A comparative analysis of TonEBP conditional knockout mouse models reveals inter-dependency between compartments of the intervertebral disc.

Interactions between notochord and sclerotome are required for normal embryonic spine patterning, but whether the postnatal derivatives of these tissues also require interactions for postnatal intervertebral disc (IVD) growth and maintenance is less established. We report here the comparative analysis of four conditional knockout mice deficient for TonEBP, a transcription factor known to allow cells to adapt to changes in extracellular osmotic pressure, in specific compartments of the IVD. We show that TonEBP deletion in nucleus pulposus (NP) cells does not affect their survival or aggrecan expression, but promoted cell proliferation in the NP and in adjacent vertebral growth plates (GPs). In cartilage end plates/GPs, TonEBP deletion induced cell death, but also structural alterations in the adjacent NP cells and vertebral bodies. Embryonic or postnatal TonEBP loss generated similar IVD changes. In addition to demonstrating the requirement of TonEBP in the different compartments of the IVD, this comparative analysis uncovers the in vivo interdependency of the different IVD compartments during the growth of the postnatal IVD-vertebral units.

Ccn2a-FGFR1-SHH signaling is necessary for intervertebral disc homeostasis and regeneration in adult zebrafish.

Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.

Nonpathological inflammation drives the development of an avian flight adaptation.

The development of modern birds provides a window into the biology of their dinosaur ancestors. We investigated avian postnatal development and found that sterile inflammation drives formation of the pygostyle, a compound structure resulting from bone fusion in the tail. Inflammation is generally induced by compromised tissue integrity, but here is involved in normal bone development. Transcriptome profiling and immuno/histochemistry reveal a robust inflammatory response that resembles bone fracture healing. The data suggest the involvement of necroptosis and multiple immune cell types, notably heterophils (the avian equivalent of neutrophils). Additionally, nucleus pulposus structures, heretofore unknown in birds, are involved in disc remodeling. Anti-inflammatory corticosteroid treatment inhibited vertebral fusion, substantiating the crucial role of inflammation in the ankylosis process. This study shows that inflammation can drive developmental skeletogenesis, in this case leading to the formation of a flight-adapted tail structure on the evolutionary path to modern avians.

Intradiscal inflammatory stimulation induces spinal pain behavior and intervertebral disc degeneration in vivo.

Degeneration of the intervertebral disc (IVD) results in a range of symptomatic (i.e., painful) and asymptomatic experiences. Components of the degenerative environment, including structural disruption and inflammatory cytokine production, often correlate with pain severity. However, the role of inflammation in the activation of pain and degenerative changes has been complex to delineate. The most common IVD injury model is puncture; however, it initiates structural damage that is not representative of the natural degenerative cascade. In this study, we utilized in vivo injection of lipopolysaccharide (LPS), a pro-inflammatory stimulus, into rat caudal IVDs using 33G needles to induce inflammatory activation without the physical tissue disruption caused by puncture using larger needles. LPS injection increased gene expression of pro-inflammatory cytokines (Tnfa, Il1b) and macrophage markers (Inos, Arg1), supported by immunostaining of macrophages (CD68, CCR7, Arg1) and systemic changes in blood cytokine and chemokine levels. Disruption of the IVD structural integrity after LPS injection was also evident through changes in histological grading, disc height, and ECM biochemistry. Ultimately, intradiscal inflammatory stimulation led to local mechanical hyperalgesia, demonstrating that pain can be initiated by inflammatory stimulation of the IVD. Gene expression of nociceptive markers (Ngf, Bdnf, Cgrp) and immunostaining for neuron ingrowth (PGP9.5) and sensitization (CGRP) in the IVD were also shown, suggesting a mechanism for the pain exhibited. To our knowledge, this rat IVD injury model is the first to demonstrate local pain behavior resulting from inflammatory stimulation of caudal IVDs. Future studies will examine the mechanistic contributions of inflammation in mediating pain.

Single cell RNA sequencing reveals emergent notochord-derived cell subpopulations in the postnatal nucleus pulposus.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-beta and phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

MIF inhibition attenuates intervertebral disc degeneration by reducing nucleus pulposus cell apoptosis and inflammation.

