Intervertebral Disc Degeneration Rat Model Research Articles

Injectable chondroitin sulfate-grafted self-antioxidant hydrogels ameliorate nucleus pulposus degeneration against overactive inflammation.

Overactive inflammatory cascade accompanied by oxidative stress in the nucleus pulposus exacerbates intervertebral disc degeneration (IVDD). Hydrogels have been demonstrated to be promising in treating IVDD, yet they remain less efficacious in the case of anti-inflammation associated with antioxidation. In this study, we designed an injectable self-antioxidant hydrogel (HA/CS) with enhanced inflammation inhibitory performance for delivering chondroitin sulfate (CS) with well-documented anti-inflammatory property to treat IVDD. The hydrogel was rapidly formed via dynamic boronate ester bonding between furan/phenylboronic acid and furan/dopamine-modified hyaluronic acid (HA), and mechanically enhanced by Diels-Alder reaction-induced secondary crosslinking, partial dopamine groups of which contribute to grafting phenylboronic acid-modified CS (CS-PBA). This hydrogel exhibits favorable injectability, mechanical property, and pH-responsive delivery behavior. The dopamine moiety endows the hydrogel with efficient antioxidative property. By sustained delivery of CS, the HA/CS hydrogel is well competent to inhibit inflammatory cytokine expression and maintain anabolic/catabolic balance in an inflammation-simulated environment. Most importantly, the HA/CS hydrogel significantly ameliorates degeneration in a puncture-induced IVDD rat model. The self-antioxidant HA/CS hydrogel designed in this work may serve as a novel and promising therapeutic platform for IVDD.

Epigenetic Changes within the Annulus Fibrosus by DNA Methylation in Rat Intervertebral Disc Degeneration Model.

Intervertebral disc degeneration (IDD) is an age-dependent progressive spinal disease that causes chronic back or neck pain. Although aging has long been presented as the main risk factor, the exact cause is not fully known. DNA methylation is associated with chronic pain, suggesting that epigenetic modulation may ameliorate disc degeneration. We examined histological changes in the DNA methylation within the discs and their association with pain-related transient receptor potential vanilloid subtype 1 (TrpV1) expression in rats subjected to IDD. Epigenetic markers (5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5Mc)), DNA methyltransferases (DNMTs), and Ten-eleven translocations (Tets) were analyzed using immunohistochemistry, real-time PCR, and DNA dot-blot following IDD. Results revealed high 5mC levels in the annulus fibrosus (AF) region within the disc after IDD and an association with TrpV1 expression. DNMT1 is mainly involved in 5mC conversion in degenerated discs. However, 5hmC levels did not differ between groups. A degenerated disc can lead to locomotor defects as assessed by ladder and tail suspension tests, no pain signals in the von Frey test, upregulated matrix metalloproteinase-3, and downregulated aggrecan levels within the disc. Thus, we found that the DNA methylation status in the AF region of the disc was mainly changed after IDD and associated with aberrant TrpV1 expression in degenerated discs.

Bone mesenchymal stem cells deliver exogenous lncRNA CAHM via exosomes to regulate macrophage polarization and ameliorate intervertebral disc degeneration

Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) were reported to have therapeutic potential in degenerative diseases. This study aimed to explore the effects of BMSC-Exos on inhibiting M1 macrophage polarization, reducing excessive nucleus pulposus cells (NPCs) apoptosis, and inhibiting ECM degradation during intervertebral disc degeneration (IDD). Rat IDD models were established by acupuncture. For the co-culture experiment, we used BMSC-Exo or human monocyte leukemia (THP-1) medium to incubate THP-1 or NPCs, respectively. BMSC-Exo was isolated from the BMSC medium, identified by TEM and NTA, and injected into the intervertebral discs of IDD rats. The macrophage infiltration in intervertebral disc tissue was evaluated by immunohistochemistry and immunofluorescence. ELISA was used to measure the levels of TNF-α, IL-6 and IL-10. The ECM degradation was analyzed by Western blot. The cell proportion and apoptosis were measured by flow cytometry. The morphological change of the intervertebral disc was analyzed by HE and safranin O fixation staining. In intervertebral disc tissues of IDD rats, we found the increased infiltration of M1 macrophages, with upregulated iNOS, TNF-α and IL-6 levels. Compared with BMSCs, the expression of CAHM in BMSC-Exo was significantly higher. Using co-cultured experiments, we proved that BMSC-Exo reduced apoptosis and ECM degradation of NPCs by inhibiting M1-type macrophage polarization by delivering CAHM. In addition, BMSC-Exo could improve IDD in vivo, including increased proteoglycan content, reduced macrophage infiltration and ECM degradation, and decrease expression of inflammatory factors by delivering CAHM. In conclusion, BMSC-Exo delivered exogenous CAHM via exosomes to regulate macrophage polarization and ameliorate IDD.

