p16 INK 4 a expression analysis. Nicotine-treated NPCs exhibited a significant increase in SA-β-gal-positive cells compared to controls, and NP tissues from heavy smokers also exhibited a comparable senescence phenotype (Fig. 7 a, b). Western blot analyses confirmed a significant upregulation of p16 INK 4 a , a canonical senescence marker, in nicotine-exposed NPCs (Fig. 7 c, d). Immunofluorescence also corroborated this finding, showing nuclear accumulation of p16 INK 4 a in treated cells (Fig. 7 e). In vivo validation using rat IVDD models showed that elevated p16 INK 4 a levels in smoke-exposed and nicotine-injected groups, with the most pronounced accumulation observed in the Puncture + Nicotine cohort (Fig. 7 f-g). Intriguingly, histological analysis revealed spatial correlation between senescence markers and regions of severe matrix degradation. RNA sequencing data corroborated that senescent NPCs displayed a robust SASP, characterized by hypersecretion of pro-inflammatory cytokines, matrix-degrading enzymes, and chemokines (Fig. 4 h, 5 a, 7 h). This SASP profile synergistically exacerbated extracellular matrix (ECM) catabolism and propagated degenerative cascades. DISCUSSION The avascular IVD depends on passive diffusion for nutrient exchange, raising questions about nicotine’s accessibility. Here, we demonstrate that nicotine and cotinine accumulate in disc tissue, challenging prior assumptions 5 , 37 . Here, we provide definitive evidence through mass spectrometry analysis that nicotine and cotinine accumulate in IVDs following both passive inhalation (smoke exposure) and systemic delivery (intraperitoneal injection). Notably, cotinine-nicotine's primary metabolite with an extended half-life (15–20 vs 1–2 h for nicotine) - showed particularly prominent accumulation, establishing its utility as a superior biomarker for chronic tobacco exposure monitoring 30 . We propose that serum cotinine levels could serve as a biomarker for early IVDD risk stratification in smokers, combined with annual MRI monitoring. This pharmacokinetic evidence fundamentally alters our understanding of tobacco's disc toxicity mechanisms, establishing that systemic nicotine exposure enables direct cellular interaction within disc tissues. Notably, our cytotoxicity assessment revealed no significant viability changes in NPCs even at 300 µg ml⁻¹ nicotine concentrations, which far exceeded typical serum levels observed in smokers (10–50 ng ml⁻¹) 30 . These finding challenges previous hypotheses attributing tobacco-related IVDD to direct cellular toxicity, highlighting its direct intradiscal action as a driver of ECM dysregulation. The evolving landscape of tobacco consumption, marked by the proliferation of alternative nicotine delivery systems including e-cigarettes and heated tobacco products, presents novel challenges in understanding IVDD pathogenesis 38 , 39 . These products, strategically marketed as reduced-risk alternatives, have gained particular traction among younger populations due to their customizable nicotine concentrations and flavor profiles 40 , 41 . These findings have immediate clinical relevance given the rising popularity of high-nicotine vaping products among adolescents, suggesting potential acceleration of IVDD in younger populations. To investigate the differential effects of various nicotine delivery methods on IVD at different pathological stages, we employed a customized smoke exposure system to simulate passive smoking and intraperitoneal nicotine injection to mimic novel nicotine delivery systems (e.g., nicotine pouches, vaping). Furthermore, we established a puncture-induced IVD degeneration model to study nicotine’s impact on already degenerated discs. While radiographic imaging failed to detect overt structural alterations in Smoking and Nicotine groups, histomorphometric and immunohistochemical analyses unveiled early degenerative signatures, including ECM disorganization and marked downregulation of aggrecan and type II collagen-hallmarks of incipient matrix destabilization 42 . Our preclinical findings reveal that nicotine disrupts disc homeostasis via biochemical dysregulation, initiating subclinical degeneration prior to structural collapse. Crucially, the synergistic acceleration of degeneration in nicotine-treated puncture models demonstrates amplified vulnerability in pathologically compromised discs, suggesting a feed-forward mechanism where pre-existing matrix damage potentiates nicotine's catabolic effects. These temporal and dose-responsive relationships highlight a critical window for smoking cessation, particularly in individuals with subclinical or early-stage IVDD, to mitigate progressive ECM catabolism and clinical deterioration. A critical hallmark of IVDD pathogenesis involves the catabolic shift in ECM homeostasis, primarily mediated by MMPs and ADAMTS family members that orchestrate collagen and proteoglycan degradation 43 , 44 . Our study mechanistically links nicotine exposure to this pathological cascade through dose- and time-dependent upregulation of MMP-13 in NPCs. RNA sequencing revealed broad activation of matrix-degrading enzymes, corroborating the pivotal role of MMP/ADAMTS families in nicotine-induced ECM catabolism. These data underscore nicotine’s dual role as both a metabolic disruptor and a catabolic amplifier, particularly in pre-degenerated discs. Crucially, in vivo experiments demonstrated that nicotine administration to puncture-induced degenerated discs markedly intensified ECM degradation compared to puncture alone. This accelerated degenerative phenotype mirrors clinical observations where smokers exhibit faster IVDD progression compared to non-smokers 9 , 45 . Inflammation is a pivotal pathological driver of IVDD 7 . Our study unveils that nicotine exacerbates inflammatory cascades in discs through synergistic activation of NF-κB and MAPK signaling pathways. Chronic low-grade inflammation, characterized by elevated pro-inflammatory cytokines such as IL-1β and IL-6, is a hallmark of IVDD pathogenesis 6 , 46 . Our experimental data demonstrated that nicotine markedly upregulated IL-1β and IL-6 expression in both in vitro and in vivo models, with amplified effects in degenerated discs. Immunohistochemical analysis revealed significantly higher IL-1β and IL-6 levels in the Puncture + Nicotine group compared to the puncture-only group, highlighting nicotine’s role in aggravating inflammatory responses within pre-damaged discs. Mechanistically, NF-κB and MAPK pathways were identified as central mediators of nicotine-induced inflammation. The rapid phosphorylation of p65 and p38 MAPK suggests nicotine initiates immediate pro-degenerative signaling, while sustained pathway activation drives progressive ECM catabolism. Besides, NF-κB exhibited higher sensitivity, suggesting its dominance in inflammatory regulation. This aligns with prior evidence: NF-κB directly governs transcription of IL-1β and MMP-13 to drive ECM degradation 47 . while MAPK not only mediates acute inflammation but also modulates long-term matrix remodeling gene 48 . Importantly, specific inhibition of NF-κB or MAPK pathways substantially attenuated nicotine-induced IL-1β and MMP-13 upregulation, confirming their indispensable roles. Furthermore, nicotine’s dual activation of these pathways establishes a self-reinforcing inflammation-catabolism axis: inflammatory cytokines directly stimulate protease production to degrade ECM, while ECM fragments in turn activate NF-κB and MAPK signaling via pattern recognition receptors, perpetuating a vicious cycle. This mechanism may explain accelerated IVDD in smokers and highlights therapeutic potential in targeting these inflammatory cascades. The role of cellular senescence as a pivotal accelerator of IVDD pathology has drawn increasing attention, with senescent NPCs accumulation correlating with degenerative severity 22 , 49 , 50 . Our study unveils that nicotine induces NPC senescence coupled with a SASP, which dynamically interacts with inflammatory pathways to disrupt disc homeostasis. Experimental evidence demonstrated that nicotine markedly upregulated the senescence marker p16 INK 4 a and enhanced SA-β-gal activity. In addition, nicotine-treated NPCs also exhibited a classic SASP profile-secreting pro-inflammatory cytokines, chemokines and proteases. These SASP factors not only directly degrade ECM but also propagate senescence to adjacent NPCs via paracrine signaling, establishing a self-amplifying “senescence-inflammation” cascade 51 , 52 . Crucially, a bidirectional crosstalk exists between nicotine-induced senescence and inflammatory pathways: chronic inflammation mediated by NF-κB and MAPK