Nucleus pulposus cells (NPCs) apoptosis and inflammation are the extremely critical factors of intervertebral disc degeneration (IVDD). Nevertheless, the underlying procedure remains mysterious. Macrophage migration inhibitory factor (MIF) is a cytokine that promotes inflammation and has been demonstrated to have a significant impact on apoptosis and inflammation. For this research, we employed a model of NPCs degeneration stimulated by lipopolysaccharides (LPS) and a rat acupuncture IVDD model to examine the role of MIF in vitro and in vivo, respectively. Initially, we verified that there was a significant rise of MIF expression in the NP tissues of individuals with IVDD, as well as in rat models of IVDD. Furthermore, this augmented expression of MIF was similarly evident in degenerated NPCs. Afterwards, it was discovered that ISO-1, a MIF inhibitor, effectively decreased the quantity of cells undergoing apoptosis and inhibited the release of inflammatory molecules (TNF-α, IL-1ß, IL-6). Furthermore, it has been shown that the PI3K/Akt pathway plays a vital part in the regulation of NPCs degeneration by MIF. Ultimately, we showcased that the IVDD process was impacted by the MIF inhibitor in the rat model. In summary, our experimental results substantiate the significant involvement of MIF in the degeneration of NPCs, and inhibiting MIF activity can effectively mitigate IVDD.

Therapeutic CRISPR epigenome editing of inflammatory receptors in the intervertebral disc.

Low back pain (LBP) ranks among the leading causes of disability worldwide and generates a tremendous socioeconomic cost. Disc degeneration, a leading contributor to LBP, can be characterized by the breakdown of the extracellular matrix of the intervertebral disc (IVD), disc height loss, and inflammation. The inflammatory cytokine tumor necrosis factor α (TNF-α) has multiple signaling pathways, including proinflammatory signaling through tumor necrosis factor receptor 1 superfamily, member 1a (TNFR1 or TNFRSF1A), and has been implicated as a primary mediator of disc degeneration. We tested our ability to regulate the TNFR1 signaling pathway in vivo, utilizing CRISPR epigenome editing to slow the progression of disc degeneration in rats. Sprague-Dawley rats were treated with TNF-α and CRISPR interference (CRISPRi)-based epigenome-editing therapeutics targeting TNFR1, showing decreased behavioral pain in a disc degeneration model. Surprisingly, while treatment with the vectors alone was therapeutic, the TNF-α injection became therapeutic after TNFR1 modulation. These results suggest direct inflammatory receptor modulation as a potent strategy for treating disc degeneration.

Dedifferentiation-like reprogramming of degenerative nucleus pulposus cells into notochordal-like cells by defined factors.

The extensive degeneration of functional somatic cells and the depletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT [OFT]) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases.

Lactic acid promotes nucleus pulposus cell senescence and corresponding intervertebral disc degeneration via interacting with Akt.

The accumulation of metabolites in the intervertebral disc is considered an important cause of intervertebral disc degeneration (IVDD). Lactic acid, which is a metabolite that is produced by cellular anaerobic glycolysis, has been proven to be closely associated with IVDD. However, little is known about the role of lactic acid in nucleus pulposus cells (NPCs) senescence and oxidative stress. The aim of this study was to investigate the effect of lactic acid on NPCs senescence and oxidative stress as well as the underlying mechanism. A puncture-induced disc degeneration (PIDD) model was established in rats. Metabolomics analysis revealed that lactic acid levels were significantly increased in degenerated intervertebral discs. Elimination of excessive lactic acid using a lactate oxidase (LOx)-overexpressing lentivirus alleviated the progression of IVDD. In vitro experiments showed that high concentrations of lactic acid could induce senescence and oxidative stress in NPCs. High-throughput RNA sequencing results and bioinformatic analysis demonstrated that the induction of NPCs senescence and oxidative stress by lactic acid may be related to the PI3K/Akt signaling pathway. Further study verified that high concentrations of lactic acid could induce NPCs senescence and oxidative stress by interacting with Akt and regulating its downstream Akt/p21/p27/cyclin D1 and Akt/Nrf2/HO-1 pathways. Utilizing molecular docking, site-directed mutation and microscale thermophoresis assays, we found that lactic acid could regulate Akt kinase activity by binding to the Lys39 and Leu52 residues in the PH domain of Akt. These results highlight the involvement of lactic acid in NPCs senescence and oxidative stress, and lactic acid may become a novel potential therapeutic target for the treatment of IVDD.

Intervertebral disc-intrinsic Hedgehog signaling maintains disc cell phenotypes and prevents disc degeneration through both cell autonomous and non-autonomous mechanisms.

