Fargesin enhances the condition of intervertebral disc degeneration by suppressing BRD4 expression and influencing autophagy in NPCs

Fargesin enhances the condition of intervertebral disc degeneration by suppressing BRD4 expression and influencing autophagy in NPCs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fargesin enhances the condition of intervertebral disc degeneration by suppressing BRD4 expression and influencing autophagy in NPCs Minqin Mao, Huan Yu, Liang Zhang, Tao Zhang, Wen Xu, Jingtang Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8530418/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Journal of Molecular Histology → Version 1 posted 9 You are reading this latest preprint version Abstract Background Intervertebral Disc Degeneration (IVDD) stands as the prevalent chronic skeletal muscle degenerative disease and the primary culprit behind low back pain, yet its underlying mechanism remains elusive. Fargesin, a novel bioactive lignan compound known for its anti-inflammatory properties, also has an unclear mechanism of action in IVDD. Methods To unravel this mechanism, we employed the CCK8 assay to determine the optimal treatment duration and concentration of Fargesin. For our in vitro experiments, we induced NPCs cells with IL-1β to mimic the IVDD model, while for in vivo studies, we established a rat IVDD model through surgical intervention coupled with an injection of 0.1mL IL-1β (1µg/mL). We utilized Elisa to measure the levels of inflammatory cytokines IL-6 and TNF-α. Western blotting and immunofluorescence techniques were harnessed to assess the expression of autophagy markers and BRD4 protein in the cells. Additionally, X-ray imaging, HE staining, and safranin o-fast green aided in evaluating the intervertebral disc lesions in our in vitro experiments. Results Our findings revealed that Fargesin significantly suppressed the inflammatory response triggered by IL-1β in NPCs and attenuated the elevated expression of BRD4. By downregulating BRD4 expression, Fargesin upregulated the proteins LC3II/I, Beclin-1, and LAMP1 related to the autophagy signaling pathway, while decreasing P62 expression. Conclusion Far activates the autophagy signaling pathway by inhibiting the expression of BRD4, enhances the activity of NPCs, and alleviates the pathological damage of intervertebral discs with ivdd lesions. Fargesin Intervertebral disc degeneration NPCs BRD4 Autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Low Back Pain (LBP) represents a widespread health concern. By 2050, it is projected that approximately 843 million people worldwide will suffer from low back pain, with the total global cases increasing by roughly 36.4% [ 1 ] . A primary factor contributing to low back pain is Intervertebral Disc Degeneration (IVDD) [ 2 ] . The Intervertebral Disc (IVD) consists of three distinct tissue types: the central gelatinous Nucleus Pulposus (NP), the encircling Annulus Fibrosus (AF), and the upper and lower Cartilage Endplates (CEP) [ 3 ] .Nucleus pulposus cells confer high hydration and elasticity to the intervertebral disc by synthesizing and secreting Extracellular Matrix (ECM) components, such as type II collagen, proteoglycans, and elastic fibers. This allows the intervertebral disc to efficiently absorb and disperse spinal pressure, performing a critical buffering and shock absorption function [ 4 , 5 ] .However, during the progression of IVDD, the nucleus pulposus cell count diminishes, leading to a gradual decrease in ECM secretion and, consequently, a reduction in the intervertebral disc's height. Simultaneously, the boundary between the annulus fibrosus and the nucleus pulposus becomes indistinct, destabilizing the overall structure of the intervertebral disc [ 6 , 7 ] . Thus, the decline in nucleus pulposus cell count is intimately linked to the advancement of IVDD and is often accompanied by excessive secretion of cellular inflammatory factors and disrupted cellular autophagy function [ 5 , 8 – 10 ] . Autophagy is essential for preserving cellular homeostasis, ensuring normal cellular operations by eliminating dysfunctional cellular elements and organelles [ 11 – 13 ] . In patients afflicted with degenerative diseases, the autophagy mechanism frequently malfunctions, leading to tissue degeneration, abnormal bone development, and even premature patient death [ 14 , 15 ] .Consequently, conducting in-depth research on the autophagy mechanism in degenerative diseases holds immense significance for the advancement of effective therapeutic approaches. Fargesin (Far), a bioactive novel lignan compound derived from Magnolia fargesii, exhibits anti-inflammatory properties and enhances lipid and glucose metabolism [ 16 , 17 ] . Research has revealed that Far attenuates myocardial ischemia/reperfusion injury in rats [ 18 ] and alleviates atherosclerosis by promoting reverse cholesterol transport and reducing inflammatory responses [ 19 ] . In THP-1 monocytes, this lignan inhibits PKC-dependent AP-1 and NF-κB signaling, exerting its anti-inflammatory effect [ 16 ] . Additionally, Far facilitates macrophage reprogramming by downregulating the MEK and NF-kB pathways, thereby benefiting osteoarthritis [ 20 ] . Bromine-containing domain protein 4 (BRD4), a member of the bromine domain and superterminal structure family, plays a role in regulating cellular gene transcription, autophagy, and inflammatory responses [ 21 – 23 ] . BRD4 has been associated with various age-related diseases such as osteoarthritis, cardiovascular diseases, and cancers. Recently, its role in the skeletal system, particularly its association with IVDD, has gained increasing attention [ 24 , 25 ] . Studies show that BRD4 expression significantly increases with higher Pfirrmann grades [ 26 ] , BRD4 influences the metabolic process of extracellular matrix in nucleus pulposus cells, promoting the development of IVDD. Inhibiting BRD4 elevates cellular autophagy levels and alleviates diabetes-induced IVDD symptoms [ 27 ] . These findings suggest that BRD4 plays a pivotal role in IVDD pathogenesis and progression, closely linked to autophagy in nucleus pulposus cells and inflammatory factor secretion. However, whether Far can modulate the autophagy level of nucleus pulposus cells and inflammatory factor secretion via BRD4, thereby enhancing cell activity in an inflammatory environment, remains to be elucidated. This study unprecedentedly demonstrates that Far modulates the autophagy level of nucleus pulposus cells by interacting with BRD4. Simultaneously, it suppresses the release of inflammatory mediators and bolsters the survival of nucleus pulposus cells amidst an inflammatory milieu, effectively mitigating the manifestations of IVDD. This finding uncovers novel putative mechanisms and therapeutic targets for managing intervertebral disc degeneration, paving the way for potential advancements in treating associated pathologies. Materials and Methods Cell culture and processing Rat intervertebral disc nucleus pulposus cells (NPCs) were sourced from Ponsure (CP-R145, Ponsure) and maintained in a 37°C, 5% CO 2 incubator. The culture medium consisted of a complete medium specifically formulated for rat intervertebral disc nucleus pulposus cells, enriched with 10% FBS and 1% penicillin-streptomycin (CM-R145, Ponsure). To investigate the impact of lignans (Far) on the viability of NPCs exposed to IL-1β, the cells were incubated with 10 ng/mL IL-1β for 24 h. Simultaneously, the cells were exposed to varying concentrations (10, 20, 50, 100 µmol/L) of Far (HY-N0719, MCE) for durations of 24, 48, and 72 h. Cell viability was assessed using the CCK-8 assay to determine the most effective concentration and exposure time. Based on these findings, the cells were categorized into the Control group, the IL-1β group, and the IL-1β + Far group for further analysis. CCK-8 testing Cells were treated with a combination of 10ng/mL IL-1β and different concentrations of Far (10, 20, 50, 100 µmol/L) for 24, 48, and 72 hours. Following treatment, the medium was replaced with fresh medium. Then, 10 µL of CCK8 reagent (C0037, Biyantian) was added to each well and incubated for 2 h. The absorbance of each well was measured at a wavelength of 450nm using a microplate reader (WD-2012B, Beijing Liuyi). Immunofluorescence The culture dishes containing crawling cells in the 24-well plate were washed thrice with PBS, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 5% BSA at 37°C for 30 min. Subsequently, LC3 (ab192890, abcam, 1:500) and LAMP1 (ab24871, abcam, 1:100) antibodies were incubated overnight at 4°C. After washing, the fluorescent secondary antibody Cy3 Goat Anti-Rabbit IgG(H + L) (AS007, ABclonal, 1:200) was added. The slide was then restained with DAPI, sealed, and examined under a fluorescence microscope (CKX53, Olympus). Western Blot A specific mass of rat intervertebral discs was taken, and RIPA lysis buffer was added. The samples were ground using a tissue grinder, and total tissue protein or cell culture medium was extracted. Total protein was isolated by ice lysis with RIPA buffer, followed by centrifugation at 12,000 r/min and 4°C for 10 min. The supernatant was