accelerates senescence via oxidative stress and DNA damage, whereas senescent NPCs perpetuate inflammation through sustained SASP secretion, reactivating NF-κB and MAPK signaling and forming a feedforward “inflammation → senescence → amplified inflammation” loop (Fig. 8 ). This synergy was most pronounced in the Puncture + Nicotine model, where combined mechanical injury and nicotine exposure synergistically elevated IL-1β and p16 INK 4 a expression compared to single-factor groups, concomitant with exacerbated ECM disruption and structural collapse. Our findings suggest that targeting the senescence-inflammation axis (e.g., via SASP inhibition or NF-κB and MAPK blockade) could disrupt this vicious cycle. Therapeutic strategies such as senolytics (eliminating senescent cells) or senomorphics (suppressing SASP) combined with anti-inflammatory agents may hold promise for mitigating smoking-related IVDD 53 , 54 . However, the molecular links bridging senescence and inflammation require further elucidation, including how specific SASP components activate NF-κB/MAPK and whether epigenetic modifications regulate this interplay. While our study provides mechanistic insights into nicotine-induced IVDD, several limitations should be acknowledged. First, we mainly focus on the role of nicotine, while tobacco smoke is a complex mixture composed of over 6,000 chemical substances 55 . Some studies have reported that cadmium, a heavy metal in tobacco, can induce apoptosis of annulus fibrosus cells 56 . Future studies should explore potential synergistic effects among these components. Second, our experimental models may not fully replicate the chronic, low-dose exposure seen in human smokers. Additionally, the complex pathophysiology of IVDD involves multiple molecular mechanisms beyond nicotine-induced inflammation and senescence, warranting further investigation. In conclusion, this study elucidates the dual-pathway mechanism by which nicotine exacerbates IVDD through NF-κB and MAPK-mediated inflammation and cellular senescence. we demonstrate for the first time that nicotine permeates and accumulates in avascular disc tissues via inhalation or systemic administration. Nicotine dose- and time-dependently suppresses aggrecan and type II collagen synthesis while upregulating various proteolytic enzymes and proinflammatory cytokines, accelerating degeneration via ECM destabilization and chronic inflammation. Notably, nicotine exposure intensifies structural damage and ECM dysregulation in pre-degenerated discs, highlighting its synergistic toxicity within compromised microenvironments. Mechanistically, nicotine activates NF-κB and MAPK pathways, driving inflammatory cytokine production and inducing cellular senescence with a pro-inflammatory SASP, establishing an “inflammation-senescence” positive feedback loop that synergistically exacerbates degeneration. Our findings emphasize smoking cessation as critical in early IVDD and propose combined NF-κB and MAPK inhibition and senolytic therapy to counteract nicotine-driven degeneration. Declarations COMPETING INTERESTS The authors have no conflicts of interest to report. Acknowledgements This work was funded by the Medical science and Technology project of Henan Province (NO. LHGJ20230080), the Key R&D and Promotion Program of Henan Science and Technology Department (NO. 242102310406), the Key R&D and Promotion Program of Henan Science and Technology Department (NO. 232102311038) and the Medical science and Technology project of Henan Province (NO. LHGJ20220031). 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Additional Declarations There is no conflict of interest Supplementary Files SupplementaryFigure.docx SupplementaryTable.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 10 Sep, 2025 Reviewer # 2 agreed at journal 29 Jul, 2025 Review # 1 received at journal 23 May, 2025 Reviewer # 1 agreed at journal 19 May, 2025 Reviewers invited by journal 19 May, 2025 Submission checks completed at journal 06 May, 2025 Editor assigned by journal 04 May, 2025 First submitted to journal 04 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6586785","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":458459383,"identity":"0ed9dbb3-87d7-4178-83d9-8416d1734e54","order_by":0,"name":"Desheng 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accumulation in intervertebral disc tissues.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Schematic representation of the in vivo experimental design. \u003cstrong\u003eb\u003c/strong\u003e Metabolomic profiles of nicotine and cotinine levels in disc tissues from smoke-exposed versus control rats. \u003cstrong\u003ec\u003c/strong\u003eNicotine and cotinine concentrations in disc tissues of intraperitoneal nicotine-injected versus control rats. \u003cstrong\u003ed\u003c/strong\u003e Plasma nicotine and cotinine levels in smoke-exposed versus control groups.\u003c/p\u003e","description":"","filename":"Fig.1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/5802fa13d7c3d001b167b2ef.jpg"},{"id":83214137,"identity":"93d5bf06-0d1f-42b7-b469-df4a31a68c4b","added_by":"auto","created_at":"2025-05-21 08:50:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":732686,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTobacco products induce IVDD.\u003c/strong\u003e​(\u003cstrong\u003ea, b)\u003c/strong\u003e X-ray imaging and disc height index (DHI) analysis of rat caudal discs. (\u003cstrong\u003ec, d\u003c/strong\u003e) T2-weighted MRI and Pfirrmann grading. (\u003cstrong\u003ee, f\u003c/strong\u003e) H\u0026amp;E and Safranin O-fast green staining (scale bar: 500 µm) with histological scoring. The values are expressed as mean ± SD. DHI, disc height index. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/36af2b13a8e523654dbd7cdb.jpg"},{"id":83214143,"identity":"4e5a3402-1899-4375-aead-9addb84429f8","added_by":"auto","created_at":"2025-05-21 08:50:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1153087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotine disrupts NPC homeostasis\u003c/strong\u003e. (\u003cstrong\u003ea-d\u003c/strong\u003e) Immunohistochemical staining and semi-quantitative analysis of aggrecan and collagen II in rat discs, Scale bars: 200 µm (low magnification), 50 µm (high magnification). \u003cstrong\u003ee\u003c/strong\u003e Cell viability of human NPCs treated with nicotine (0–300 µg/mL). (\u003cstrong\u003ef-i\u003c/strong\u003e) Dose- and time-dependent suppression of aggrecan and collagen II mRNA by nicotine. (\u003cstrong\u003ej-l)\u003c/strong\u003e Western blot analysis of aggrecan and collagen II protein levels. (m, n) Immunofluorescence confirming reduced aggrecan and collagen II expression (200× magnification). Data are presented as mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/a84a945c57474419f2e35672.jpg"},{"id":83215240,"identity":"fd780f28-7c20-4a27-9429-789a7f0b1a5c","added_by":"auto","created_at":"2025-05-21 08:58:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":878202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotine exacerbates ECM catabolism\u003c/strong\u003e. (\u003cstrong\u003ea, b)\u003c/strong\u003eImmunohistochemistry and quantification of MMP-13 in rat discs (scale bar, 200µm in low magnification and 50 µm in high magnification). (\u003cstrong\u003ec, d\u003c/strong\u003e) Dose- and time-dependent upregulation of MMP-13 mRNA in nicotine-treated NPCs. \u003cstrong\u003ee\u003c/strong\u003eImmunofluorescence showing MMP-13 protein overexpression (200×). (\u003cstrong\u003ef, g\u003c/strong\u003e) Western blot analysis of MMP-13 protein levels. h Heatmap of protease-related mRNA upregulation from RNA sequencing. Data: mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/239b7dfb3cbf021db3d5f3eb.jpg"},{"id":83214141,"identity":"718addc9-7eb9-4976-8369-d1fe7e97eab7","added_by":"auto","created_at":"2025-05-21 08:50:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":881093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotine-driven inflammatory responses in IVDD\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e Heatmap of pro-inflammatory mRNA expression in nicotine-treated NPCs. (\u003cstrong\u003eb, c\u003c/strong\u003e) Dose- and time-dependent IL-1β mRNA upregulation. d IL-1β immunofluorescence (200×). (\u003cstrong\u003ee, f)\u003c/strong\u003e Western blot analysis of IL-1β protein levels. (\u003cstrong\u003eg-i\u003c/strong\u003e) Immunohistochemistry of IL-1β and IL-6 in rat discs (scale bar, 200µm in low magnification and 50 µm in high magnification). Data: mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/2d3b3fe6c12783be85f4d4b2.jpg"},{"id":83215789,"identity":"f5802668-d167-463a-8b6f-5e5a22e0f686","added_by":"auto","created_at":"2025-05-21 09:06:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":449342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotine activates NF-κB and MAPK signaling\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e KEGG pathway enrichment analysis of RNA-seq data. (\u003cstrong\u003eb, c\u003c/strong\u003e) Immunofluorescence showing nuclear translocation of P65 (NF-κB) and P38 (MAPK) (arrows indicate nuclear localization, 200×). (\u003cstrong\u003ed, e\u003c/strong\u003e) Time-dependent phosphorylation of P65 and P38 by Western blot. (\u003cstrong\u003ef-h\u003c/strong\u003e) NF-κB (JSH-23) and MAPK (BIRB796) pathway inhibitors attenuate IL-1β/MMP-13 induction. Data: mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/6155006ddbb2f1e9ae2d5394.jpg"},{"id":83215243,"identity":"aca542d0-21e5-410a-9bb7-136d26e3d76e","added_by":"auto","created_at":"2025-05-21 08:58:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":898993,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNicotine induces NPC senescence.