NiHHTP Attenuates Intervertebral Disc Degeneration via Necroptosis Inhibition: Mechanistic Insights from Integrated In Vitro and In Vivo Models

NiHHTP Attenuates Intervertebral Disc Degeneration via Necroptosis Inhibition: Mechanistic Insights from Integrated In Vitro and In Vivo Models | 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 NiHHTP Attenuates Intervertebral Disc Degeneration via Necroptosis Inhibition: Mechanistic Insights from Integrated In Vitro and In Vivo Models Feng Jiang, Yun Sun, Wei Wu, Xiang Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6543877/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Intervertebral disc degeneration (IVDD) is a leading cause of low back pain, with necroptosis playing a pivotal role in its pathogenesis. This study investigates the potential of Material NIHHTP in mitigating IVDD by inhibiting necroptosis. Nucleus pulposus (NP) cell cultures and a lumbar puncture-induced IVDD mouse model were employed to assess the effects of NIHHTP. Western blot (WB) analysis was conducted to evaluate necroptosis markers, including receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), mixed lineage kinase domain-like protein (MLKL), and their phosphorylated forms. Additionally, histological analyses, including hematoxylin-eosin (HE) staining, safranin O-fast green staining, and immunofluorescence, were performed to assess tissue integrity and protein expression. Our results demonstrate that NIHHTP confers protection against intervertebral disc degeneration by targeting and inhibiting necroptotic signaling pathways, underscoring its promise as a potential therapeutic approach for treating disc degeneration. Health sciences/Anatomy/Cells/Chondrocytes Health sciences/Diseases/Rheumatic diseases/Osteoarthritis Biological sciences/Cell biology/Cell death Biological sciences/Cell biology/Cell death/Necroptosis Intervertebral disc degeneration necroptosis NIHHTP nucleus pulposus cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The Low back pain (LBP) is a major global health issue, significantly contributing to disability and reduced quality of life, with a lifetime prevalence of up to 84% [ 1 ]. A key contributor to LBP is intervertebral disc degeneration (IVDD), a complex disorder influenced by genetic predisposition, aging, mechanical stress, and inflammation[ 2 – 4 ]. IVDD involves extracellular matrix (ECM) breakdown, increased apoptosis and senescence of nucleus pulposus (NP) cells, and chronic inflammation, ultimately leading to structural and functional deterioration of the intervertebral disc[ 5 , 6 ].As the intervertebral disc is avascular, nutrient exchange occurs primarily through passive diffusion, making it particularly susceptible to metabolic stress. Degeneration increases catabolic activity, causing depletion of ECM components such as aggrecan and type II collagen, while simultaneously impairing anabolic processes essential for maintaining tissue integrity[ 7 ]. Pro-inflammatory cytokines like IL-1β and TNF-α further aggravate ECM degradation and oxidative stress. Current IVDD treatments, including physiotherapy, NSAIDs, epidural steroid injections, and surgical interventions such as spinal fusion or artificial disc replacement, primarily focus on symptom relief rather than addressing underlying degenerative processes[ 8 ]. Thus, identifying key signaling pathways associated with NP cell survival, inflammation, and ECM metabolism is crucial for developing regenerative therapies aimed at targeting IVDD at the molecular level. Metal-organic frameworks (MOFs) are highly tunable materials with large surface areas and versatile chemical properties, making them valuable for catalysis, energy storage, and biomedicine[ 9 , 10 ]. Among them, two-dimensional (2D) MOFs offer unique layered architectures, enhanced surface accessibility, and superior stability[ 11 , 12 ]. NiHHTP is a nickel-based MOF synthesized via the coordination of Ni²⁺ ions with 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP). It features a highly ordered hexagonal honeycomb lattice and a porous, graphene-like structure[ 13 , 14 ]. Its interconnected nanorods create uniform nanopores, ensuring high surface area and structural stability through strong Ni–O coordination. These properties make NiHHTP a promising material for biomedical applications, including antioxidative therapy, biosensing, and drug delivery[ 15 , 16 ]. Its high porosity enables efficient molecular transport, while its nickel composition enhances catalytic functionality. Further studies are needed to fully explore its potential in biological systems. Necroptosis is a regulated form of cell death that is caspase-independent and is mediated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), along with the phosphorylation of mixed lineage kinase domain-like protein (MLKL)[ 17 ]. During this process, activated MLKL forms membrane pores, leading to the leakage of intracellular contents and eventual cell death [ 18 ]. Necroptosis plays a crucial role in various physiological and pathological processes, including host defense, inflammatory responses, and neurodegenerative diseases[ 19 , 20 ]. Understanding the molecular mechanisms of necroptosis provides valuable insights for developing therapeutic strategies against related diseases. This study provides a comprehensive assessment of the NiHHTP for mitigating IVDD. The findings reveal that NiHHTP effectively scavenges reactive oxygen species and reduces inflammation through its antioxidative and anti-inflammatory properties, while also substantially inhibiting the necroptosis pathway mediated by RIPK1, RIPK3, and MLKL. As a result, it alleviates extracellular matrix degradation and NP cell apoptosis[ 21 ]. Both in vivo and in vitro experiments demonstrate that NiHHTP treatment significantly preserves disc structural integrity and promotes NP cell viability, thereby slowing the progression of disc degeneration. These insights highlight the promise of employing functionalized MOFs to precisely target necroptosis signaling, offering a novel therapeutic strategy for IVDD. Methods/Experimental The Materials The reagents and antibodies used in this study included DMEM/F12 medium, fetal bovine serum (FBS), antibiotics, NIHHTP compound, and primary antibodies for RIPK1, RIPK3, MLKL, ACAN, and MMP3. Laboratory equipment included a CO₂ incubator, inverted fluorescence microscope, Western blot system, and real-time PCR system. Cell Culture Human immortalized nucleus pulposus cells were obtained from Pricella Biotechnology Co., Ltd., China. The cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 medium from Gibco, USA, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, also from Gibco, USA. Cultures were kept at 37°C in a humidified environment containing 5% carbon dioxide. Upon reaching 80–90% confluence, cells were detached using 0.25% trypsin-EDTA from Gibco, USA, and subsequently passaged. Experiments utilized cells between passages three and nine. Animal Model Male Sprague-Dawley rats (8 weeks old) were purchased from Shanghai Leigen Biotechnology Co., Ltd. Upon arrival, the animals were housed in a specific pathogen-free (SPF) facility under controlled environmental conditions (constant temperature of 27°C and relative humidity of 50–55%). To facilitate acclimatization, minimal environmental disturbance was maintained except for essential experimental procedures. Prior to surgery, rats were anesthetized by intraperitoneal injection of 2.5% Avertin working solution at a dose of 1.5 mL/100 g body weight. Anesthesia took effect within approximately 5 minutes. Upon confirmation of adequate anesthesia, the tail was disinfected with 75% ethanol and the rat was placed in the prone position. A needle puncture-induced intervertebral disc degeneration model was established as previously described. A 20-gauge sterile needle was vertically inserted into the center of the Co5/6, Co6/7, and Co7/8 intervertebral discs. Each disc was punctured once to a depth of approximately 5 mm, held in place for 5 seconds, and then withdrawn. Rats in the control group did not undergo puncture. Animals were randomly assigned into three groups (n = 6 per group): control group, degeneration model group, and treatment group. The treatment group received intraperitoneal injections