Unlocking the Therapeutic Potential of 1,25(OH)₂D₃: Targeting Ferroptosis to Alleviate Lumbar Intervertebral Disc Degeneration

Unlocking the Therapeutic Potential of 1,25(OH)₂D₃: Targeting Ferroptosis to Alleviate Lumbar Intervertebral Disc Degeneration | 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 Unlocking the Therapeutic Potential of 1,25(OH)₂D₃: Targeting Ferroptosis to Alleviate Lumbar Intervertebral Disc Degeneration Qiang Li, Jing Peng, Fan Ding This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5612461/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Lumbar intervertebral disc degeneration (LIDD) is a primary cause of low back pain, a condition with significant global health and socioeconomic impacts. Recent studies have highlighted the role of ferroptosis, an iron-dependent form of programmed cell death, in nucleus pulposus (NP) cells degeneration. This study investigates the protective effects of 1,25(OH)₂D₃, the active form of Vitamin D, on LIDD by targeting ferroptosis. Our findings demonstrate that 1,25(OH)₂D₃ effectively suppresses ferroptosis in nucleus pulposus cells by reducing lipid peroxidation, restoring glutathione levels, and enhancing antioxidant defenses. Mechanistically, 1,25(OH)₂D₃ exerts its effects through activation of the Vitamin D receptor (VDR) signaling pathway, which regulates key ferroptosis-associated molecules such as GPX4 and SLC7A11. These results reveal the therapeutic potential of 1,25(OH)₂D₃ in mitigating LIDD, offering a novel approach to suppress ferroptosis and preserve intervertebral disc function. Biological sciences/Biochemistry Biological sciences/Cell biology Lumbar Intervertebral Disc Degeneration 1 25(OH)₂D₃ ferroptosis nucleus pulposus cells Vitamin D Receptor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Low back pain is a progressively critical global health concern, impacting over 80% of the global population and imposing substantial social and economic expenses 1,2 . Intervertebral disc degeneration is a significant contributor to low back pain 3 . LIDD encompasses a range of structural and tissue alterations, including decreased disc height, NP fissures, tears in the annulus fibrosus (AF), calcification of the cartilaginous endplate, and disrupted extracellular matrix (ECM) metabolism 4,5 . The precise mechanisms underlying LIDD remain unclear. Nonetheless, it is commonly accepted that its progression begins and speeds up due to the depletion of NP cells and the degradation of the ECM 5 . NP cells play a key role in the synthesis and breakdown of the ECM, serving as essential components of the intervertebral disc, which is important for preserving its structure and physiological functions. The deterioration and impaired function of NP cells play crucial roles in the development of LIDD 6 . Consequently, examining NP cells to clarify the pathogenesis of LIDD holds considerable clinical significance 7 . Previous studies indicate that various factors of disc degeneration could be associated with apoptosis, as a decline in cell numbers results in lower ECM protein production and disturbances in cellular energy metabolism along with other physiological functions 8 . As a result, treatment for LIDD mainly aims to avert cell loss caused by programmed or regulated cell death. Recent studies show that different types of cell death, such as apoptosis, pyroptosis, necroptosis, autophagy, ferroptosis, and senescence, play a role in the progression of LIDD, providing fresh avenues for treatment 9 . Ferroptosis, first introduced in 2012 as a new type of programmed cell death, is unique due to its reliance on iron and is marked by a significant buildup of lipid reactive oxygen species (ROS) 10 . It can be distinguished morphologically and biochemically from apoptosis, necrosis, autophagy-dependent cell death, and pyroptosis 11 . Ferroptosis is associated with the pathologies of several diseases, including cancer, stroke, cerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and degenerative diseases such as Alzheimer's and Parkinson's 12 . Morphologically, ferroptosis is characterized by the early preservation of cell membrane integrity, lipid peroxidation within cell membranes, alterations in mitochondria (including shrinkage, diminished mitochondrial cristae, and rupture of the outer membrane), lack of chromatin condensation, cytosolic vesiculation, and a reduction in cell volume or atrophy 11,13 . Comprehensive studies have shown a notable connection between ferroptosis and LIDD, highlighting the potential for effective therapies in the treatment of LIDD 12,14,15 . Research on disc degeneration has shown that ferroptosis inhibitors like ferrostatin-1 and deferoxamine can mitigate degeneration in a TBHP-induced rat model. Furthermore, ferroptosis inducers TBHP and RSL3 have comparable effects on cell death in AFCs and NP cells, which can be blocked by ferroptosis inhibitors, highlighting the significance of ferroptosis in the mechanism of LIDD 15 . Vitamin D is essential for regulating serum calcium and phosphorus levels and is found mainly in two forms, D2 and D3 16 . Vitamin D3 (VD3), or cholecalciferol, is produced from 7-dehydrocholesterol in the skin when exposed to UV radiation or obtained from animal-derived foods. VD3 undergoes a two-step hydroxylation process to be converted into its active form, calcitriol (1,25(OH)₂D₃) 17 . In addition to its traditional function in calcium balance, VD influences cell growth, differentiation, and programmed cell death 18 . 1,25(OH)₂D₃ acts as an antioxidant, possibly mitigating ferroptosis induced by oxidative stress by regulating intracellular glutathione (GSH) and superoxide dismutase (SOD) 19 . Furthermore, numerous clinical studies suggest that a deficiency in vitamin D could hasten the progression of lumbar disc degeneration and elevate the risk of lumbar disc herniation 20,21 . Conversely, vitamin D supplementation may mitigate the degradation process and reduce the incidence of low back discomfort 22 . 1,25(OH)₂D₃ positively affects LIDD by diminishing inflammation, oxidative stress, and NP cell apoptosis, while also postponing NP cell aging 23 . The VDR plays a crucial role in the biological effects of 1,25(OH)₂D₃, being prevalent in cells of the intervertebral disc and linked to disc degeneration 24 . Genetic variations in VDR, including TaqI (rs731236), FokI (rs2228570), and ApaI (rs7975232), are associated with a heightened risk of LIDD 25,26 . Ferroptosis is crucial in the progression of LIDD, and 1,25(OH)₂D₃, a variant of Vitamin D, might impede this process. This study investigates the potential of 1,25(OH)₂D₃ to mitigate LIDD by inhibiting ferroptosis in NP cells through VDR, aiming to reveal novel preventative approaches for LIDD. Materials and Methods Cell Culture and Maintenance Rat-derived NP cells, selected for their relevance in lumbar disc studies, were obtained from SAIBAIKANG Biosciences, Shanghai. Authenticated and mycoplasma-free, these cells were initially thawed at 37 °C and cultured in a medium supplemented with 10% fetal bovine serum (FBS). The cells were cultivated in T25 flasks within a CO2 incubator under conditions optimal for NP cell growth. Cells were passaged when confluence exceeded 85%, including washing with PBS and detachment using trypsin-EDTA. Cell growth was monitored using an inverted microscope. For long-term preservation, cells were cryopreserved in a solution of 90% FBS and 10% DMSO, following a controlled-rate freezing protocol. The cells were initially stored at -80°C and transferred to a liquid nitrogen tank. Cell viability and concentration were assessed post-thawing using Trypan blue staining and a hemocytometer. VDR Gene Silencing For the inhibition of the rat VDR gene (Gene ID: 24873), three siRNAs (si-VDR-1, si-VDR-2, si-VDR-3) alongside a non-targeting control (si-Control) were employed. These were designed and synthesized by RiboBio Co., Ltd., with their sequences detailed in Supplementary Table S1. Bioinformatic analyses informed selection to optimize specificity and efficiency, and RT-qPCR and Western blot analysis confirmed gene silencing. The transfection protocol used cells seeded in 6-well plates to 60-70% confluence. siRNAs, diluted to 20 μM in DEPC-treated water, were mixed with Opti-MEM and Lipofectamine 2000 to form the transfection complex. This complex was incubated with the cells for 6 hours at 37 °C in a 5% CO2 atmosphere, followed by a medium replacement with a growth medium. The cells were then cultured for 72 hours to facilitate gene silencing, with post-transfection assessments confirming the effectiveness. Cell Viability Assessment with CCK-8 To inhibit the rat VDR gene (Gene ID: 24873), three siRNAs (si-VDR-1, si-VDR-2, si-VDR-3) and a non-targeting control (si-Control) were used. RiboBio Co., Ltd. designed and synthesized these siRNAs, with their sequences detailed in Supplementary Table S1. The selection was based on bioinformatic analyses to optimize specificity and efficiency, and RT-qPCR and Western blot analysis confirmed gene silencing. The transfection protocol seeded cells in 6-well plates to 60-70% confluence. The siRNAs, diluted to 20 μM in DEPC-treated water, were mixed with Opti-MEM and Lipofectamine 2000 to form the transfection complex. This complex was incubated with the cells for 6 hours at 37 °C in a 5% CO2 atmosphere, then replaced with a growth medium. The cells were cultured for 72 hours to facilitate gene silencing, and post-transfection assessments confirmed the effectiveness. Assessment of Biochemical Markers Post-treatment, cells (2×10 5 per well) were cultured in 6-well plates for 48 hours. For LDH activity assessment, supernatants collected after centrifugation (2000g, 4 °C, 10 minutes) were evaluated using an LDH assay kit (Jiancheng Bioengineering Institute). Intracellular MDA, SOD, and GSH levels were quantified after cell ultrasonication and centrifugation. Protein concentration was determined using a BCA protein assay kit (Aspen Biological) for normalization. MDA content, SOD activity, and total GSH levels (both oxidized and reduced forms) were quantified using their respective assay kits (Jiancheng Bioengineering Institute), following the manufacturer's protocols. Quantitative Assessment of Gene Expression via RT-qPCR RNA was extracted from cells using TRIzol-based TRIpure reagent (ELK Biotechnology), which involved chloroform-induced phase separation and isopropanol precipitation. The purified RNA was converted to cDNA using the EntiLink™ 1st Strand cDNA Synthesis Super Mix (ELK Biotechnology). Quantitative PCR was performed using a QuantStudio 6 Flex Real-Time PCR System (Life Technologies) with