Exosome-targeted injectable hydrogels for sustained DEPTOR delivery and delay of IDD via the mTORC1/SASP pathway.

This study aimed to determine the molecular mechanisms by which the DEP domain-containing mTOR-interacting protein (DEPTOR) regulates the senescence of nucleus pulposus (NP) cells (NPCs), alleviating intervertebral disk degeneration (IDD). This study investigated how DEPTOR regulates the mechanistic target of rapamycin complex 1 (mTORC1)/S6 kinase beta-1 (S6K1)/autophagy-related gene 1 (ATG1) pathway to regulate senescence-associated secretory phenotype (SASP) and cellular autophagy in NPCs. Isobaric tags for relative and absolute quantitation was used to measure the differences in protein expression between degenerated and normal intervertebral disk tissues. Western blotting and immunofluorescence were used to quantify DEPTOR levels in NP tissues. DEPTOR was overexpressed in vitro, and changes in autophagy and SASP were monitored to determine its effects on NPCs. Moreover, lentiviral overexpression of S6K1 (LV-S6K1) and siRNA-mediated knockdown of ATG1 (ATG1-ShRNA) in both in vitro and in vivo models were used to verify whether DEPTOR stimulates autophagy in NPCs via ATG1 and inhibits SASP through S6K1. The results demonstrated that degenerated intervertebral disks had lower DEPTOR levels. Matrix metalloproteinases, inflammatory cytokines, chemokines, and aging-related proteins were downregulated when DEPTOR was overexpressed in NPCs. Furthermore, autophagic activity was stimulated, SASP secretion was inhibited, and extracellular matrix synthesis was increased. ATG1 knockdown decreased the capacity of DEPTOR to promote cellular autophagy and inhibit SASP, whereas S6K1 overexpression diminished DEPTOR-mediated SASP inhibition. DEPTOR attenuates IDD by inhibiting SASP secretion via the mTORC1/S6K1 pathway and promoting autophagy in NPCs via the mTORC1/ATG1 pathway.

Exosomes inhibit ferroptosis to alleviate intervertebral disc degeneration via the p62-KEAP1-NRF2 pathway.

Background: Intervertebral disc (IVD) degeneration is a common degenerative disease that can lead to collapse or herniation of the nucleus pulposus (NP) and result in radiculopathy in patients.Methods: NP tissue and cells were isolated from patients and mice, and the expression profile of cortistatin (CST) was analysed. In addition, ageing of the NP was compared between 6-month-old WT and CST-knockout (CST-/-) mice. Furthermore, NP tissues and cells were cultured to validate the role of CST in TNF-α-induced IVD degeneration. Moreover, in vitro and in vivo experiments were performed to identify the potential role of CST in mitochondrial dysfunction, mitochondrial ROS generation and activation of the NLRP3 inflammasome during IVD degeneration. In addition, NF-κB signalling pathway activity was tested in NP tissues and cells from CST-/- mice.Results: The expression of CST in NP cells was diminished in the ageing- and TNF-α-induced IVD degeneration process. In addition, compared with WT mice, aged CST-/- mice displayed accelerated metabolic imbalance and enhanced apoptosis, and these mice showed a disorganized NP tissue structure. Moreover, TNF-α-mediated catabolism and apoptosis were alleviated by exogenous CST treatment. Furthermore, CST inhibited mitochondrial dysfunction in NP cells through IVD degeneration and suppressed activation of the NLRP3 inflammasome. In vitro and ex vivo experiments indicated that increased NF-κB pathway activity might have been associated with the IVD degeneration observed in CST-/- mice.Conclusion: This study suggests the role of CST in mitochondrial ROS and activation of the NLRP3 inflammasome in IVD degeneration, which might shed light on therapeutic targets for IVD degeneration.

Bardoxolone methyl breaks the vicious cycle between M1 macrophages and senescent nucleus pulposus cells through the Nrf2/STING/NF-κB pathway