Activation of Nrf2 signaling by 4-octyl itaconate attenuates the cartilaginous endplate degeneration by inhibiting E3 ubiquitin ligase ZNF598

Cartilaginous endplate (CEP) degeneration is the main early manifestations of intervertebral disc degeneration (IVDD), and is closely related to the oxidative stress. Nrf2 (nuclear factor E2-related factor 2, NFE2L2) is a vital transcriptional factor of cellular antioxidant and anti-inflammatory responses. We aimed to illustrate whether the Nrf2 which was increased in expression by 4-octyl itaconate (4OI) could attenuate intervertebral disc degeneration through suppressing macrophage associated inflammation and catabolism of cartilaginous endplate. Firstly, we detected the expression of Nrf2 in human degenerative CEPs. Then, we performed in vitro, ex vivo and in vivo (a rat-tail puncture model) experiments to explore the role of 4OI in IVDD. Also, by cell co-culture experiments, we demonstrated 4OI restrained the macrophage-associated inflammatory responses. Finally, through western blotting and immunoprecipitation (IP) assay, we clarified the ZNF598-mediated ubiquitination of Nrf2. We found decreased expression of Nrf2 in human degenerative CEPs. Using a rat IVDD model(n=6), 4OI significantly ameliorated the progression of IVDD by MR images and histological analysis. Immunofluorescence results reveal that catabolism of CEPs and macrophage-associated inflammation are suppressed by 4OI treatment. Mechanistically, the 4OI increases Nrf2 expression and inhibits the secretion of inflammatory factors (IL-1β) by Lipopolysaccharide (LPS)-induced macrophages, thus preventing the inflammatory-related CEP degeneration. Meanwhile, 4OI suppresses the reactive oxygen species (ROS) production and catabolism of LPS-induced rat CEP cells. In addition, 4OI inhibits the ZNF598-dependent ubiquitination of Nrf2 in LPS-induced rat CEP cells. 4OI may alleviate IVDD by suppressing CEP degeneration and macrophage-associated inflammation. 4OI may be an alternative therapy for degenerative CEPs/IVDs.

DPSCs Protect Architectural Integrity and Alleviate Intervertebral Disc Degeneration by Regulating Nucleus Pulposus Immune Status.

Intervertebral disc (IVD) degeneration is the primary cause for low back pain that has a high prevalence in modern society and poses enormous economic burden on patients. Few effective therapeutic strategies are available for IVD degeneration treatment. To understand the biological effects of dental pulp stem cells (DPSCs) on nucleus pulposus (NP) cells, we carried out RNA sequencing, bioinformatic analysis which unveiled gene expression differences, and pathway variation in primarily isolated patients' NP cells after treatment with DPSCs supernatant. Western blot and immunofluorescence were used to verify these molecular alterations. Besides, to evaluate the therapeutic effect of DPSCs in IVD degeneration treatment, DPSCs were injected into a degeneration rat model in situ, with treatment outcome measured by micro-CT and histological analysis. RNA sequencing and in vitro experiments demonstrated that DPSCs supernatant could downregulate NP cells' inflammation-related NF-κB and JAK-STAT pathways, reduce IL-6 production, increase collagen II expression, and mitigate apoptosis. In vivo results showed that DPSCs treatment protected the integrity of the disc structure, alleviated extracellular matrix degradation, and increased collagen fiber expression. In this study, we verified the therapeutic effect of DPSCs in an IVD degeneration rat model and elucidated the underlying molecular mechanism of DPSCs treatment, which provides a foundation for the application of DPSCs in IVD degeneration treatment.