collected, and the total protein was quantified using a BCA protein quantification kit (E-BC-K318-M, Elabscience). After denaturing the protein samples, electrophoresis was performed using sodium dodecyl sulfate gel (SDS-PAGE) for 1.5 h. The proteins were then transferred to a PVDF membrane (millipore) and blocked at room temperature with 5% skimmed milk powder for 1 hour. LC3 (ab192890, abcam, 1:2000), Beclin 1 (ab62557, abcam, 1:1000), P62 (ab109012, abcam, 1:5000), and BRD4 (ab75898, abcam, 1:2000) antibodies were incubated overnight at 4°C. The next day, the corresponding HRP-labeled secondary antibody was incubated at room temperature for 1 hour. The PVDF membrane was then moistened with ECL luminescent solution (RJ239676, Thermo Fisher) and developed using an ultra-high sensitivity chemiluminescence imaging system (Tanon-5200, Shanghai Tianneng Technology Co., LTD). Establish of the rat intervertebral disc degeneration model All animal experiments were conducted in compliance with institutional ethical guidelines and approved by the Experimental Animal Ethics Committee (No.20241209-001), and the experiment was carried out in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. In this study, experimental rat were anesthetized via intraperitoneal injection of 2% sodium pentobarbital solution at a dosage of 40 mg/kg. Animal euthanasia was performed through anesthetic overdose, achieved by intraperitoneal administration at a dosage of 100 mg/kg of 2% sodium pentobarbital solution. This procedure complies with the ethical guidelines for treating laboratory animals. 6-week-old SD rats (License Number: SCXK (Beijing) 2024-0001) were purchased from Spafu (Beijing) Biotechnology Co., LTD and housed under controlled conditions of 20–26℃ and 40%-70% humidity, receiving a standard rat diet. Following a 7-day adaptation period, the rats were randomly assigned to the sham operation group, IVDD + Sh-NC group, IVDD + Sh-BRD4 group, IVDD + Far group, and IVDD + Far + Sh-IVDD group. After anesthetizing the rats, they were positioned on the operating table. A 2 cm incision was made in the left abdomen, and the muscle and fat were carefully separated to expose the L4-5 and L5-6 intervertebral discs. A 1 mm diameter needle was inserted 2 mm into the center of each disc, rotated 360°, and held for 30 s before removal. Subsequently, 0.1 mL of IL-1β (1 µg/mL) was injected into the discs, the wounds were closed in layers, and the rats were returned to their cages for a 6-week period to establish the intervertebral disc degeneration model. On the 4th week following modeling, shRNA-NC and shRNA-BRD4 (provided by Suzhou Zhongke Zhihui Biotechnology Co., LTD) were administered once into the central space of the NP tissues. Additionally, Far was injected weekly via the tail vein at a dose of 15 µmol/kg for a total of 6 weeks. X-ray inspection The rats were anesthetized using isoflurane, ensuring their complete immobility during the experiment. They were then positioned on the imaging platform to maintain a natural extension of their spine. Imaging parameters were set according to the equipment manual, typically using a voltage of 26 kV and an exposure time of 10 s to capture lateral X-rays of the rats' caudal vertebrae, specifically focusing on intervertebral disc changes. Calculate the intervertebral disc height index (DHI%) according to the described method [ 28 ] . HE staining Following the baking, dewaxing, and hydration of paraffin sections, they were stained with hematoxylin solution for 3 to 5 min. After rinsing under running water, the sections were differentiated with 1% hydrochloric acid alcohol and inverted blue solution, subsequently stained with eosin for another 3 to 5 min. The stained sections were then dehydrated, sealed, and examined under a microscope (BX43, Olympus). Safranin O-fast green dyeing Paraffin sections underwent baking, dewaxing, and hydration before being stained with bone tissue green fixation solution for 5 min. Excess staining solution was rinsed away with running tap water until the cartilage appeared colorless. The sections were then briefly treated with 1% hydrochloric acid for 10 s, followed by staining with bone tissue muscose solution for 1 min. After rapid dehydration in four consecutive tanks of anhydrous ethanol (3 s each), the sections were examined under a microscope (BX43, Olympus) until the cartilage turned red with a colorless background. The slices were then made transparent with xylene for 5 min, followed by fresh xylene for another 5 min, and finally sealed with neutral gum for observation. evaluated the condition of the intervertebral disc using the grading scale as described previously [ 28 ] . Immunohistochemistry Sections of rat intervertebral disc tissue underwent baking, dewaxing, and hydration procedures, followed by antigen retrieval using a citric acid buffer. Blocking was performed with 5% BSA, and the primary antibodies—LC3 (ab192890, abcam, 1:200), Beclin 1 (ab62557, abcam, 1:100), and P62 (ab109012, abcam, 1:1000)—were incubated. After an overnight incubation at 4°C, the sections were treated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:100), developed with DAB, counterstained with hematoxylin and anti-blue, dehydrated, cleared, sealed, and finally observed under a microscope (BX43, Olympus). Data analysis Data visualization and statistical analysis were conducted using Graphpad Prism 8.0 software. Quantitative results are presented as mean ± standard deviation (X ± S). For comparisons among multiple groups, one-way analysis of variance was employed, with post hoc testing performed using the Tukey method, * P < 0.05,** P < 0.01, *** P < 0.001。 Results Far can reduce the toxicity to NPCs cells in the presence of IL-1β IL-1β-induced NPCs have been utilized in investigations of IVDD [ 29 ] . To determine the optimal concentration and duration of Far treatment in the presence of 10 ng/mL IL-1β, NPCs cells were stimulated with IL-1β for 24 h and simultaneously treated with various concentrations of Far (10, 20, 50, 100 µmol/L) for 24, 48, and 72 h. Cell viability was assessed using CCK-8. Notably, IL-1β significantly reduced cell viability compared to the control group. However, Far treatment at different concentrations and durations partially reversed this effect, leading to increased cell viability. Specifically, at 24 and 48 h of Far treatment, cell viability exhibited a dose-dependent increase (Fig. 1 A-C). The highest cell viability was observed with 50 µmol/L Far for 48 h, and these conditions were chosen for subsequent experiments. Cells were then treated under the identified optimal conditions and grouped as Control, IL-1β, and IL-1β + Far. Elisa was used to measure the levels of inflammatory factors IL-6 and TNF-α. The results indicated that Far significantly reduced the levels of IL-6 and TNF-α elevated by IL-1β treatment (Fig. 1 D-E). In summary, IL-1β treatment reduced cell viability and increased inflammatory factor levels, while Far attenuated these effects, suggesting its protective role against IL-1β-induced cell damage. Far reverses the inhibition of autophagy in NPCs cells induced by IL-1β To assess the impact of Far on autophagy in NPCs cells exposed to IL-1β, we established three experimental groups: Control, IL-1β, and IL-1β + Far. Western blot analysis was used to evaluate the expression levels of autophagy-related proteins LC3II/I, Beclin-1, and P62. Our findings revealed that IL-1β treatment elevated LC3II/I and Beclin-1 levels while reducing P62 levels compared to the Control group. Notably, Far treatment further augmented the autophagy level in NPCs cells (refer to Fig. 2 A-D). Immunofluorescence staining corroborated these results, showing a marked decrease in LC3 and LAMP1 fluorescence after IL-1β exposure, which was subsequently restored by Far treatment, indicating a significant increase in autophagy (Fig. 2 E-F). To further validate the role of Far in modulating autophagy, we pretreated NPCs cells with the autophagy inhibitor 3-MA (10 µM) and compared the effects in IL-1β, IL-1β + Far, and IL-1β + Far + 3-MA groups. Western blot results demonstrated that Far treatment led to a significant increase in LC3II/I and Beclin-1 levels, accompanied by a decrease in P62 protein levels (Fig. 2 G-J). Importantly, 3-MA effectively blocked the autophagy-activating effects of Far. Cellular immunofluorescence provided additional support for these findings, revealing that while Far restored LC3 and LAMP1 fluorescence intensities, this effect was reversed by the addition of 3-MA (Fig. 2 K-L). Far modulates autophagy in NPCs by regulating BRD4 Our findings indicate that IL-1β treatment notably elevates the autophagy level in NPCs. Additionally, this treatment leads to a significant increase in BRD4 protein expression (Fig. 3 A). We hypothesize that Far influences NPC autophagy via BRD4. To test this hypothesis, we constructed a BRD4 interference vector and transfected it into IL-1β-treated NPCs. Western blot analysis confirmed the successful transfection of BRD4 into NPCs (Fig. 3 B). We divided the cells into four groups: IL-1β + si-NC, IL-1β + si-BRD4, IL-1β + Far, and IL-1β + Far + si-BRD4 ((provided by Suzhou Zhongke Zhihui Biotechnology Co., LTD). In comparison to the IL-1β + si-NC group, both BRD4 interference and Far treatment led to a marked decrease in inflammatory factors IL-6 and TNF-α (Fig. 3 C-D). Western blot analysis revealed a significant downregulation of LC3II/I and Beclin-1 protein expressions, along with an increase in P62 protein expression (Fig. 3 E-H). Immunofluorescence staining further showed enhanced fluorescence intensification of LC3 and LAMP1 (Fig. 3 I-J). When BRD4 interference was combined with Far treatment, there was a further downregulation of inflammatory factors and autophagy-related protein markers. Thus, our results suggest that Far regulates autophagy in cells through its interaction with BRD4. Far significantly mitigates the degenerative damage to intervertebral discs in rats by suppressing BRD4 To substantiate through in vivo experiments that Far modulates BRD4 to stimulate autophagy and consequently retard IVDD progression, we adaptively reared SD rats and randomly assigned them to the Sham, IVDD + Sh-NC, IVDD + Sh-BRD4, IVDD + Far, and IVDD + Far + Sh-IVDD groups, treating each group accordingly. X-ray imaging revealed (Fig. 4 A) that the intervertebral discs of the IVDD + Sh-NC group exhibited swelling, loss and blurring of endplate boundary height, and compromised structural integrity, with the disc height index (DHI%) significantly lower than that in the Sham group. However, upon shRNA-BRD4 and Far administration, the degenerative modifications in the intervertebral disc structure were ameliorated, without notable endplate blurring or structural compromise, and the DHI% values also indicated that shRNA-BRD4 could prevent the occurrence of IVDD. (Fig. 4 B). Silencing BRD4 significantly attenuates the inflammatory response in rat IVD and mitigates the progression of IVDD Far exhibits potent efficacy in mitigating IVDD damage. HE staining (Fig. 5 A) and safranin O-fast green staining (Fig. 5 B) revealed bleeding in the bone marrow cavity, chondrocyte degeneration, altered bone trabeculae structure, and pronounced tissue damage in the IVDD + Sh-NC group. The intervertebral disc histological score markedly increased (Fig. 5 C) alongside elevated levels of inflammatory factors IL-6 and TNF-α (Fig. 5 D-E). Both shRNA-BRD4 and Far interventions substantially reduced bone tissue hemorrhage and chondrocyte degeneration while lowering the histological score. IL-6 and TNF-α levels decreased significantly, with the combined shRNA-BRD4 and Far treatment showing the most pronounced effect. These findings indicate that Far mitigates IVDD by suppressing cellular inflammatory factors and ameliorating bone tissue structural damage. Far effectively slows down IVDD injury by suppressing BRD4 activity and enhancing autophagy in rat IVD Both in vitro and in vivo experiments have demonstrated Far's ability to modulate autophagy in NPCs through BRD4 regulation. Western blot analysis revealed significant upregulation of LC3II/I and Beclin-1 protein levels, along with a notable decrease in P62 levels, in the intervertebral discs of IVDD rats. The activation of autophagy was further evident after the introduction of shRNA-BRD4 or Far, leading to the recovery of related protein expressions (Fig. 6 A-D). Notably, the combined treatment of shRNA-BRD4 and Far exhibited the most pronounced effect on autophagy activation, as confirmed by immunohistochemical analysis (Fig. 6 E-H). Discussion IVDD is a degenerative spinal disease strongly associated with age, featuring a relatively high incidence rate. It severely impairs patients' occupational function and quality of life, while also imposing a considerable economic burden on families and society [ 30 , 31 ] . As IVDD progresses chronically, it may give rise to various spine-related pathological changes, including intervertebral disc protrusion, spinal forward displacement, spinal canal stenosis, and degenerative scoliosis [ 32 , 33 ] . These conditions often manifest as acute or chronic LBP, a widespread public health concern globally. Notably, approximately 40% of LBP cases are attributed to IVDD [ 33 ] . Although therapeutic approaches for IVDD have been documented, its precise pathogenesis, as well as the therapeutic role and mechanism of Far in IVDD, remain incompletely understood. This study novelly investigated how Far mitigates IVDD damage by suppressing BRD4 expression in NPCs, diminishing cellular inflammatory responses, activating autophagy, and enhancing the activity of nucleus pulposus cells. Our findings elucidate the mechanism of Far in IVDD and offer innovative therapeutic insights for its future treatment. Far exhibits anti-inflammatory properties and holds significant value in treating cardiovascular and inflammatory diseases [ 16 , 17 ] . This study explored Far's effect on the viability of IL-1β-induced nucleus pulposus cells. Far demonstrated a capacity to restore the reduced cell viability caused by IL-1β stimulation, showing a dose-dependent trend. In vitro experiments revealed that IL-1β stimulation of NPCs led to increased levels of IL-6 and TNF-α, aligning with the notable elevation of these inflammatory factors in IVDD [ 10 ] . During IVDD progression, inflammatory factors rise significantly, triggering local autoimmune inflammatory responses that disrupt the normal metabolic process of the extracellular matrix (ECM), ultimately leading to IVDD occurrence [ 34 ] . In this study, IL-1β was used to induce NPCs and mimic the pathophysiological phenomena of IVDD. However, the specific role of Far in IVDD remained unexplored. Current research suggests that Far exerts an anti-inflammatory effect in OA development [ 20 ] , corroborating our experimental findings. Following Far treatment, inflammatory factor levels in NPCs decreased significantly. Similarly, in vivo experimental results indicated that Far could notably reduce inflammatory factor levels in intervertebral disc tissue, suggesting a potent anti-inflammatory role of Far in IVDD. Furthermore, our study revealed that Far significantly suppresses BRD4 expression and activates autophagy in cells, a finding that has not been previously reported. BRD4, a key member of the BET protein family, plays a pivotal role in regulating the transcription of genes involved in various cellular processes, including senescence, autophagy, inflammation, cell death, and extracellular matrix (EC) metabolism [ 26 , 27 , 35 ] . In recent years, research has intensified on BRD4's specific functions in the skeletal system, particularly in IVDD [ 27 , 36 ] . Our findings reveal that under the influence of IL-1β, BRD4 expression escalates, leading to a surge in cellular inflammatory factors and a concurrent inhibition of autophagy. However, with the introduction of Far treatment, BRD4 becomes silenced, resulting in the recovery of inflammatory factor levels and the reactivation of autophagy. These observations implicate BRD4 as a critical factor in the interplay between cellular inflammation and autophagy during the progression of IVDD. Furthermore, our data suggest that Far exerts its effects by modulating BRD4 expression. This aligns with previous reports indicating that BRD4 inhibition can ameliorate IVDD by regulating cellular inflammatory signaling and autophagy pathways [ 27 , 36 ] . Additionally, our in vitro studies demonstrate that silencing BRD4 significantly mitigates intervertebral disc injury in IVDD. Based on these findings, we hypothesize that Far's beneficial effects on intervertebral disc degeneration, cellular inflammation, and autophagy are mediated through BRD4 inhibition. Autophagy is a self-protection mechanism of eukaryotic cells, which can digest and degrade their own damaged, degenerated or senescent biological macromolecules and organelles, and maintain cellular homeostasis [ 11 – 13 ] . In IVDD diseases, autophagy is associated with cell senescence and cell death of NPCs [ 37 ] . Studies have reported that 3-MA inhibiting autophagy can eliminate the anti-cell death and senescence effects of SIRT6 on NP cells [ 29 ] . In addition, autophagy controls the senescence of bone marpe-derived mesenchymal stem cells during bone senescence [ 38 ] , and autophagy is closely related to the development of IVDD. Our research results revealed that in the treatment of IVDD, Far, in addition to its anti-inflammatory effect, autophagy also plays a key role. The results indicated that Far could significantly promote the expression of autophagy-related proteins LC3II/I and Beclin-1. The use of 3-MA to inhibit autophagy counteracted the autophagy-activating effect of Far on NPCs cells. Moreover, Far can inhibit the expression of BRD4. Therefore, Far can exert therapeutic effects on IVDD by inhibiting BRD4-activated autophagy and suppressing inflammatory responses, which is consistent with the research findings that BRD4 knockdown activates autophagy and inhibits the activity of NLRP3 inflammasome through the NF-kB pathway to alleviate the degradation of extracellular substances in NP cells and exert anti-IVDD effects [ 39 ] . Conclusion In summary, our study demonstrates that Far mitigates intervertebral disc lesions