Intervertebral disc degeneration is closely related to abnormal phenotypic changes in disc cells. However, the mechanism by which disc cell phenotypes are maintained remains poorly understood. Here, Hedgehog-responsive cells were found to be specifically localized in the inner annulus fibrosus and cartilaginous endplate of postnatal discs, likely activated by Indian Hedgehog. Global inhibition of Hedgehog signaling using a pharmacological inhibitor or Agc1-CreERT2-mediated deletion of Smo in disc cells of juvenile mice led to spontaneous degenerative changes in annulus fibrosus and cartilaginous endplate accompanied by aberrant disc cell differentiation in adult mice. In contrast, Krt19-CreER-mediated deletion of Smo specifically in nucleus pulposus cells led to healthy discs and normal disc cell phenotypes. Similarly, age-related degeneration of nucleus pulposus was accelerated by genetic inactivation of Hedgehog signaling in all disc cells, but not in nucleus pulposus cells. Furthermore, inactivation of Gli2 in disc cells resulted in partial loss of the vertebral growth plate but otherwise healthy discs, whereas deletion of Gli3 in disc cells largely corrected disc defects caused by Smo ablation in mice. Taken together, our findings not only revealed for the first time a direct role of Hedgehog-Gli3 signaling in maintaining homeostasis and cell phenotypes of annuls fibrosus and cartilaginous endplate, but also identified disc-intrinsic Hedgehog signaling as a novel non-cell-autonomous mechanism to regulate nucleus pulposus cell phenotype and protect mice from age-dependent nucleus pulposus degeneration. Thus, targeting Hedgehog signaling may represent a potential therapeutic strategy for the prevention and treatment of intervertebral disc degeneration.

Intervertebral disc repair and regeneration: Insights from the notochord.

The vertebrate notochord plays an essential role in patterning multiple structures during embryonic development. In the early 2000s, descendants of notochord cells were demonstrated to form the entire nucleus pulposus of the intervertebral disc in addition to their key role in embryonic patterning. The nucleus pulposus undergoes degeneration during postnatal life, which can lead to back pain. Recently, gene and protein profiles of notochord and nucleus pulposus cells have been identified. These datasets, coupled with the ability to differentiate human induced pluripotent stem cells (iPSCs) into cells that resemble nucleus pulposus cells, provide the possibility of generating a cell-based therapy to halt and/or reverse disc degeneration.

SEPHS1 attenuates intervertebral disc degeneration by delaying nucleus pulposus cell senescence through the Hippo-Yap/Taz pathway.

Nucleus pulposus cell (NPC) senescence is a major cause of intervertebral disc degeneration (IVDD). Oxidative stress and reactive oxygen species (ROS) play critical roles in regulating cell senescence. Selenophosphate synthetase 1 (SEPHS1) was reported to play an important role in mitigating oxidative stress in an osteoarthritis (OA) model by reducing the production of ROS, thereby, delaying the occurrence and development of osteoarthritis. In this study, we explored the, hitherto unknown, role of SEPHS1 in IVDD in vitro and in vivo using an interleukin-1ß (IL-1ß)-induced NPC senescence model and a rat needle puncture IVDD model, respectively. SEPHS1 delayed NPC senescence in vitro by reducing ROS production. Age-related dysfunction was also ameliorated by the overexpression of SEPHS1 and inhibition of the Hippo-Yap/Taz signaling pathway. In vivo experiments revealed that the overexpression of SEPHS1 and inhibition of Hippo-Yap/Taz alleviated IVDD in rats. Moreover, a selenium (Se)-deficient diet and lack of SEPHS1 synergistically aggravated IVDD progression. Taken together, our results demonstrate that SEPHS1 plays a significant role in NPC senescence. Overexpression of SEPHS1 and inhibition of Hippo-Yap/Taz can delay NPC senescence, restore the balance of extracellular matrix metabolism, and attenuate IVDD. SEPHS1 could be a promising therapeutic target for IVDD.NEW & NOTEWORTHY Selenophosphate synthetase 1 (SEPHS1) deficiency leads to an increase in reactive oxygen species levels and in the subsequent activation of the Hippo-Yap/Taz signaling pathway. In the rat model of intervertebral disc degeneration (IVDD), overexpression of SEPHS1 and inhibition of Hippo-YAP/Taz mitigated the progression of disc degeneration indicating the involvement of SEPHS1 in IVDD. SEPHS1 is a promising therapeutic target for IVDD.