Nanofiber reinforced alginate hydrogel for leak-proof delivery and higher stress loading in nucleus pulposus

Injectable hydrogels effectively remodel degenerative nucleus pulposus (NP) with a resemblance to the in vivo microenvironment. However, the pressure within the intervertebral disc requires load-bearing implants. The hydrogel must undergo a rapid phase transition upon injection to avoid leakage. In this study, an injectable sodium alginate hydrogel was reinforced with silk fibroin nanofibers with core-shell structures. The nanofiber-embedded hydrogel provided support to adjacent tissues and facilitated cell proliferation. Platelet-rich plasma (PRP) was incorporated into the core-shell nanofibers for sustained release and enhanced NP regeneration. The composite hydrogel exhibited excellent compressive strength and enabled leak-proof delivery of PRP. In rat intervertebral disc degeneration models, radiography and MRI signal intensities were significantly reduced after 8 weeks of injections with the nanofiber-reinforced hydrogel. The biomimetic fiber gel-like structure was constructed in situ, providing mechanical support for NP repair, promoting the reconstruction of the tissue microenvironment, and finally realizing the regeneration of NP.

Dual-dynamic-bond cross-linked injectable hydrogel of multifunction for intervertebral disc degeneration therapy

Developing smart hydrogels with integrated and suitable properties to treat intervertebral disc degeneration (IVDD) by minimally invasive injection is of high desire in clinical application and still an ongoing challenge. In this work, an extraordinary injectable hydrogel PBNPs@OBG (Prussian blue nanoparticles@oxidized hyaluronic acid/borax/gelatin) with promising antibacterial, antioxidation, rapid gelation, and self-healing characteristics was designed via dual-dynamic-bond cross-linking among the oxidized hyaluronic acid (OHA), borax, and gelatin. The mechanical performance of the hydrogel was studied by dynamic mechanical analysis. Meanwhile, the swelling ratio and degradation level of the hydrogel was explored. Benefiting from its remarkable mechanical properties, sufficient tissue adhesiveness, and ideal shape-adaptability, the injectable PBNPs containing hydrogel was explored for IVDD therapy. Astoundingly, the as-fabricated hydrogel was able to alleviate H2O2-induced excessive ROS against oxidative stress trauma of nucleus pulposus, which was further revealed by theoretical calculations. Rat IVDD model was next established to estimate therapeutic effect of this PBNPs@OBG hydrogel for IVDD treatment in vivo. On the whole, combination of the smart multifunctional hydrogel and nanotechnology-mediated antioxidant therapy can serve as a fire-new general type of therapeutic strategy for IVDD and other oxidative stress-related diseases.

Ion elemental-optimized layered double hydroxide nanoparticles promote chondrogenic differentiation and intervertebral disc regeneration of mesenchymal stem cells through focal adhesion signaling pathway.

Chronic low back pain and dyskinesia caused by intervertebral disc degeneration (IDD) are seriously aggravated and become more prevalent with age. Current clinical treatments do not restore the biological structure and inherent function of the disc. The emergence of tissue engineering and regenerative medicine has provided new insights into the treatment of IDD. We synthesized biocompatible layered double hydroxide (LDH) nanoparticles and optimized their ion elemental compositions to promote chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). The chondrogenic differentiation of LDH-treated MSCs was validated using Alcian blue staining, qPCR, and immunofluorescence analyses. LDH-pretreated hUC-MSCs were differentiated prior to transplantation into the degenerative site of a needle puncture IDD rat model. Repair and regeneration evaluated using X-ray, magnetic resonance imaging, and tissue immunostaining 4–12 weeks after transplantation showed recovery of the disc space height and integrated tissue structure. Transcriptome sequencing revealed significant regulatory roles of the extracellular matrix (ECM) and integrin receptors of focal adhesion signaling pathway in enhancing chondrogenic differentiation and thus prompting tissue regeneration. The construction of ion-specific LDH nanomaterials for in situ intervertebral disc regeneration through the focal adhesion signaling pathway provides theoretical basis for clinical transformation in IDD treatment.

Dihydroartemisinin Attenuated Intervertebral Disc Degeneration via Inhibiting PI3K/AKT and NF-κB Signaling Pathways.