of NIHHTP at a predetermined dose according to the experimental protocol. After model induction, rats were maintained under standard housing conditions and evaluated at 8 weeks post-operation using magnetic resonance imaging (MRI) and histological analysis to assess the extent of disc degeneration and therapeutic outcomes. At the end of the experimental period, rats were euthanized using a gradual-fill carbon dioxide (CO₂) inhalation method in accordance with institutional animal welfare guidelines and the ARRIVE reporting standards. CO₂ gas, sourced from a certified compressed gas cylinder, was delivered into a transparent euthanasia chamber at a flow rate not exceeding 30% of the chamber volume per minute (e.g., ≤ 6.5 L/min for a standard rat cage), ensuring a slow and controlled rise in CO₂ concentration. To minimize animal distress, animals were placed in the chamber without overcrowding, ensuring that each animal's limbs could contact the chamber floor. Different species or sizes of animals were not euthanized together. After cessation of visible respiration, animals were kept in the chamber for an additional 2–3 minutes to ensure complete loss of consciousness and death. Western Blot Analysis Nucleus pulposus cells and intervertebral disc tissues were lysed using radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified via the bicinchoninic acid assay. Equal amounts of protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies targeting receptor-interacting serine/threonine-protein kinase 1, receptor-interacting serine/threonine-protein kinase 3, mixed lineage kinase domain-like protein, aggrecan, and matrix metallopeptidase 3. Following incubation with horseradish peroxidase-conjugated secondary antibodies, protein bands were visualized using an enhanced chemiluminescence detection system. Hematoxylin and Eosin (HE) Staining Lumbar spine specimens were immersed in a 4% paraformaldehyde solution for 48 hours to achieve fixation, followed by decalcification using a 10% ethylenediaminetetraacetic acid solution. Post-decalcification, the tissues underwent dehydration through a graded series of ethanol concentrations, were embedded in paraffin wax, and sectioned into slices of 5 micrometers thickness.​ The paraffin-embedded sections were deparaffinized, rehydrated, and stained with hematoxylin for five minutes, then counterstained with eosin for two minutes. Subsequently, the slides were dehydrated through ascending ethanol concentrations, cleared with xylene, and coverslipped.​ Histological evaluations of the nucleus pulposus and annulus fibrosus regions were conducted using light microscopy. Safranin O-Fast Green Staining To assess proteoglycan content and extracellular matrix integrity, paraffin-embedded lumbar intervertebral disc sections underwent Safranin O and Fast Green staining. Following deparaffinization and rehydration, sections were stained with Weigert's iron hematoxylin for five minutes, then with Fast Green for five minutes. Excess dye was removed using one percent acetic acid, and sections were counterstained with Safranin O for ten minutes. After dehydration, slides were mounted for microscopic examination. Proteoglycan-rich areas appeared red, whereas collagen fibers were stained green. Immunofluorescence Analysis Nucleus pulposus cells were cultured on sterilized glass coverslips and exposed to NIHHTP for a duration of 24 hours. Post-treatment, the cells underwent fixation using a 4% paraformaldehyde solution, followed by permeabilization with 0.1% Triton X-100. To prevent nonspecific antibody binding, a blocking step was performed using 5% bovine serum albumin.​ Subsequently, the cells were incubated overnight at 4°C with primary antibodies targeting aggrecan and matrix metallopeptidase 3. After thorough washing, appropriate Alexa Fluor-conjugated secondary antibodies were applied. Nuclear staining was achieved using 4',6-diamidino-2-phenylindole. Finally, fluorescence microscopy was employed to capture images, facilitating the assessment of protein localization and expression levels. Results Structure and Properties of NIHHTP NiHHTP is a nickel-based MOF synthesized via the coordination-driven self-assembly of nickel ions (Ni²⁺) with the organic ligand 2,3,6,7,10,11-hexahydroxytriphenylene (Fig. 1 A). Structural characterization through X-ray diffraction (XRD) confirms the highly ordered crystalline nature of NiHHTP, as evidenced by distinct diffraction peaks observed at approximately 4.7°, 9.5°, 12.6°, and 27.4°, which are indicative of a well-defined hexagonal honeycomb lattice (Fig. 1 B). The pronounced peak intensity and sharpness further highlight the excellent crystallinity and structural uniformity of the material[ 13 , 22 , 23 ]. Scanning electron microscopy (SEM) analysis further elucidates that NiHHTP exhibits a two-dimensional (2D) layered nanostructure, primarily composed of uniformly dispersed nanorods (Fig. 1 C). These nanorods, approximately 100 nm in length and 20 nm in width, are interconnected, forming a porous, graphene-like network with uniform nanopores averaging 1.8 nm in diameter. The well-organized porosity and high surface area of NiHHTP provide a favorable structural framework for biomedical applications, enhancing potential biological interactions and functionalization. Additionally, X-ray photoelectron spectroscopy (XPS) analysis verifies the chemical stability of NiHHTP, demonstrating that the nickel ions predominantly exist in a stable Ni²⁺ oxidation state. The strong coordination between Ni²⁺ and oxygen atoms from the HHTP ligand, facilitated by robust Ni–O bonds, contributes to the material’s overall stability and durability (Fig. 1 D). The combination of high crystallinity, structural uniformity, and chemical stability underscores the reproducibility and reliability of NiHHTP for biomedical applications. These intrinsic properties suggest that NiHHTP is a promising candidate for therapeutic applications, particularly in the treatment of oxidative stress-associated disorders such as IVDD. Biocompatibility assessments using the CCK-8 assay confirmed minimal cytotoxicity at the selected experimental concentration, indicating excellent biological safety for NiHHTP usage. Notably, NiHHTP also enhanced cell viability, further supporting its biocompatibility[ 24 ]. These structural and biological characteristics collectively highlight the significant potential of NiHHTP as a therapeutic material, especially for oxidative stress-associated conditions such as IVDD. NIHHTP Promotes NP Cell Survival and Regulates ECM-Associated Proteins in IVDD The aim of this study was to investigate the protective effects of NIHHTP on NP cells in the context of IVDD Initially, biocompatibility assessments utilizing the Cell Counting Kit-8 (CCK-8) assay demonstrated that NIHHTP exhibited minimal cytotoxicity across a range of concentrations, with 10 µg/ml identified as the optimal, non-toxic concentration selected for subsequent experiments. NP cell viability was then evaluated under different treatment conditions: untreated controls, cells subjected to inflammatory and oxidative stress conditions (TSZ: TNF-α, Smac mimetic, and Z-VAD), and TSZ combined with NIHHTP at the established concentration (Fig. 1 A). Cells exposed to TSZ alone exhibited significantly reduced viability, indicating severe cellular injury. However, NIHHTP treatment markedly improved NP cell viability under these stress conditions, suggesting a robust protective effect against inflammatory and oxidative damage[ 25 , 26 ]. Additionally, the influence of NIHHTP on ECM components, which are essential for intervertebral disc function, was evaluated. Western blot analysis (Fig. 1 B) showed that TSZ treatment significantly decreased the expression levels of aggrecan (ACAN), a key ECM anabolic component, and increased matrix metalloproteinase 3 (MMP3), an indicator of ECM degradation. Importantly, NIHHTP reversed these changes, significantly restoring ACAN expression and suppressing MMP3 expression, thereby protecting ECM integrity. Immunofluorescence staining further corroborated these findings (Figs. 1 C and 1 D), visually confirming enhanced ACAN-positive staining and diminished MMP3-positive signals in NIHHTP-treated cells compared to those treated with TSZ alone. Collectively, these