\u003c/strong\u003e (\u003cstrong\u003ea, b\u003c/strong\u003e) SA-β-gal staining of NPCs (blue: senescent cells; scale bar: 200 µm). (\u003cstrong\u003ec, d\u003c/strong\u003e) Western blot analysis of p16\u003csup\u003eINK4a\u003c/sup\u003e expression. e Immunofluorescence of p16\u003csup\u003eINK4a\u003c/sup\u003e (200×). (\u003cstrong\u003ef-g\u003c/strong\u003e) Immunohistochemical analysis of p16\u003csup\u003eINK4a\u003c/sup\u003e in rat discs (scale bars: 200 µm and 50 µm). \u003cstrong\u003eh\u003c/strong\u003e Heatmap of SASP-related mRNA upregulation. Data: mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/f3b5d0035b77d7df9cabbbf9.jpg"},{"id":83214154,"identity":"cbc26d70-6b2b-4907-92a9-74305bd28af4","added_by":"auto","created_at":"2025-05-21 08:50:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":638819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the role of Nicotine in IVDD progression\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Fig.8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/bbffd31731479fa2487a8181.jpg"},{"id":83216712,"identity":"3a582aef-6583-4fb2-8c3e-3929a1b4a15c","added_by":"auto","created_at":"2025-05-21 09:14:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7560515,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/18fc1d53-3b52-4ffd-8442-9870623854b6.pdf"},{"id":83215788,"identity":"27e1c38f-ae4e-4271-a760-46ed2237d32d","added_by":"auto","created_at":"2025-05-21 09:06:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1522546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/244f3f773c69aa15ee445ae7.docx"},{"id":83215238,"identity":"79697fa8-2d6c-40cf-a024-6ac342cd55f4","added_by":"auto","created_at":"2025-05-21 08:58:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-6586785/v1/c93f5d43e1dd577499817a45.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Nicotine exacerbates intervertebral disc degeneration via NF-κB/MAPK-dependent inflammatory activation and cellular senescence","fulltext":[{"header":"​INTRODUCTION","content":"\u003cp\u003eLow back pain (LBP) affects over 540\u0026nbsp;million people globally, representing a leading cause of disability and imposing significant socioeconomic burdens​\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. As the leading cause of disability worldwide, LBP imposes substantial socioeconomic burdens on individuals, families, and healthcare systems\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Intervertebral disc degeneration (IVDD) is a primary pathological contributor to LBP, often precipitating secondary spinal disorders such as disc herniation, spinal stenosis, and segmental instability\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe intervertebral disc (IVD) comprises three distinct structures: the gelatinous nucleus pulposus (NP), the concentric lamellae of the annulus fibrosus (AF), and the cartilaginous endplates. The NP, populated by nucleus pulposus cells (NPCs) embedded in an extracellular matrix (ECM) rich in type II collagen and aggrecan, maintains disc hydration, mechanical resilience, and nutrient exchange\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Notably, the IVD is an avascular tissue, relying on diffusion through the endplates and peripheral AF capillaries for metabolic sustenance\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. A hallmark of IVDD is the dysregulation of ECM homeostasis, characterized by enhanced catabolism mediated by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family enzymes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Concurrently, IVDD is associated with elevated pro-inflammatory cytokine expression, which exacerbates ECM degradation, oxidative stress, and cellular senescence, collectively accelerating IVDD\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLarge-scale epidemiological studies unequivocally identify smoking as a modifiable risk factor for IVDD\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. A longitudinal study of 5,180 forestry workers demonstrated that smoking significantly correlated with radicular neck and sciatic pain\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, while another large-scale investigation (n\u0026thinsp;=\u0026thinsp;25,610) confirmed its association with chronic LBP\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Notably, MRI-based analyses in monozygotic twins revealed an 18% higher IVDD severity in smokers compared to non-smokers\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Meta-analyses further indicate that smokers exhibit a 27% increased risk of IVDD, with disease severity correlating positively with smoking intensity and duration\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Additionally, smoking adversely impacts spinal surgical outcomes, increasing non-union rates in anterior cervical discectomy/fusion and postoperative airway complications\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious mechanistic studies highlight nicotine-the primary addictive component of tobacco-as a key mediator of smoking-induced IVDD. Nicotine compromises disc microcirculation, impairing nutrient supply and metabolic waste clearance\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Furthermore, tobacco smoke or nicotine extracts upregulate IL-1β and ADAMTS5 expression while suppressing aggrecan and collagen synthesis, thereby disrupting ECM equilibrium\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEmerging evidence implicates cellular senescence as a pivotal driver​of IVDD\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Senescent NPCs acquire a senescence-associated secretory phenotype (SASP), releasing pro-inflammatory cytokines, chemokines, and proteases that amplify ECM catabolism, propagate senescence bystander effects, and sustain a degenerative microenvironment\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite epidemiological associations, the direct pathogenic role​of nicotine in IVDD remains poorly define. With the global smoking population projected to reach 1.6\u0026nbsp;billion by 2025 (WHO), elucidating nicotine\u0026rsquo;s pathological role in IVDD is clinically imperative\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Here, this study systematically delineates nicotine\u0026rsquo;s penetration into avascular disc tissue, its activation of NF-κB and MAPK-mediated inflammation, and induction of senescence-mechanisms that collectively exacerbate IVDD.