Targeting cellular senescence and senescence associated secretory phenotype (SASP) through autophagy has emerged as a promising intervertebral disc (IVD) degeneration (IDD) treatment strategy in recent years. This study aimed to clarify the role and mechanism of autophagy in preventing IVD SASP. Methods involved in vitro experiments with nucleus pulposus (NP) tissues from normal and IDD patients, as well as an in vivo IDD animal model. GATA4's regulatory role in SASP was validated both in vitro and in vivo, while autophagy modulators were employed to assess their impact on GATA4 and SASP. Transcriptomic sequencing identified oxidized low-density lipoprotein receptor 1 (OLR1) as a key regulator of autophagy and GATA4. A series of experiments manipulated OLR1 expression to investigate associated effects. Results demonstrated significantly increased senescent NP cells (NPCs) and compromised autophagy in IDD patients and animal models, with SASP closely linked to IDD progression. The aged disc milieu impeded autophagic GATA4 degradation, leading to elevated SASP expression in senescent NPCs. Restoring autophagy reversed senescence by degrading GATA4, hence disrupting the SASP cascade. Moreover, OLR1 was identified for its regulation of autophagy and GATA4 in senescent NPCs. Silencing OLR1 enhanced autophagic activity, suppressing GATA4-induced senescence, and SASP expression in senescent NPCs. In conclusion, OLR1 was found to control autophagy-GATA4 and SASP, with targeted OLR1 inhibition holding promise in alleviating GATA4-induced senescence and SASP expression while delaying extracellular matrix degradation, offering a novel therapeutic approach for IDD management.

Duhuo Jisheng decoction alleviates intervertebral disc degeneration by inhibiting nucleus pulposus cell ferroptosis via the exosome-mediated miR-19b-3p/ACSL4 axis.

BQU57 suppresses IL-1β-induced apoptosis and extracellular matrix degradation in nucleus pulposus cells by blocking the NF-κB signaling pathway.

Injectable self-healing hydrogel with siRNA delivery property for sustained STING silencing and enhanced therapy of intervertebral disc degeneration

Intervertebral disc (IVD) degeneration (IDD) is a worldwide spinal degenerative disease. Low back pain (LBP) is frequently caused by a variety of conditions brought on by IDD, including IVD herniation and spinal stenosis, etc. These conditions bring substantial physical and psychological pressure and economic burden to patients. IDD is closely tied with the structural or functional changes of the IVD tissue and can be caused by various complex factors like senescence, genetics, and trauma. The IVD dysfunction and structural changes can result from extracellular matrix (ECM) degradation, differentiation, inflammation, oxidative stress, mechanical stress, and senescence of IVD cells. At present, the treatment of IDD is basically to alleviate the symptoms, but not from the pathophysiological changes of IVD. Interestingly, the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway is involved in many processes of IDD, including inflammation, ECM degradation, apoptosis, senescence, proliferation, oxidative stress, and autophagy. These activities in degenerated IVD tissue are closely relevant to the development trend of IDD. Hence, the p38 MAPK signaling pathway may be a fitting curative target for IDD. In order to better understand the pathophysiological alterations of the intervertebral disc tissue during IDD and offer potential paths for targeted treatments for intervertebral disc degeneration, this article reviews the purpose of the p38 MAPK signaling pathway in IDD.

DEP domain-containing mTOR-interacting protein (DEPTOR) has been recently discovered as an endogenous regulator of the mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2. mTORC1 is present in the brain, and there is growing evidence that its dysregulation contributes to several brain alterations. This suggests the involvement of mTOR signaling and its modulators in neurobiological controls. Here, we characterized and mapped the expression of DEPTOR in the rat brain. We show that DEPTOR was widely expressed from the forebrain to the hindbrain, including the hippocampus, the mediobasal hypothalamus, and the circumventricular organs (CVOs). In the hippocampus, DEPTOR protein and Deptor mRNA were highly expressed in the dendate gyrus and CA3 field. In the CVOs, DEPTOR was expressed in the subfornical organ, the median eminence, and the area postrema. In the mediobasal hypothalamus, DEPTOR was expressed in neurons of the ventromedial nucleus (VMH) and colocalized with proopiomelanocortin (POMC) in the arcuate nucleus (ARC). The hypothalamic distribution suggested a role for DEPTOR in energy balance. Supporting this possibility, we observed that Deptor hypothalamic expression was modulated by the nutritional status in a context of diet-induced and genetic obesity; food deprivation increased Deptor mRNA in both the ARC and VMH of obese rats. In conclusion, the present results illustrate the presence of DEPTOR in the rat brain and suggest a role for DEPTOR in the hypothalamic regulation of energy balance, which further supports the role of mTOR in energy homeostasis. J. Comp. Neurol. 523:93-107, 2015. © 2014 Wiley Periodicals, Inc.