in IVDD by modulating BRD4 expression, suppressing cellular inflammatory markers IL-6 and TNF-α, elevating LC3II/I and Beclin-1 protein levels, and activating cellular autophagy signaling. This offers novel insights and potential therapeutic targets for addressing intervertebral disc degeneration. Nevertheless, our investigation has its constraints. Besides focusing on NPCs, the progression of IVDD is tied to AF degeneration, and it remains unclear if BRD4 engages in IVDD via alternate signaling pathways. Hence, further examination of BRD4's role and mechanisms in IVDD pathogenesis is warranted. Declarations Compliance with ethical standards Conflict of interest The authors declare no competing financial interests. Clinical trial number Not applicable. Funding This research was supported by grants from Science and Technology Program of Jiangxi Provincial Administration of Traditional Chinese Medicine (2024A0152). Author Contribution Minqin Mao: Conceptualization, Data curation, Visualization, Methodology, Writing - original draft, Writing - review & editing; Huan Yu and Liang Zhang: Data curation, Validation, Investigation; Tao Zhang, Wen Xu and Jingtang Li: Investigation, Methodology, Formal Analysis, Conceptualization, Supervision; Yongxing Peng: Conceptualization, Methodology, Writing - review & editing. All authors reviewed the manuscript. Data Availability The data supporting the findings are available within the article materials. References Collaborators GLBP (2023) Global, regional, and national burden of other musculoskeletal disorders, 1990–2020, and projections to 2050: a systematic analysis of the Global Burden of Disease Study 2021.[J]. 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Aging Cell. 17(1) Junmin H et al (2020) Bromodomain-containing protein 4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity in NP cells.[J]. J Cell Physiol. 235(0) Additional Declarations No competing interests reported. Supplementary Files WB.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Journal of Molecular Histology → Version 1 posted Editorial decision: Revision requested 14 Jan, 2026 Reviews received at journal 14 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviews received at journal 11 Jan, 2026 Reviewers agreed at journal 10 Jan, 2026 Reviewers invited by journal 08 Jan, 2026 Editor assigned by journal 07 Jan, 2026 Submission checks completed at journal 07 Jan, 2026 First submitted to journal 06 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":2173657,"visible":true,"origin":"","legend":"\u003cp\u003eFar can reduce the toxicity to NPCs cells in the presence of IL-1β. (\u003cstrong\u003eA\u003c/strong\u003e) CCK8 was used to detect the 24h cell viability. (\u003cstrong\u003eB\u003c/strong\u003e) Cell viability was detected by CCK8 at 48 h. (\u003cstrong\u003eC\u003c/strong\u003e) CCK8 was used to detect the cell viability at 72 h. (\u003cstrong\u003eD\u003c/strong\u003e) Elisa was used to detect the level of IL-6 in NPCs. (\u003cstrong\u003eE\u003c/strong\u003e) Elisa was used to detect the level of TNF-α in NPCs. ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001. ## \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ### \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8530418/v1/f91430798f2e9fd1414ea173.png"},{"id":100378027,"identity":"a60f0c77-130b-440f-a433-65c0b82edce1","added_by":"auto","created_at":"2026-01-16 08:49:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":694871,"visible":true,"origin":"","legend":"\u003cp\u003eFar reverses the inhibition of autophagy in NPCs cells induced by IL-1β. (\u003cstrong\u003eA\u003c/strong\u003e) Western blot detection strip. (\u003cstrong\u003eB\u003c/strong\u003e) Beclin-1 protein expression. (\u003cstrong\u003eC\u003c/strong\u003e) LC3II/I protein expression. (\u003cstrong\u003eD\u003c/strong\u003e) P62 protein expression. (\u003cstrong\u003eE\u003c/strong\u003e) LC3 protein expression was detected by immunofluorescence (400×). (\u003cstrong\u003eF\u003c/strong\u003e) LAMP1 protein expression was detected by immunofluorescence (400×). (\u003cstrong\u003eG\u003c/strong\u003e) Western blot detection strip. (\u003cstrong\u003eH\u003c/strong\u003e) Beclin-1 protein expression. (\u003cstrong\u003eI\u003c/strong\u003e) LC3II/I protein expression. (\u003cstrong\u003eJ\u003c/strong\u003e) P62 protein expression. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001. # \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ### \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8530418/v1/ed9152bf51bb495ed6b5f34e.png"},{"id":100342727,"identity":"4251fbcb-4778-4f19-aee0-1a8bae83a11a","added_by":"auto","created_at":"2026-01-16 00:07:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":723078,"visible":true,"origin":"","legend":"\u003cp\u003eFar modulates autophagy in NPCs by regulating BRD4. (\u003cstrong\u003eA\u003c/strong\u003e) The expression of BRD4 protein in NPCs was detected by WB. (\u003cstrong\u003eB\u003c/strong\u003e) WB verification of BRD4 interference efficiency. (\u003cstrong\u003eC\u003c/strong\u003e) Elisa was used to detect the level of IL-6 in NPCs supernatant. (\u003cstrong\u003eD\u003c/strong\u003e) Elisa was used to detect the level of TNF-α in NPCs supernatant. (\u003cstrong\u003eE\u003c/strong\u003e) \u0026nbsp;Western blot detection strip. (\u003cstrong\u003eF\u003c/strong\u003e) Beclin-1 protein expression. (\u003cstrong\u003eG\u003c/strong\u003e) LC3II/I protein expression. (\u003cstrong\u003eH\u003c/strong\u003e) P62 protein expression. (\u003cstrong\u003eI\u003c/strong\u003e) LC3 protein expression was detected by immunofluorescence (400×). (\u003cstrong\u003eJ\u003c/strong\u003e) LAMP1 protein expression was detected by immunofluorescence (400×). * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001. ### \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. @@@\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8530418/v1/eb73244784508225c517ae81.png"},{"id":100342729,"identity":"25a126ee-9496-4ba4-8180-1f4fe678abbf","added_by":"auto","created_at":"2026-01-16 00:07:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":172218,"visible":true,"origin":"","legend":"\u003cp\u003eFar significantly mitigates the degenerative damage to intervertebral discs in rats by suppressing BRD4. (\u003cstrong\u003eB\u003c/strong\u003e) Score of disc height index. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001. # \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ### \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. @@@\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001. \u0026amp;\u0026amp;\u0026amp;\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8530418/v1/5b1556cb8294a56ee844fa09.png"},{"id":100373187,"identity":"870692a2-ce21-4fcd-ae17-524ec8acbbba","added_by":"auto","created_at":"2026-01-16 08:13:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":516083,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing BRD4 significantly attenuates the inflammatory response in rat IVD and mitigates the progression of IVDD. (\u003cstrong\u003eA\u003c/strong\u003e) HE staining (100×). (\u003cstrong\u003eB\u003c/strong\u003e) Safranin o-fast green dyeing (200×). (\u003cstrong\u003eC\u003c/strong\u003e) Histological scores. (\u003cstrong\u003eD\u003c/strong\u003e) Elisa was used to detect the levels of IL-6 in IVD tissues. (\u003cstrong\u003eE\u003c/strong\u003e) Elisa was used to detect the levels of TNF-α in IVD tissues. *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. ### \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. @@@\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001. \u0026amp;\u0026amp;\u0026amp;\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8530418/v1/6937c42bb8ff7aa9a03d550d.png"},{"id":100373325,"identity":"495b45e6-b3b6-4088-a394-0f8649aebc7c","added_by":"auto","created_at":"2026-01-16 08:14:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":717975,"visible":true,"origin":"","legend":"\u003cp\u003eFar effectively slows down IVDD injury by suppressing BRD4 activity and enhancing autophagy in rat IVD. (\u003cstrong\u003eA\u003c/strong\u003e) Western blot detection strip. (\u003cstrong\u003eB\u003c/strong\u003e) Beclin-1 protein expression. (\u003cstrong\u003eC\u003c/strong\u003e) LC3II/I protein expression. (\u003cstrong\u003eD\u003c/strong\u003e) P62 protein expression. (\u003cstrong\u003eE\u003c/strong\u003e) Immunohistochemical detection (100×). (\u003cstrong\u003eF\u003c/strong\u003e) Beclin-1 protein expression. (\u003cstrong\u003eG\u003c/strong\u003e) LC3 protein expression. (\u003cstrong\u003eH\u003c/strong\u003e) P62 protein expression. ***\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001. ### \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. @@@\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. \u0026amp;\u0026amp;\u0026amp;\u003cem\u003e P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8530418/v1/d50e982586d6c5fb3e4d9087.png"},{"id":105223355,"identity":"53f77fb4-c35f-4364-b225-2ce9b80b2fa3","added_by":"auto","created_at":"2026-03-23 