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eEthics statement​\u003c/b\u003e​\u003c/p\u003e \u003cp\u003e All protocols were approved by the Institutional Ethics Committee of Henan Provincial People\u0026rsquo;s Hospital, Zhengzhou University, in compliance with the Declaration of Helsinki. Animal procedures strictly followed the guidelines of Zhengzhou University\u0026rsquo;s Animal Care and Use Committee (Ethical Application Ref: No. 1-233). Human NP tissues were obtained from non-smoking patients undergoing discectomy, with informed consent. IVDD status was classified using preoperative MRI (Pfirrmann grade I-III: control; IV-V: IVDD)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReagents and antibodies\u003c/b\u003e​​\u003c/p\u003e \u003cp\u003eNicotine was purchased from Sigma-Aldrich. Cigarettes (Double Happiness; Shanghai Tobacco Group) contained 15 mg tar, 1.3 mg nicotine, and 14 mg CO per unit. Fetal bovine serum (FBS), trypsin and penicillin-streptomycin were purchased from Gibco (Grand Island, NY, USA). Phosphate buffered solution (PBS) and Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/F12 (DMEM/F12) were purchased from Hyclone (South Logan, UT, USA). The type II collagenase were purchased from (St. Louis, MO, USA). The primary antibody of Collagen II, MMP-13, IL-1β and P16INK4a were acquired from Abcam (Cambridge, MA, USA). The antibodies against GAPDH, P65, P38 and P-p38 were purchased from CST (Danvers, MA, USA). The antibodies for P-p65 and IL-6 were purchased from SANTA (Dallas, TX, USA). The antibody against Aggrecan was purchased from Thermo Fisher (Waltham, MA, USA). NF-κB inhibitor JSH-23\u003csup\u003e25\u003c/sup\u003e and MAPK inhibitor BIRB796\u003csup\u003e26\u003c/sup\u003e were purchased from APExBIO (Houston, TX, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental design\u003c/b\u003e​​\u003c/p\u003e \u003cp\u003eFor in vitro studies, human NPCs were serum-starved overnight in DMESM/F12 containing 2% FBS prior to experimentation. Nicotine solutions (25, 100, and 200 \u0026micro;g ml⁻\u0026sup1;) were prepared in complete medium (10% FBS, 1.5% penicillin/streptomycin). To investigate nicotine's temporal effects on ECM metabolism, NPCs were treated with 100 \u0026micro;g ml⁻\u0026sup1; nicotine for 0, 3, 5, and 7 days. Concentration-dependent responses were assessed using 0-200 \u0026micro;g ml⁻\u0026sup1; nicotine over 5 days. mRNA expression of ECM components (Aggrecan, Collagen II) and catabolic enzymes (MMP-13, ADAMTS-4) was quantified by RT-qPCR, with protein levels analyzed via western blot. NF-κB and MAPK pathway activation was evaluated by immunofluorescence and phosphorylation status of p65 and p38 at 15 min, 30 min, and 2 h post-treatment (100 \u0026micro;g ml⁻\u0026sup1; nicotine). Pharmacological inhibition studies employed JSH-23 (30 \u0026micro;M, NF-κB inhibitor) and BIRB796 (400 nM, p38 inhibitor) pre-treated 1 h before nicotine exposure. Cellular senescence was assessed by β-galactosidase (β-gal) staining and p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e expression in nicotine-treated NPCs and surgical specimens from heavy smokers (\u0026gt;\u0026thinsp;2 packs/day, \u0026gt;\u0026thinsp;10 years). RNA sequencing of nicotine-treated NPCs (100 \u0026micro;g ml⁻\u0026sup1;, 48 h) was performed to identify global transcriptomic changes.\u003c/p\u003e \u003cp\u003eFor in vivo studies, 8-week-old SD rats (Shanghai SLAC Laboratory Animal Co.) were randomized into five experimental groups: Control group, Smoking group, Nicotine group (Intraperitoneal nicotine injection), Puncture group, Puncture\u0026thinsp;+\u0026thinsp;Nicotine group. Metabolic profiling of serum and disc tissues was conducted at week 1 post-intervention. Intervertebral disc degeneration grading was evaluated using X-ray and MRI. Histological assessment of intervertebral disc tissues was performed through Hematoxylin \u0026amp; Eosin (H\u0026amp;E) staining and Safranine O-Fast Green staining. Immunohistochemical analysis was employed to examine the expression of extracellular matrix proteins and evaluate inflammatory status in the disc tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHuman nucleus pulposus cells culture\u003c/b\u003e​​\u003c/p\u003e \u003cp\u003eMicroscopically, NP tissues were meticulously dissected and minced into 1\u0026ndash;2 mm\u0026sup3; fragments. Tissue fragments underwent sequential enzymatic digestion: first with 0.25% trypsin-EDTA for 30 min at 37\u0026deg;C, followed by 0.1% type II collagenase for 4 h at 37\u0026deg;C with gentle agitation. The resulting cell suspension was centrifuged at 300 \u0026times; g for 5 min, and pelleted NPCs were resuspended in complete growth medium composed of DMEM/F12 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine. Primary cells were maintained under standard hypoxic conditions (2% O₂, 5% CO₂, 37\u0026deg;C) to mimic the native intervertebral disc microenvironment. The culture medium was refreshed every 72 h until 80\u0026ndash;90% confluence, at which point cells were passaged using 0.25% trypsin-EDTA at a 1:2 split ratio. All experiments utilized passage 2\u0026ndash;3 cells to preserve phenotypic stability and minimize dedifferentiation artifacts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability assay\u003c/b\u003e​​\u003c/p\u003e \u003cp\u003eThe viability of NPCs was assayed with the cell counting kit-8 (CCK-8; Dojindo Co, Kumamoto, Japan) according to the manufacturer\u0026rsquo;s protocol. NPCs were uniformly seeded in 96-well plates at a density of 1.5 \u0026times; 10⁴ cells/well. The cells were then treated with nicotine at concentrations of 0, 0.2, 0.5, 1, 10, 50, 100, 200, and 300 \u0026micro;g ml⁻\u0026sup1; for 24 h. Following treatment, 100 \u0026micro;L of DMEM containing CCK-8 reagent was added to each well, followed by incubation at 37\u0026deg;C for 2 h. Optical density values were measured at 450 nm using a microplate reader, and cell viability was calculated accordingly.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eThe total protein of NPCs was extracted using the total protein extraction kit (Beyotime Biotechnology, Shanghai, China). The determination of the protein concentration was conducted using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal aliquots of protein from each sample were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to 22 \u0026micro;m polyvinylidene fluoride membranes (Millipore, USA) using a wet-blotting method. Then, the membranes were blocked by 5% nonfat milk in TBST buffer and incubated overnight at 4\u0026deg;C with primary antibodies, including GAPDH (1:5,000), Collagen II (1:5,000), Aggrecan (1:500), MMP-13 (1:3,000), IL-1β (1:1,000), P65 (1:1,000y), P-p65 (1:200, SANTA), P38 (1:1,000), P-p38 (1:1,000), p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e (1:1,000). Afterwards, the membranes were incubated with secondary antibodies for 1 h at 37\u0026deg;C. After washing with PBS three times, the bands were observed in a darkroom and quantified using the ImageJ software (National Institutes of Health USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eNPCs were cultured in 24-well plates (4x104 cells/well), treated as mentioned above and fixed with 4% paraformaldehyde for 30 min. Following washing with PBS that contained 0.1% Tween-20 (PBST), the cells were incubated with 0.2% Triton X-100 for 20 min. Then, the samples were blocked for 30 min in 5% goat serum albumin. Subsequently, the samples were incubated with primary antibodies against Aggrecan (1:50), Collagen II (1:100), MMP-13 (1:500), IL-1β (1:1,00), P65 (1:400), P38 (1:50) and p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e (1:200) overnight at 4\u0026deg;C. The samples were then were washed and incubated with FITC-conjugated secondary antibodies for 1 h in the dark and labeled with DAPI for 5 min. Finally, the fluorescent images were obtained using a fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR\u003c/h3\u003e\n\u003cp\u003eThe RNA/DNA/Protein Isolation Kit was used to extract total RNA from NPCs, and RNA and qRT SuperMix were then used to synthesize cDNA, following to the manufacturer\u0026rsquo;s instructions (Vazyme-innovation in enzyme technology, China). GAPDH was used to standardize the cycle thresholds (Ct) of the transcripts that were obtained. The △△Ct method was used to calculate the relative mRNA levels of each target gene. Primer sequences are presented in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003emRNA sequencing and data analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from rat NP cells using TRIzol reagent, and the BGI (Shenzhen, China) was commissioned to perform mRNA transcriptome sequencing. Differential expression analyses were conducted using the DESeq2 package, with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, to identify differentially expressed genes. Gene ontology (GO) terms for enriched genes were determined using GOSeq, and in-house scripts were used to analyze significantly differentially expressed genes in KEGG pathways. The threshold was set as follows: | log2 (fold change) | \u0026gt;1 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003ch3\u003eSA-β-gal staining\u003c/h3\u003e\n\u003cp\u003eThe level of senescence was measured by SA-β-gal staining kit (Beyotime, Shanghai, China) according to the instruction. Cells were seeded in a six-well plate and then washed twice with PBS. Cells were fixed with 0.2% glutaraldehyde for 15 min at room, washed three times with PBS and then stained with X-gal staining solution at pH 6.0 overnight. SA-β-gal-positive cells were counted in six randomly selected images under a microscope, and the percentages of SA-β-gal-positive cells were averaged and quantified.