findings clearly illustrate that NIHHTP significantly enhances NP cell survival and maintains ECM integrity under degenerative conditions, emphasizing its therapeutic potential for IVDD management. NIHHTP Protects Intervertebral Disc Structure and ECM Integrity in an IVDD Rat Model In the present study, a rat tail puncture model was established to investigate the protective effects of NIHHTP on IVDD through comprehensive histological and immunofluorescence analyses (Fig. 2 ). HE staining (Fig. 2 A) revealed significant degeneration of the NP and pronounced disorganization of the annulus fibrosus (AF) in the IVDD group. Conversely, the IVDD + NIHHTP group exhibited improved preservation of NP morphology and reduced fragmentation of the AF, indicating a protective effect conferred by NIHHTP treatment[ 27 ]. Additionally, Safranin O-Fast Green staining (Fig. 2 B) demonstrated marked proteoglycan depletion in the IVDD group, while NIHHTP-treated animals retained higher proteoglycan content, suggesting that NIHHTP effectively preserves extracellular matrix (ECM) integrity. Immunofluorescence staining for aggrecan (ACAN) (Fig. 2 C) showed significantly decreased ACAN expression in the IVDD group, reflecting heightened ECM degradation; however, ACAN expression levels were substantially preserved following NIHHTP intervention, underscoring the compound’s role in promoting ECM synthesis. Furthermore, immunofluorescence analysis of matrix metalloproteinase-3 (MMP3) (Fig. 2 D) indicated elevated MMP3 expression in the IVDD group, indicative of increased ECM breakdown. Notably, NIHHTP-treated rats exhibited markedly reduced MMP3 expression, highlighting the compound’s efficacy in attenuating ECM degradation and disc degeneration. Collectively, these findings demonstrate that NIHHTP mitigates IVDD progression by preserving the structural integrity of NP and AF tissues, maintaining ECM homeostasis, and inhibiting matrix degradation[ 28 ]. These observations align closely with the molecular findings presented in Fig. 3 , further confirming NIHHTP's therapeutic potential in managing IVDD. NIHHTP Inhibits Necroptosis to Protect NP Cells in IVDD To elucidate the protective role of NIHHTP against necroptosis-induced nucleus pulposus cell injury, cellular viability and death were evaluated through fluorescence staining. As shown in Fig. 4 A, Calcein AM/propidium iodide (PI) staining demonstrated predominantly green fluorescence signals in control cells, indicative of high cell viability. In contrast, cells treated with TSZ exhibited a marked increase in PI-positive cells, reflecting elevated necroptotic cell death. Importantly, treatment with NIHHTP significantly reduced the number of PI-positive cells, highlighting its inhibitory effect on TSZ-induced necroptosis. To further investigate the molecular mechanisms through which NIHHTP inhibits necroptosis, Western blot analysis was conducted to evaluate phosphorylation levels of key signaling proteins, including RIPK1, RIPK3, and MLKL (Fig. 4 B). The results indicated that TSZ treatment significantly increased the phosphorylation of RIPK1, RIPK3, and MLKL, confirming activation of the necroptotic signaling pathway. Conversely, NIHHTP treatment markedly attenuated the phosphorylation levels of RIPK1 and MLKL, suggesting effective suppression of necroptosis. Quantitative analyses further supported these observations (Fig. 4 C). Compared with the TSZ-treated group, NIHHTP administration significantly decreased the phosphorylation ratios of p-RIPK1/RIPK1 and p-MLKL/MLKL. Although the phosphorylation ratio of p-RIPK3/RIPK3 displayed a decreasing trend following NIHHTP treatment, this difference was not statistically significant. Collectively, these findings suggest that NIHHTP exerts protective effects against necroptosis-mediated NP cell injury primarily by attenuating the phosphorylation of key signaling proteins in the necroptotic pathway, highlighting its therapeutic potential for intervertebral disc degeneration. Discussion MOFs have gained considerable attention in biomedical research due to their tunable structures, high surface areas, and versatile physicochemical properties, enabling applications in drug delivery, biosensing, and antioxidative therapy[ 10 ]. Recent studies have demonstrated the potential of MOFs in modulating oxidative stress and inflammatory responses, which are critical factors in various degenerative diseases [ 29 ]. In particular, NiHHTP, a two-dimensional nickel-based MOF, has shown promise in biomedical applications owing to its highly porous architecture and catalytic capabilities [ 30 , 31 ]. Guo L et al. developed a radical-scavenging MOF for targeted siRNA delivery, synergistically treating rheumatoid arthritis by reducing ROS and silencing pro-inflammatory genes. Zhang B et al. showcased immunomodulatory MOFs for biomedical applications by regulating immune cells, delivering immunotherapeutic agents, and modulating the inflammatory microenvironment to facilitate anti-inflammatory therapies, vaccine development, and immunotherapy[ 32 , 33 ]. Inspired by the rapidly expanding research on MOFs, this study investigates their potential in alleviating IVDD [ 34 ]. The findings reveal that NiHHTP significantly slows IVDD progression in both in vivo and in vitro models. Recent research has identified necroptosis as a crucial driver of NP cell loss and extracellular matrix degradation in degenerative discs [ 35 ]. Recent evidence indicates that necroptotic signaling can trigger the activation of ADAM metalloproteinases, which in turn promote the cleavage and release of extracellular domains from membrane-bound proteins, including E-cadherin. The resulting soluble fragments subsequently trigger inflammatory pathways like NF-κB, revealing a novel mechanism through which necroptosis directly initiates inflammation[ 36 ]. Building on this, Gong Y et al. found that limonin delays IVDD progression by suppressing the MAPK/NF-κB and necroptosis pathways, thereby reducing inflammatory cytokine release and cell death while preserving ECM homeostasis[ 37 , 38 ]. In light of the pathological relevance of necroptosis, the present study reveals that NiHHTP mitigates intervertebral disc degeneration by effectively inhibiting necroptotic cell death, an effect attributed to its strong antioxidative and anti-inflammatory capacities, which collectively contribute to the preservation of disc tissue structure and function. In this study, we highlight the potential therapeutic application of NiHHTP in IVDD by demonstrating its ability to modulate necroptotic pathways. Our findings suggest that NiHHTP exerts protective effects on NP cells, likely through its antioxidative and catalytic properties, which contribute to the suppression of necroptosis-mediated disc degeneration. By inhibiting key necroptotic signaling molecules, NiHHTP may serve as an effective biomaterial for mitigating IVDD progression. Further in-depth mechanistic studies and preclinical evaluations are necessary to validate these findings and explore the translational potential of NiHHTP-based interventions in IVDD therapy. Conclusions In conclusion, our findings demonstrate that NIHHTP exerts protective effects against IVDD by inhibiting necroptosis, preserving ECM integrity, and improving NP cell viability. These results provide a novel perspective on IVDD treatment and suggest that targeting necroptosis could be a promising therapeutic strategy. Future studies should focus on optimizing NIHHTP dosage, evaluating its long-term safety, and exploring potential clinical applications in human IVDD patients. Collectively, this study advances our understanding of necroptosis in IVDD pathogenesis and highlights NIHHTP as a potential candidate for IVDD therapy. Abbreviations IVDD Intervertebral Disc Degeneration NP Nucleus Pulposus RIPK1 Receptor-Interacting Protein Kinase 1 RIPK3 Receptor-Interacting Protein Kinase 3 MLKL Mixed Lineage Kinase Domain-Like Protein ACAN Aggrecan MMP3 Matrix Metalloproteinase 3 XRD X-ray Diffraction SEM Scanning Electron Microscopy XPS X-ray Photoelectron Spectroscopy HE Hematoxylin and Eosin TSZ TNF-α, Smac mimetic, and Z-VAD CCK-8 Cell Counting Kit-8 DAPI 4′,6-Diamidino-2-phenylindole PI Propidium Iodide BCA Bicinchoninic Acid SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis PVDF Polyvinylidene Fluoride HRP Horseradish Peroxidase NF-κB Nuclear Factor kappa-light-chain-enhancer of activated B cells Declarations Ethical approval and consent to participate All procedures involving animals were performed in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) and the National Research Council’s Guide for the Care and Use of Laboratory Animals . The study protocol was approved by the Ethics Committee of Huaibei City People’s Hospital (Approval No. 2024-079), and all experiments were carried out in compliance with relevant guidelines and regulations. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files. Competing interests The authors declare that they have no competing interests. Funding This research was funded by the Development Fund of the Department of Science and Technology, The Affiliated Hospital of Xuzhou Medical University (Grant No. ZX202426). Authors’ contributions F.J.: Writing—review & editing, Writing—original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization; W.W. and Y.S.: Writing—review & editing, Supervision, Software, Resources, Funding acquisition, Data curation, Conceptualization; X.L. : Supervision, Software, Funding acquisition. All authors have read and agreed to the published version of the manuscript. Acknowledgments The authors would like to express their sincere gratitude to the Experimental Center of the University of Shanghai for Science and Technology for providing essential facilities and technical support. We also thank the technical staff of Shanghai Changzheng Hospital for their valuable assistance during the course of this study. References McKee, M. D., Addison, W. N. & Kaartinen, M. T. Hierarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton. Cells Tissues Organs. 181 , 176–188 (2005). 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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-6543877","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472636086,"identity":"2b5e3a8f-9754-4886-b2ab-253d6d670fd2","order_by":0,"name":"Feng Jiang","email":"","orcid":"","institution":"University of Shanghai for Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Jiang","suffix":""},{"id":472636087,"identity":"f0068f18-49e9-4ddf-b377-dd5faec1282f","order_by":1,"name":"Yun Sun","email":"","orcid":"","institution":"The People's Hospital of Huaibei City","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Sun","suffix":""},{"id":472636088,"identity":"980f8df0-5bcb-4913-8a42-f8218b411d69","order_by":2,"name":"Wei Wu","email":"","orcid":"","institution":"The People's Hospital of Huaibei City","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wu","suffix":""},{"id":472636089,"identity":"dd823a1f-9811-48ea-81dc-0ba2a8f5699d","order_by":3,"name":"Xiang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDACZhBRICGHzCVGi4GEMZBkbCBOCxgYMCQ2EK3F4Djzs4dfDCzSN5w//vwBQ4V1YgP72QN4tUg2s5kbyxhI5G64kWPYwHAmPbGBJy8BrxZ+ZgYzaQmwFh7GBsa2w4kNEjwGeLWwMbN/A2lJNzh//GED4z8itPAz85hJfjCQSDA4kGDYwNhAhBbJZp4yaWAgG84E+mVGwrF04zaeHPxagO7ZJvmjok6e7/zxBx8+1FjL9rOfwa8FBJh5YKwEkO8IqgcCxh/EqBoFo2AUjIKRCwDVsT7WpE1THQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Shanghai for Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-04-28 04:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6543877/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6543877/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84995479,"identity":"270a10c3-2cb6-4347-b913-f0d8f05af218","added_by":"auto","created_at":"2025-06-19 16:09:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":342172,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis and characterization of NiHHTP.(A) Schematic illustration of the synthesis process of NiHHTP via coordination-driven self-assembly of Ni²⁺ ions with the HHTP ligand.(B) X-ray diffraction (XRD) pattern of NiHHTP, displaying distinct diffraction peaks at approximately 4.7°, 9.5°, 12.6°, and 27.4°, confirming its highly ordered hexagonal honeycomb lattice structure.(C) Scanning electron microscopy (SEM) image of NiHHTP, showing its 2D layered nanostructure composed of uniformly dispersed nanorods.(D) X-ray photoelectron spectroscopy (XPS) spectrum of NiHHTP, confirming the presence of Ni²⁺ and the stability of Ni–O coordination bonds.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6543877/v1/fed53abd623f68c598fb94b7.png"},{"id":84996013,"identity":"4dcf0d8f-de71-4d29-809b-fc7b290fbce3","added_by":"auto","created_at":"2025-06-19 16:17:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":516054,"visible":true,"origin":"","legend":"\u003cp\u003eNIHHTP delays intervertebral disc degeneration.(A) Cell viability (%) across various treatment groups (Control, TSZ, TSZ+NIHHTP). Statistical significance is indicated as follows: **** (p \u0026lt; 0.0001); ns (not significant).(B) Western blot analysis of ACAN (250 kDa), MMP3 (60 kDa), and GAPDH (36 kDa) under different conditions (Control, TSZ, TSZ+NIHHTP), with three replicates per condition.(C) Immunofluorescence staining showing ACAN (red) and MMP3 (green) expression in different experimental groups. Nuclei are counterstained with DAPI (blue). The images are arranged in three rows: ACAN, MMP3, and merged images.(D) 3D surface plots illustrating fluorescence intensity for ACAN (top row) and MMP3 (bottom row) under the indicated conditions\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6543877/v1/e22f8d9ba46b2fff7116d251.png"},{"id":84997114,"identity":"f4031902-e051-4621-b88f-0e44911a4cf3","added_by":"auto","created_at":"2025-06-19 16:25:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":735044,"visible":true,"origin":"","legend":"\u003cp\u003eNIHHTP attenuates IVDD in a needle-puncture SD rat model.(A) Hematoxylin-eosin (HE) staining illustrating tissue morphology in the Control, IVDD at 4 and 8 weeks, and IVDD+NIHHTP groups at 4 and 8 weeks. The left column shows images at a 200 µm scale, while the right column presents magnified views at a 50 µm scale.(B) Safranin O-Fast Green staining displaying proteoglycan content in rat tail intervertebral disc tissue under different conditions. The left column presents images at 200 µm scale, while the right column shows magnified images at 50 µm scale.(C) Immunohistochemical staining for ACAN (red) in different experimental groups, indicating extracellular matrix preservation. The left column represents images at 200 µm scale, and the right column provides higher magnification at 50 µm scale.(D) Immunohistochemical staining for MMP3 (green), demonstrating extracellular matrix degradation across different groups. The left column displays images at 200 µm scale, and the right column presents magnified images at 50 µm scale.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6543877/v1/3a4bd8a9e0ecd57b0ac86b71.png"},{"id":84997115,"identity":"56c5ac1c-d2eb-4f41-aaec-1873d7d00e97","added_by":"auto","created_at":"2025-06-19 16:25:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":406980,"visible":true,"origin":"","legend":"\u003cp\u003eNIHHTP inhibits necroptosis in intervertebral disc degeneration.(A) Fluorescence microscopy images showing nucleus pulposus cell viability across different treatment groups (Control, TSZ, TSZ+NIHHTP). Green fluorescence (Calcein AM) represents live cells, while red fluorescence (PI) indicates dead cells.(B) Western blot analysis of p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL, and GAPDH expression levels in nucleus pulposus cells under different conditions (Control, TSZ, TSZ+NIHHTP), with three replicates per condition.(C) Quantitative analysis of Western blot results comparing p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL expression levels. Statistical significance is represented as follows: **** (p \u0026lt; 0.0001), ** (p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6543877/v1/0a7f660c34ca26c9934e9207.png"},{"id":85632042,"identity":"4db0c823-5301-42de-ab48-1cdbc4ef58e1","added_by":"auto","created_at":"2025-06-30 04:08:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10358432,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6543877/v1/f6836623-87c8-4848-ada9-4bc460801a60.pdf"},{"id":84995475,"identity":"cb279820-9d0d-454f-8dd0-3249f013d779","added_by":"auto","created_at":"2025-06-19 16:09:39","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4462231,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalImagesforBlots.zip","url":"https://assets-eu.researchsquare.com/files/rs-6543877/v1/54a4333dde53517f2c297852.