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Isoginkgetin-loaded reactive oxygen species scavenging nanoparticles ameliorate intervertebral disc degeneration via enhancing autophagy in nucleus pulposus cells.

ABSTRACT: Excessive reactive oxygen species (ROS) in nucleus pulposus cells (NPCs) promote extracellular matrix (ECM) degradation and cellular inflammatory responses by activating a variety of cellular pathways, ultimately inducing cell apoptosis and leading to the development of low back pain. Here, we designed and fabricated an isoginkgetin-loaded ROS-responsive delivery system (IGK@SeNP) based on diselenide block copolymers. Successfully encapsulated IGK was released intelligently and rapidly in a microenvironment with high ROS levels in degenerative disc. Controlled-release IGK not only efficiently scavenged ROS from the intervertebral disc together with diselenide block copolymers but also effectively enhanced autophagy in NPCs to inhibit ECM degradation and cell apoptosis, and showed significant therapeutic effects in the rat intervertebral disc degeneration (IDD) model. Overall, the synergistic effects of IGK@SeNP in ROS scavenging and autophagy enhancement endowed it with an attractive therapeutic strategy for IDD treatment.

SUBMITTER: Yu H

PROVIDER: S-EPMC10029295 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Isoginkgetin-loaded reactive oxygen species scavenging nanoparticles ameliorate intervertebral disc degeneration via enhancing autophagy in nucleus pulposus cells.

Yu Hao H Teng Yun Y Ge Jun J Yang Ming M Xie Haifeng H Wu Tianyi T Yan Qi Q Jia Mengting M Zhu Qing Q Shen Yanping Y Zhang Lianxue L Zou Jun J

Journal of nanobiotechnology 20230321 1

Excessive reactive oxygen species (ROS) in nucleus pulposus cells (NPCs) promote extracellular matrix (ECM) degradation and cellular inflammatory responses by activating a variety of cellular pathways, ultimately inducing cell apoptosis and leading to the development of low back pain. Here, we designed and fabricated an isoginkgetin-loaded ROS-responsive delivery system (IGK@SeNP) based on diselenide block copolymers. Successfully encapsulated IGK was released intelligently and rapidly in a micr ...[more]

PMID: 36941611

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Project description:Background/objectiveIntervertebral disc (IVD) degeneration (IVDD) that greatly affected by regional biomechanical environment is a major cause of low back pain. Injectable hydrogels have been commonly studied for treatment of IVDD due to their capability of mimicking extracellular matrix structure to support cellular behavior and clinical prospects in minimally invasive treatment. However, most hydrogels suffer from complicated chemistry, potential uncertainty and toxicity from in-situ gelation, and mismatch with IVD mechanical environment that limit their therapeutic effects or clinical translation in IVDD or intervertebral disc defect repair. For IVD lesion repair, the study aims to develop a novel hydrogel with shear-thinning enabled injectability, high bio-safety, and mechanical properties adaptable to the IVD environment, using a simple chemistry and method. And therapeutic efficacy of the novel hydrogel in the treatment of IVDD or intervertebral disc defect will be revealed.MethodsA glycerol cross-linked PVA gel (GPG) was synthesized based on multiple H-bonds formation between glycerol molecules and PVA chains. The rheological and mechanical properties were tested. The swelling ratio was measured. The micro-architecture was observed through scanning and transmission electron microscopes. Nucleus pulposus (NP) cells were cultured in GPG-coated plates or silicone chambers treated under hydrostatic or dynamic loading in vitro, and examined for proliferation, vitality, apoptosis, expression of catabolic and anabolic markers. GPG was injected in needle puncture (IDD) or NP discectomy (NPD) models in vivo, and examined through magnetic resonance imaging, micro-computed tomography scanning and histological staining.ResultsGPG had a highly porous structure consisting of interconnected pores. Meanwhile, the GPG had NP-like viscoelastic property, and was able to withstand the cyclic deformation while exhibiting a prominent energy-dissipating capability. In vitro cell tests demonstrated that, the hydrogel significantly down-regulated the expression of catabolic markers, maintained the level of anabolic markers, preserved cell proliferation and vitality, reduced apoptotic rate of NP cells under pathologically hydrostatic and dynamic loading environments compared to cells cultured on untreated plate or silicone chamber. In vivo animal studies revealed that injection of GPG efficiently maintained NP structural integrity, IVD height and relative water content in IDD models, and stimulated the fibrous repair in NPD models.ConclusionThis study showed that GPG, with high injectability, NP-like viscoelastic characteristics, good energy-dissipating properties and swelling capacities, preserved NP cells vitality against pathological loading, and had therapeutic effects on IVD repair in IDD and NPD models.The translational potential of this articleEffective clinical strategy for treatment of intervertebral disc degeneration (IVDD) is still lacking. This study demonstrates that injection of a hydrogel with nucleus pulposus-matched viscoelastic property could remarkably prevent the IVDD progress. Prepared with simple chemistry and procedure, the cell/drug-free GPG with high bio-safety and shear-thinning enabled injectability bears great translational potential for the clinical treatment of IVDD via a minimally invasive approach.