16:04:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5850137,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8530418/v1/6417ef1b-1276-4f50-9ab7-9114bd15a212.pdf"},{"id":100373011,"identity":"850a21f5-4ae6-4cd7-86e3-dee0c5212c00","added_by":"auto","created_at":"2026-01-16 08:13:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1454060,"visible":true,"origin":"","legend":"","description":"","filename":"WB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8530418/v1/2cb4eefd8bf11b885e24232e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fargesin enhances the condition of intervertebral disc degeneration by suppressing BRD4 expression and influencing autophagy in NPCs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLow Back Pain (LBP) represents a widespread health concern. By 2050, it is projected that approximately 843\u0026nbsp;million people worldwide will suffer from low back pain, with the total global cases increasing by roughly 36.4%\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. A primary factor contributing to low back pain is Intervertebral Disc Degeneration (IVDD)\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The Intervertebral Disc (IVD) consists of three distinct tissue types: the central gelatinous Nucleus Pulposus (NP), the encircling Annulus Fibrosus (AF), and the upper and lower Cartilage Endplates (CEP) \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.Nucleus pulposus cells confer high hydration and elasticity to the intervertebral disc by synthesizing and secreting Extracellular Matrix (ECM) components, such as type II collagen, proteoglycans, and elastic fibers. This allows the intervertebral disc to efficiently absorb and disperse spinal pressure, performing a critical buffering and shock absorption function\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.However, during the progression of IVDD, the nucleus pulposus cell count diminishes, leading to a gradual decrease in ECM secretion and, consequently, a reduction in the intervertebral disc's height. Simultaneously, the boundary between the annulus fibrosus and the nucleus pulposus becomes indistinct, destabilizing the overall structure of the intervertebral disc\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Thus, the decline in nucleus pulposus cell count is intimately linked to the advancement of IVDD and is often accompanied by excessive secretion of cellular inflammatory factors and disrupted cellular autophagy function\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAutophagy is essential for preserving cellular homeostasis, ensuring normal cellular operations by eliminating dysfunctional cellular elements and organelles \u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In patients afflicted with degenerative diseases, the autophagy mechanism frequently malfunctions, leading to tissue degeneration, abnormal bone development, and even premature patient death\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.Consequently, conducting in-depth research on the autophagy mechanism in degenerative diseases holds immense significance for the advancement of effective therapeutic approaches.\u003c/p\u003e \u003cp\u003eFargesin (Far), a bioactive novel lignan compound derived from Magnolia fargesii, exhibits anti-inflammatory properties and enhances lipid and glucose metabolism\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Research has revealed that Far attenuates myocardial ischemia/reperfusion injury in rats\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e and alleviates atherosclerosis by promoting reverse cholesterol transport and reducing inflammatory responses\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In THP-1 monocytes, this lignan inhibits PKC-dependent AP-1 and NF-κB signaling, exerting its anti-inflammatory effect\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Additionally, Far facilitates macrophage reprogramming by downregulating the MEK and NF-kB pathways, thereby benefiting osteoarthritis\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBromine-containing domain protein 4 (BRD4), a member of the bromine domain and superterminal structure family, plays a role in regulating cellular gene transcription, autophagy, and inflammatory responses\u003csup\u003e[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. BRD4 has been associated with various age-related diseases such as osteoarthritis, cardiovascular diseases, and cancers. Recently, its role in the skeletal system, particularly its association with IVDD, has gained increasing attention\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Studies show that BRD4 expression significantly increases with higher Pfirrmann grades\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, BRD4 influences the metabolic process of extracellular matrix in nucleus pulposus cells, promoting the development of IVDD. Inhibiting BRD4 elevates cellular autophagy levels and alleviates diabetes-induced IVDD symptoms\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. These findings suggest that BRD4 plays a pivotal role in IVDD pathogenesis and progression, closely linked to autophagy in nucleus pulposus cells and inflammatory factor secretion. However, whether Far can modulate the autophagy level of nucleus pulposus cells and inflammatory factor secretion via BRD4, thereby enhancing cell activity in an inflammatory environment, remains to be elucidated.\u003c/p\u003e \u003cp\u003eThis study unprecedentedly demonstrates that Far modulates the autophagy level of nucleus pulposus cells by interacting with BRD4. Simultaneously, it suppresses the release of inflammatory mediators and bolsters the survival of nucleus pulposus cells amidst an inflammatory milieu, effectively mitigating the manifestations of IVDD. This finding uncovers novel putative mechanisms and therapeutic targets for managing intervertebral disc degeneration, paving the way for potential advancements in treating associated pathologies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and processing\u003c/h2\u003e \u003cp\u003eRat intervertebral disc nucleus pulposus cells (NPCs) were sourced from Ponsure (CP-R145, Ponsure) and maintained in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The culture medium consisted of a complete medium specifically formulated for rat intervertebral disc nucleus pulposus cells, enriched with 10% FBS and 1% penicillin-streptomycin (CM-R145, Ponsure). To investigate the impact of lignans (Far) on the viability of NPCs exposed to IL-1β, the cells were incubated with 10 ng/mL IL-1β for 24 h. Simultaneously, the cells were exposed to varying concentrations (10, 20, 50, 100 \u0026micro;mol/L) of Far (HY-N0719, MCE) for durations of 24, 48, and 72 h. Cell viability was assessed using the CCK-8 assay to determine the most effective concentration and exposure time. Based on these findings, the cells were categorized into the Control group, the IL-1β group, and the IL-1β\u0026thinsp;+\u0026thinsp;Far group for further analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCCK-8 testing\u003c/h3\u003e\n\u003cp\u003eCells were treated with a combination of 10ng/mL IL-1β and different concentrations of Far (10, 20, 50, 100 \u0026micro;mol/L) for 24, 48, and 72 hours. Following treatment, the medium was replaced with fresh medium. Then, 10 \u0026micro;L of CCK8 reagent (C0037, Biyantian) was added to each well and incubated for 2 h. The absorbance of each well was measured at a wavelength of 450nm using a microplate reader (WD-2012B, Beijing Liuyi).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eThe culture dishes containing crawling cells in the 24-well plate were washed thrice with PBS, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 5% BSA at 37\u0026deg;C for 30 min. Subsequently, LC3 (ab192890, abcam, 1:500) and LAMP1 (ab24871, abcam, 1:100) antibodies were incubated overnight at 4\u0026deg;C. After washing, the fluorescent secondary antibody Cy3 Goat Anti-Rabbit IgG(H\u0026thinsp;+\u0026thinsp;L) (AS007, ABclonal, 1:200) was added. The slide was then restained with DAPI, sealed, and examined under a fluorescence microscope (CKX53, Olympus).\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eA specific mass of rat intervertebral discs was taken, and RIPA lysis buffer was added. The samples were ground using a tissue grinder, and total tissue protein or cell culture medium was extracted. Total protein was isolated by ice lysis with RIPA buffer, followed by centrifugation at 12,000 r/min and 4\u0026deg;C for 10 min. The supernatant was collected, and the total protein was quantified using a BCA protein quantification kit (E-BC-K318-M, Elabscience). After denaturing the protein samples, electrophoresis was performed using sodium dodecyl sulfate gel (SDS-PAGE) for 1.5 h. The proteins were then transferred to a PVDF membrane (millipore) and blocked at room temperature with 5% skimmed milk powder for 1 hour. LC3 (ab192890, abcam, 1:2000), Beclin 1 (ab62557, abcam, 1:1000), P62 (ab109012, abcam, 1:5000), and BRD4 (ab75898, abcam, 1:2000) antibodies were incubated overnight at 4\u0026deg;C. The next day, the corresponding HRP-labeled secondary antibody was incubated at room temperature for 1 hour. The PVDF membrane was then moistened with ECL luminescent solution (RJ239676, Thermo Fisher) and developed using an ultra-high sensitivity chemiluminescence imaging system (Tanon-5200, Shanghai Tianneng Technology Co., LTD).