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"NiHHTP Attenuates Intervertebral Disc Degeneration via Necroptosis Inhibition: Mechanistic Insights from Integrated In Vitro and In Vivo Models","fulltext":[{"header":"Background","content":"\u003cp\u003eThe Low back pain (LBP) is a major global health issue, significantly contributing to disability and reduced quality of life, with a lifetime prevalence of up to 84% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A key contributor to LBP is intervertebral disc degeneration (IVDD), a complex disorder influenced by genetic predisposition, aging, mechanical stress, and inflammation[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. IVDD involves extracellular matrix (ECM) breakdown, increased apoptosis and senescence of nucleus pulposus (NP) cells, and chronic inflammation, ultimately leading to structural and functional deterioration of the intervertebral disc[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].As the intervertebral disc is avascular, nutrient exchange occurs primarily through passive diffusion, making it particularly susceptible to metabolic stress. Degeneration increases catabolic activity, causing depletion of ECM components such as aggrecan and type II collagen, while simultaneously impairing anabolic processes essential for maintaining tissue integrity[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Pro-inflammatory cytokines like IL-1β and TNF-α further aggravate ECM degradation and oxidative stress. Current IVDD treatments, including physiotherapy, NSAIDs, epidural steroid injections, and surgical interventions such as spinal fusion or artificial disc replacement, primarily focus on symptom relief rather than addressing underlying degenerative processes[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, identifying key signaling pathways associated with NP cell survival, inflammation, and ECM metabolism is crucial for developing regenerative therapies aimed at targeting IVDD at the molecular level.\u003c/p\u003e \u003cp\u003eMetal-organic frameworks (MOFs) are highly tunable materials with large surface areas and versatile chemical properties, making them valuable for catalysis, energy storage, and biomedicine[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among them, two-dimensional (2D) MOFs offer unique layered architectures, enhanced surface accessibility, and superior stability[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. NiHHTP is a nickel-based MOF synthesized via the coordination of Ni\u0026sup2;⁺ ions with 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP). It features a highly ordered hexagonal honeycomb lattice and a porous, graphene-like structure[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Its interconnected nanorods create uniform nanopores, ensuring high surface area and structural stability through strong Ni\u0026ndash;O coordination. These properties make NiHHTP a promising material for biomedical applications, including antioxidative therapy, biosensing, and drug delivery[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Its high porosity enables efficient molecular transport, while its nickel composition enhances catalytic functionality. Further studies are needed to fully explore its potential in biological systems.\u003c/p\u003e \u003cp\u003eNecroptosis is a regulated form of cell death that is caspase-independent and is mediated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), along with the phosphorylation of mixed lineage kinase domain-like protein (MLKL)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. During this process, activated MLKL forms membrane pores, leading to the leakage of intracellular contents and eventual cell death [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Necroptosis plays a crucial role in various physiological and pathological processes, including host defense, inflammatory responses, and neurodegenerative diseases[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Understanding the molecular mechanisms of necroptosis provides valuable insights for developing therapeutic strategies against related diseases.\u003c/p\u003e \u003cp\u003eThis study provides a comprehensive assessment of the NiHHTP for mitigating IVDD. The findings reveal that NiHHTP effectively scavenges reactive oxygen species and reduces inflammation through its antioxidative and anti-inflammatory properties, while also substantially inhibiting the necroptosis pathway mediated by RIPK1, RIPK3, and MLKL. As a result, it alleviates extracellular matrix degradation and NP cell apoptosis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Both in vivo and in vitro experiments demonstrate that NiHHTP treatment significantly preserves disc structural integrity and promotes NP cell viability, thereby slowing the progression of disc degeneration. These insights highlight the promise of employing functionalized MOFs to precisely target necroptosis signaling, offering a novel therapeutic strategy for IVDD.\u003c/p\u003e "},{"header":"Methods/Experimental","content":"\u003cp\u003e \u003cstrong\u003eThe Materials\u003c/strong\u003e \u003cp\u003eThe reagents and antibodies used in this study included DMEM/F12 medium, fetal bovine serum (FBS), antibiotics, NIHHTP compound, and primary antibodies for RIPK1, RIPK3, MLKL, ACAN, and MMP3. Laboratory equipment included a CO₂ incubator, inverted fluorescence microscope, Western blot system, and real-time PCR system.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell Culture\u003c/strong\u003e \u003cp\u003eHuman immortalized nucleus pulposus cells were obtained from Pricella Biotechnology Co., Ltd., China. The cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 medium from Gibco, USA, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, also from Gibco, USA. Cultures were kept at 37\u0026deg;C in a humidified environment containing 5% carbon dioxide. Upon reaching 80\u0026ndash;90% confluence, cells were detached using 0.25% trypsin-EDTA from Gibco, USA, and subsequently passaged. Experiments utilized cells between passages three and nine.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAnimal Model\u003c/strong\u003e \u003cp\u003eMale Sprague-Dawley rats (8 weeks old) were purchased from Shanghai Leigen Biotechnology Co., Ltd. Upon arrival, the animals were housed in a specific pathogen-free (SPF) facility under controlled environmental conditions (constant temperature of 27\u0026deg;C and relative humidity of 50\u0026ndash;55%). To facilitate acclimatization, minimal environmental disturbance was maintained except for essential experimental procedures.\u003c/p\u003e \u003c/p\u003e \u003cp\u003ePrior to surgery, rats were anesthetized by intraperitoneal injection of 2.5% Avertin working solution at a dose of 1.5 mL/100 g body weight. Anesthesia took effect within approximately 5 minutes. Upon confirmation of adequate anesthesia, the tail was disinfected with 75% ethanol and the rat was placed in the prone position. A needle puncture-induced intervertebral disc degeneration model was established as previously described. A 20-gauge sterile needle was vertically inserted into the center of the Co5/6, Co6/7, and Co7/8 intervertebral discs. Each disc was punctured once to a depth of approximately 5 mm, held in place for 5 seconds, and then withdrawn. Rats in the control group did not undergo puncture.\u003c/p\u003e \u003cp\u003eAnimals were randomly assigned into three groups (n\u0026thinsp;=\u0026thinsp;6 per group): control group, degeneration model group, and treatment group. The treatment group received intraperitoneal injections of NIHHTP at a predetermined dose according to the experimental protocol. After model induction, rats were maintained under standard housing conditions and evaluated at 8 weeks post-operation using magnetic resonance imaging (MRI) and histological analysis to assess the extent of disc degeneration and therapeutic outcomes.