hASCs-derived exosomal miR-155-5p targeting TGFβR2 promotes autophagy and reduces pyroptosis to alleviate intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a major cause of lower back pain. The high morbidity associated with this disease diminishes the quality of life of those who are affected. MicroRNAs (miRs) play crucial roles in various diseases, including IDD. However, the mechanism via which miR-200c-3p plays a role in the development of IDD remains unknown. The present study aimed to investigate the effect of miR-200c-3p on the progression of IDD and the underlying mechanism. The expression level of miR-200c-3p was evaluated in intervertebral disc tissues from patients with IDD. To construct the IDD cell model, the nucleus pulposus (NP) cells were treated with lipopolysaccharide (LPS) 24 h following transfection with miR-200c-3p mimic or inhibitor. A luciferase activity assay was performed, while reverse transcription-quantitative PCR and western blotting were conducted to determine the RNA and protein expression levels, respectively. The expression level of miR-200c-3p in the intervertebral disc tissues of patients with IDD was lower than that of normal subjects. LPS treatment reduced the expression level of miR-200c-3p in NP cells. Moreover, miR-200c-3p mimic inhibited LPS-induced NP cell apoptosis. It was found that miR-200c-3p attenuated inflammatory cytokine levels and extracellular matrix (ECM) degradation in NP cells. Furthermore, miR-200c-3p targeted Ras-related protein 2C (RAP2C) in NP cells. RAP2C promoted apoptosis, inflammatory cytokine levels and ECM degradation by activating ERK signaling. Knockdown of RAP2C and inhibition of ERK signaling by SCH772984 partially reversed the proinflammatory effect of the miR-200c-3p inhibitor on LPS-treated NP cells. Thus, miR-200c-3p inhibits NP cell apoptosis, inflammatory cytokine levels and ECM degradation in IDD by targeting RAP2C/ERK signaling.

Experimental analysis of circular RNA in intervertebral disk degeneration (IDD). This study aimed to explore the roles of hsa_circ_0001946 (circ-CDR1as) in mechanical stress-induced nucleus pulposus cell injury in IDD. Mechanical stress is an important pathogenic factor for IDD. Excessive compression stress leads to nucleus pulposus (NP) cell apoptosis and extracellular matrix (ECM) degradation and accelerated IDD. Circ-CDR1as is associated with various degenerative conditions, but its role in IDD is not clear. Herein, we explored the roles and mechanisms of circ-CDR1as in IDD in vitro. An in vitro model of IDD was constructed by treating NP cells with 1.0MPa compression stress. Quantitative real-time polymerase chain reaction assay was used for detecting the expression of circ-CDR1as and miR-432-5p. Immunofluorescent analysis was performed for MMP13 detection. Western blot assay was performed for detecting apoptosis and ECM-related protein expression. Flow cytometry analysis was used for cell apoptosis analysis. The dual-luciferase reporter was used to analyze the interaction between miR-432-5p and circ-CDR1as or SOX9. Differences in means between groups were evaluated using the Student t test or one-way analysis of variance. In compression-treated human NP cells, we found that circ-CDR1as was significantly downregulated. Functional experiments showed that circ-CDR1as overexpression reduced the compression-induced apoptosis and ECM degradation in NP cells. Further research indicated that circ-CDR1as could act as a molecular sponge for miR-432-5p, a miRNA that enhanced compression-induced damage of NP cells by inhibiting the expression of SOX9. The luciferase reporter experiments also showed that the mutual dialogue between circ-CDR1as and miR-432-5p regulated the expression of SOX9. Circ-CDR1as binds to miR-432-5p and plays a protective role in mitigating compression-induced NP cell apoptosis and ECM degradation by targeting SOX9. Circ-CDR1as may provide a novel therapeutic target for the clinical management of IDD in the future.

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.

BackgroundCircular RNAs (circRNAs) play an important role in the progression of intervertebral disc (IVD) degeneration (IVDD). Using bioinformatics analysis, we have found that the expression of circRNA hsa_circ_0059955 was significantly downregulated in IVDD tissues. However, the relevant mechanism of hsa_circ_0059955 in the progression of IVDD remains unclear.MethodsCCK-8 and flow cytometry assays were used to evaluate cell proliferation and apoptosis. In addition, Western blot assay was used to detect the expressions of ITCH, p73, CDK2 in nucleus pulposus (NP) cells. Moreover, a puncture-induced IVDD rat model was established to explore the role of hsa_circ_0059955 in IVDD.ResultsThe level of hsa_circ_0059955 was significantly decreased in IVDD tissues from IVDD patients. Itchy E3 ubiquitin protein ligase (ITCH) is the host gene of hsa_circ_0059955, and downregulation of hsa_circ_0059955 significantly decreased the expression of ITCH in NP cells. In addition, downregulation of hsa_circ_0059955 markedly inhibited proliferation and induced apoptosis and cell cycle arrest in NP cells. Moreover, in vivo study illustrated that overexpression of hsa_circ_0059955 ameliorated IVDD in rats.ConclusionDownregulation of hsa_circ_0059955 could induce apoptosis and cell cycle arrest in NP cells in vitro, while overexpression of hsa_circ_0059955 attenuated the IVDD in a puncture-induced rat model in vivo. Therefore, hsa_circ_0059955 might serve as a therapeutic target for the treatment of IVDD.