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePassive smoking model in rats\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eWe established a chronic smoke exposure system using a custom-designed chamber equipped with real-time CO/O₂/PM₂.₅ monitoring and automated ventilation control (Supplementary Fig.\u0026nbsp;1). Rats were exposed to mainstream smoke from two combusted cigarettes (1.3 mg nicotine each) per session, with CO concentrations maintained at 180\u0026ndash;208 ppm through feedback-regulated airflow. Exposure cycles consisted of 1 h smoke inhalation followed by 20 min clearance, repeated twice daily (6 total hours/day). The protocol ran 5 days/week for 12 weeks, as previously optimized in smoke-induced COPD models\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Food and water were available ad libitum during exposures.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNicotine intraperitoneal injection model\u003c/h2\u003e \u003cp\u003eNicotine solution was prepared in normal saline at a concentration of 1 mg ml⁻\u0026sup1;, protected from light and stored at 4\u0026deg;C. Prior to injection, solutions were equilibrated to room temperature for 30 minutes. Daily intraperitoneal injections (2 mg kg⁻\u0026sup1; body weight) were administered in the lower abdominal quadrant, with a regimen of 5 consecutive days per week over 8 weeks, as previously validated\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Dose selection and administration protocols were based on pharmacokinetic studies demonstrating sustained nicotine exposure mimicking chronic smoking patterns in human\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe annulus fibrosus needle puncture model\u003c/h3\u003e\n\u003cp\u003eThe puncture procedure was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Briefly, rats were anesthetized via intraperitoneal injection of 2% (w/v) pentobarbital sodium (40 mg kg⁻\u0026sup1;)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The target coccygeal intervertebral discs (Co7/8) were identified by manual palpation and confirmed radiographically through vertebral counting from the sacral region. A 26-gauge needle was percutaneously inserted through the full thickness of the AF under aseptic conditions. The needle was rotated 360\u0026deg; and maintained within the disc space for 1 minute to induce controlled annular injury. Puncture depth (about 5 mm) was standardized based on preliminary radiographic measurements of AF and NP dimensions in coccygeal discs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolomic profiling and analysis\u003c/b\u003e​​\u003c/p\u003e \u003cp\u003eUpon completion of modeling, blood and intervertebral disc specimens were collected from experimental rats. Metabolomic analysis was conducted by Shanghai Biotree Biotech Co., Ltd. (Shanghai, China) using ultra-performance liquid chromatography (UPLC, Shimadzu Nexera X2) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS, Sciex X500R). Raw data processing included peak detection, extraction, alignment, and integration using MetaboAnalyst 5.0. Compound annotation was performed against the BiotreeDB (v2.1) in-house library with a matching score threshold of 0.3. Multivariate statistical analysis (PCA and OPLS-DA) was conducted to identify differential metabolites (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by Student's t-test).\u003c/p\u003e\n\u003ch3\u003eX-ray and MRI assays\u003c/h3\u003e\n\u003cp\u003eThe X-ray films of the rat tails were captured using an X-ray system (uDR 588i, United Imaging, Shanghai, China) to evaluate disc gross appearance and disc height status. The Disc Height Index (DHI) was adopted to assess disc height loss using the method as previously described\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Magnetic resonance imaging was performed on all rats to evaluate the signal and structural changes in sagittal T2-weighted images using a 3.0 T MRI system (uMR 770, United Imaging, Shanghai, China). IVDD grade was assessed using the Pfirrmann system.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistopathologic analysis\u003c/h2\u003e \u003cp\u003eThe isolated rat spines and adjacent vertebral bodies were first preserved for 24 h in 4% paraformaldehyde before being decalcified in a 10% EDTA solution. Following decalcification, the tissues were immersed in paraffin and the blocks of paraffin were sectioned into homogeneous slices with a thickness of 5 \u0026micro;m. Histopathological characteristics were assessed using hematoxylin and eosin (H\u0026amp;E) staining and Safranin O-fast green staining. Histological grading of the intervertebral discs was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eThe sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity for 10 min and 5% bovine serum albumin was used to block nonspecific binding sites for 30 min at 37\u0026deg;C. Subsequently, sections were incubated overnight at 4\u0026deg;C with primary antibodies Aggrecan (1:100), Collagen II (1:100), MMP-13 (1:100), IL-1β (1:1,00), IL-6 (1:50) and p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e (1:1,00). Following this, sections were incubated with appropriate secondary antibodies for 1 h at ambient temperature. The DAB detection system (Sangon Biotech, Shanghai, China) was employed to visualize the immunoreactivity. Finally, sections were dehydrated, sealed, and digitally scanned using a slide scanner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted utilizing SPSS 22.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 9 (GraphPad Software, USA). The collected data were tested for normality by Shapiro-Wilk normality test. The independent samples t-test assessed the statistical difference between two groups, while one-way analysis of variance (ANOVA) was employed for multiple data group comparison. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eNicotine and its metabolites accumulate in IVD\u003c/b\u003e ​​\u003c/p\u003e \u003cp\u003eWhile nicotine distribution has been documented in vascularized organs, its penetration into avascular IVD tissues remained unclear. Our multi-modal metabolomic investigation using liquid chromatography-tandem mass spectrometry provides first evidence of nicotine accumulation in disc tissues through diverse exposure routes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Plasma analysis revealed significantly elevated nicotine and cotinine (nicotine's primary metabolite) levels in smoke-exposed rats compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), validating our exposure model. Strikingly, mass spectrometry analysis of disc tissues demonstrated marked nicotine accumulation in the nicotine injection group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) and elevated cotinine levels in smoke-exposed animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). These findings establish that nicotine penetrates the avascular disc microenvironment and accumulates at biologically relevant concentrations, regardless of administration route.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNicotine/tobacco exposure induces IVDD\u003c/h2\u003e \u003cp\u003eTo investigate tobacco-related IVDD pathogenesis, we established three experimental models: passive smoke exposure simulating conventional cigarette use, intraperitoneal nicotine injection mimicking novel delivery systems (e.g., vaping and nicotine pouches), and AF puncture with nicotine administration to assess combined mechanical-chemical injury. X-ray imaging revealed the Smoking and Nicotine groups exhibited slight increases in vertebral translucency but no significant structural alterations. In contrast, the Puncture group displayed marked disc height reduction, blurred bony endplates, and localized defects. The Puncture\u0026thinsp;+\u0026thinsp;Nicotine group demonstrated exacerbated degeneration, with further disc height loss, severe endplate erosion resembling moth-eaten defects, and peri-endplate osteophyte formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). MRI analysis of smoking and nicotine groups exhibited no overt degeneration. In contrast, the Puncture group displayed reduced disc height, structural disorganization, and diminished T2 signal. while, the Puncture\u0026thinsp;+\u0026thinsp;Nicotine group exhibited near-complete structural collapse, extreme disc space narrowing, and Pfirrmann grade 5 degeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). Histological evaluation revealed distinct degeneration patterns: Smoking and Nicotine groups showed early matrix disorganization with reduced NP volume, partial annular disarray, and fibrotic folding, while Puncture\u0026thinsp;+\u0026thinsp;Nicotine specimens