\u003c/p\u003e \u003cp\u003e At the end of the experimental period, rats were euthanized using a gradual-fill carbon dioxide (CO₂) inhalation method in accordance with institutional animal welfare guidelines and the ARRIVE reporting standards. CO₂ gas, sourced from a certified compressed gas cylinder, was delivered into a transparent euthanasia chamber at a flow rate not exceeding 30% of the chamber volume per minute (e.g., \u0026le;\u0026thinsp;6.5 L/min for a standard rat cage), ensuring a slow and controlled rise in CO₂ concentration.\u003c/p\u003e \u003cp\u003eTo minimize animal distress, animals were placed in the chamber without overcrowding, ensuring that each animal's limbs could contact the chamber floor. Different species or sizes of animals were not euthanized together. After cessation of visible respiration, animals were kept in the chamber for an additional 2\u0026ndash;3 minutes to ensure complete loss of consciousness and death.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e \u003cp\u003eNucleus pulposus cells and intervertebral disc tissues were lysed using radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified via the bicinchoninic acid assay. Equal amounts of protein were resolved by sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk and incubated overnight at 4\u0026deg;C with primary antibodies targeting receptor-interacting serine/threonine-protein kinase 1, receptor-interacting serine/threonine-protein kinase 3, mixed lineage kinase domain-like protein, aggrecan, and matrix metallopeptidase 3. Following incubation with horseradish peroxidase-conjugated secondary antibodies, protein bands were visualized using an enhanced chemiluminescence detection system.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHematoxylin and Eosin (HE) Staining\u003c/strong\u003e \u003cp\u003eLumbar spine specimens were immersed in a 4% paraformaldehyde solution for 48 hours to achieve fixation, followed by decalcification using a 10% ethylenediaminetetraacetic acid solution. Post-decalcification, the tissues underwent dehydration through a graded series of ethanol concentrations, were embedded in paraffin wax, and sectioned into slices of 5 micrometers thickness.​\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe paraffin-embedded sections were deparaffinized, rehydrated, and stained with hematoxylin for five minutes, then counterstained with eosin for two minutes. Subsequently, the slides were dehydrated through ascending ethanol concentrations, cleared with xylene, and coverslipped.​\u003c/p\u003e \u003cp\u003eHistological evaluations of the nucleus pulposus and annulus fibrosus regions were conducted using light microscopy.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSafranin O-Fast Green Staining\u003c/strong\u003e \u003cp\u003eTo assess proteoglycan content and extracellular matrix integrity, paraffin-embedded lumbar intervertebral disc sections underwent Safranin O and Fast Green staining. Following deparaffinization and rehydration, sections were stained with Weigert's iron hematoxylin for five minutes, then with Fast Green for five minutes. Excess dye was removed using one percent acetic acid, and sections were counterstained with Safranin O for ten minutes. After dehydration, slides were mounted for microscopic examination. Proteoglycan-rich areas appeared red, whereas collagen fibers were stained green.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eImmunofluorescence Analysis\u003c/strong\u003e \u003cp\u003eNucleus pulposus cells were cultured on sterilized glass coverslips and exposed to NIHHTP for a duration of 24 hours. Post-treatment, the cells underwent fixation using a 4% paraformaldehyde solution, followed by permeabilization with 0.1% Triton X-100. To prevent nonspecific antibody binding, a blocking step was performed using 5% bovine serum albumin.​\u003c/p\u003e \u003c/p\u003e \u003cp\u003eSubsequently, the cells were incubated overnight at 4\u0026deg;C with primary antibodies targeting aggrecan and matrix metallopeptidase 3. After thorough washing, appropriate Alexa Fluor-conjugated secondary antibodies were applied. Nuclear staining was achieved using 4',6-diamidino-2-phenylindole. Finally, fluorescence microscopy was employed to capture images, facilitating the assessment of protein localization and expression levels.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eStructure and Properties of NIHHTP\u003c/p\u003e \u003cp\u003eNiHHTP is a nickel-based MOF synthesized via the coordination-driven self-assembly of nickel ions (Ni\u0026sup2;⁺) with the organic ligand 2,3,6,7,10,11-hexahydroxytriphenylene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Structural characterization through X-ray diffraction (XRD) confirms the highly ordered crystalline nature of NiHHTP, as evidenced by distinct diffraction peaks observed at approximately 4.7\u0026deg;, 9.5\u0026deg;, 12.6\u0026deg;, and 27.4\u0026deg;, which are indicative of a well-defined hexagonal honeycomb lattice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The pronounced peak intensity and sharpness further highlight the excellent crystallinity and structural uniformity of the material[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM) analysis further elucidates that NiHHTP exhibits a two-dimensional (2D) layered nanostructure, primarily composed of uniformly dispersed nanorods (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These nanorods, approximately 100 nm in length and 20 nm in width, are interconnected, forming a porous, graphene-like network with uniform nanopores averaging 1.8 nm in diameter. The well-organized porosity and high surface area of NiHHTP provide a favorable structural framework for biomedical applications, enhancing potential biological interactions and functionalization.\u003c/p\u003e \u003cp\u003eAdditionally, X-ray photoelectron spectroscopy (XPS) analysis verifies the chemical stability of NiHHTP, demonstrating that the nickel ions predominantly exist in a stable Ni\u0026sup2;⁺ oxidation state. The strong coordination between Ni\u0026sup2;⁺ and oxygen atoms from the HHTP ligand, facilitated by robust Ni\u0026ndash;O bonds, contributes to the material\u0026rsquo;s overall stability and durability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The combination of high crystallinity, structural uniformity, and chemical stability underscores the reproducibility and reliability of NiHHTP for biomedical applications. These intrinsic properties suggest that NiHHTP is a promising candidate for therapeutic applications, particularly in the treatment of oxidative stress-associated disorders such as IVDD.\u003c/p\u003e \u003cp\u003eBiocompatibility assessments using the CCK-8 assay confirmed minimal cytotoxicity at the selected experimental concentration, indicating excellent biological safety for NiHHTP usage. Notably, NiHHTP also enhanced cell viability, further supporting its biocompatibility[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These structural and biological characteristics collectively highlight the significant potential of NiHHTP as a therapeutic material, especially for oxidative stress-associated conditions such as IVDD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNIHHTP Promotes NP Cell Survival and Regulates ECM-Associated Proteins in IVDD\u003c/p\u003e \u003cp\u003eThe aim of this study was to investigate the protective effects of NIHHTP on NP cells in the context of IVDD Initially, biocompatibility assessments utilizing the Cell Counting Kit-8 (CCK-8) assay demonstrated that NIHHTP exhibited minimal cytotoxicity across a range of concentrations, with 10 \u0026micro;g/ml identified as the optimal, non-toxic concentration selected for subsequent experiments. NP cell viability was then evaluated under different treatment conditions: untreated controls, cells subjected to inflammatory and oxidative stress conditions (TSZ: TNF-α, Smac mimetic, and Z-VAD), and TSZ combined with NIHHTP at the established concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Cells exposed to TSZ alone exhibited significantly reduced viability, indicating severe cellular injury. However, NIHHTP treatment markedly improved NP cell viability under these stress conditions, suggesting a robust protective effect against inflammatory and oxidative damage[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, the influence of NIHHTP on ECM components, which are essential for intervertebral disc function, was evaluated. Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) showed that TSZ treatment significantly decreased the expression levels of aggrecan (ACAN), a key ECM anabolic component, and increased matrix metalloproteinase 3 (MMP3), an indicator of ECM degradation. Importantly, NIHHTP reversed these changes, significantly restoring ACAN expression and suppressing MMP3 expression, thereby protecting ECM integrity. Immunofluorescence staining further corroborated these findings (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), visually confirming enhanced ACAN-positive staining and diminished MMP3-positive signals in NIHHTP-treated cells compared to those treated with TSZ alone.\u003c/p\u003e \u003cp\u003eCollectively, these findings clearly illustrate that NIHHTP significantly enhances NP cell survival and maintains ECM integrity under degenerative conditions, emphasizing its therapeutic potential for IVDD management.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNIHHTP Protects Intervertebral Disc Structure and ECM Integrity in an IVDD Rat Model\u003c/p\u003e \u003cp\u003eIn the present study, a rat tail puncture model was established to investigate the protective effects of NIHHTP on IVDD through comprehensive histological and immunofluorescence analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). HE staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) revealed significant degeneration of the NP and pronounced disorganization of the annulus fibrosus (AF) in the IVDD group. Conversely, the IVDD\u0026thinsp;+\u0026thinsp;NIHHTP group exhibited improved preservation of NP morphology and reduced fragmentation of the AF, indicating a protective effect conferred by NIHHTP treatment[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, Safranin O-Fast Green staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) demonstrated marked proteoglycan depletion in the IVDD group, while NIHHTP-treated animals retained higher proteoglycan content, suggesting that NIHHTP effectively preserves extracellular matrix (ECM) integrity. Immunofluorescence staining for aggrecan (ACAN) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) showed significantly decreased ACAN expression in the IVDD group, reflecting heightened ECM degradation; however, ACAN expression levels were substantially preserved following NIHHTP intervention, underscoring the compound\u0026rsquo;s role in promoting ECM synthesis. Furthermore, immunofluorescence analysis of matrix metalloproteinase-3 (MMP3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) indicated elevated MMP3 expression in the IVDD group, indicative of increased ECM breakdown. Notably, NIHHTP-treated rats exhibited markedly reduced MMP3 expression, highlighting the compound\u0026rsquo;s efficacy in attenuating ECM degradation and disc degeneration. Collectively, these findings demonstrate that NIHHTP mitigates IVDD progression by preserving the structural integrity of NP and AF tissues, maintaining ECM homeostasis, and inhibiting matrix degradation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These observations align closely with the molecular findings presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, further confirming NIHHTP's therapeutic potential in managing IVDD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNIHHTP Inhibits Necroptosis to Protect NP Cells in IVDD\u003c/p\u003e \u003cp\u003eTo elucidate the protective role of NIHHTP against necroptosis-induced nucleus pulposus cell injury, cellular viability and death were evaluated through fluorescence staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Calcein AM/propidium iodide (PI) staining demonstrated predominantly green fluorescence signals in control cells, indicative of high cell viability. In contrast, cells treated with TSZ exhibited a marked increase in PI-positive cells, reflecting elevated necroptotic cell death. Importantly, treatment with NIHHTP significantly reduced the number of PI-positive cells, highlighting its inhibitory effect on TSZ-induced necroptosis.\u003c/p\u003e \u003cp\u003eTo further investigate the molecular mechanisms through which NIHHTP inhibits necroptosis, Western blot analysis was conducted to evaluate phosphorylation levels of key signaling proteins, including RIPK1, RIPK3, and MLKL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The results indicated that TSZ treatment significantly increased the phosphorylation of RIPK1, RIPK3, and MLKL, confirming activation of the necroptotic signaling pathway. Conversely, NIHHTP treatment markedly attenuated the phosphorylation levels of RIPK1 and MLKL, suggesting effective suppression of necroptosis.\u003c/p\u003e \u003cp\u003eQuantitative analyses further supported these observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Compared with the TSZ-treated group, NIHHTP administration significantly decreased the phosphorylation ratios of p-RIPK1/RIPK1 and p-MLKL/MLKL. Although the phosphorylation ratio of p-RIPK3/RIPK3 displayed a decreasing trend following NIHHTP treatment, this difference was not statistically significant.\u003c/p\u003e \u003cp\u003eCollectively, these findings suggest that NIHHTP exerts protective effects against necroptosis-mediated NP cell injury primarily by attenuating the phosphorylation of key signaling proteins in the necroptotic pathway, highlighting its therapeutic potential for intervertebral disc degeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMOFs have gained considerable attention in biomedical research due to their tunable structures, high surface areas, and versatile physicochemical properties, enabling applications in drug delivery, biosensing, and antioxidative therapy[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Recent studies have demonstrated the potential of MOFs in modulating oxidative stress and inflammatory responses, which are critical factors in various degenerative diseases [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In particular, NiHHTP, a two-dimensional nickel-based MOF, has shown promise in biomedical applications owing to its highly porous architecture and catalytic capabilities [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Guo L et al. developed a radical-scavenging MOF for targeted siRNA delivery, synergistically treating rheumatoid arthritis by reducing ROS and silencing pro-inflammatory genes. Zhang B et al. showcased immunomodulatory MOFs for biomedical applications by regulating immune cells, delivering immunotherapeutic agents, and modulating the inflammatory microenvironment to facilitate anti-inflammatory therapies, vaccine development, and immunotherapy[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Inspired by the rapidly expanding research on MOFs, this study investigates their potential in alleviating IVDD [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The findings reveal that NiHHTP significantly slows IVDD progression in both in vivo and in vitro models.\u003c/p\u003e \u003cp\u003eRecent research has identified necroptosis as a crucial driver of NP cell loss and extracellular matrix degradation in degenerative discs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Recent evidence indicates that necroptotic signaling can trigger the activation of ADAM metalloproteinases, which in turn promote the cleavage and release of extracellular domains from membrane-bound proteins, including E-cadherin. The resulting soluble fragments subsequently trigger inflammatory pathways like NF-κB, revealing a novel mechanism through which necroptosis directly initiates inflammation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Building on this, Gong Y et al. found that limonin delays IVDD progression by suppressing the MAPK/NF-κB and necroptosis pathways, thereby reducing inflammatory cytokine release and cell death while preserving ECM homeostasis[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In light of the pathological relevance of necroptosis, the present study reveals that NiHHTP mitigates intervertebral disc degeneration by effectively inhibiting necroptotic cell death, an effect attributed to its strong antioxidative and anti-inflammatory capacities, which collectively contribute to the preservation of disc tissue structure and function.