Project description:The nucleus pulposus (NP) plays a vital role in intervertebral disc degeneration (IVDD). Previous studies have revealed cellular heterogeneity in NP tissue during IVDD progression. However, the cellular and molecular alterations of diverse cell clusters during IVDD remain to be fully elucidated. NP tissues were isolated from patients with different grades of IVDD undergoing discectomy, and then subjected to single-cell RNA sequencing (scRNA-seq). Cell subsets were identified based on unbiased clustering of gene expression profiles. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine the molecular features of diverse cell clusters. Monocle analysis was used to illustrate the differentiation trajectories of chondrocytes. Additionally, CellPhoneDB analysis revealed potential interactions between chondrocytes and other cells during IVDD. Based on the expression profiles of 47,610 individual cells, eight putative clusters including chondrocytes, endothelial cells, fibroblasts, macrophages, mural cells, osteoclasts, proliferating stromal cells and T cells were identified. The chondrocyte cluster was classified into three subsets, C1-C3, which were associated with stress-resistance, fibrosis and inflammatory responses, respectively. Pseudo-time trajectories suggested that chondrocytes gradually differentiated into fibroblasts during IVDD. Immune cells including cDC2s, macrophages and monocytes were identified. Further analysis showed that chondrocytes might communicate with immune cells via the MIF, TNFSF9, SPP1 and CCL4L2 signaling pathways. In addition, we found that invading endothelial cells might interact with chondrocytes through the COL4A1, CXCL12, VEGFA and SEMA3E signaling pathways. Our results reveal the cellular complexity and phenotypic characteristics of NP tissues at single-cell resolution, which will contribute to the in-depth investigation of preventative and regenerative strategies for IVDD.

Project description:Background contextLow back pain, affecting nearly 40% of adults, mainly results from intervertebral disc degeneration (IVDD), while the pathogenesis of IVDD is still not fully elucidated. Recently, some researches have revealed that necroptosis, a programmed necrosis, participated in the progression of IVDD, nevertheless, the underlying mechanism remains unclear.PurposeTo study the mechanism of necroptosis of Nucleus Pulposus (NP) cells in IVDD, focusing on the role of MyD88 signaling.Study designThe expression and co-localization of necroptotic indicators and MyD88 were examined in vivo, and MyD88 inhibitor was applied to determine the role of MyD88 signaling in necroptosis of NP cells in vitro.MethodsHuman disc specimens were collected from patients receiving diskectomy for lumbar disc herniation (LDH) or traumatic lumbar fractures after MRI scanning. According to the Pfirrmann grades, they were divided into normal (Grades 1, 2) and degenerated groups (4, 5). Tissue slides were prepared for immunofluorescence to assess the co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 histologically. The combination of TNFα, LPS and Z-VAD-FMK was applied to induce necroptosis of NP cells. Level of ATP, reactive oxygen species (ROS), live-cell staining and electron microscope study were employed to study the role of MyD88 signaling in necroptosis of NP cells.ResultsIn vivo, the increased expression and co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 were found in NP cells of degenerated disc, while very l low fluorescence intensity in tissue of traumatic lumbar fractures. In vitro, the MyD88 inhibitor effectively rescued the necroptosis of NP cells, accompanied by increased viability, ATP level, and decreased ROS level. The effect of MyD88 inhibition on necroptosis of NP cells was further confirmed by ultrastructure of mitochondria shown by Transmission Electron Microscope (TEM).ConclusionOur results indicated that the involvement of MyD88 signaling in the necroptosis of NP cells in IVDD, which will replenish the pathogenesis of IVDD and provide a novel potential therapeutic target for IVDD.