\u003c/p\u003e\n\u003ch3\u003eEstablish of the rat intervertebral disc degeneration model\u003c/h3\u003e\n\u003cp\u003e All animal experiments were conducted in compliance with institutional ethical guidelines and approved by the Experimental Animal Ethics Committee (No.20241209-001), and the experiment was carried out in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. In this study, experimental rat were anesthetized via intraperitoneal injection of 2% sodium pentobarbital solution at a dosage of 40 mg/kg. Animal euthanasia was performed through anesthetic overdose, achieved by intraperitoneal administration at a dosage of 100 mg/kg of 2% sodium pentobarbital solution. This procedure complies with the ethical guidelines for treating laboratory animals.\u003c/p\u003e \u003cp\u003e6-week-old SD rats (License Number: SCXK (Beijing) 2024-0001) were purchased from Spafu (Beijing) Biotechnology Co., LTD and housed under controlled conditions of 20\u0026ndash;26℃ and 40%-70% humidity, receiving a standard rat diet. Following a 7-day adaptation period, the rats were randomly assigned to the sham operation group, IVDD\u0026thinsp;+\u0026thinsp;Sh-NC group, IVDD\u0026thinsp;+\u0026thinsp;Sh-BRD4 group, IVDD\u0026thinsp;+\u0026thinsp;Far group, and IVDD\u0026thinsp;+\u0026thinsp;Far\u0026thinsp;+\u0026thinsp;Sh-IVDD group.\u003c/p\u003e \u003cp\u003eAfter anesthetizing the rats, they were positioned on the operating table. A 2 cm incision was made in the left abdomen, and the muscle and fat were carefully separated to expose the L4-5 and L5-6 intervertebral discs. A 1 mm diameter needle was inserted 2 mm into the center of each disc, rotated 360\u0026deg;, and held for 30 s before removal. Subsequently, 0.1 mL of IL-1β (1 \u0026micro;g/mL) was injected into the discs, the wounds were closed in layers, and the rats were returned to their cages for a 6-week period to establish the intervertebral disc degeneration model. On the 4th week following modeling, shRNA-NC and shRNA-BRD4 (provided by Suzhou Zhongke Zhihui Biotechnology Co., LTD) were administered once into the central space of the NP tissues. Additionally, Far was injected weekly via the tail vein at a dose of 15 \u0026micro;mol/kg for a total of 6 weeks.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eX-ray inspection\u003c/h2\u003e \u003cp\u003eThe rats were anesthetized using isoflurane, ensuring their complete immobility during the experiment. They were then positioned on the imaging platform to maintain a natural extension of their spine. Imaging parameters were set according to the equipment manual, typically using a voltage of 26 kV and an exposure time of 10 s to capture lateral X-rays of the rats' caudal vertebrae, specifically focusing on intervertebral disc changes. Calculate the intervertebral disc height index (DHI%) according to the described method\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHE staining\u003c/h3\u003e\n\u003cp\u003eFollowing the baking, dewaxing, and hydration of paraffin sections, they were stained with hematoxylin solution for 3 to 5 min. After rinsing under running water, the sections were differentiated with 1% hydrochloric acid alcohol and inverted blue solution, subsequently stained with eosin for another 3 to 5 min. The stained sections were then dehydrated, sealed, and examined under a microscope (BX43, Olympus).\u003c/p\u003e\n\u003ch3\u003eSafranin O-fast green dyeing\u003c/h3\u003e\n\u003cp\u003eParaffin sections underwent baking, dewaxing, and hydration before being stained with bone tissue green fixation solution for 5 min. Excess staining solution was rinsed away with running tap water until the cartilage appeared colorless. The sections were then briefly treated with 1% hydrochloric acid for 10 s, followed by staining with bone tissue muscose solution for 1 min. After rapid dehydration in four consecutive tanks of anhydrous ethanol (3 s each), the sections were examined under a microscope (BX43, Olympus) until the cartilage turned red with a colorless background. The slices were then made transparent with xylene for 5 min, followed by fresh xylene for another 5 min, and finally sealed with neutral gum for observation. evaluated the condition of the intervertebral disc using the grading scale as described previously\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eSections of rat intervertebral disc tissue underwent baking, dewaxing, and hydration procedures, followed by antigen retrieval using a citric acid buffer. Blocking was performed with 5% BSA, and the primary antibodies\u0026mdash;LC3 (ab192890, abcam, 1:200), Beclin 1 (ab62557, abcam, 1:100), and P62 (ab109012, abcam, 1:1000)\u0026mdash;were incubated. After an overnight incubation at 4\u0026deg;C, the sections were treated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:100), developed with DAB, counterstained with hematoxylin and anti-blue, dehydrated, cleared, sealed, and finally observed under a microscope (BX43, Olympus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData visualization and statistical analysis were conducted using Graphpad Prism 8.0 software. Quantitative results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (X\u0026thinsp;\u0026plusmn;\u0026thinsp;S). For comparisons among multiple groups, one-way analysis of variance was employed, with post hoc testing performed using the Tukey method, * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05,** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001。\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFar can reduce the toxicity to NPCs cells in the presence of IL-1β\u003c/h2\u003e \u003cp\u003eIL-1β-induced NPCs have been utilized in investigations of IVDD\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. To determine the optimal concentration and duration of Far treatment in the presence of 10 ng/mL IL-1β, NPCs cells were stimulated with IL-1β for 24 h and simultaneously treated with various concentrations of Far (10, 20, 50, 100 \u0026micro;mol/L) for 24, 48, and 72 h. Cell viability was assessed using CCK-8. Notably, IL-1β significantly reduced cell viability compared to the control group. However, Far treatment at different concentrations and durations partially reversed this effect, leading to increased cell viability. Specifically, at 24 and 48 h of Far treatment, cell viability exhibited a dose-dependent increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). The highest cell viability was observed with 50 \u0026micro;mol/L Far for 48 h, and these conditions were chosen for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCells were then treated under the identified optimal conditions and grouped as Control, IL-1β, and IL-1β\u0026thinsp;+\u0026thinsp;Far. Elisa was used to measure the levels of inflammatory factors IL-6 and TNF-α. The results indicated that Far significantly reduced the levels of IL-6 and TNF-α elevated by IL-1β treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). In summary, IL-1β treatment reduced cell viability and increased inflammatory factor levels, while Far attenuated these effects, suggesting its protective role against IL-1β-induced cell damage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFar reverses the inhibition of autophagy in NPCs cells induced by IL-1β\u003c/h2\u003e \u003cp\u003eTo assess the impact of Far on autophagy in NPCs cells exposed to IL-1β, we established three experimental groups: Control, IL-1β, and IL-1β\u0026thinsp;+\u0026thinsp;Far. Western blot analysis was used to evaluate the expression levels of autophagy-related proteins LC3II/I, Beclin-1, and P62. Our findings revealed that IL-1β treatment elevated LC3II/I and Beclin-1 levels while reducing P62 levels compared to the Control group. Notably, Far treatment further augmented the autophagy level in NPCs cells (refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Immunofluorescence staining corroborated these results, showing a marked decrease in LC3 and LAMP1 fluorescence after IL-1β exposure, which was subsequently restored by Far treatment, indicating a significant increase in autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the role of Far in modulating autophagy, we pretreated NPCs cells with the autophagy inhibitor 3-MA (10 \u0026micro;M) and compared the effects in IL-1β, IL-1β\u0026thinsp;+\u0026thinsp;Far, and IL-1β\u0026thinsp;+\u0026thinsp;Far\u0026thinsp;+\u0026thinsp;3-MA groups. Western blot results demonstrated that Far treatment led to a significant increase in LC3II/I and Beclin-1 levels, accompanied by a decrease in P62 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-J). Importantly, 3-MA effectively blocked the autophagy-activating effects of Far. Cellular immunofluorescence provided additional support for these findings, revealing that while Far restored LC3 and LAMP1 fluorescence intensities, this effect was reversed by the addition of 3-MA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-L).