\u003c/p\u003e \u003cp\u003eIn this study, we highlight the potential therapeutic application of NiHHTP in IVDD by demonstrating its ability to modulate necroptotic pathways. Our findings suggest that NiHHTP exerts protective effects on NP cells, likely through its antioxidative and catalytic properties, which contribute to the suppression of necroptosis-mediated disc degeneration. By inhibiting key necroptotic signaling molecules, NiHHTP may serve as an effective biomaterial for mitigating IVDD progression. Further in-depth mechanistic studies and preclinical evaluations are necessary to validate these findings and explore the translational potential of NiHHTP-based interventions in IVDD therapy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our findings demonstrate that NIHHTP exerts protective effects against IVDD by inhibiting necroptosis, preserving ECM integrity, and improving NP cell viability. These results provide a novel perspective on IVDD treatment and suggest that targeting necroptosis could be a promising therapeutic strategy. Future studies should focus on optimizing NIHHTP dosage, evaluating its long-term safety, and exploring potential clinical applications in human IVDD patients. Collectively, this study advances our understanding of necroptosis in IVDD pathogenesis and highlights NIHHTP as a potential candidate for IVDD therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eIVDD\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eIntervertebral Disc Degeneration\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNP\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eNucleus Pulposus\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRIPK1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eReceptor-Interacting Protein Kinase 1\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRIPK3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eReceptor-Interacting Protein Kinase 3\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eMLKL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eMixed Lineage Kinase Domain-Like Protein\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eACAN\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAggrecan\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eMMP3\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eMatrix Metalloproteinase 3\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eXRD\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eX-ray Diffraction\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSEM\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eScanning Electron Microscopy\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eXPS\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eX-ray Photoelectron Spectroscopy\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eHE\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eHematoxylin and Eosin\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eTSZ\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTNF-α, Smac mimetic, and Z-VAD\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCCK-8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eCell Counting Kit-8\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDAPI\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u0026prime;,6-Diamidino-2-phenylindole\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePI\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePropidium Iodide\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eBCA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eBicinchoninic Acid\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSDS-PAGE\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePVDF\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePolyvinylidene Fluoride\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eHRP\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eHorseradish Peroxidase\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNF-κB\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eNuclear Factor kappa-light-chain-enhancer of activated B cells\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll procedures involving animals were performed in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) and the National Research Council\u0026rsquo;s \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e. The study protocol was approved by the Ethics Committee of Huaibei City People\u0026rsquo;s Hospital (Approval No. 2024-079), and all experiments were carried out in compliance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Development Fund of the Department of Science and Technology, The Affiliated Hospital of Xuzhou Medical University (Grant No. ZX202426).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; contributions\u003c/p\u003e\n\u003cp\u003eF.J.: Writing\u0026mdash;review \u0026amp; editing, Writing\u0026mdash;original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization; W.W. and Y.S.: Writing\u0026mdash;review \u0026amp; editing, Supervision, Software, Resources, Funding acquisition, Data curation, Conceptualization; X.L. : Supervision, Software, Funding acquisition. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to the Experimental Center of the University of Shanghai for Science and Technology for providing essential facilities and technical support. We also thank the technical staff of Shanghai Changzheng Hospital for their valuable assistance during the course of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcKee, M. D., Addison, W. N. \u0026amp; Kaartinen, M. T. Hierarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton. \u003cem\u003eCells Tissues Organs.\u003c/em\u003e \u003cb\u003e181\u003c/b\u003e, 176\u0026ndash;188 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRisbud, M. V. \u0026amp; Shapiro, I. M. 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Pharmacol.\u003c/em\u003e \u003cb\u003e75\u003c/b\u003e (2), 233\u0026ndash;244 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Intervertebral disc degeneration, necroptosis, NIHHTP, nucleus pulposus cells","lastPublishedDoi":"10.21203/rs.3.rs-6543877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6543877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntervertebral disc degeneration (IVDD) is a leading cause of low back pain, with necroptosis playing a pivotal role in its pathogenesis. This study investigates the potential of Material NIHHTP in mitigating IVDD by inhibiting necroptosis. Nucleus pulposus (NP) cell cultures and a lumbar puncture-induced IVDD mouse model were employed to assess the effects of NIHHTP. Western blot (WB) analysis was conducted to evaluate necroptosis markers, including receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), mixed lineage kinase domain-like protein (MLKL), and their phosphorylated forms. Additionally, histological analyses, including hematoxylin-eosin (HE) staining, safranin O-fast green staining, and immunofluorescence, were performed to assess tissue integrity and protein expression. Our results demonstrate that NIHHTP confers protection against intervertebral disc degeneration by targeting and inhibiting necroptotic signaling pathways, underscoring its promise as a potential therapeutic approach for treating disc degeneration.\u003c/p\u003e","manuscriptTitle":"NiHHTP Attenuates Intervertebral Disc Degeneration via Necroptosis Inhibition: Mechanistic Insights from Integrated In Vitro and In Vivo Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 16:09:34","doi":"10.21203/rs.3.rs-6543877/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6080cf64-826c-4dda-8507-7458878ccb19","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50193764,"name":"Health sciences/Anatomy/Cells/Chondrocytes"},{"id":50193765,"name":"Health sciences/Diseases/Rheumatic diseases/Osteoarthritis"},{"id":50193766,"name":"Biological sciences/Cell biology/Cell death"},{"id":50193767,"name":"Biological sciences/Cell biology/Cell death/Necroptosis"}],"tags":[],"updatedAt":"2025-06-30T04:08:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-19 16:09:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6543877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6543877","identity":"rs-6543877","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}