displayed complete architectural collapse featuring fibrotic nuclear replacement and annular lamellar disintegration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). These findings establish that nicotine exposure induces progressive IVDD through both direct biochemical effects and mechanical vulnerability potentiation. The accelerated degeneration in combined mechanical-chemical injury models underscores nicotine's synergistic toxicity in compromised discs, suggesting heightened clinical risk for smokers with pre-existing disc pathology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTobacco products exposure disrupt intervertebral discs matrix homeostasis\u003c/h2\u003e \u003cp\u003eOur investigations revealed substantial nicotine-induced disruption of ECM homeostasis in both in vivo and in vitro models. Immunohistochemical analysis demonstrated marked reductions in aggrecan and collagen II expression within tobacco or nicotine-exposed discs compared to controls. The most severe depletion was observed in the Puncture\u0026thinsp;+\u0026thinsp;Nicotine group, highlighting nicotine\u0026rsquo;s synergistic exacerbation of ECM degradation in pre-injured discs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). Cellular viability assays revealed no significant cytotoxicity across a broad nicotine concentration range (0.2\u0026ndash;300 \u0026micro;g mL⁻\u0026sup1;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), suggesting nicotine's degenerative effects operate through mechanisms beyond direct cellular toxicity. Subsequent experiments employing pathologically relevant concentrations (25\u0026ndash;200 \u0026micro;g mL⁻\u0026sup1;) demonstrated dose- and time-dependent suppression of ECM synthesis. RT-qPCR analysis showed progressive downregulation of aggrecan and collagen II mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-i). Western blot analysis confirmed dose-dependent suppression of aggrecan and collagen II protein expression, with progressive reduction over extended exposure periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej-l). Immunofluorescence further confirmed diminished aggrecan and collagen II immunoreactivity in nicotine-treated NPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em, n).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings establish that nicotine penetrates avascular disc tissues and exerts suppressive effects on critical ECM components through non-cytotoxic mechanisms. Notably, while imaging studies showed subtle changes in Smoking and Nicotine groups, histological and molecular analyses revealed early ECM destabilization, confirming ECM disorder prior to structural failure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNicotine Disrupts ECM Homeostasis via Catabolic Enzyme Activation​\u003c/h2\u003e \u003cp\u003eEnhanced ECM catabolism is a hallmark of IVDD\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Histopathological evaluation revealed distinct ECM degradation patterns across experimental groups. Immunohistochemical analysis revealed elevated MMP-13 expression in smoking and nicotine groups compared to controls, with the most pronounced upregulation observed in the Puncture\u0026thinsp;+\u0026thinsp;Nicotine group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). RT-qPCR demonstrated that nicotine exposure upregulated MMP-13 mRNA levels in human NPCs in a concentration and time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). Immunofluorescence confirmed increased MMP-13 protein expression in nicotine-treated NPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Western blot analysis further validated these findings, showing progressive MMP-13 protein accumulation over 3\u0026ndash;7 days of nicotine exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-g). RNA sequencing of nicotine-exposed NPCs revealed broad activation of matrix-degrading enzymes, including MMP family (e.g., MP -3, -9, -13) and ADAMTS family (e.g., ADAMTS-4, -5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). This catabolic signature was accompanied by downregulation of ECM maintenance genes (Collagen II, Aggrecan), establishing nicotine's dual disruption of matrix homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNicotine induces inflammatory responses in IVD\u003c/h2\u003e \u003cp\u003eChronic low-grade inflammation mediated by proinflammatory cytokines represents a core pathological mechanism of IVDD\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. RNA sequencing revealed significant enrichment of inflammation-related pathways, with multiple pro-inflammatory cytokines implicated in nicotine-induced functional impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). RT-qPCR demonstrated dose- and time-dependent upregulation of IL-1β mRNA expression in nicotine-exposed NPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). Immunofluorescence confirmed elevated IL-1β protein levels following nicotine treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), with Western blot analysis showing progressive accumulation of IL-1β protein over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f). In vivo validation through immunohistochemical analysis revealed striking nicotine-mediated inflammatory amplification. While control discs exhibited minimal IL-1β and IL-6 expression, Smoking and Nicotine groups showed marked increases in cytokine-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-i). Notably, punctured discs receiving nicotine injections demonstrated exacerbated IL-1β and IL-6 expression compared to puncture-only controls, establishing nicotine's synergistic inflammatory effects in degenerated discs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNicotine activates NF-κB and MAPK signaling pathways to drive IVDD\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanisms underlying nicotine-induced ECM dysregulation and inflammation, we performed transcriptomic analysis of nicotine-exposed NPCs. KEGG pathway enrichment revealed significant upregulation of genes associated with NF-κB and MAPK signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), both pivotal regulators of inflammatory cascades in IVDD. Immunofluorescence assays demonstrated rapid nuclear translocation of the NF-κB subunit p65 and MAPK component p38 within 20 minutes of nicotine exposure, with p65 activation being more pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c). Western blot analysis confirmed time-dependent phosphorylation of p65 and p38, peaking at 15\u0026ndash;30 minutes post-exposure and declining by 2 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). This transient activation pattern suggests nicotine initiates acute signaling events that trigger downstream degenerative processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo functionally validate these pathways, we employed specific inhibitors of NF-κB (JSH-23) and p38 MAPK (BIRB796). Pharmacological blockade significantly attenuated nicotine-induced upregulation of IL-1β and MMP-13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef-h). The greater efficacy of NF-κB inhibition aligns with its predominant activation pattern observed in our earlier experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eNicotine Disrupts ECM Homeostasis via Catabolic Enzyme Activation\u003c/h2\u003e \u003cp\u003eCellular senescence is increasingly recognized as a pivotal mechanism in IVDD pathogenesis. Transcriptomic KEGG pathway analysis revealed significant enrichment of senescence-associated signaling pathways in nicotine-treated NPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). To validate this finding, we performed SA-β-gal staining and p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e expression analysis. Nicotine-treated NPCs exhibited a significant increase in SA-β-gal-positive cells compared to controls, and NP tissues from heavy smokers also exhibited a comparable senescence phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). Western blot analyses confirmed a significant upregulation of p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e, a canonical senescence marker, in nicotine-exposed NPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, d). Immunofluorescence also corroborated this finding, showing nuclear accumulation of p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e in treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). In vivo validation using rat IVDD models showed that elevated p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e levels in smoke-exposed and nicotine-injected groups, with the most pronounced accumulation observed in the Puncture\u0026thinsp;+\u0026thinsp;Nicotine cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef-g). Intriguingly, histological analysis revealed spatial correlation between senescence markers and regions of severe matrix degradation. RNA sequencing data corroborated that senescent NPCs displayed a robust SASP, characterized by hypersecretion of pro-inflammatory cytokines, matrix-degrading enzymes, and chemokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). This SASP profile synergistically exacerbated extracellular matrix (ECM) catabolism and propagated degenerative cascades.