EnTurbo™ SYBR Green PCR SuperMix. The protocol included initial denaturation, 40 amplification cycles, and melt curve analysis for product verification. Gene expression was analyzed using the comparative ΔΔCT method, normalizing the expression of R-SLC7A11, R-SLC40A1, R-VDR, and R-GPX4 to R-β-actin. The primer sequences listed in Supplemental Table S2 were designed to span exon-exon junctions to avoid amplification of genomic DNA. Flow Cytometry for Lipid ROS Detection Flow cytometry was employed for intracellular lipid ROS quantification. Following trypsinization and centrifugation, cells were stained with DCFH-DA, a ROS-sensitive fluorogenic dye. After a 20-minute incubation at 37 °C with intermittent inversion for uniform dye distribution, cells were washed three times with serum-free medium to remove excess dye and then resuspended for analysis. Flow cytometric analysis was conducted using a BD flow cytometer with a 488 nm excitation and 525 nm emission filter. Stained and unstained controls were included for calibration. Fluorescence intensities, indicative of lipid ROS levels, were measured in triplicate. Data acquisition and analysis were performed using cytometry software to evaluate oxidative stress levels in response to different treatments. Western Blot Analysis for Protein Expression Profiling For Western blotting, adherent cells were rinsed with ice-cold PBS and lysed. The supernatant, after centrifugation, was used for protein quantification using a BCA assay. Proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were then blocked to prevent non-specific binding. Membranes were incubated with primary antibodies (Supplementary Table S3) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (Supplementary Table S4) for 30 minutes at room temperature. Bands were visualized using an ECL kit and imaged on X-ray film. Band intensities were quantified using AlphaEaseFC software and normalized against a housekeeping protein. To ensure specificity, membranes were re-probed, involving stripping and re-blocking. Transmission Electron Microscopy for Mitochondrial Morphology NP cells were fixed overnight at 4 °C in a fixative solution, followed by PBS washing. Cells were stained with osmium tetroxide for two hours at room temperature to enhance membrane contrast. Cells underwent a graduated dehydration process using ethanol and acetone, were then embedded in epoxy resin, and polymerized. Ultra-thin sections (~70 nm) were prepared and stained with uranyl acetate and lead citrate for ultrastructural visualization. Mitochondrial morphology was examined using a transmission electron microscope (TEM), which provided detailed insights into the cellular environment of the NP cells. Statistical Analysis Data were analyzed using SPSS software (version 23.0) and GraphPad Prism to ensure analytical robustness and reproducibility. Results are presented as mean ± standard deviation (SD). A one-way Analysis of Variance (ANOVA) or Student's t-test was employed to assess group differences, contingent upon the data's distribution. Post-hoc tests were conducted where necessary for detailed analysis. Statistical significance was indicated as *p < 0.05, **p < 0.01, and ***p < 0.001, with adjustments for multiple comparisons to minimize type I errors. Data visualization included bar graphs and scatter plots, with a predefined significance threshold of p < 0.05. Results Effect of 1,25(OH)₂D₃ on the Viability of NP Cells Rat NP cells were subjected to treatment with different concentrations of 1,25(OH)₂D₃ (0, 0.01, 0.1, 1, 10, 100, and 1000 nmol/L) for durations of 6, 12, 24, and 48 hours. The CCK8 assay was employed to evaluate cell viability. The results showed that NP cells exhibited the highest viability at 48 hours when exposed to a concentration of 10 nmol/L of 1,25(OH)₂D₃ (Supplementary Fig. S1). This indicates that 1,25(OH)₂D₃ has the potential to enhance NP cell proliferation, with the optimal concentration and duration being 10 nmol/L and 48 hours, respectively. The chosen conditions were set for the subsequent experiments. Impact of 1,25(OH)₂D₃ on the Viability of NP Cells treated with Erastin Erastin was employed to replicate an in vivo ferroptosis environment and to trigger ferroptosis in NP cells 27 . The study had four groups: control, 1,25(OH)₂D₃, Erastin, and 1,25(OH)₂D₃ combined with Erastin. The Erastin cohort received 10 μmol/L Erastin for a duration of 24 hours. The 1,25(OH)₂D₃+Erastin cohort comprised NP cells that were pre-treated with 10 nmol/L 1,25(OH)₂D₃ for 48 hours, then receiving treatment with 10 μmol/L Erastin for 24 hours. Microscopic examination revealed that NP cells in the Erastin group exhibited spindle-shaped, fusiform, and irregular morphologies, with sparse distribution and evidence of cell death, in contrast to the normal polygonal cells observed in the control group. Conversely, the 1,25(OH)₂D₃+Erastin group exhibited considerable enhancements in cell proliferation, mirroring the appearance of normal NP cells observed in the control group (Fig. 1). In a CCK8 experiment assessing cell viability across the four NP cell groups, the 1,25(OH)₂D₃+Erastin group exhibited a considerably higher number of viable cells than the Erastin group (Fig. 2a). Lactate dehydrogenase (LDH) activity in cell supernatants was elevated in the Erastin group relative to the control, while a significant reduction was observed in the 1,25(OH)₂D₃+Erastin group compared to the Erastin group (Fig. 2b). This suggests that 1,25(OH)₂D₃ promotes NP cell growth and viability in ferroptosis conditions. Effect of 1,25(OH)₂D₃ on Ferroptosis in NP Cells To assess whether 1,25(OH)₂D₃ could mitigate ferroptosis in NP cells, the research evaluated intracellular levels of GPX4, lipid ROS, MDA, SOD, and GSH in NP cells subjected to Erastin and following 1,25(OH)₂D₃ administration. The findings indicated that pretreatment with 1,25(OH)₂D₃ led to a significant increase in GPX4, SOD and GSH (Fig. 3 and 4b-c) levels, while markedly reducing lipid ROS (Fig. 4a) and MDA (Fig. 4d) levels in NP cells when compared to the Erastin group, indicating reduced oxidative stress in cells treated with 1,25(OH)₂D₃. This suggests a reduction in oxidative stress in cells treated with 1,25(OH)₂D₃. RT-qPCR results indicated that, in comparison to the Erastin group, the 1,25(OH)₂D₃+Erastin group exhibited a notable increase in the expression levels of SLC7A11 and SLC40A1 (Fig. 4e-f). Furthermore, transmission electron microscopy observations of mitochondrial morphology in NP cells revealed that in the Erastin group, NP cells exhibited increased mitochondrial membrane density and decreased cristae. The mitochondrial morphology in the 1,25(OH)₂D₃+Erastin group was similar to that observed in both the blank control group and the 1,25(OH)₂D₃ group (Fig. 5). In conclusion, 1,25(OH)₂D₃ has the ability to reduce ferroptosis in NP cells when exposed to ferroptotic conditions. Influence of 1,25(OH)₂D₃ on VDR Expression in NP Cells RT-qPCR results demonstrated a significant elevation of VDR in the 1,25(OH)₂D₃-treated group relative to the control, while VDR was substantially diminished in the Erastin-treated group (Fig. 6a). NP cells pre-treated with 1,25(OH)₂D₃ demonstrated a significant enhancement in VDR protein expression relative to untreated cells (Fig. 6b), underscoring a possible mechanism through VDR activation. Western blot examination confirmed these findings, revealing a marked reduction in VDR protein expression in NP cells under ferroptotic conditions, which was reinstated by 1,25(OH)₂D₃ treatment (Fig. 6c). The findings indicate that VDR activation is essential for the suppression of ferroptosis in NP cells mediated by 1,25(OH)₂D₃. Effect of 1,25(OH)₂D₃ on Ferroptosis in NP Cells Induced by Erastin Following VDR Gene Silencing To validate that 1,25(OH)₂D₃ promotes NP cell survival via VDR activation, three siRNA sequences were designed by RiboBio Co., Ltd. (Guangzhou, China) to silence the VDR gene in NP cells. Subsequent to transfection, Western blot analysis confirmed the efficacy of RNA interference. siRNA-2 had the greatest efficacy, significantly reducing VDR gene expression (Supplementary Fig. S2), and was selected for subsequent investigation. The experiment had four groups: control siRNA+Erastin, control siRNA+1,25(OH)₂D₃+Erastin, VDR siRNA+Erastin and VDR siRNA+1,25(OH)₂D₃+Erastin. The study examined the concentrations of GPX4, lipid ROS, MDA, SOD, GSH, SLC7A11, and SLC40A1 in NP cells that were pre-treated with 1,25(OH)₂D₃, assessing the results both prior to and following siRNA interference. The data indicated that, in contrast to the VDR siRNA+Erastin group, the VDR siRNA+1,25(OH)₂D₃+Erastin group had elevated expression of GPX4, SLC7A11, SLC40A1, SOD and GSH (Fig. 7, 8b-c and 8e-f), alongside a significant reduction in lipid ROS and MDA (Fig. 8a and 8d). This demonstrates that 1,25(OH)₂D₃ maintains its suppressive influence on NP cell ferroptosis despite VDR gene silencing. In comparison to the control siRNA+1,25(OH)₂D₃+Erastin group, the VDR siRNA+1,25(OH)₂D₃+Erastin group exhibited substantial decreases in GPX4, SLC7A11, SLC40A1, SOD and GSH, alongside elevated lipid ROS and MDA. This suggests that VDR gene silencing reduces the suppressive influence of 1,25(OH)₂D₃ on NP cell ferroptosis. The results substantiate that the anti-ferroptotic effect of 1,25(OH)₂D₃ in NP cells is partially contingent upon the activation of VDR. Discussion The Relationship Between LIDD and NP Cells Ferroptosis The manifestation and progression of LIDD are affected by factors including oxidative stress, trauma, infection, and inflammation 28,29 . Oxidative stress plays a pivotal role in the ageing of nuclear NP cells, inducing premature cellular senescence via DNA damage and telomere attrition resulting from replicative ageing 30,31 . As degeneration progresses, levels of lipid reactive oxygen species in NP cells markedly elevate, including superoxide anion, hydroxyl radical, hydrogen peroxide, and nitric oxide 32,33 . Elevated ROS levels accelerate LIDD through key signaling pathways, such as MAPK and NF-κB pathways. Increased ROS levels expedite LIDD via critical signalling pathways, including MAPK and NF-κB pathways. In oxidative stress circumstances, exemplified by a TBHP model, diminished expression of iron transporter proteins results in intracellular iron buildup, facilitating ferroptosis in NP cells and the subsequent progression of LIDD 34 . Homocysteine can elevate oxidative stress and ferroptosis in NP cells by augmenting GPX4 methylation, hence contributing to LIDD 14 . These investigations demonstrate that oxidative stress-induced ferroptosis is closely