Brachyury promotes extracellular matrix synthesis through transcriptional regulation of Smad3 in nucleus pulposus.

Metabolic dysfunction of the extracellular matrix (ECM) is one of the primary causes of intervertebral disc degeneration (IVDD). Previous studies have demonstrated that the transcription factor Brachyury (Bry) has the potential to promote the synthesis of collagen II and aggrecan, while the specific mechanism is still unknown. In this study, we used a lipopolysaccharide (LPS)-induced model of nucleus pulposus cell (NPC) degeneration and a rat acupuncture IVDD model to elucidate the precise mechanism through which Bry affects collagen II and aggrecan synthesis in vitro and in vivo. First, we confirmed Bry expression decreased in degenerated human nucleus pulposus (NP) cells (NPCs). Knockdown of Bry exacerbated the decrease in collagen II and aggrecan expression in the lipopolysaccharide (LPS)-induced NPCs degeneration in vitro model. Bioinformatic analysis indicated that Smad3 may participate in the regulatory pathway of ECM synthesis regulated by Bry. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays demonstrated that Bry enhances the transcription of Smad3 by interacting with a specific motif on the promoter region. In addition, Western blot and reverse transcription-qPCR assays demonstrated that Smad3 positively regulates the expression of aggrecan and collagen II in NPCs. The following rescue experiments revealed that Bry-mediated regulation of ECM synthesis is partially dependent on Smad3 phosphorylation. Finally, the findings from the in vivo rat acupuncture-induced IVDD model were consistent with those obtained from in vitro assays. In conclusion, this study reveals that Bry positively regulates the synthesis of collagen II and aggrecan in NP through transcriptional activation of Smad3.NEW & NOTEWORTHY Mechanically, in the nucleus, Bry enhances the transcription of Smad3, leading to increased expression of Smad3 protein levels; in the cytoplasm, elevated substrate levels further lead to an increase in the phosphorylation of Smad3, thereby regulating collagen II and aggrecan expression. Further in vivo experiments provide additional evidence that Bry can alleviate IVDD through this mechanism.

DNMT3a-mediated methylation of PPARγ promote intervertebral disc degeneration by regulating the NF-κB pathway.

Intervertebral disc degeneration (IVDD) is a common chronic musculoskeletal disease that causes chronic low back pain and imposes an immense financial strain on patients. The pathological mechanisms underlying IVDD have not been fully elucidated. The development of IVDD is closely associated with abnormal epigenetic changes, suggesting that IVDD progression may be controlled by epigenetic mechanisms. Consequently, this study aimed to investigate the role of epigenetic regulation, including DNA methyltransferase 3a (DNMT3a)-mediated methylation and peroxisome proliferator-activated receptor γ (PPARγ) inhibition, in IVDD development. The expression of DNMT3a and PPARγ in early and late IVDD of nucleus pulposus (NP) tissues was detected using immunohistochemistry and western blotting analyses. Cellularly, DNMT3a inhibition significantly inhibited IL-1ß-induced apoptosis and extracellular matrix (ECM) degradation in rat NP cells. Pretreatment with T0070907, a specific inhibitor of PPARγ, significantly reversed the anti-apoptotic and ECM degradation effects of DNMT3a inhibition. Mechanistically, DNMT3a modified PPARγ promoter hypermethylation to activate the nuclear factor-κB (NF-κB) pathway. DNMT3a inhibition alleviated IVDD progression. Conclusively, the results of this study show that DNMT3a activates the NF-κB pathway by modifying PPARγ promoter hypermethylation to promote apoptosis and ECM degradation. Therefore, we believe that the ability of DNMT3a to mediate the PPARγ/NF-κB axis may provide new ideas for the potential pathogenesis of IVDD and may become an attractive target for the treatment of IVDD.

microRNA-365 attenuated intervertebral disc degeneration through modulating nucleus pulposus cell apoptosis and extracellular matrix degradation by targeting EFNA3.