Intervertebral disc degeneration (IDD) is the leading cause of low back pain (LBP). However, effective therapeutic drugs for IDD remain to be further explored. Inflammatory cytokines play a pivotal role in the onset and progression of IDD. Dihydroartemisinin (DHA) has been well reported to have powerful anti-inflammatory effects, but whether DHA could ameliorate the development of IDD remained unclear. In this study, the effects of DHA on extracellular matrix (ECM) metabolism and cellular senescence were firstly investigated in nucleus pulposus cells (NPCs) under tumor necrosis factor alpha (TNFα)-induced inflammation. Meanwhile, AKT agonist sc-79 was used to determine whether DHA exerted its actions through regulating PI3K/AKT and NF-κB signaling pathways. Next, the therapeutic effects of DHA were tested in a puncture-induced rat IDD model. Finally, we detected the activation of PI3K/AKT and NF-κB signaling pathways in clinical degenerative nucleus pulposus specimens. We demonstrated that DHA ameliorated the imbalance between anabolism and catabolism of extracellular matrix and alleviated NPCs senescence induced by TNFα in vitro. Further, we illustrated that DHA mitigated the IDD progression in a puncture-induced rat model. Mechanistically, DHA inhibited the activation of PI3K/AKT and NF-κB signaling pathways induced by TNFα, which was undermined by AKT agonist sc-79. Molecular docking predicted that DHA bound to the PI3K directly. Intriguingly, we also verified the activation of PI3K/AKT and NF-κB signaling pathways in clinical degenerative nucleus pulposus specimens, suggesting that DHA may qualify itself as a promising drug for mitigating IDD.

O-GlcNAc transferase regulates intervertebral disc degeneration by targeting FAM134B-mediated ER-phagy

Both O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) and endoplasmic reticulum-phagy (ER-phagy) are well-characterized conserved adaptive regulatory mechanisms that maintain cellular homeostasis and function in response to various stress conditions. Abnormalities in O-GlcNAcylation and ER-phagy have been documented in a wide variety of human pathologies. However, whether O-GlcNAcylation or ER-phagy is involved in the pathogenesis of intervertebral disc degeneration (IDD) is largely unknown. In this study, we investigated the function of O-GlcNAcylation and ER-phagy and the related underlying mechanisms in IDD. We found that the expression profiles of O-GlcNAcylation and O-GlcNAc transferase (OGT) were notably increased in degenerated NP tissues and nutrient-deprived nucleus pulposus (NP) cells. By modulating the O-GlcNAc level through genetic manipulation and specific pharmacological intervention, we revealed that increasing O-GlcNAcylation abundance substantially enhanced cell function and facilitated cell survival under nutrient deprivation (ND) conditions. Moreover, FAM134B-mediated ER-phagy activation was regulated by O-GlcNAcylation, and suppression of ER-phagy by FAM134B knockdown considerably counteracted the protective effects of amplified O-GlcNAcylation. Mechanistically, FAM134B was determined to be a potential target of OGT, and O-GlcNAcylation of FAM134B notably reduced FAM134B ubiquitination-mediated degradation. Correspondingly, the protection conferred by modulating O-GlcNAcylation homeostasis was verified in a rat IDD model. Our data demonstrated that OGT directly associates with and stabilizes FAM134B and subsequently enhances FAM134B-mediated ER-phagy to enhance the adaptive capability of cells in response to nutrient deficiency. These findings may provide a new option for O-GlcNAcylation-based therapeutics in IDD prevention.

Macrophage polarization regulates intervertebral disc degeneration by modulating cell proliferation, inflammation mediator secretion, and extracellular matrix metabolism.

Macrophage infiltration and polarization have been increasingly observed in intervertebral disc (IVD) degeneration (IDD). However, their biological roles in IDD are still unrevealed. We harvested conditioned media (CM) derived from a spectrum of macrophages induced from THP-1 cells, and examined how they affect nucleus pulposus cells (NPCs) in vitro, by studying cell proliferation, extracellular matrix (ECM) synthesis, and pro-inflammation expression; and in vivo by injection CM in a rat IDD model. Then, high-throughput sequencing was used to detect differentially expressed genes (DEGs). Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks were used to further analysis. Higher CCR7+ (M1 marker) and CD206+ (M2 marker) cell counts were found in the degenerated human IVD tissues as compared with the control. Furthermore, the cell co-culture model showed M1CM attenuated NPC proliferation, downregulated the expression of ECM anabolic genes encoding aggrecan and collagen IIα1, upregulated the expression of ECM catabolic genes encoding MMP-13, and inflammation-related genes encoding IL-1β, IL-6, and IL-12, while M2CM showed contrasting trends. In IDD model, higher histological scores and lower disc height index were found following M1CM treatment, while M2CM exhibited opposite results. M1CM injection decreased ECM anabolic and increased ECM catabolic, as well as the upregulation of inflammation-related genes after 8 weeks treatment, while M2CM slowed down these trends. Finally, a total of 637 upregulated and 655 downregulated genes were detected in M1CM treated NPCs, and 975 upregulated genes and 930 downregulated genes in the M2CM groups. The top 30 GO terms were shown and the most significant KEGG pathway was cell cycle in both groups. Based on the PPI analysis, the five most significant hub genes were PLK1, KIF20A, RRM2, CDC20, and UBE2C in the M1CM groups and RRM2, CCNB1, CDC20, PLK1, and UBE2C in the M2CM groups. In conclusion, macrophage polarization exhibited diverse roles in IDD progression, with M1CM exacerbating cell proliferation suppression and IVD degeneration, while M2CM attenuated IDD development. These findings may facilitate the further elucidation of the role of macrophage polarization in IDD, and provide novel insights into the therapeutic potential of macrophages.