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe avascular IVD depends on passive diffusion for nutrient exchange, raising questions about nicotine\u0026rsquo;s accessibility. Here, we demonstrate that nicotine and cotinine accumulate in disc tissue, challenging prior assumptions \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Here, we provide definitive evidence through mass spectrometry analysis that nicotine and cotinine accumulate in IVDs following both passive inhalation (smoke exposure) and systemic delivery (intraperitoneal injection). Notably, cotinine-nicotine's primary metabolite with an extended half-life (15\u0026ndash;20 vs 1\u0026ndash;2 h for nicotine) - showed particularly prominent accumulation, establishing its utility as a superior biomarker for chronic tobacco exposure monitoring \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We propose that serum cotinine levels could serve as a biomarker for early IVDD risk stratification in smokers, combined with annual MRI monitoring. This pharmacokinetic evidence fundamentally alters our understanding of tobacco's disc toxicity mechanisms, establishing that systemic nicotine exposure enables direct cellular interaction within disc tissues. Notably, our cytotoxicity assessment revealed no significant viability changes in NPCs even at 300 \u0026micro;g ml⁻\u0026sup1; nicotine concentrations, which far exceeded typical serum levels observed in smokers (10\u0026ndash;50 ng ml⁻\u0026sup1;)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. These finding challenges previous hypotheses attributing tobacco-related IVDD to direct cellular toxicity, highlighting its direct intradiscal action as a driver of ECM dysregulation.\u003c/p\u003e \u003cp\u003eThe evolving landscape of tobacco consumption, marked by the proliferation of alternative nicotine delivery systems including e-cigarettes and heated tobacco products, presents novel challenges in understanding IVDD pathogenesis\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These products, strategically marketed as reduced-risk alternatives, have gained particular traction among younger populations due to their customizable nicotine concentrations and flavor profiles\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These findings have immediate clinical relevance given the rising popularity of high-nicotine vaping products among adolescents, suggesting potential acceleration of IVDD in younger populations. To investigate the differential effects of various nicotine delivery methods on IVD at different pathological stages, we employed a customized smoke exposure system to simulate passive smoking and intraperitoneal nicotine injection to mimic novel nicotine delivery systems (e.g., nicotine pouches, vaping). Furthermore, we established a puncture-induced IVD degeneration model to study nicotine\u0026rsquo;s impact on already degenerated discs. While radiographic imaging failed to detect overt structural alterations in Smoking and Nicotine groups, histomorphometric and immunohistochemical analyses unveiled early degenerative signatures, including ECM disorganization and marked downregulation of aggrecan and type II collagen-hallmarks of incipient matrix destabilization\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Our preclinical findings reveal that nicotine disrupts disc homeostasis via biochemical dysregulation, initiating subclinical degeneration prior to structural collapse. Crucially, the synergistic acceleration of degeneration in nicotine-treated puncture models demonstrates amplified vulnerability in pathologically compromised discs, suggesting a feed-forward mechanism where pre-existing matrix damage potentiates nicotine's catabolic effects. These temporal and dose-responsive relationships highlight a critical window for smoking cessation, particularly in individuals with subclinical or early-stage IVDD, to mitigate progressive ECM catabolism and clinical deterioration.\u003c/p\u003e \u003cp\u003eA critical hallmark of IVDD pathogenesis involves the catabolic shift in ECM homeostasis, primarily mediated by MMPs and ADAMTS family members that orchestrate collagen and proteoglycan degradation\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Our study mechanistically links nicotine exposure to this pathological cascade through dose- and time-dependent upregulation of MMP-13 in NPCs. RNA sequencing revealed broad activation of matrix-degrading enzymes, corroborating the pivotal role of MMP/ADAMTS families in nicotine-induced ECM catabolism. These data underscore nicotine\u0026rsquo;s dual role as both a metabolic disruptor and a catabolic amplifier, particularly in pre-degenerated discs. Crucially, in vivo experiments demonstrated that nicotine administration to puncture-induced degenerated discs markedly intensified ECM degradation compared to puncture alone. This accelerated degenerative phenotype mirrors clinical observations where smokers exhibit faster IVDD progression compared to non-smokers\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInflammation is a pivotal pathological driver of IVDD\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Our study unveils that nicotine exacerbates inflammatory cascades in discs through synergistic activation of NF-κB and MAPK signaling pathways. Chronic low-grade inflammation, characterized by elevated pro-inflammatory cytokines such as IL-1β and IL-6, is a hallmark of IVDD pathogenesis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Our experimental data demonstrated that nicotine markedly upregulated IL-1β and IL-6 expression in both in vitro and in vivo models, with amplified effects in degenerated discs. Immunohistochemical analysis revealed significantly higher IL-1β and IL-6 levels in the Puncture\u0026thinsp;+\u0026thinsp;Nicotine group compared to the puncture-only group, highlighting nicotine\u0026rsquo;s role in aggravating inflammatory responses within pre-damaged discs. Mechanistically, NF-κB and MAPK pathways were identified as central mediators of nicotine-induced inflammation. The rapid phosphorylation of p65 and p38 MAPK suggests nicotine initiates immediate pro-degenerative signaling, while sustained pathway activation drives progressive ECM catabolism. Besides, NF-κB exhibited higher sensitivity, suggesting its dominance in inflammatory regulation. This aligns with prior evidence: NF-κB directly governs transcription of IL-1β and MMP-13 to drive ECM degradation\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. while MAPK not only mediates acute inflammation but also modulates long-term matrix remodeling gene\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Importantly, specific inhibition of NF-κB or MAPK pathways substantially attenuated nicotine-induced IL-1β and MMP-13 upregulation, confirming their indispensable roles. Furthermore, nicotine\u0026rsquo;s dual activation of these pathways establishes a self-reinforcing inflammation-catabolism axis: inflammatory cytokines directly stimulate protease production to degrade ECM, while ECM fragments in turn activate NF-κB and MAPK signaling via pattern recognition receptors, perpetuating a vicious cycle. This mechanism may explain accelerated IVDD in smokers and highlights therapeutic potential in targeting these inflammatory cascades.