associated with LIDD. Mitigating NP cell ferroptosis may be a unique preventive approach to intervertebral disc degeneration. The Effects of 1,25(OH)₂D₃ on Normal NP Cells Our work shown that 1,25(OH)₂D₃ significantly enhances the proliferation of rat NP cells, with the optimal concentration being 10 nmol/L at 48 hours. This discovery highlights the possible function of 1,25(OH)₂D₃ in facilitating NP cell proliferation. Notably, the cytoprotective effects of vitamin D3 seem to be unaffected by its concentration. Subsequent observations indicate that elevated doses may induce detrimental effects, undermining its protective attributes. Increased concentrations of 1,25(OH)₂D₃ have been shown to enhance the ability of macrophages infected with Mycobacterium TB to synthesise the antibiotic peptide LL37 35 . The primary mechanism by which vitamin D prevents LIDD is its suppression of the NF-κB signalling pathway 36 . This pathway modulates the transcription of several genes, including pro-inflammatory cytokines such as IL-6, cell cycle regulators like cyclin D1, anti-apoptotic factors such as bcl-2, and extracellular proteases like MMP3. The mammalian NF-κB family, comprising RelA/p65, c-Rel, RelB, p50, and p52, produces many dimers, including the prevalent p50/RelA heterodimer, which activates multiple genes by binding to κB sites within gene promoters 37 . Insufficient 1,25(OH)₂D₃ hinders matrix protein synthesis and hastens disc degeneration, a phenomenon associated with diminished SIRT1 expression in NP tissue. Augmenting SIRT1 inhibits the NF-κB pathway, hence averting degeneration. Vitamin D or resveratrol, through the activation of SIRT1, can mitigate these degenerative alterations 38 . Efficacy of 1,25(OH)₂D₃ in mitigating intervertebral disc degeneration. This impact is mainly accomplished by blocking the NF-κB pathway, resulting in diminished inflammation, reduced oxidative stress, suppressed apoptosis, and delayed cellular ageing in the intervertebral discs 39 . These results, corroborated by in vivo and in vitro studies, underscore the potential of vitamin D as an effective therapeutic agent for the treatment and management of disc degeneration. The Impact of 1,25(OH)₂D₃ on NP Cells in a Ferroptotic Environment Since 2012, Erastin has been identified as a principal inducer of cellular ferroptosis and was originally employed to target neoplastic cells. The pathophysiological mechanisms are analogous to those in numerous injury-related diseases, resulting in its extensive application for treatment and prognostic evaluation of significant illnesses 11,12 . This study noted a substantial rise in LDH activity following Erastin induction, signifying reduced viability in NP cells. Erastin decreased intracellular levels of GPX4, SOD, and GSH, while elevating lipid ROS and MDA, so efficiently promoting ferroptosis. Treatment with 1,25(OH)₂D₃ significantly improved the growth, viability, and proliferation of Erastin-treated NP cells, increased levels of GPX4, SOD, and GSH, and reduced lipid ROS and MDA. SLC7A11 is a crucial subunit of the Xc system, an intracellular antioxidant mechanism vital for sustaining GPX4 activity, whereas SLC40A1 facilitates cellular iron efflux and inhibits intracellular iron accumulation, both of which are proteins that counteract ferroptosis 9 . The findings indicated that the expression levels of SLC7A11 and SLC40A1 were markedly elevated in the 1,25(OH)₂D₃+Erastin group relative to the Erastin group. Consequently, 1,25(OH)₂D₃ administration mitigated oxidative stress in NP cells. The mitochondria serve as the primary site of cellular oxidative stress, making mitochondrial alterations a significant aspect of the ferroptosis process 40 . This research employed Transmission Electron Microscopy (TEM) to examine the mitochondrial morphology of NP cells across several groups. The research indicated that the Erastin group exhibited heightened mitochondrial membrane density and reduced cristae in NP cells. Conversely, NP cells subjected to 1,25(OH)₂D₃ exhibited mitochondrial traits akin to those of the control group. This verifies that 1,25(OH)₂D₃ efficiently mitigates ferroptosis in NP cells under oxidative stress conditions. GPX4, SLC7A11, and SLC40A1, recognised as indicators of ferroptosis, have garnered interest in conditions including cancer, neurodegeneration, and ischemia-reperfusion injury 41 . GPX4, an essential antioxidant enzyme, reduces lipid peroxidation, hence averting cellular apoptosis. SLC7A11 is an essential element of the cellular antioxidant defence mechanism, regulating intracellular cysteine concentrations and glutathione production, both of which are necessary for GPX4 function. SLC40A1 modulates iron export, inhibiting iron buildup and the production of detrimental lipid radicals. Alterations in the expression or function of these proteins may induce ferroptosis, underscoring their potential as therapeutic targets. In cancer cells, the overexpression of SLC7A11 correlates with chemoresistance, whereas the inhibition of GPX4 is proposed as a potential anticancer approach 42 . Modulating SLC40A1 expression influences iron metabolism in several illnesses, presenting a novel treatment approach. The downregulation of this mechanism in ferroptosis is associated with iron overload in streptozotocin-induced type 1 diabetes, indicating its crucial function in iron homeostasis. This downregulation may result in iron buildup and ferroptosis, perhaps contributing to cognitive impairment associated with diabetes 43 . Modulating SLC40A1 may offer innovative therapies for certain illnesses. The examination of biomarkers such as GPX4, SLC7A11, and SLC40A1 is essential, as it elucidates the mechanisms of ferroptosis and provides novel diagnostic and therapeutic approaches for disorders profoundly influenced by ferroptosis. Consequently, continuous investigation of these biomarkers is crucial for enhancing our comprehension and management of cellular death and survival mechanisms. The Expression of VDR and Its Relationship to the Effects of 1,25(OH)₂D₃ on NP Cells Under Ferroptotic Conditions The VDR is a nuclear receptor that predominantly operates via the genomic pathway, facilitating transcriptional activities within the cell nucleus. Moreover, VDR enables fast signalling through non-genomic routes, including the enhancement of transmembrane calcium transport 44 . In this investigation, VDR expression diminished in NP cells under ferroptosis conditions (Erastin group). Treatment with 1,25(OH)₂D₃ markedly enhanced VDR expression in NP cells, indicating that 1,25(OH)₂D₃ upregulates VDR and activates additional VDR. Pre-treatment with 1,25(OH)₂D₃ reinstated diminished VDR levels under ferroptosis conditions, suggesting that VDR activation may be essential in mitigating ferroptosis in NP cells via 1,25(OH)₂D₃. Oxidative stress can trigger ferroptosis in NP cells via an autophagy-dependent mechanism. 1,25(OH)₂D₃ suppressed autophagy in disc cells through VDR activation and mTOR/p70S6k pathway suppression 18 . Decreased VDR levels may lead to age-related LIDD, while VDR activation could protect disc cells from oxidative stress-induced death by maintaining mitochondrial activity. Activating the VDR enhances mitochondrial autophagy and protection, thereby decreasing apoptosis in NP cells. This activation augments the expression of the PINK1/Parkin pathway, essential for mitochondrial autophagy. Inhibiting this pathway diminishes VDR's protective effects on mitochondria, indicating that VDR activation may mitigate apoptosis in NP cells by enhancing PINK1/Parkin-dependent mitochondrial autophagy during oxidative stress 45 . In summary, autophagy is a multifaceted intracellular degradation mechanism that has a dual function in modulating cellular survival and death, potentially competing with or facilitating apoptosis 46 . In early phases, it may inhibit ferroptosis in NP cells, however in the latter stages of the disease, excessive autophagy may result in cell death. Vitamin D3 may influence the autophagy process by activating the VDR, hence serving a protective function in LIDD. To further ascertain whether 1,25(OH)₂D₃ influences the viability and ferroptosis of Erastin-induced NP cells via VDR activation, we employed siRNA to inhibit VDR gene expression in NP cells. The findings indicated that siRNA effectively inhibited VDR expression. Erastin induction led to VDR gene silencing, which resulted in heightened NP cell mortality, as evidenced by a decrease in viable cells and an elevation in LDH activity. Although 1,25(OH)₂D₃ continued to enhance NP cell proliferation, its efficacy was reduced by VDR inhibition, indicating that 1,25(OH)₂D₃ may partially augment NP cell viability and stimulate proliferation through VDR activation. Furthermore, we examined the concentrations of GPX4, lipid ROS, MDA, SOD, GSH, SLC7A11, and SLC40A1 in cells pre-treated and non-pre-treated with 1,25(OH)₂D₃ following siRNA interference. The findings indicated that 1,25(OH)₂D₃ continued to prevent ferroptosis following VDR gene silencing, as seen by the increase of GPX4, SLC7A11, and SLC40A1 expression, alongside a decrease in lipid ROS and MDA levels. The inhibitory effect of 1,25(OH)₂D₃ was diminished following VDR gene silencing compared to before VDR inhibition, indicating that the suppression of ferroptosis in NP cells by 1,25(OH)₂D₃ is partially reliant on VDR activation. The research underscores that the VDR substantially affects the impact of 1,25(OH)₂D₃ on NP cells in terms of viability and ferroptosis. Inhibition of VDR diminishes the protective function of 1,25(OH)₂D₃, elucidating the complex mechanism by which 1,25(OH)₂D₃ combats NP cell ferroptosis and highlighting the essential involvement of VDR in this process. Conclusions In conclusion, this study effectively established the optimal conditions under which 1,25(OH)₂D₃ enhances the proliferation of rat NP cells. Utilizing Erastin to mimic ferroptosis conditions, it was demonstrated that 1,25(OH)₂D₃ reduces ferroptosis in NP cells by upregulating ferroptosis-protective proteins and antioxidants while decreasing oxidative stress markers. Notably, a reduction in VDR expression was observed under ferroptotic conditions. The subsequent VDR gene silencing experiments revealed that while 1,25(OH)₂D₃ continues to mitigate ferroptosis, its efficacy is diminished without VDR activation, highlighting the significance of VDR in its protective mechanism (Fig. 9). This opens avenues for further exploration into additional pathways by which 1,25(OH)₂D₃ may exert its therapeutic effects. Declarations Data a vailability The data supporting the study results are available from the corresponding author upon reasonable request. Author contributions LQ contributed to project design, performed the experiments, and provided financial support. PJ contributed to the experiments, collected analysis data, and wrote the manuscript. DF reviewed the manuscript and contributed to its preparation. All authors read and approved the final manuscript. Funding This research has been supported by the National Natural Science Foundation of China (Grant No.: 81601943), and the China Hubei Provincial Health Commission Research Project(Grant No.: WJ2019M023). Conflict of interest The authors declare no conflict of interest. References Maher, C., Underwood, M. & Buchbinder, R. Non-specific low back pain. Lancet 389 , 736–747 (2017). Knezevic, N. N., Candido, K. D., Vlaeyen, J. W. S., Zundert, J. & Cohen, S. P. Low back pain. Lancet 398 , 78–92 (2021). Cao, C. et al. Bone marrow mesenchymal stem cells slow intervertebral disc degeneration through the NF-κB pathway. Spine J 15 , 530–538 (2015). Millecamps, M. & Stone, L. S. Delayed onset of persistent discogenic axial and radiating pain after a single-level lumbar intervertebral disc injury in mice. Pain 159 , 1843–1855 (2018). Liu, J., Yu, J., Jiang, W., He, M. & Zhao, J. Targeting of CDKN1B by miR-222-3p may contribute to the development of intervertebral disc degeneration. 