Berberine Attenuates IL-1β-Induced Damage of Nucleus Pulposus Cells via Activating the AMPK/mTOR/Ulk1 Pathway.

Intervertebral disc degeneration (IDD) is a chronic progressive condition mainly caused by excessive inflammatory cytokines. Berberine (BBR) exerts anti-inflammatory effect on diseases and protective effect against IDD. However, the mechanism is not uncertain. This study is aimed at investigating the molecular mechanism of BBR on IDD. Nucleus pulposus (NP) cells were treated with BBR at different concentrations. The IDD rat model was established by acupuncture. The effect of BBR on interleukin- (IL-) 1β-induced cell proliferation was measured by CCK-8 assay and BrdU staining. The role of BBR in IL-1β-induced apoptosis, autophagy repression, and extracellular matrix (ECM) degradation was measured by Annexin/PI staining, immunofluorescence, and immunoblot. The effect of BBR on IDD was investigated in rat. Our findings showed that BBR restored cell growth and attenuated apoptosis in IL-1β-induced NP cells. BBR also prevented the IL-1β-induced ECM degradation through regulating ECM-related enzymes and factors. Additionally, BBR significantly activated autophagy repressed by IL-1β. Autophagy stimulated by BBR was diminished by the inhibition of the AMPK/mTOR/Ulk1 signaling pathway. In vivo study also showed BBR attenuated intervertebral disc degeneration. BBR could attenuate NP cells apoptosis and ECM degradation induced by IL-1β through autophagy by the AMPK/mTOR/Ulk1 pathway. This study suggests BBR might function as an AMPK activator to alleviate IDD progression.

Irisin Ameliorates Intervertebral Disc Degeneration by Activating LATS/YAP/CTGF Signaling.

Unbalanced metabolism of an extracellular matrix (ECM) in nucleus pulposus cells (NPCs) is widely acknowledged as the primary cause of intervertebral disc degeneration (IDD). Irisin, a novel myokine, is cleaved from fibronectin type III domain-containing 5 (FNDC5) and has recently been proven to regulate the metabolism of ECM. However, little is known about its potential on NPCs and the development of IDD. Therefore, this study sought to examine the protective effects and molecular mechanism of irisin on IDD in vivo and in vitro. Decreased expression levels of FNDC5 and anabolism markers (COL2A1 and ACAN) but increased levels of catabolism markers (ADAMTS4) were found in degenerative nucleus pulposus (NP) tissues. In a punctured-induced rat IDD model, irisin treatment was found to significantly slow the development of IDD, and in TNF-α-stimulated NPCs, irisin treatment partly reversed the disorder of ECM metabolism. In mechanism, RNA-seq results suggested that irisin treatment affected the Hippo signaling pathway. Further studies revealed that with irisin treatment, the phosphorylation levels of key factors (LATS and YAP) were downregulated, while the expression level of CTGF was upregulated. Moreover, CTGF knockdown partially eliminated the protective effects of irisin on the metabolism of ECM in NPCs, including inhibiting the anabolism and promoting the catabolism. Taken together, this study demonstrated that the expression levels of FNDC5 were decreased in degenerative NP tissues, while irisin treatment promoted the anabolism, inhibited the catabolism of the ECM in NPCs, and delayed the progression of IDD via LATS/YAP/CTGF signaling. These results shed light on the protective actions of irisin on NPCs, leading to the development of a novel therapeutic target for treating IDD.