This present study is aimed to investigate the role of microRNA-365 (miR-365) in the development of intervertebral disc degeneration (IDD). Nucleus pulposus (NP) cells were transfected by miR-365 mimic and miR-365 inhibitor, respectively. Concomitantly, the transfection efficiency and the expression level of miRNA were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Meanwhile, NP cells apoptosis was measured through propidium iodide (PI)-AnnexinV-fluorescein isothiocyanate (FITC) apoptosis detection kit. Subsequently, immunofluorescence (IF) staining was performed to assess the expression of collagen II, aggrecan and matrix metalloproteinase 13 (MMP-13). In addition, bioinformatic prediction and Luciferase reporter assay were used to reveal the target gene of miR-365. Finally, we isolated the primary NP cells from rats and injected NP-miR-365 in rat IDD models. The results showed that overexpression of miR-365 could effectively inhibit NP cells apoptosis and MMP-13 expression and upregulate the expression of collagen II and aggrecan. Conversely, suppression of miR-365 enhanced NP cell apoptosis and elevated MMP-13 expression, but decreased the expression of collagen II and aggrecan. Moreover, the further data demonstrated that miR-365 mediated NP cell degradation through targeting ephrin-A3 (EFNA3). In addition, the cells apoptosis and catabolic markers were increased in NP cells when EFNA3 upregulated. More importantly, the vivo data supported that miR-365-NP cells injection ameliorated IDD in rats models. miR-365 could alleviate the development of IDD by regulating NP cell apoptosis and ECM degradation, which is likely mediated by targeting EFNA3. Therefore, miR-365 may be a promising therapeutic avenue for treatment IDD through EFNA3.

Identifying the protective effects of miR-874-3p/ATF3 axis in intervertebral disc degeneration by single-cell RNA sequencing and validation.

Intervertebral disc degeneration (IVDD) severely affects the work and the quality of life of people. We previously demonstrated that silencing activation transcription factor 3 (ATF3) blocked the IVDD pathological process by regulating nucleus pulposus cell (NPC) ferroptosis, apoptosis, inflammation, and extracellular matrix (ECM) metabolism. Nevertheless, whether miR-874-3p mediated the IVDD pathological process by targeting ATF3 remains unclear. We performed single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis to identify ATF3 as a key ferroptosis gene in IVDD. Then, Western blotting, flow cytometry, ELISA, and animal experiments were performed to validate the roles and regulatory mechanisms of miR-874-3p/ATF3 signalling axis in IVDD. ATF3 was highly expressed in IVDD patients and multiple cell types of IVDD rat, as revealed by scRNA-seq and bioinformatics analysis. GO analysis unveiled the involvement of ATF3 in regulating cell apoptosis and ECM metabolism. Furthermore, we verified that miR-874-3p might protect against IVDD by inhibiting NPC ferroptosis, apoptosis, ECM degradation, and inflammatory response by targeting ATF3. In vivo experiments displayed the protective effect of miR-874-3p/ATF3 axis on IVDD. These findings propose the potential of miR-874-3p and ATF3 as biomarkers of IVDD and suggest that targeting the miR-874-3p/ATF3 axis may be a therapeutic target for IVDD.

Regulatory mechanism of FCGR2A in macrophage polarization and its effects on intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) poses a significant health burden, necessitating a deeper understanding of its molecular underpinnings. Transcriptomic analysis reveals 485 differentially expressed genes (DEGs) associated with IDD, underscoring the importance of immune regulation. Weighted gene co-expression network analysis (WGCNA) identifies a yellow module strongly correlated with IDD, intersecting with 197 DEGs. Protein-protein interaction (PPI) analysis identifies ITGAX, MMP9 and FCGR2A as hub genes, predominantly expressed in macrophages. Functional validation through in vitro and in vivo experiments demonstrates the pivotal role of FCGR2A in macrophage polarization and IDD progression. Mechanistically, FCGR2A knockdown suppresses M1 macrophage polarization and NF-κB phosphorylation while enhancing M2 polarization and STAT3 activation, leading to ameliorated IDD in animal models. This study sheds light on the regulatory function of FCGR2A in macrophage polarization, offering novel insights for IDD intervention strategies. KEY POINTS: This study unveils the role of FCGR2A in intervertebral disc (IVD) degeneration (IDD). FCGR2A knockdown mitigates IDD in cellular and animal models. Single-cell RNA-sequencing uncovers diverse macrophage subpopulations in degenerated IVDs. This study reveals the molecular mechanism of FCGR2A in regulating macrophage polarization. This study confirms the role of the NF-κB/STAT3 pathway in regulating macrophage polarization in IDD.

The role of sirtuins in intervertebral disc degeneration: Mechanisms and therapeutic potential.