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Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell 171 , 273–285 (2017). Xiong, Y. et al. The Regulatory Role of Ferroptosis in Bone Homeostasis. Stem Cells Int 2022 , (2022). Zhang, X. et al. Homocysteine induces oxidative stress and ferroptosis of nucleus pulposus via enhancing methylation of GPX4. Free Radic Biol Med 160 , 552–565 (2020). Yang, R. Z. et al. Involvement of oxidative stress-induced annulus fibrosus cell and nucleus pulposus cell ferroptosis in intervertebral disc degeneration pathogenesis. J Cell Physiol 236 , 2725–2739 (2021). Misof, B. M. et al. No Role of Osteocytic Osteolysis in the Development and Recovery of the Bone Phenotype Induced by Severe Secondary Hyperparathyroidism in Vitamin D Receptor Deficient Mice. Int J Mol Sci 21 , (2020). Rosen, C. J. et al. The nonskeletal effects of vitamin D: an Endocrine Society scientific statement. Endocr Rev 33 , 456–492 (2012). Tong, T. et al. Age-dependent expression of the vitamin D receptor and the protective effect of vitamin D receptor activation on H(2)O(2)-induced apoptosis in rat intervertebral disc cells. J Steroid Biochem Mol Biol 190 , 126–138 (2019). Dong, J. et al. Calcitriol protects renovascular function in hypertension by down-regulating angiotensin II type 1 receptors and reducing oxidative stress. Eur Heart J 33 , 2980–2990 (2012). Withanage, N. D., Perera, S., Peiris, H. & Athiththan, L. V. Serum 25-hydroxyvitamin D, serum calcium and vitamin D receptor (VDR) polymorphisms in a selected population with lumbar disc herniation-A case control study. PLoS One 13 , (2018). Xu, H. W. et al. Does vitamin D status influence lumbar disc degeneration and low back pain in postmenopausal women? A retrospective single-center study. Menopause 27 , 586–592 (2020). Krasowska, K. et al. The Preoperative Supplementation With Vitamin D Attenuated Pain Intensity and Reduced the Level of Pro-inflammatory Markers in Patients After Posterior Lumbar Interbody Fusion. Front Pharmacol 10 , (2019). Zhang, C., Tong, T., Miao, D. C. & Wang, L. F. Vitamin D inhibits TNF-α induced apoptosis of human nucleus pulposus cells through regulation of NF-kB signaling pathway. J Orthop Surg Res 16 , (2021). Videman, T. et al. Determinants of the progression in lumbar degeneration: a 5-year follow-up study of adult male monozygotic twins. Spine 31 , 671–678 (2006). Biczo, A., Szita, J., McCall, I., Varga, P. P. & Lazary, A. Association of vitamin D receptor gene polymorphisms with disc degeneration. Eur Spine J 29 , 596–604 (2020). Pękala, P. A. et al. Vitamin D receptor gene polymorphism influence on lumbar intervertebral disc degeneration. Clin Anat 35 , 738–744 (2022). Li, Z. et al. Notch3 regulates ferroptosis via ROS-induced lipid peroxidation in NSCLC cells. FEBS Open Bio 12 , 1197–1205 (2022). Vo, N. et al. An overview of underlying causes and animal models for the study of age-related degenerative disorders of the spine and synovial joints. J Orthop Res 31 , 831–837 (2013). Feng, C. et al. Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways. Cell Cycle 15 , 1674–1684 (2016). Cheng, F. et al. The role of oxidative stress in intervertebral disc cellular senescence. Front Endocrinol (Lausanne 13 , (2022). Wen, P. et al. The role of ageing and oxidative stress in intervertebral disc degeneration. Front Mol Biosci 9 , (2022). Valgimigli, L. Lipid Peroxidation and Antioxidant Protection. Biomolecules 13 , (2023). Wang, Y. et al. Oxidative stress in intervertebral disc degeneration: Molecular mechanisms, pathogenesis and treatment. Cell Proliferation 56 , e13448 (2023). Lu, S. et al. Ferroportin-Dependent Iron Homeostasis Protects against Oxidative Stress-Induced Nucleus Pulposus Cell Ferroptosis and Ameliorates Intervertebral Disc Degeneration In Vivo. Oxid Med Cell Longev 2021 , (2021). Herrera, M. T. et al. High Vitamin D Concentrations Restore the Ability to Express LL37 by M. tuberculosis-Infected Human Macrophages. Biomolecules 12 , (2022). Zhang, L. et al. Effects of the NF‐κB/p53 signaling pathway on intervertebral disc nucleus pulposus degeneration. Mol Med Rep 22 , 1821–1830 (2020). Zhang, Y. et al. A20 regulates inflammation through autophagy mediated by NF-κB pathway in human nucleus pulposus cells and ameliorates disc degeneration in vivo. Biochem Biophys Res Commun 549 , 179–186 (2021). Wang, P. et al. Sirt1 protects against intervertebral disc degeneration induced by 1,25-dihydroxyvitamin D insufficiency in mice by inhibiting the NF-κB inflammatory pathway. J Orthop Translat 40 , 13–26 (2023). Huang, H. et al. Vitamin D retards intervertebral disc degeneration through inactivation of the NF-κB pathway in mice. Am J Transl Res 11 , 2496–2506 (2019). Gao, M. et al. Role of Mitochondria in Ferroptosis. Mol Cell 73 , 354–363 (2019). Jiang, X., Stockwell, B. R. & Conrad, M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 22 , 266–282 (2021). Janssen, E. M. et al. Analysis of Patient Preferences in Lung Cancer - Estimating Acceptable Tradeoffs Between Treatment Benefit and Side Effects. Patient Prefer Adherence 14 , 927–937 (2020). Hao, L. et al. SLC40A1 Mediates Ferroptosis and Cognitive Dysfunction in Type 1 Diabetes. Neuroscience 463 , 216–226 (2021). Bikle, D. D. Vitamin D metabolism, mechanism of action, and clinical applications. Chem Biol 21 , 319–329 (2014). Lan, T., Yan, B., Guo, W., Shen, Z. & Chen, J. VDR promotes nucleus pulposus cell mitophagy as a protective mechanism against oxidative stress injury. Free Radic Res 56 , 316–327 (2022). Gump, J. M. & Thorburn, A. Autophagy and apoptosis: what is the connection? Trends Cell Biol 21 , 387–392 (2011). Additional Declarations No competing interests reported. Supplementary Files supplementary.pdf Cite Share Download PDF Status: Published Journal Publication published 07 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 01 Jan, 2025 Reviews received at journal 29 Dec, 2024 Reviews received at journal 27 Dec, 2024 Reviewers agreed at journal 22 Dec, 2024 Reviewers agreed at journal 21 Dec, 2024 Reviewers invited by journal 20 Dec, 2024 Editor assigned by journal 20 Dec, 2024 Editor invited by journal 20 Dec, 2024 Submission checks completed at journal 19 Dec, 2024 First submitted to journal 09 Dec, 2024 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. 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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-5612461","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":393420951,"identity":"be3a0fee-6cb3-49c7-9c88-3ee92af4ef61","order_by":0,"name":"Qiang Li","email":"","orcid":"","institution":"Department of Spine Surgery, Wuhan Puren Hospital, Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Li","suffix":""},{"id":393420955,"identity":"eec485a3-a1c1-495f-a770-ae78be6526b0","order_by":1,"name":"Jing Peng","email":"","orcid":"","institution":"Department of Orthopedics, The Third Affiliated Hospital of Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Peng","suffix":""},{"id":393420957,"identity":"b295e60b-ca36-433d-99a3-45601a8a59d4","order_by":2,"name":"Fan Ding","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACeWb+Dwc+/pGw42dvIFKLYXuD4cGZDTbJkj0HiLXmzAHjw7wNaYwbbiQQqYNxRkLCwZk7DjMb3Hy88QZDjU00QS3sEgkHDnw8c5hP8nZasQXDsbTcBsK2JDYcnMF2mJnvdo6ZBGPDYcJaGG4kMxzmYTvM2HDzDLFazhxjOMzblsY44QYPkVoM23sYDs44AwpkoF8SiPGLPDMP84cPFaCoPLzxxocaGyIchgQMJBJIUQ7RQqqOUTAKRsEoGBkAAIIvR+6fsIbeAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Spine Surgery, Wuhan Puren Hospital, Wuhan University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Fan","middleName":"","lastName":"Ding","suffix":""}],"badges":[],"createdAt":"2024-12-10 02:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5612461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5612461/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-92405-x","type":"published","date":"2025-03-07T15:57:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72146097,"identity":"db967b3f-46c3-40fe-9850-6ed81aaf90e1","added_by":"auto","created_at":"2024-12-23 07:29:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":745582,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic Comparison of Cell Number and Growth Conditions in Experimental and Control Groups. (a) Cell morphology in the blank control group, (b) Cell morphology in the 1,25(OH)₂D₃ treated group, (c) Cell morphology in the Erastin-treated group, (d) Cell morphology in the 1,25(OH)₂D₃ and Erastin-treated group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/4a0cf912742f20b26b6f0ad3.png"},{"id":72147875,"identity":"99e679fc-d35d-4a1f-ba79-f444c4f03ca2","added_by":"auto","created_at":"2024-12-23 07:45:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41713,"visible":true,"origin":"","legend":"\u003cp\u003eEnhancement of NP cells Proliferation by 1,25(OH)₂D₃ under Ferroptotic Conditions. (a) Cell viability assessed by CCK8 assay across different groups, (b) LDH activity measured by assay kit (Mean±SD, n=3, ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/51a2a89d4b3433095883c98b.png"},{"id":72146098,"identity":"8cfbdbc3-7876-4795-9613-b63724f8319e","added_by":"auto","created_at":"2024-12-23 07:29:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73625,"visible":true,"origin":"","legend":"\u003cp\u003eAugmentation of GPX4 Expression in NP cells by 1,25(OH)₂D₃ under Ferroptotic Conditions. (a) GPX4 expression levels evaluated by RT-qPCR, (b) Western blot quantification of GXP4 protein levels, (C) representative Western blot images. (Mean±SD, n=3, ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/fbe148c33d0312ca2262bd13.png"},{"id":72146101,"identity":"918b3aed-0469-4310-a795-2be52f3fa7a2","added_by":"auto","created_at":"2024-12-23 07:29:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":88171,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 1,25(OH)₂D₃ on Alleviating Intracellular Oxidative Stress and Regulating Ferroptosis-Related Gene Expression in Ferroptotic Environment. (a) Flow cytometry analysis of intracellular lipid ROS levels, (b) Intracellular SOD levels measured using an assay kit, (c) Intracellular GSH levels measured using an assay kit, (d) Intracellular MDA levels measured using an assay kit, (e) Relative mRNA expression levels of SLC7A11 analyzed by RT-qPCR, (f) Relative mRNA expression levels of SLC40A1 analyzed by RT-qPCR. (Mean±SD, n=3. **P\u0026lt;0.01, ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/76652564e9f917686e310c46.png"},{"id":72146115,"identity":"e5962f8e-68dc-41c2-a198-986e028a65e6","added_by":"auto","created_at":"2024-12-23 07:29:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":408905,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission Electron Microscopy Observation of Mitochondrial Morphology in NP cells.