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFar modulates autophagy in NPCs by regulating BRD4\u003c/h2\u003e \u003cp\u003eOur findings indicate that IL-1β treatment notably elevates the autophagy level in NPCs. Additionally, this treatment leads to a significant increase in BRD4 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We hypothesize that Far influences NPC autophagy via BRD4. To test this hypothesis, we constructed a BRD4 interference vector and transfected it into IL-1β-treated NPCs. Western blot analysis confirmed the successful transfection of BRD4 into NPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe divided the cells into four groups: IL-1β\u0026thinsp;+\u0026thinsp;si-NC, IL-1β\u0026thinsp;+\u0026thinsp;si-BRD4, IL-1β\u0026thinsp;+\u0026thinsp;Far, and IL-1β\u0026thinsp;+\u0026thinsp;Far\u0026thinsp;+\u0026thinsp;si-BRD4 ((provided by Suzhou Zhongke Zhihui Biotechnology Co., LTD). In comparison to the IL-1β\u0026thinsp;+\u0026thinsp;si-NC group, both BRD4 interference and Far treatment led to a marked decrease in inflammatory factors IL-6 and TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). Western blot analysis revealed a significant downregulation of LC3II/I and Beclin-1 protein expressions, along with an increase in P62 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). Immunofluorescence staining further showed enhanced fluorescence intensification of LC3 and LAMP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J). When BRD4 interference was combined with Far treatment, there was a further downregulation of inflammatory factors and autophagy-related protein markers. Thus, our results suggest that Far regulates autophagy in cells through its interaction with BRD4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFar significantly mitigates the degenerative damage to intervertebral discs in rats by suppressing BRD4\u003c/h2\u003e \u003cp\u003e To substantiate through in vivo experiments that Far modulates BRD4 to stimulate autophagy and consequently retard IVDD progression, we adaptively reared SD rats and randomly assigned them to the Sham, IVDD\u0026thinsp;+\u0026thinsp;Sh-NC, IVDD\u0026thinsp;+\u0026thinsp;Sh-BRD4, IVDD\u0026thinsp;+\u0026thinsp;Far, and IVDD\u0026thinsp;+\u0026thinsp;Far\u0026thinsp;+\u0026thinsp;Sh-IVDD groups, treating each group accordingly. X-ray imaging revealed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) that the intervertebral discs of the IVDD\u0026thinsp;+\u0026thinsp;Sh-NC group exhibited swelling, loss and blurring of endplate boundary height, and compromised structural integrity, with the disc height index (DHI%) significantly lower than that in the Sham group. However, upon shRNA-BRD4 and Far administration, the degenerative modifications in the intervertebral disc structure were ameliorated, without notable endplate blurring or structural compromise, and the DHI% values also indicated that shRNA-BRD4 could prevent the occurrence of IVDD. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing BRD4 significantly attenuates the inflammatory response in rat IVD and mitigates the progression of IVDD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFar exhibits potent efficacy in mitigating IVDD damage. HE staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and safranin O-fast green staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) revealed bleeding in the bone marrow cavity, chondrocyte degeneration, altered bone trabeculae structure, and pronounced tissue damage in the IVDD\u0026thinsp;+\u0026thinsp;Sh-NC group. The intervertebral disc histological score markedly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) alongside elevated levels of inflammatory factors IL-6 and TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). Both shRNA-BRD4 and Far interventions substantially reduced bone tissue hemorrhage and chondrocyte degeneration while lowering the histological score. IL-6 and TNF-α levels decreased significantly, with the combined shRNA-BRD4 and Far treatment showing the most pronounced effect. These findings indicate that Far mitigates IVDD by suppressing cellular inflammatory factors and ameliorating bone tissue structural damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFar effectively slows down IVDD injury by suppressing BRD4 activity and enhancing autophagy in rat IVD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBoth in vitro and in vivo experiments have demonstrated Far's ability to modulate autophagy in NPCs through BRD4 regulation. Western blot analysis revealed significant upregulation of LC3II/I and Beclin-1 protein levels, along with a notable decrease in P62 levels, in the intervertebral discs of IVDD rats. The activation of autophagy was further evident after the introduction of shRNA-BRD4 or Far, leading to the recovery of related protein expressions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). Notably, the combined treatment of shRNA-BRD4 and Far exhibited the most pronounced effect on autophagy activation, as confirmed by immunohistochemical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIVDD is a degenerative spinal disease strongly associated with age, featuring a relatively high incidence rate. It severely impairs patients' occupational function and quality of life, while also imposing a considerable economic burden on families and society\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. As IVDD progresses chronically, it may give rise to various spine-related pathological changes, including intervertebral disc protrusion, spinal forward displacement, spinal canal stenosis, and degenerative scoliosis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. These conditions often manifest as acute or chronic LBP, a widespread public health concern globally. Notably, approximately 40% of LBP cases are attributed to IVDD\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Although therapeutic approaches for IVDD have been documented, its precise pathogenesis, as well as the therapeutic role and mechanism of Far in IVDD, remain incompletely understood. This study novelly investigated how Far mitigates IVDD damage by suppressing BRD4 expression in NPCs, diminishing cellular inflammatory responses, activating autophagy, and enhancing the activity of nucleus pulposus cells. Our findings elucidate the mechanism of Far in IVDD and offer innovative therapeutic insights for its future treatment.\u003c/p\u003e \u003cp\u003eFar exhibits anti-inflammatory properties and holds significant value in treating cardiovascular and inflammatory diseases\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. This study explored Far's effect on the viability of IL-1β-induced nucleus pulposus cells. Far demonstrated a capacity to restore the reduced cell viability caused by IL-1β stimulation, showing a dose-dependent trend. In vitro experiments revealed that IL-1β stimulation of NPCs led to increased levels of IL-6 and TNF-α, aligning with the notable elevation of these inflammatory factors in IVDD\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. During IVDD progression, inflammatory factors rise significantly, triggering local autoimmune inflammatory responses that disrupt the normal metabolic process of the extracellular matrix (ECM), ultimately leading to IVDD occurrence\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. In this study, IL-1β was used to induce NPCs and mimic the pathophysiological phenomena of IVDD. However, the specific role of Far in IVDD remained unexplored. Current research suggests that Far exerts an anti-inflammatory effect in OA development\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, corroborating our experimental findings. Following Far treatment, inflammatory factor levels in NPCs decreased significantly. Similarly, in vivo experimental results indicated that Far could notably reduce inflammatory factor levels in intervertebral disc tissue, suggesting a potent anti-inflammatory role of Far in IVDD. Furthermore, our study revealed that Far significantly suppresses BRD4 expression and activates autophagy in cells, a finding that has not been previously reported.\u003c/p\u003e \u003cp\u003eBRD4, a key member of the BET protein family, plays a pivotal role in regulating the transcription of genes involved in various cellular processes, including senescence, autophagy, inflammation, cell death, and extracellular matrix (EC) metabolism\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. In recent years, research has intensified on BRD4's specific functions in the skeletal system, particularly in IVDD\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Our findings reveal that under the influence of IL-1β, BRD4 expression escalates, leading to a surge in cellular inflammatory factors and a concurrent inhibition of autophagy. However, with the introduction of Far treatment, BRD4 becomes silenced, resulting in the recovery of inflammatory factor levels and the reactivation of autophagy. These observations implicate BRD4 as a critical factor in the interplay between cellular inflammation and autophagy during the progression of IVDD. Furthermore, our data suggest that Far exerts its effects by modulating BRD4 expression. This aligns with previous reports indicating that BRD4 inhibition can ameliorate IVDD by regulating cellular inflammatory signaling and autophagy pathways \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Additionally, our in vitro studies demonstrate that silencing BRD4 significantly mitigates intervertebral disc injury in IVDD. Based on these findings, we hypothesize that Far's beneficial effects on intervertebral disc degeneration, cellular inflammation, and autophagy are mediated through BRD4 inhibition.