BMSC-Derived Exosomes Alleviate Intervertebral Disc Degeneration by Modulating AKT/mTOR-Mediated Autophagy of Nucleus Pulposus Cells.

The pathogenesis of intervertebral disc degeneration (IDD) is still unclear. It has been shown that the pathological process of IDD is most closely related to inflammation of nucleus pulposus cells (NPCs), in which inflammatory factors play an important role. Exosomes are the main paracrine mediators and are microvesicles with biological functions similar to those of the cells from which they are derived. Studies have shown that bone mesenchymal stem cells (BMSCs) can inhibit apoptosis of NPCs by sending exosomes as anti-inflammatory and antioxidant, which has been proved to be effective on IDD. However, the specific mechanism of inhibiting apoptosis of NPCs is still unclear. In our study, BMSC-derived exosomes (BMSC-Exo) were isolated from the BMSC culture medium, and their antiapoptotic effects were evaluated in cells and rat models to explore the possible mechanisms. We observed that BMSC-Exo promotes autophagy in NPCs and inhibits the release of inflammatory factors such as IL-1β and TNF-α in LPS-treated NPCs and inhibits apoptosis in NPCs. Further studies showed that BMSC-Exo inhibited the Akt-mTOR pathway. Intramuscular injection of BMSC-Exo alleviates disc degeneration in rat IDD models. In conclusion, our results suggest that BMSC-Exo can reduce NPC apoptosis and alleviate IDD by promoting autophagy by inhibiting the Akt-mTOR pathway. Our study confers a promising therapeutic strategy for IDD.

Development of a Novel Rat Intervertebral Disc Degeneration Model by Surgical Multifidus Resection–Induced Instability

This study aimed to investigate whether surgical resection of multifidus in rats could generate a reliable model of intervertebral disc degeneration (IVDD). Instability of the lumbar spine in Sprague-Dawley rats was induced by multifidus resection. Longissimus changes were examined by hematoxylin and eosin staining and immunohistochemistry. Specific protein and mRNA changes in the nucleus pulposus (NP) were quantified by Western blot and reverse transcription-polymerase chain reaction. Bone alterations were assessed using X-ray imaging, and disc changes were evaluated by hematoxylin and eosin staining, immunofluorescence, and immunohistochemistry. Fat infiltration and increased tumor necrosis factor-α expression in the longissimus were detected following surgery. Reverse transcription-polymerase chain reaction and Western blot results demonstrated that the inflammation and catabolism in the NP were increased after the surgical intervention. Moreover, X-ray imaging showed that the disc height had decreased and bone spurs had formed at the vertebral rims. Histological analyses further revealed degeneration of the annulus fibrosus, endplate, and NP. Furthermore, in contrast to the sham group, the collagen II expression was reduced, while matrix metalloproteinase-13 was increased in the surgery group. Surgical resection of the multifidus in rats resulted in a reproducible IVDD model. Because the present procedure does not impart direct injury to the intervertebral disc, it can better imitate the pathological states in humans. Therefore, our rat multifidus resection model might help us further understand the intrinsic pathophysiology of IVDD.

Controlled Release of TGF-β3 for Effective Local Endogenous Repair in IDD Using Rat Model.

IntroductionIntervertebral disc (IVD) degeneration (IDD) is one of the most widespread musculoskeletal diseases worldwide and remains an intractable clinical challenge. Currently, regenerative strategies based on biomaterials and biological factors to facilitate IVD repair have been widely explored. However, the harsh microenvironment, such as increased ROS and acidity, of the degenerative region impedes the efficiency of IVD repair. Here, an intelligent biodegradable nanoplatform using hollow manganese dioxide (H-MnO2) was developed to modulate the degenerative microenvironment and release transforming growth factor beta-3 (TGF-β3), which may achieve good long-term therapeutic effects on needle puncture-induced IDD.MethodsSurface morphology and elemental analysis of the MnO2 nanoparticles (NPs) were performed by transmission electron microscopy and an energy-dispersive X-ray spectroscopy detector system, respectively. The biological effects of MnO2 loaded with TGF-β3 (TGF-β3/MnO2) on nucleus pulposus cells (NPCs) were assessed via cytoskeleton staining, EdU staining, qPCR and immunofluorescence. The efficacy of TGF-β3/MnO2 on needle puncture-induced IDD was further examined using MRI and histopathological and immunohistochemical staining.ResultsThe MnO2 NPs had a spherical morphology and hollow structure that dissociated in the setting of a low pH and H2O2 to release loaded TGF-β3 molecules. In the oxidative stress environment, TGF-β3/MnO2 was superior to TGF-β3 and MnO2 NPs in the suppression of H2O2-induced matrix degradation, ROS, and apoptosis in NPCs. When injected into the IVDs of a rat IDD model, TGF-β3/MnO2 was able to prevent the degeneration and promote self-regeneration.ConclusionUse of an MnO2 nanoplatform for biological factors release to regulate the IDD microenvironment and promote endogenous repair may be an effective approach for treating IDD.