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/3fd0e3cc5a615d6f69b3765d.png"},{"id":72146114,"identity":"05eb327d-c747-444c-9451-75fc95658c61","added_by":"auto","created_at":"2024-12-23 07:29:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59572,"visible":true,"origin":"","legend":"\u003cp\u003eRestoration of VDR Expression by 1,25(OH)₂D₃ in Ferroptotic Environment. (a) RT-qPCR analysis of VDR expression, (b) Western blot quantification of VDR protein levels, (c) representative Western blot images (Mean±SD, n=3. **P\u0026lt;0.01; ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/fc19ffde69025543f1c3d50b.png"},{"id":72146105,"identity":"449499e5-0987-414c-9a15-1fc9c3aaef7f","added_by":"auto","created_at":"2024-12-23 07:29:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":61607,"visible":true,"origin":"","legend":"\u003cp\u003e1,25(OH)₂D₃ Potentially Increases Intracellular GPX4 Expression Partially Through VDR Activation. (a) RT-qPCR analysis of GPX4 expression, (b) Western blot quantification of GXP4 protein levels, (c) representative Western blot images. (Mean±SD, n=3. *P\u0026lt;0.05; ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/b131e41e452874acd2e2abc1.png"},{"id":72146116,"identity":"af196756-faaf-4e17-9907-b614eaf8e29e","added_by":"auto","created_at":"2024-12-23 07:29:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":81219,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 1,25(OH)₂D₃ on Alleviating Intracellular Oxidative Stress and Regulating Ferroptosis-Related Gene Expression in Ferroptotic Environment. (a) Flow cytometry analysis of intracellular lipid ROS levels, (b) Intracellular SOD levels measured using an assay kit, (c) Intracellular GSH levels measured using an assay kit, (d) Intracellular MDA levels measured using an assay kit, (e) Relative mRNA expression levels of SLC7A11 analyzed by RT-qPCR, (f) Relative mRNA expression levels of SLC40A1 analyzed by RT-qPCR. (Mean±SD, n=3. **P\u0026lt;0.01, ***P\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/ddf2216d40ce95337a3bc006.png"},{"id":72146348,"identity":"b9f1f695-0ab3-4571-93b2-34178c5504e8","added_by":"auto","created_at":"2024-12-23 07:37:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":124086,"visible":true,"origin":"","legend":"\u003cp\u003e1,25(OH)₂D₃ inhibit Ferroptosis in NP cells (PDB 2HAS).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/33ac9d57978e4d31aaf55700.png"},{"id":78181506,"identity":"c864663a-e92f-4d93-8e8b-5a7f2703a6cf","added_by":"auto","created_at":"2025-03-10 17:46:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2794800,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/151da1d2-ba57-420d-a95c-23413b8cf497.pdf"},{"id":72146104,"identity":"8fa7d45e-499a-4e6a-b562-d1f282c35bfc","added_by":"auto","created_at":"2024-12-23 07:29:57","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":356824,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5612461/v1/7f35c572060cfc5d31c1621f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unlocking the Therapeutic Potential of 1,25(OH)₂D₃: Targeting Ferroptosis to Alleviate Lumbar Intervertebral Disc Degeneration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLow back pain is a progressively critical global health concern, impacting over 80% of the global population and imposing substantial social and economic expenses\u003csup\u003e1,2\u003c/sup\u003e. Intervertebral disc degeneration is a significant contributor to low back pain\u003csup\u003e3\u003c/sup\u003e. LIDD encompasses a range of structural and tissue alterations, including decreased disc height, NP fissures, tears in the annulus fibrosus\u0026nbsp;(AF), calcification of the cartilaginous endplate, and disrupted extracellular matrix\u0026nbsp;(ECM)\u0026nbsp;metabolism\u003csup\u003e4,5\u003c/sup\u003e. The precise mechanisms underlying LIDD remain unclear. Nonetheless, it is commonly accepted that its progression begins and speeds up due to the depletion of NP cells and the degradation of the ECM\u003csup\u003e5\u003c/sup\u003e. NP cells play a key role in the synthesis and breakdown of the ECM, serving as essential components of the intervertebral disc, which is important for preserving its structure and physiological functions. The deterioration and impaired function of NP cells play crucial roles in the development of LIDD\u003csup\u003e6\u003c/sup\u003e. Consequently, examining NP cells to clarify the pathogenesis of LIDD holds considerable clinical significance\u003csup\u003e7\u003c/sup\u003e. Previous studies indicate that various factors of disc degeneration could be associated with apoptosis, as a decline in cell numbers results in lower ECM protein production and disturbances in cellular energy metabolism along with other physiological functions\u003csup\u003e8\u003c/sup\u003e. As a result, treatment for LIDD mainly aims to avert cell loss caused by programmed or regulated cell death. Recent studies show that different types of cell death, such as apoptosis, pyroptosis, necroptosis, autophagy, ferroptosis, and senescence, play a role in the progression of LIDD, providing fresh avenues for treatment\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFerroptosis, first introduced in 2012 as a new type of programmed cell death, is unique due to its reliance on iron and is marked by a significant buildup of lipid reactive oxygen species\u0026nbsp;(ROS)\u003csup\u003e10\u003c/sup\u003e. It can be distinguished morphologically and biochemically from apoptosis, necrosis, autophagy-dependent cell death, and pyroptosis\u003csup\u003e11\u003c/sup\u003e. Ferroptosis is associated with the pathologies of several diseases, including cancer, stroke, cerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and degenerative diseases such as Alzheimer's and Parkinson's\u003csup\u003e12\u003c/sup\u003e. Morphologically, ferroptosis is characterized by the early preservation of cell membrane integrity, lipid peroxidation within cell membranes, alterations in mitochondria\u0026nbsp;(including shrinkage, diminished mitochondrial cristae, and rupture of the outer membrane), lack of chromatin condensation, cytosolic vesiculation, and a reduction in cell volume or atrophy\u003csup\u003e11,13\u003c/sup\u003e. Comprehensive studies have shown a notable connection between ferroptosis and LIDD, highlighting the potential for effective therapies in the treatment of LIDD\u003csup\u003e12,14,15\u003c/sup\u003e. Research on disc degeneration has shown that ferroptosis inhibitors like ferrostatin-1 and deferoxamine can mitigate degeneration in a TBHP-induced rat model. Furthermore, ferroptosis inducers TBHP and RSL3 have comparable effects on cell death in AFCs and NP cells, which can be blocked by ferroptosis inhibitors, highlighting the significance of ferroptosis in the mechanism of LIDD\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eVitamin D is essential for regulating serum calcium and phosphorus levels and is found mainly in two forms, D2 and D3\u003csup\u003e16\u003c/sup\u003e. Vitamin D3\u0026nbsp;(VD3), or cholecalciferol, is produced from 7-dehydrocholesterol in the skin when exposed to UV radiation or obtained from animal-derived foods. VD3 undergoes a two-step hydroxylation process to be converted into its active form, calcitriol\u0026nbsp;(1,25(OH)₂D₃)\u003csup\u003e17\u003c/sup\u003e. In addition to its traditional function in calcium balance, VD influences cell growth, differentiation, and programmed cell death\u003csup\u003e18\u003c/sup\u003e.\u0026nbsp;1,25(OH)₂D₃ acts as an antioxidant, possibly mitigating ferroptosis induced by oxidative stress by regulating intracellular glutathione (GSH) and superoxide dismutase (SOD)\u003csup\u003e19\u003c/sup\u003e. Furthermore, numerous clinical studies suggest that a deficiency in vitamin D could hasten the progression of lumbar disc degeneration and elevate the risk of lumbar disc herniation\u003csup\u003e20,21\u003c/sup\u003e. Conversely, vitamin D supplementation may mitigate the degradation process and reduce the incidence of low back discomfort\u003csup\u003e22\u003c/sup\u003e.