Project description:Intervertebral Disc degeneration (IDD) is one of the leading causes of disability, and current therapies are ineffective. Phosphodiesterase 4B (PDE4B) plays an essential role in regulating the activation of nuclear factor E2-related factor 2 (Nrf2), while Nrf2 regulates ferroptosis. However, it is still unknown whether PDE4B is involved in the development of IDD. In this study, we explored the role of PDE4B on ferroptosis and Nrf2 in IDD pathogenesis by in vivo and in vitro experiments. The findings suggested that the expressions of PDE4B, ASCL4, and TRFC were significantly upregulated, and the expression of Nrf2 was significantly downregulated in nucleus pulposus (NP) tissues from human IDD patients dependent on IDD degeneration. Overexpression of PDE4B (PDE4B-OE) in NP cells upregulated the expression of ASCL4 and TRFC, and downregulated the expression of Nrf2. Meanwhile, the level of cytokine and oxidative stress were upregulated. Ferroptosis inhibitor Fer-1 or Nrf2 activator dimethyl fumarate (DMF) suppressed the effect of PDE4B-OE, while ferroptosis inducer elastin enhanced the effect of PDE4B-OE. In the IDD rat model, PDE4 inhibitor roflumilast, ferroptosis inhibitor Fer-1, or Nrf2 activator dimethyl fumarate (DMF) delayed IDD pathogenesis. While administration of ferroptosis inducer elastin enhanced IDD pathogenesis. Combination with PDE4B inhibitor and ferroptosis inhibitor Fer-1 significantly synergistic reversed IDD pathogenesis. While combination with PDE4B inhibitor or Nrf2 activator and elastin also decreased the degree of the IDD. The IHC suggested PDE4 inhibitor downregulated the expression of ASCL4 and TRFC. However, the combination effect of the Nrf2 activator was not obvious. Our study suggested that aberrant PDE4B activation in NP tissues induces pathological changes in IDD mediated by ferroptosis, and PDE4 inhibitor reveres the process of IDD by suppressing ferroptosis, and has a synergic effect with ferroptosis inhibitor. So PDE4B inhibition may be a potential therapeutic strategy for IDD.

Project description:Intervertebral disc (IVD) degeneration is a complicated physiological change involving cellular senescence, inflammation and the degradation of the extracellular matrix. Long non‑coding RNAs (lncRNAs) have been identified as new players in IVD degeneration. The present study aimed to identify lncRNAs implicated in IVD degeneration via the regulation of cellular senescence. In the present study, nucleus pulposus (NP) cells isolated from moderately degenerated IVD tissues exhibited a senescent phenotype with increased senescence rates, detected by senescence‑associated β‑galactosidase (SA‑β‑gal) staining, and reduced growth and migratory abilities. Microarray and target prediction analyses identified 353 differentially expressed lncRNAs, and 251 cis‑ and 2,170 trans‑acting targets in degenerated NP cells. Bioinformatic analyses revealed that these predicted targets were enriched in the regulation of response to DNA damage stimulus, positive regulation of cell cycle processes and interferon‑β production. In addition, a network of the top 10 upregulated and top 10 downregulated lncRNA targets was constructed, and two trans‑acting targets, C‑C motif chemokine ligand 5 (CCL5) and polyribonucleotide nucleotidyltransferase 1 (PNPT1) involved in aging or senescence, and their corresponding lncRNAs, lnc‑ST8SIA5‑1:2 and lnc‑HRK‑2:1, were identified. Reverse transcription‑quantitative PCR validation demonstrated that the two targets and two candidate lncRNAs were significantly upregulated in degenerated NP cells. Overexpression of lnc‑HRK‑2:1, with validated higher expression levels, in normal NP cells induced a senescent phenotype, with enhanced rates of senescence detected by SA‑β‑gal staining in cells, decreased growth and migratory abilities and improved expression levels of CCL5 and PNPT1. Collectively, these results suggested that upregulation of lnc‑HRK‑2:1 prompted NP cell senescence in IVD degeneration, which may be associated with increased expression levels of CCL5 and PNPT1.

Dataset Information

Isoginkgetin-loaded reactive oxygen species scavenging nanoparticles ameliorate intervertebral disc degeneration via enhancing autophagy in nucleus pulposus cells.

Publications

Isoginkgetin-loaded reactive oxygen species scavenging nanoparticles ameliorate intervertebral disc degeneration via enhancing autophagy in nucleus pulposus cells.

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