\u003c/p\u003e \u003cp\u003eAutophagy is a self-protection mechanism of eukaryotic cells, which can digest and degrade their own damaged, degenerated or senescent biological macromolecules and organelles, and maintain cellular homeostasis\u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In IVDD diseases, autophagy is associated with cell senescence and cell death of NPCs\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Studies have reported that 3-MA inhibiting autophagy can eliminate the anti-cell death and senescence effects of SIRT6 on NP cells\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. In addition, autophagy controls the senescence of bone marpe-derived mesenchymal stem cells during bone senescence\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, and autophagy is closely related to the development of IVDD. Our research results revealed that in the treatment of IVDD, Far, in addition to its anti-inflammatory effect, autophagy also plays a key role. The results indicated that Far could significantly promote the expression of autophagy-related proteins LC3II/I and Beclin-1. The use of 3-MA to inhibit autophagy counteracted the autophagy-activating effect of Far on NPCs cells. Moreover, Far can inhibit the expression of BRD4. Therefore, Far can exert therapeutic effects on IVDD by inhibiting BRD4-activated autophagy and suppressing inflammatory responses, which is consistent with the research findings that BRD4 knockdown activates autophagy and inhibits the activity of NLRP3 inflammasome through the NF-kB pathway to alleviate the degradation of extracellular substances in NP cells and exert anti-IVDD effects\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study demonstrates that Far mitigates intervertebral disc lesions in IVDD by modulating BRD4 expression, suppressing cellular inflammatory markers IL-6 and TNF-α, elevating LC3II/I and Beclin-1 protein levels, and activating cellular autophagy signaling. This offers novel insights and potential therapeutic targets for addressing intervertebral disc degeneration. Nevertheless, our investigation has its constraints. Besides focusing on NPCs, the progression of IVDD is tied to AF degeneration, and it remains unclear if BRD4 engages in IVDD via alternate signaling pathways. Hence, further examination of BRD4's role and mechanisms in IVDD pathogenesis is warranted.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompliance with ethical standards\u003c/h2\u003e \u003cp\u003eConflict of interest The authors declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by grants from Science and Technology Program of Jiangxi Provincial Administration of Traditional Chinese Medicine (2024A0152).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMinqin Mao: Conceptualization, Data curation, Visualization, Methodology, Writing - original draft, Writing - review \u0026amp; editing; Huan Yu and Liang Zhang: Data curation, Validation, Investigation; Tao Zhang, Wen Xu and Jingtang Li: Investigation, Methodology, Formal Analysis, Conceptualization, Supervision; Yongxing Peng: Conceptualization, Methodology, Writing - review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings are available within the article materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCollaborators GLBP (2023) Global, regional, and national burden of other musculoskeletal disorders, 1990\u0026ndash;2020, and projections to 2050: a systematic analysis of the Global Burden of Disease Study 2021.[J]. Lancet Rheumatol. 5(11)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikolay LM et al (2016) Genetic Alterations in Intervertebral Disc Disease.[J]. Front Surg. 3(0)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM D H, et al. Human intervertebral disc: structure and function.[J]. Anat Rec. (1988) 220(4)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD R E, et al. Types I and II collagens in intervertebral disc. 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Biomark Res 12(1):92\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRebecca LO et al (2019) JNK signalling mediates aspects of maternal immune activation: importance of maternal genotype in relation to schizophrenia risk.[J]. J Neuroinflammation. 16(1)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhan S et al (2020) lncRNA HOTAIR upregulates autophagy to promote apoptosis and senescence of nucleus pulposus cells.[J]. J Cell Physiol 235(3):2195\u0026ndash;2208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa Y et al (2018) Autophagy controls mesenchymal stem cell properties and senescence during bone aging.[J]. Aging Cell. 17(1)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJunmin H et al (2020) Bromodomain-containing protein 4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity in NP cells.[J]. J Cell Physiol. 235(0)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fargesin, Intervertebral disc degeneration, NPCs, BRD4, Autophagy","lastPublishedDoi":"10.21203/rs.3.rs-8530418/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8530418/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIntervertebral Disc Degeneration (IVDD) stands as the prevalent chronic skeletal muscle degenerative disease and the primary culprit behind low back pain, yet its underlying mechanism remains elusive. Fargesin, a novel bioactive lignan compound known for its anti-inflammatory properties, also has an unclear mechanism of action in IVDD.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo unravel this mechanism, we employed the CCK8 assay to determine the optimal treatment duration and concentration of Fargesin. For our in vitro experiments, we induced NPCs cells with IL-1β to mimic the IVDD model, while for in vivo studies, we established a rat IVDD model through surgical intervention coupled with an injection of 0.1mL IL-1β (1\u0026micro;g/mL). We utilized Elisa to measure the levels of inflammatory cytokines IL-6 and TNF-α. Western blotting and immunofluorescence techniques were harnessed to assess the expression of autophagy markers and BRD4 protein in the cells. Additionally, X-ray imaging, HE staining, and safranin o-fast green aided in evaluating the intervertebral disc lesions in our in vitro experiments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur findings revealed that Fargesin significantly suppressed the inflammatory response triggered by IL-1β in NPCs and attenuated the elevated expression of BRD4. By downregulating BRD4 expression, Fargesin upregulated the proteins LC3II/I, Beclin-1, and LAMP1 related to the autophagy signaling pathway, while decreasing P62 expression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFar activates the autophagy signaling pathway by inhibiting the expression of BRD4, enhances the activity of NPCs, and alleviates the pathological damage of intervertebral discs with ivdd lesions.\u003c/p\u003e","manuscriptTitle":"Fargesin enhances the condition of intervertebral disc degeneration by suppressing BRD4 expression and influencing autophagy in NPCs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 00:07:11","doi":"10.21203/rs.3.rs-8530418/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-14T21:26:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-14T14:47:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97374040721458520231588490663629432972","date":"2026-01-14T13:19:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T14:28:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312436875754597414657412677350598439725","date":"2026-01-10T14:27:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-08T13:44:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-08T01:00:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-07T07:44:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Histology","date":"2026-01-06T10:49:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"93e53c18-46e7-4c26-8b03-0e79c3164da6","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:01:27+00:00","versionOfRecord":{"articleIdentity":"rs-8530418","link":"https://doi.org/10.1007/s10735-026-10777-4","journal":{"identity":"journal-of-molecular-histology","isVorOnly":false,"title":"Journal of Molecular Histology"},"publishedOn":"2026-03-17 15:58:12","publishedOnDateReadable":"March 17th, 2026"},"versionCreatedAt":"2026-01-16 00:07:11","video":"","vorDoi":"10.1007/s10735-026-10777-4","vorDoiUrl":"https://doi.org/10.1007/s10735-026-10777-4","workflowStages":[]},"version":"v1","identity":"rs-8530418","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8530418","identity":"rs-8530418","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}