\u0026nbsp;1,25(OH)₂D₃ positively affects LIDD by diminishing inflammation, oxidative stress, and NP cell apoptosis, while also postponing NP cell aging\u003csup\u003e23\u003c/sup\u003e. The VDR plays a crucial role in the biological effects of 1,25(OH)₂D₃, being prevalent in cells of the intervertebral disc and linked to disc degeneration\u003csup\u003e24\u003c/sup\u003e. Genetic variations in VDR, including TaqI\u0026nbsp;(rs731236), FokI\u0026nbsp;(rs2228570), and ApaI\u0026nbsp;(rs7975232), are associated with a heightened risk of LIDD\u003csup\u003e25,26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFerroptosis is crucial in the progression of LIDD, and 1,25(OH)₂D₃, a variant of Vitamin D, might impede this process. This study investigates the potential of 1,25(OH)₂D₃ to mitigate LIDD by inhibiting ferroptosis in NP cells through VDR, aiming to reveal novel preventative approaches for LIDD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Culture and Maintenance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRat-derived NP cells, selected for their relevance in lumbar disc studies, were obtained from SAIBAIKANG Biosciences, Shanghai. Authenticated and mycoplasma-free, these cells were initially thawed at 37\u0026nbsp;°C and cultured in a medium supplemented with 10% fetal bovine serum\u0026nbsp;(FBS). The cells were cultivated in T25 flasks within a CO2 incubator under conditions optimal for NP cell growth. Cells were passaged when confluence exceeded 85%, including washing with PBS and detachment using trypsin-EDTA. Cell growth was monitored using an inverted microscope. For long-term preservation, cells were cryopreserved in a solution of 90% FBS and 10% DMSO, following a controlled-rate freezing protocol. The cells were initially stored at -80°C and transferred to a liquid nitrogen tank. Cell viability and concentration were assessed post-thawing using Trypan blue staining and a hemocytometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVDR Gene Silencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the inhibition of the rat VDR gene (Gene ID: 24873), three siRNAs (si-VDR-1, si-VDR-2, si-VDR-3) alongside a non-targeting control (si-Control) were employed. These were designed and synthesized by RiboBio Co., Ltd., with their sequences detailed in Supplementary Table S1. Bioinformatic analyses informed selection to optimize specificity and efficiency, and RT-qPCR and Western blot analysis confirmed gene silencing. The transfection protocol used cells seeded in 6-well plates to 60-70% confluence. siRNAs, diluted to 20 μM in DEPC-treated water, were mixed with Opti-MEM and Lipofectamine 2000 to form the transfection complex. This complex was incubated with the cells for 6 hours at 37\u0026nbsp;°C in a 5% CO2 atmosphere, followed by a medium replacement with a growth medium. The cells were then cultured for 72 hours to facilitate gene silencing, with post-transfection assessments confirming the effectiveness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Viability Assessment with CCK-8\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo inhibit the rat VDR gene (Gene ID: 24873), three siRNAs (si-VDR-1, si-VDR-2, si-VDR-3) and a non-targeting control (si-Control) were used. RiboBio Co., Ltd. designed and synthesized these siRNAs, with their sequences detailed in Supplementary Table S1. The selection was based on bioinformatic analyses to optimize specificity and efficiency, and RT-qPCR and Western blot analysis confirmed gene silencing. The transfection protocol seeded cells in 6-well plates to 60-70% confluence. The siRNAs, diluted to 20 μM in DEPC-treated water, were mixed with Opti-MEM and Lipofectamine 2000 to form the transfection complex. This complex was incubated with the cells for 6 hours at 37 °C in a 5% CO2 atmosphere, then replaced with a growth medium. The cells were cultured for 72 hours to facilitate gene silencing, and post-transfection assessments confirmed the effectiveness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Biochemical Markers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePost-treatment, cells\u0026nbsp;(2×10\u003csup\u003e5\u003c/sup\u003e per well)\u0026nbsp;were cultured in 6-well plates for 48 hours. For LDH activity assessment, supernatants collected after centrifugation\u0026nbsp;(2000g, 4\u0026nbsp;°C, 10 minutes)\u0026nbsp;were evaluated using an LDH assay kit\u0026nbsp;(Jiancheng Bioengineering Institute). Intracellular MDA, SOD, and GSH levels were quantified after cell ultrasonication and centrifugation. Protein concentration was determined using a BCA protein assay kit\u0026nbsp;(Aspen Biological)\u0026nbsp;for normalization. MDA content, SOD activity, and total GSH levels\u0026nbsp;(both oxidized and reduced forms)\u0026nbsp;were quantified using their respective assay kits\u0026nbsp;(Jiancheng Bioengineering Institute), following the manufacturer's protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Assessment of Gene Expression via RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted from cells using TRIzol-based TRIpure reagent\u0026nbsp;(ELK Biotechnology), which involved chloroform-induced phase separation and isopropanol precipitation. The purified RNA was converted to cDNA using the EntiLink™ 1st Strand cDNA Synthesis Super Mix\u0026nbsp;(ELK Biotechnology). Quantitative PCR was performed using a QuantStudio 6 Flex Real-Time PCR System\u0026nbsp;(Life Technologies)\u0026nbsp;with EnTurbo™ SYBR Green PCR SuperMix. The protocol included initial denaturation, 40 amplification cycles, and melt curve analysis for product verification. Gene expression was analyzed using the comparative ΔΔCT method, normalizing the expression of R-SLC7A11, R-SLC40A1, R-VDR, and R-GPX4 to R-β-actin. The primer sequences listed in Supplemental Table S2 were designed to span exon-exon junctions to avoid amplification of genomic DNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry for Lipid ROS Detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry was employed for intracellular lipid ROS quantification. Following trypsinization and centrifugation, cells were stained with DCFH-DA, a ROS-sensitive fluorogenic dye. After a 20-minute incubation at 37\u0026nbsp;°C with intermittent inversion for uniform dye distribution, cells were washed three times with serum-free medium to remove excess dye and then resuspended for analysis. Flow cytometric analysis was conducted using a BD flow cytometer with a 488 nm excitation and 525 nm emission filter. Stained and unstained controls were included for calibration. Fluorescence intensities, indicative of lipid ROS levels, were measured in triplicate. Data acquisition and analysis were performed using cytometry software to evaluate oxidative stress levels in response to different treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis for Protein Expression Profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor Western blotting, adherent cells were rinsed with ice-cold PBS and lysed. The supernatant, after centrifugation, was used for protein quantification using a BCA assay. Proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were then blocked to prevent non-specific binding. Membranes were incubated with primary antibodies\u0026nbsp;(Supplementary Table S3)\u0026nbsp;overnight at 4\u0026nbsp;°C, followed by HRP-conjugated secondary antibodies\u0026nbsp;(Supplementary Table S4)\u0026nbsp;for 30 minutes at room temperature. Bands were visualized using an ECL kit and imaged on X-ray film. Band intensities were quantified using AlphaEaseFC software and normalized against a housekeeping protein. To ensure specificity, membranes were re-probed, involving stripping and re-blocking.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy for Mitochondrial Morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNP cells were fixed overnight at 4\u0026nbsp;°C in a fixative solution, followed by PBS washing. Cells were stained with osmium tetroxide for two hours at room temperature to enhance membrane contrast. Cells underwent a graduated dehydration process using ethanol and acetone, were then embedded in epoxy resin, and polymerized. Ultra-thin sections\u0026nbsp;(~70 nm)\u0026nbsp;were prepared and stained with uranyl acetate and lead citrate for ultrastructural visualization. Mitochondrial morphology was examined using a transmission electron microscope\u0026nbsp;(TEM), which provided detailed insights into the cellular environment of the NP cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using SPSS software (version 23.0) and GraphPad Prism to ensure analytical robustness and reproducibility. Results are presented as mean ± standard deviation (SD). A one-way Analysis of Variance (ANOVA) or Student's t-test was employed to assess group differences, contingent upon the data's distribution. Post-hoc tests were conducted where necessary for detailed analysis. Statistical significance was indicated as *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001, with adjustments for multiple comparisons to minimize type I errors. Data visualization included bar graphs and scatter plots, with a predefined significance threshold of p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffect of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1,25(OH)₂D₃\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;on the Viability of NP Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRat NP cells were subjected to treatment with different concentrations of 1,25(OH)₂D₃\u0026nbsp;(0, 0.01, 0.1, 1, 10, 100, and 1000 nmol/L)\u0026nbsp;for durations of 6, 12, 24, and 48 hours. The CCK8 assay was employed to evaluate cell viability. The results showed that NP cells exhibited the highest viability at 48 hours when exposed to a concentration of 10 nmol/L of 1,25(OH)₂D₃\u0026nbsp;(Supplementary\u0026nbsp;Fig.\u0026nbsp;S1). This indicates that 1,25(OH)₂D₃ has the potential to enhance NP cell proliferation, with the optimal concentration and duration being 10 nmol/L and 48 hours, respectively. The chosen conditions were set for the subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1,25(OH)₂D₃ on the Viability of NP Cells\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etreated with Erastin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eErastin was employed to replicate an in vivo ferroptosis environment and to trigger ferroptosis in NP cells\u003csup\u003e27\u003c/sup\u003e. The study had four groups: control,\u0026nbsp;1,25(OH)₂D₃, Erastin, and\u0026nbsp;1,25(OH)₂D₃\u0026nbsp;combined with Erastin. The Erastin cohort received 10 μmol/L Erastin for a duration of 24 hours. The\u0026nbsp;1,25(OH)₂D₃+Erastin cohort comprised NP cells that were pre-treated with 10 nmol/L\u0026nbsp;1,25(OH)₂D₃\u0026nbsp;for 48 hours, then receiving treatment with 10 μmol/L Erastin for 24 hours. Microscopic examination revealed that NP cells in the Erastin group exhibited spindle-shaped, fusiform, and irregular morphologies, with sparse distribution and evidence of cell death, in contrast to the normal polygonal cells observed in the control group. Conversely, the\u0026nbsp;1,25(OH)₂D₃+Erastin group exhibited considerable enhancements in cell proliferation, mirroring the appearance of normal NP cells observed in the control group\u0026nbsp;(Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eIn a CCK8 experiment assessing cell viability across the four NP cell groups, the\u0026nbsp;1,25(OH)₂D₃+Erastin group exhibited a considerably higher number of viable cells than the Erastin group\u0026nbsp;(Fig.\u0026nbsp;2a). Lactate dehydrogenase\u0026nbsp;(LDH)\u0026nbsp;activity in cell supernatants was elevated in the Erastin group relative to the control, while a significant reduction was observed in the\u0026nbsp;1,25(OH)₂D₃+Erastin group compared to the Erastin group\u0026nbsp;(Fig.