Inhibition of miR-130b-3p restores autophagy and attenuates intervertebral disc degeneration through mediating ATG14 and PRKAA1.

Oxidative stress-induced autophagy dysfunction is involved in the pathogenesis of intervertebral disc degeneration (IVDD). MicroRNAs (miRNAs) not only have been regarded as important regulators of IVDD but also reported to be related to autophagy. This research was aimed to explore the role of miR-130b-3p in IVDD and its regulation on autophagy mechanism. The miR-130b-3p expression in the patient's degenerative nucleus pulposus (NP) samples and rat NP tissues was detected by qRT-PCR and FISH assay. The miR-130b-3p was knocked down or overexpressed in the human NP cells by lentivirus transfection. TBHP was used to induce oxidative stress in the human NP cells. Apoptosis, senescence, and autophagy were evaluated by flow cytometry, β-gal staining, immunofluorescence, electron microscopy, and Western blot in the miR-130b-3p knocked down human NP cells under TBHP treatment. The relationship between the miR-130b-3p and ATG14 or PRKAA1 was confirmed by luciferase assay. The siRNA transfection was used to knock down the ATG14 and PRKAA1 expression, and then the human NP cells functions were further determined. In the in vivo experiment, the IVDD rat model was constructed and an adeno-associated virus (AAV)-miR-130b-3p inhibitor was intradiscally injected. After that, MRI and histological staining were conducted to evaluate the role of miR-130b-3p inhibition in the IVDD rat model. We found that the miR-130b-3p was upregulated in the degenerative NP samples from humans and rats. Interestingly, the inhibition of miR-130b-3p rescued oxidative stress-induced dysfunction of the human NP cells, and miR-130b-3p inhibition upregulated autophagy. Mechanistically, we confirmed that the miR-130b-3p regulated the ATG14 and PRKAA1 directly and the knockdown of the ATG14 or PRKAA1 as well as the treatment of autophagy inhibitor blockaded the autophagic flux and reversed the protective effects of miR-130b-3p inhibition in the TBHP-induced human NP cells. Furthermore, the inhibition of the miR-130b-3p via AAV- miR-130b-3p injection ameliorated the IVDD in a rat model. These data demonstrated that the miR-130b-3p inhibition could upregulate the autophagic flux and alleviate the IVDD via targeting ATG14 and PRKAA1.The translational potential of this article: The suppression of miR-130b-3p may become an effective therapeutic strategy for IVDD.

Effect of resveratrol on high mobility group box-1 protein signaling pathway in cartilage endplate degeneration caused by inflammation

To investigate the effect of resveratrol (RES) on inflammation-induced cartilage endplate (CEP) degeneration, and its regulatory mechanism on high mobility group box-1 protein (HMGB1) signaling pathway. The intervertebral CEP cells of Sprague Dawley (SD) rats aged 3 weeks were extracted and identified by toluidine blue staining and immunofluorescence staining of rabbit anti-rat collagen type Ⅱ. The cell counting kit 8 (CCK-8) method was used to screen the optimal concentration of RES on intervertebral CEP cells. Gene chip analysis was used to determine the target of RES on intervertebral CEP cells. Interleukin 1β (IL-1β) was used to construct the intervertebral CEP cell degeneration model caused by inflammation and the 7-8-week-old SD rat intervertebral disc degeneration model, and pcDNA3.1-HMGB1 (pcDNA3.1) was used as the control of RES effect. Flow cytometry and TUNEL staining were used to detect the apoptotic rate of intervertebral CEP cells and rat intervertebral disc tissue cells, respectively. ELISA kit was used to detect the content of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in the cell supernatant and rat serum. Western blot was used to detect the expressions of HMGB1, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (p-ERK), B cell lymphoma/leukemia 2 gene (Bcl-2), and Bcl-2-associated X protein (Bax). The extracted cells were identified as rat intervertebral CEP cells. CCK-8 method screened out the highest activity of intervertebral CEP cells treated with 30 μmol/L RES. The gene chip analysis confirmed that the HMGB1-ERK signal was the target of RES. Both cell experiments and animal experiments showed that RES treatment can significantly down-regulate the apoptosis rate of intervertebral CEP cells, inhibit the release of TNF-α, and increase the content of IL-10; and down-regulate the expressions of HMGB1, p-ERK, and Bax, and increase Bcl-2; and pcDNA3.1 could partially reverse these effects of RES, and the differences were all significant (P<0.05). RES can significantly inhibit the apoptosis of intervertebral CEP cells induced by inflammation, which is related to inhibiting the expression of HMGB1.