\u003c/p\u003e \u003cp\u003eThe role of cellular senescence as a pivotal accelerator of IVDD pathology has drawn increasing attention, with senescent NPCs accumulation correlating with degenerative severity \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Our study unveils that nicotine induces NPC senescence coupled with a SASP, which dynamically interacts with inflammatory pathways to disrupt disc homeostasis. Experimental evidence demonstrated that nicotine markedly upregulated the senescence marker p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e and enhanced SA-β-gal activity. In addition, nicotine-treated NPCs also exhibited a classic SASP profile-secreting pro-inflammatory cytokines, chemokines and proteases. These SASP factors not only directly degrade ECM but also propagate senescence to adjacent NPCs via paracrine signaling, establishing a self-amplifying \u0026ldquo;senescence-inflammation\u0026rdquo; cascade\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Crucially, a bidirectional crosstalk exists between nicotine-induced senescence and inflammatory pathways: chronic inflammation mediated by NF-κB and MAPK accelerates senescence via oxidative stress and DNA damage, whereas senescent NPCs perpetuate inflammation through sustained SASP secretion, reactivating NF-κB and MAPK signaling and forming a feedforward \u0026ldquo;inflammation \u0026rarr; senescence \u0026rarr; amplified inflammation\u0026rdquo; loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This synergy was most pronounced in the Puncture\u0026thinsp;+\u0026thinsp;Nicotine model, where combined mechanical injury and nicotine exposure synergistically elevated IL-1β and p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e expression compared to single-factor groups, concomitant with exacerbated ECM disruption and structural collapse. Our findings suggest that targeting the senescence-inflammation axis (e.g., via SASP inhibition or NF-κB and MAPK blockade) could disrupt this vicious cycle. Therapeutic strategies such as senolytics (eliminating senescent cells) or senomorphics (suppressing SASP) combined with anti-inflammatory agents may hold promise for mitigating smoking-related IVDD\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. However, the molecular links bridging senescence and inflammation require further elucidation, including how specific SASP components activate NF-κB/MAPK and whether epigenetic modifications regulate this interplay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile our study provides mechanistic insights into nicotine-induced IVDD, several limitations should be acknowledged. First, we mainly focus on the role of nicotine, while tobacco smoke is a complex mixture composed of over 6,000 chemical substances\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Some studies have reported that cadmium, a heavy metal in tobacco, can induce apoptosis of annulus fibrosus cells\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Future studies should explore potential synergistic effects among these components. Second, our experimental models may not fully replicate the chronic, low-dose exposure seen in human smokers. Additionally, the complex pathophysiology of IVDD involves multiple molecular mechanisms beyond nicotine-induced inflammation and senescence, warranting further investigation.\u003c/p\u003e \u003cp\u003eIn conclusion, this study elucidates the dual-pathway mechanism by which nicotine exacerbates IVDD through NF-κB and MAPK-mediated inflammation and cellular senescence. we demonstrate for the first time that nicotine permeates and accumulates in avascular disc tissues via inhalation or systemic administration. Nicotine dose- and time-dependently suppresses aggrecan and type II collagen synthesis while upregulating various proteolytic enzymes and proinflammatory cytokines, accelerating degeneration via ECM destabilization and chronic inflammation. Notably, nicotine exposure intensifies structural damage and ECM dysregulation in pre-degenerated discs, highlighting its synergistic toxicity within compromised microenvironments. Mechanistically, nicotine activates NF-κB and MAPK pathways, driving inflammatory cytokine production and inducing cellular senescence with a pro-inflammatory SASP, establishing an \u0026ldquo;inflammation-senescence\u0026rdquo; positive feedback loop that synergistically exacerbates degeneration. Our findings emphasize smoking cessation as critical in early IVDD and propose combined NF-κB and MAPK inhibition and senolytic therapy to counteract nicotine-driven degeneration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to report.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was funded by the Medical science and Technology project of Henan Province (NO. LHGJ20230080), the Key R\u0026amp;D and Promotion Program of Henan Science and Technology Department (NO. 242102310406), the Key R\u0026amp;D and Promotion Program of Henan Science and Technology Department (NO. 232102311038) and the Medical science and Technology project of Henan Province (NO. LHGJ20220031).\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGBD 2015 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. \u003cem\u003eLancet\u003c/em\u003e 2016; \u003cstrong\u003e388\u003c/strong\u003e: 1545\u0026ndash;1602.\u003c/li\u003e\n\u003cli\u003eGoodchild M, Nargis N, Tursan d\u0026rsquo;Espaignet E. 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Toxic elements in tobacco and in cigarette smoke: inflammation and sensitization. \u003cem\u003eMetallomics\u003c/em\u003e 2011; \u003cstrong\u003e3\u003c/strong\u003e: 1181\u0026ndash;1198.\u003c/li\u003e\n\u003cli\u003eJing D, Wu W, Deng X, Peng Y, Yang W, Huang D \u003cem\u003eet al.\u003c/em\u003e FoxO1a mediated cadmium-induced annulus fibrosus cells apoptosis contributes to intervertebral disc degeneration in smoking. \u003cem\u003eJ Cell Physiol\u003c/em\u003e 2021; \u003cstrong\u003e236\u003c/strong\u003e: 677\u0026ndash;687.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6586785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6586785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cigarette smoking constitutes a major modifiable risk factor for intervertebral disc degeneration (IVDD), yet the mechanistic underpinnings remain incompletely elucidated. This study aimed to elucidate the pathological effects of nicotine-the primary addictive component in tobacco-on IVDD, focusing on its role in inflammation, extracellular matrix (ECM) disruption, and cellular senescence. Utilizing in vivo models (smoke exposure, nicotine injection, and disc puncture) and in vitro human nucleus pulposus cell (NPC) cultures, we employed multi-omics approaches (metabolomics, transcriptomics, imaging, and functional assays) to investigate nicotine's impact on ECM metabolism, inflammatory signaling, and senescence. Nicotine and its primary metabolite, cotinine, accumulated in avascular disc tissue following inhalation or systemic administration. Nicotine dose- and time-dependently inhibited aggrecan and collagen II synthesis, concomitant with MMP-13 upregulation, indicating a catabolic shift in ECM homeostasis. In vivo, nicotine exacerbated puncture-induced IVDD, with synergistic ECM degradation and inflammation. RNA-seq revealed NF-κB and MAPK pathway activation, confirmed by rapid p65 and p38 phosphorylation and elevated IL-1β and IL-6 expression. Pharmacological inhibition of these pathways attenuated nicotine-induced ECM degradation and inflammation. Notably, nicotine triggered NPC senescence with a pro-inflammatory senescence-associated secretory phenotypes (SASP), synergizing with pre-existing disc injury to accelerate IVDD. Our study uncovers a dual-pathway mechanism in which nicotine activates NF-κB and MAPK signaling to amplify inflammatory cascades and SASP, synergistically accelerating IVDD progression. These insights underscore the urgency of smoking cessation, particularly in early-stage IVDD, and propose targeted inhibition of these pathways as a therapeutic strategy.","manuscriptTitle":"Nicotine exacerbates intervertebral disc degeneration via NF-κB/MAPK-dependent inflammatory activation and cellular senescence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-21 08:50:35","doi":"10.21203/rs.3.rs-6586785/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-09-10T07:30:37+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-29T07:25:42+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-05-23T13:37:29+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-19T12:18:15+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-05-19T05:59:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-07T01:47:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-04T05:39:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2025-05-04T05:39:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b5a507bf-2f08-4e10-bc75-1f076005337c","owner":[],"postedDate":"May 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":48703059,"name":"Health sciences/Diseases/Rheumatic diseases/Osteoarthritis"},{"id":48703060,"name":"Health sciences/Medical research/Genetics research"}],"tags":[],"updatedAt":"2026-02-23T07:16:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-21 08:50:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6586785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6586785","identity":"rs-6586785","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}