\u0026nbsp;2b). This suggests that\u0026nbsp;1,25(OH)₂D₃\u0026nbsp;promotes NP cell growth and viability in ferroptosis conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1,25(OH)₂D₃ on Ferroptosis in NP Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess whether 1,25(OH)₂D₃ could mitigate ferroptosis in NP cells, the research evaluated intracellular levels of GPX4, lipid ROS, MDA, SOD, and GSH in NP cells subjected to Erastin\u0026nbsp;and following 1,25(OH)₂D₃ administration. The findings indicated that pretreatment with 1,25(OH)₂D₃ led to a significant increase in GPX4, SOD\u0026nbsp;and GSH\u0026nbsp;(Fig.\u0026nbsp;3 and 4b-c)\u0026nbsp;levels, while markedly reducing lipid ROS\u0026nbsp;(Fig.\u0026nbsp;4a)\u0026nbsp;and MDA\u0026nbsp;(Fig.\u0026nbsp;4d)\u0026nbsp;levels in NP cells when compared to the Erastin group, indicating reduced oxidative stress in cells treated with\u0026nbsp;1,25(OH)₂D₃.\u0026nbsp;This suggests a reduction in oxidative stress in cells treated with 1,25(OH)₂D₃. RT-qPCR results indicated that, in comparison to the Erastin group, the 1,25(OH)₂D₃+Erastin group exhibited a notable increase in the expression levels of SLC7A11\u0026nbsp;and SLC40A1\u0026nbsp;(Fig.\u0026nbsp;4e-f). Furthermore, transmission electron microscopy observations of mitochondrial morphology in NP cells revealed that in the Erastin group, NP cells exhibited increased mitochondrial membrane density and decreased cristae. The mitochondrial morphology in the 1,25(OH)₂D₃+Erastin group was similar to that observed in both the blank control group and the 1,25(OH)₂D₃ group (Fig.\u0026nbsp;5). In conclusion, 1,25(OH)₂D₃ has the ability to reduce ferroptosis in NP cells when exposed to ferroptotic conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfluence of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1,25(OH)₂D₃ on VDR Expression in NP Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR results demonstrated a significant elevation of VDR in the 1,25(OH)₂D₃-treated group relative to the control, while VDR was substantially diminished in the Erastin-treated group (Fig. 6a). NP cells pre-treated with 1,25(OH)₂D₃ demonstrated a significant enhancement in VDR protein expression relative to untreated cells (Fig. 6b), underscoring a possible mechanism through VDR activation. Western blot examination confirmed these findings, revealing a marked reduction in VDR protein expression in NP cells under ferroptotic conditions, which was reinstated by 1,25(OH)₂D₃ treatment (Fig. 6c). The findings indicate that VDR activation is essential for the suppression of ferroptosis in NP cells mediated by 1,25(OH)₂D₃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1,25(OH)₂D₃ on Ferroptosis in NP Cells Induced by Erastin Following VDR Gene Silencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate that 1,25(OH)₂D₃ promotes NP cell survival via VDR activation, three siRNA sequences were designed by RiboBio Co., Ltd. (Guangzhou, China) to silence the VDR gene in NP cells. Subsequent to transfection, Western blot analysis confirmed the efficacy of RNA interference. siRNA-2 had the greatest efficacy, significantly reducing VDR gene expression (Supplementary Fig. S2), and was selected for subsequent investigation. The experiment had four groups: control siRNA+Erastin, control siRNA+1,25(OH)₂D₃+Erastin, VDR siRNA+Erastin and VDR siRNA+1,25(OH)₂D₃+Erastin. The study examined the concentrations of GPX4, lipid ROS, MDA, SOD, GSH, SLC7A11, and SLC40A1 in NP cells that were pre-treated with 1,25(OH)₂D₃, assessing the results both prior to and following siRNA interference. \u0026nbsp;The data indicated that, in contrast to the VDR siRNA+Erastin group, the VDR siRNA+1,25(OH)₂D₃+Erastin group had elevated expression of GPX4, SLC7A11, SLC40A1, SOD and GSH (Fig. 7, 8b-c and 8e-f), alongside a significant reduction in lipid ROS and MDA (Fig. 8a and 8d). This demonstrates that 1,25(OH)₂D₃ maintains its suppressive influence on NP cell ferroptosis despite VDR gene silencing. In comparison to the control siRNA+1,25(OH)₂D₃+Erastin group, the VDR siRNA+1,25(OH)₂D₃+Erastin group exhibited substantial decreases in GPX4, SLC7A11, SLC40A1, SOD and GSH, alongside elevated lipid ROS and MDA. This suggests that VDR gene silencing reduces the suppressive influence of 1,25(OH)₂D₃ on NP cell ferroptosis. The results substantiate that the anti-ferroptotic effect of 1,25(OH)₂D₃ in NP cells is partially contingent upon the activation of VDR.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eThe Relationship Between LIDD and NP Cells Ferroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manifestation and progression of LIDD are affected by factors including oxidative stress, trauma, infection, and inflammation\u003csup\u003e28,29\u003c/sup\u003e. Oxidative stress plays a pivotal role in the ageing of nuclear NP cells, inducing premature cellular senescence via DNA damage and telomere attrition resulting from replicative ageing\u003csup\u003e30,31\u003c/sup\u003e. As degeneration progresses, levels of lipid reactive oxygen species in NP cells markedly elevate, including superoxide anion, hydroxyl radical, hydrogen peroxide, and nitric oxide\u003csup\u003e32,33\u003c/sup\u003e. Elevated ROS levels accelerate LIDD through key signaling pathways, such as MAPK and NF-κB pathways. Increased ROS levels expedite LIDD via critical signalling pathways, including MAPK and NF-κB pathways. In oxidative stress circumstances, exemplified by a TBHP model, diminished expression of iron transporter proteins results in intracellular iron buildup, facilitating ferroptosis in NP cells and the subsequent progression of LIDD\u003csup\u003e34\u003c/sup\u003e. Homocysteine can elevate oxidative stress and ferroptosis in NP cells by augmenting GPX4 methylation, hence contributing to LIDD\u003csup\u003e14\u003c/sup\u003e. These investigations demonstrate that oxidative stress-induced ferroptosis is closely associated with LIDD. Mitigating NP cell ferroptosis may be a unique preventive approach to intervertebral disc degeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Effects of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1,25(OH)₂D₃ on Normal NP Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur work shown that 1,25(OH)₂D₃ significantly enhances the proliferation of rat NP cells, with the optimal concentration being 10 nmol/L at 48 hours. This discovery highlights the possible function of 1,25(OH)₂D₃ in facilitating NP cell proliferation. Notably, the cytoprotective effects of vitamin D3 seem to be unaffected by its concentration.\u0026nbsp;Subsequent observations indicate that elevated doses may induce detrimental effects, undermining its protective attributes. Increased concentrations of 1,25(OH)₂D₃ have been shown to enhance the ability of macrophages infected with Mycobacterium TB to synthesise the antibiotic peptide LL37\u003csup\u003e35\u003c/sup\u003e. The primary mechanism by which vitamin D prevents LIDD is its suppression of the NF-κB signalling pathway\u003csup\u003e36\u003c/sup\u003e. This pathway modulates the transcription of several genes, including pro-inflammatory cytokines such as IL-6, cell cycle regulators like cyclin D1, anti-apoptotic factors such as bcl-2, and extracellular proteases like MMP3. The mammalian NF-κB family, comprising RelA/p65, c-Rel, RelB, p50, and p52, produces many dimers, including the prevalent p50/RelA heterodimer, which activates multiple genes by binding to κB sites within gene promoters\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eInsufficient 1,25(OH)₂D₃ hinders matrix protein synthesis and hastens disc degeneration, a phenomenon associated with diminished SIRT1 expression in NP tissue. Augmenting SIRT1 inhibits the NF-κB pathway, hence averting degeneration. Vitamin D or resveratrol, through the activation of SIRT1, can mitigate these degenerative alterations\u003csup\u003e38\u003c/sup\u003e.\u0026nbsp;Efficacy of 1,25(OH)₂D₃ in mitigating intervertebral disc degeneration. This impact is mainly accomplished by blocking the NF-κB pathway, resulting in diminished inflammation, reduced oxidative stress, suppressed apoptosis, and delayed cellular ageing in the intervertebral discs\u003csup\u003e39\u003c/sup\u003e. These\u0026nbsp;results, corroborated by in vivo and in vitro studies, underscore the potential of vitamin D as an effective therapeutic agent for the treatment and management of disc degeneration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Impact of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1,25(OH)₂D₃ on NP Cells in a Ferroptotic Environment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince 2012, Erastin has been identified as a principal inducer of cellular ferroptosis and was originally employed to target neoplastic cells. The pathophysiological mechanisms are analogous to those in numerous injury-related diseases, resulting in its extensive application for treatment and prognostic evaluation of significant illnesses\u003csup\u003e11,12\u003c/sup\u003e. This study noted a substantial rise in LDH activity following Erastin induction, signifying reduced viability in NP cells. Erastin decreased intracellular levels of GPX4, SOD, and GSH, while elevating lipid ROS and MDA, so efficiently promoting ferroptosis. Treatment with 1,25(OH)₂D₃ significantly improved the growth, viability, and proliferation of Erastin-treated NP cells, increased levels of GPX4, SOD, and GSH, and reduced lipid ROS and MDA. SLC7A11 is a crucial subunit of the Xc system, an intracellular antioxidant mechanism vital for sustaining GPX4 activity, whereas SLC40A1 facilitates cellular iron efflux and inhibits intracellular iron accumulation, both of which are proteins that counteract ferroptosis\u003csup\u003e9\u003c/sup\u003e. The findings indicated that the expression levels of SLC7A11 and SLC40A1 were markedly elevated in the 1,25(OH)₂D₃+Erastin group relative to the Erastin group. Consequently, 1,25(OH)₂D₃ administration mitigated oxidative stress in NP cells. The mitochondria serve as the primary site of cellular oxidative stress, making mitochondrial alterations a significant aspect of the ferroptosis process\u003csup\u003e40\u003c/sup\u003e. This research employed Transmission Electron Microscopy (TEM) to examine the mitochondrial morphology of NP cells across several groups. The research indicated that the Erastin group exhibited heightened mitochondrial membrane density and reduced cristae in NP cells. Conversely, NP cells subjected to 1,25(OH)₂D₃ exhibited mitochondrial traits akin to those of the control group. This verifies that 1,25(OH)₂D₃ efficiently mitigates ferroptosis in NP cells under oxidative stress conditions.