Selenium Attenuates TBHP-Induced Apoptosis of Nucleus Pulposus Cells by Suppressing Mitochondrial Fission through Activating Nuclear Factor Erythroid 2-Related Factor 2.

Intervertebral disc (IVD) degeneration (IDD), the leading cause of low back pain (LBP), remains intractable due to a lack of effective therapeutic strategies. Several lines of studies have documented that nucleus pulposus cell (NPC) death induced by excessive oxidative stress is a crucial contributor to IDD. However, the concrete role and regulation mechanisms have not been fully clarified. Selenium (Se), a vital prosthetic group of antioxidant enzymes, is indispensable for maintaining redox homeostasis and promoting cell survival. However, no light was shed on the role of Se on IDD progression, especially regulation on mitochondrial dynamics and homeostasis. To fill this research gap, the current study focuses on the effects of Se, including sodium selenite (SS) and selenomethionine (Se-Met), on IDD progression and the underlying mechanisms. In vitro, we found that both SS and Se-Met alleviated tert-butyl hydroperoxide- (TBHP-) induced oxidative stress, protected mitochondrial function, and inhibited apoptosis of NPCs. Further experiments indicated that Se suppressed TBHP-induced mitochondrial fission and rescued the imbalance of mitochondrial dynamics. Promoting mitochondrial fission by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) partially counteracted the cytoprotective effects of Se. Moreover, blocking nuclear factor erythroid 2-related factor 2 (Nrf2) with ML385 proved that the effect of Se on regulating mitochondrial dynamics was attributed to the activation of the Nrf2 pathway. In the puncture-induced rat IDD model, a supplement of Se-Met ameliorated degenerative manifestations. Taken together, our results demonstrated that Se suppressed TBHP-induced oxidative stress and mitochondrial fission by activating the Nrf2 pathway, thereby inhibiting the apoptosis of NPCs and ameliorating IDD. Regulation of mitochondrial dynamics by Se may have a potential application value in attenuating the pathological process of IDD.

Pulsed Electromagnetic Field Alleviates Intervertebral Disc Degeneration by Activating Sirt1-Autophagy Signaling Network.

Intervertebral disc (IVD) degeneration is regarded as a major contributor to low back pain (LBP), causing serious economic burden on individuals and society. Unfortunately, there are limited effective treatment for IVD degeneration. Pulsed electromagnetic field (PEMF) is an economical and effective physical therapy method, with reduced side-effects. It offers certain protection to a number of degenerative diseases. Therefore, understanding the underlying mechanism of PEMF on IVD is important for improving the PEMF therapeutic efficiency. In this study, PEMF up-regulated extracellular matrix (ECM) related genes in degenerated nucleus pulposus (NP) cells. It also increased SIRT1 expression and promoted autophagy in degenerated NP cells. In contrast, the autophagy suppressor 3-methyladenine (3-MA) reversed the beneficial effect of PEMF on ECM production. Similarly, the SIRT1 enzyme activity suppressor EX 527 also inhibited the effect of PEMF on autophagy and ECM production in NP cells, thereby suggesting that PEMF regulated ECM related genes expression through SIRT1-autophagy signaling pathway. Lastly, PEMF significantly reduced IVD degeneration in a rat model of IVD degeneration in vivo. In summary, our study uncovers a critical role of SIRT1-dependent autophagy signaling pathway in ECM protection and thus in the establishment of therapeutic effect of PEMF on IVD degeneration.