Intervertebral disc degeneration (IDD) is one of the main causes of low back pain, which affects the patients' quality of life and health and imposes a significant socioeconomic burden. Despite great efforts made by researchers to understand the pathogenesis of IDD, effective strategies for preventing and treating this disease remain very limited. Sirtuins are a highly conserved family of (NAD+)-dependent deacetylases in mammals that are involved in a variety of metabolic processes in vivo. In recent years, sirtuins have attracted much attention owing to their regulatory roles in IDD on physiological activities such as inflammation, apoptosis, autophagy, aging, oxidative stress, and mitochondrial function. At the same time, many studies have explored the therapeutic effects of sirtuins-targeting activators or micro-RNA in IDD. This review summarizes the molecular pathways of sirtuins involved in IDD, and summarizes the therapeutic role of activators or micro-RNA targeting Sirtuins in IDD, as well as the current limitations and challenges, with a view to provide possible solutions for the treatment of IDD.

Abnormal stress promotes intervertebral disc degeneration through WTAP/YTHDF2-dependent TIMP3 m6A modification.

Mechanical environment worsening is an important predisposing factor that accelerates intervertebral disc degeneration (IDD), but its specific regulatory mechanisms remain unclear. In this study, we reveal the molecular mechanisms of WTAP/YTHDF2-mediated m6A modification in abnormal stress-induced intervertebral disc (IVD) matrix degradation. WTAP expression in human nucleus pulposus cells was elevated under tension. Similarly, high WTAP expression was detected in severe degenerated human and rat nucleus pulposus tissues. Functionally, WTAP was found to increase the TIMP3 transcript methylation level under tension, resulting in YTHDF2 recognition, binding, and induction of its degradation. Reduction in TIMP3 caused increases in active matrix metalloproteinases, ultimately inducing extracellular matrix degradation in nucleus pulposus cells. Macroscopically, this promotes IDD. Additionally, in vitro and in vivo inhibition of WTAP expression or TIMP3 overexpression significantly increased stress resistance in the nucleus pulposus, thereby alleviating IDD. Our results show that abnormal stress disrupts IVD matrix stability through WTAP/YTHDF2-dependent TIMP3 m6A modification.

Protectin D1 ameliorates non-compressive lumbar disc herniation through SIRT1-mediated CGRP signaling.

Background. Neuro-inflammatory response promotes the initiation and sustenance of lumbar disc herniation (LDH). Protectin D1 (PD1), as a new type of specialized pro-resolving mediator (SPM), can improve the prognosis of various inflammatory diseases. Recent studies have shown that over representation of calcitonin gene-related peptides (CGRP) may activate nociceptive signaling following nerve injury. Silent information regulator 1 (SIRT1) is ubiquitously expressed in the dorsal horn of the spinal cord and plays a role in the pathogenesis of LDH. In this study, we investigated the analgesic effects of PD1 and elucidated the impact of neurogenic inflammation in the pathogenesis of neuropathic pain induced by non-compressive lumbar disc herniation (NCLDH) in a rat model. Methods. NCLDH models were established by applying protruding autologous nucleus pulposus to the L5 Dorsal root ganglion (DRG). PD1, SIRT1 antagonist or agonist, CGRP or antagonist were administered as daily intrathecal injections for three consecutive days postoperatively. Behavioral tests were conducted to assess mechanical and thermal hyperalgesia. The ipsilateral lumbar (L4-6) segment of the spinal dorsal horn was isolated for further analysis. Alterations in the release of SIRT1 and CGRP were explored using western blot and immunofluorescence. Results. Application of protruded nucleus (NP) materials to the DRG induced mechanical and thermal allodynia symptoms, and deregulated the expression of pro-inflammatory and anti-inflammatory cytokines in rats. Intrathecal delivery of PD1 significantly reversed the NCLDH-induced imbalance in neuro-inflammatory response and alleviated the symptoms of mechanical and thermal hyperalgesia. In addition, NP application to the DGRs resulted the spinal upregulation of CGRP and SIRT1 expression, which was almost restored by intrathecal injection of PD1 in a dose-dependent manner. SIRT1 antagonist or agonist and CGRP or antagonist treatment further confirmed the result. Conclusion. Our findings indicate PD1 has a potent analgesic effect, and can modulate neuro-inflammation by regulating SIRT1-mediated CGRP signaling in NCLDH.