\u003c/p\u003e\n\u003cp\u003eGPX4, SLC7A11, and SLC40A1, recognised as indicators of ferroptosis, have garnered interest in conditions including cancer, neurodegeneration, and ischemia-reperfusion injury\u003csup\u003e41\u003c/sup\u003e. GPX4, an essential antioxidant enzyme, reduces lipid peroxidation, hence averting cellular apoptosis. SLC7A11 is an essential element of the cellular antioxidant defence mechanism, regulating intracellular cysteine concentrations and glutathione production, both of which are necessary for GPX4 function. SLC40A1 modulates iron export, inhibiting iron buildup and the production of detrimental lipid radicals. Alterations in the expression or function of these proteins may induce ferroptosis, underscoring their potential as therapeutic targets. In cancer cells, the overexpression of SLC7A11 correlates with chemoresistance, whereas the inhibition of GPX4 is proposed as a potential anticancer approach\u003csup\u003e42\u003c/sup\u003e. Modulating SLC40A1 expression influences iron metabolism in several illnesses, presenting a novel treatment approach. The downregulation of this mechanism in ferroptosis is associated with iron overload in streptozotocin-induced type 1 diabetes, indicating its crucial function in iron homeostasis. This downregulation may result in iron buildup and ferroptosis, perhaps contributing to cognitive impairment associated with diabetes\u003csup\u003e43\u003c/sup\u003e. Modulating SLC40A1 may offer innovative therapies for certain illnesses. The examination of biomarkers such as GPX4, SLC7A11, and SLC40A1 is essential, as it elucidates the mechanisms of ferroptosis and provides novel diagnostic and therapeutic approaches for disorders profoundly influenced by ferroptosis. Consequently, continuous investigation of these biomarkers is crucial for enhancing our comprehension and management of cellular death and survival mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Expression of VDR and Its Relationship to the Effects of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1,25(OH)₂D₃ on NP Cells Under Ferroptotic Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe VDR is a nuclear receptor that predominantly operates via the genomic pathway, facilitating transcriptional activities within the cell nucleus. Moreover, VDR enables fast signalling through non-genomic routes, including the enhancement of transmembrane calcium transport\u003csup\u003e44\u003c/sup\u003e. In this investigation, VDR expression diminished in NP cells under ferroptosis conditions (Erastin group). Treatment with 1,25(OH)₂D₃ markedly enhanced VDR expression in NP cells, indicating that 1,25(OH)₂D₃ upregulates VDR and activates additional VDR. Pre-treatment with 1,25(OH)₂D₃ reinstated diminished VDR levels under ferroptosis conditions, suggesting that VDR activation may be essential in mitigating ferroptosis in NP cells via 1,25(OH)₂D₃. Oxidative stress can trigger ferroptosis in NP cells via an autophagy-dependent mechanism. 1,25(OH)₂D₃ suppressed autophagy in disc cells through VDR activation and mTOR/p70S6k pathway suppression\u003csup\u003e18\u003c/sup\u003e. Decreased VDR levels may lead to age-related LIDD, while VDR activation could protect disc cells from oxidative stress-induced death by maintaining mitochondrial activity. Activating the VDR enhances mitochondrial autophagy and protection, thereby decreasing apoptosis in NP cells. This activation augments the expression of the PINK1/Parkin pathway, essential for mitochondrial autophagy. Inhibiting this pathway diminishes VDR's protective effects on mitochondria, indicating that VDR activation may mitigate apoptosis in NP cells by enhancing PINK1/Parkin-dependent mitochondrial autophagy during oxidative stress\u003csup\u003e45\u003c/sup\u003e. In summary, autophagy is a multifaceted intracellular degradation mechanism that has a dual function in modulating cellular survival and death, potentially competing with or facilitating apoptosis\u003csup\u003e46\u003c/sup\u003e. In early phases, it may inhibit ferroptosis in NP cells, however in the latter stages of the disease, excessive autophagy may result in cell death. Vitamin D3 may influence the autophagy process by activating the VDR, hence serving a protective function in LIDD. To further ascertain whether 1,25(OH)₂D₃ influences the viability and ferroptosis of Erastin-induced NP cells via VDR activation, we employed siRNA to inhibit VDR gene expression in NP cells. The findings indicated that siRNA effectively inhibited VDR expression. Erastin induction led to VDR gene silencing, which resulted in heightened NP cell mortality, as evidenced by a decrease in viable cells and an elevation in LDH activity. Although 1,25(OH)₂D₃ continued to enhance NP cell proliferation, its efficacy was reduced by VDR inhibition, indicating that 1,25(OH)₂D₃ may partially augment NP cell viability and stimulate proliferation through VDR activation. Furthermore, we examined the concentrations of GPX4, lipid ROS, MDA, SOD, GSH, SLC7A11, and SLC40A1 in cells pre-treated and non-pre-treated with 1,25(OH)₂D₃ following siRNA interference. The findings indicated that 1,25(OH)₂D₃ continued to prevent ferroptosis following VDR gene silencing, as seen by the increase of GPX4, SLC7A11, and SLC40A1 expression, alongside a decrease in lipid ROS and MDA levels. The inhibitory effect of 1,25(OH)₂D₃ was diminished following VDR gene silencing compared to before VDR inhibition, indicating that the suppression of ferroptosis in NP cells by 1,25(OH)₂D₃ is partially reliant on VDR activation. The research underscores that the VDR substantially affects the impact of 1,25(OH)₂D₃ on NP cells in terms of viability and ferroptosis. Inhibition of VDR diminishes the protective function of 1,25(OH)₂D₃, elucidating the complex mechanism by which 1,25(OH)₂D₃ combats NP cell ferroptosis and highlighting the essential involvement of VDR in this process.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study effectively established the optimal conditions under which 1,25(OH)₂D₃ enhances the proliferation of rat NP cells. Utilizing Erastin to mimic ferroptosis conditions, it was demonstrated that 1,25(OH)₂D₃ reduces ferroptosis in NP cells by upregulating ferroptosis-protective proteins and antioxidants while decreasing oxidative stress markers. Notably, a reduction in VDR expression was observed under ferroptotic conditions. The subsequent VDR gene silencing experiments revealed that while 1,25(OH)₂D₃ continues to mitigate ferroptosis, its efficacy is diminished without VDR activation, highlighting the significance of VDR in its protective mechanism \u0026nbsp;(Fig. 9). This opens avenues for further exploration into additional pathways by which 1,25(OH)₂D₃ may exert its therapeutic effects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData a\u003c/strong\u003e\u003cstrong\u003evailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the study results are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLQ contributed to project design, performed the experiments, and provided financial support. PJ contributed to the experiments, collected analysis data, and wrote the manuscript. DF reviewed the manuscript and contributed to its preparation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis\u0026nbsp;research has been supported by\u0026nbsp;the National Natural Science Foundation of China\u0026nbsp;(Grant No.: 81601943), and the China Hubei Provincial Health Commission Research Project(Grant No.: WJ2019M023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaher, C., Underwood, M. \u0026amp; Buchbinder, R. Non-specific low back pain. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e389\u003c/strong\u003e, 736\u0026ndash;747 (2017).\u003c/li\u003e\n\u003cli\u003eKnezevic, N. N., Candido, K. D., Vlaeyen, J. W. S., Zundert, J. \u0026amp; Cohen, S. P. 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Recent studies have highlighted the role of ferroptosis, an iron-dependent form of programmed cell death, in nucleus pulposus (NP) cells degeneration. This study investigates the protective effects of 1,25(OH)₂D₃, the active form of Vitamin D, on LIDD by targeting ferroptosis. Our findings demonstrate that 1,25(OH)₂D₃ effectively suppresses ferroptosis in nucleus pulposus cells by reducing lipid peroxidation, restoring glutathione levels, and enhancing antioxidant defenses. Mechanistically, 1,25(OH)₂D₃ exerts its effects through activation of the Vitamin D receptor (VDR) signaling pathway, which regulates key ferroptosis-associated molecules such as GPX4 and SLC7A11. These results reveal the therapeutic potential of 1,25(OH)₂D₃ in mitigating LIDD, offering a novel approach to suppress ferroptosis and preserve intervertebral disc function.","manuscriptTitle":"Unlocking the Therapeutic Potential of 1,25(OH)₂D₃: Targeting Ferroptosis to Alleviate Lumbar Intervertebral Disc Degeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 07:29:52","doi":"10.21203/rs.3.rs-5612461/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-01T17:08:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-30T00:15:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-27T09:51:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311445936200473112235885297145063898239","date":"2024-12-22T20:54:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258226646712276500662519687907306566678","date":"2024-12-21T10:04:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-21T01:21:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-21T00:48:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-12-20T13:13:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-19T13:22:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-12-10T02:28:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"54aedc3a-a723-4601-bb4b-d0929c285f5d","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41936913,"name":"Biological sciences/Biochemistry"},{"id":41936914,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-03-10T17:10:14+00:00","versionOfRecord":{"articleIdentity":"rs-5612461","link":"https://doi.org/10.1038/s41598-025-92405-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-07 15:57:41","publishedOnDateReadable":"March 7th, 2025"},"versionCreatedAt":"2024-12-23 07:29:52","video":"","vorDoi":"10.1038/s41598-025-92405-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-92405-x","workflowStages":[]},"version":"v1","identity":"rs-5612461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5612461","identity":"rs-5612461","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}