Therapeutic Target For Intervertebral Disc Degeneration Research Articles

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. Invivo 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.

Identification of Biomarkers, Pathways, Immune Properties of Mitophagy Genes, and Prediction Models for Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is the leading cause of low back pain (LBP). The mechanism of IDD development and progression is not fully understood. Peripheral biomarkers are increasingly vital non-radioactive methods in early detection and diagnosis for IDD. Nevertheless, less attention has been paid to the role of mitophagy genes in the progress of IDD. This study aimed to identify the mitophagy disease-causing genes in the process of IDD and mitophagy diagnostic biomarkers for IDD. Mitophagy-related differentially expressed genes (MRDEGs) related to IDD were investigated by analyzing the microarray datasets of IDD cases from GEO, PathCards and Molecular Signatures Databases. We used R software, WGCNA, PPI, mRNA-miRNA, mRNA-TF, GO, KEGG, GSEA, GSVA and Cytoscape to analyze and visualize the data. We further used ssGSEA for immunoinfiltration analysis to obtain different immune cell infiltration. LASSO model was developed to screen for genes that met the diagnostic gene model requirements. Finally, qRT-PCR, Western blotting and HE were used to verify hub genes and their expression from clinical IDD samples. We identified 14 MRDEGs and 12 hub genes. GO, KEGG, GSEA and GSVA analyses demonstrated that hub genes were critical for the development of IDD. LASSO diagnostic model consisted of six hub genes, among which SQSTM1, ATG7 and OPTN were significantly different between the two IDD disease subtypes. At the same time, SQSTM1 also had a high correlation with immune characteristic subtypes. The results of qRT-PCR and Western blotting also indicated that these genes were significantly differentially expressed in nucleus pulposus cells (NPCs) of the IDD group. We explored an association between MRDEGs-associated signature in IDD and validated that hub genes like SQSTM1 might serve as biomarkers for diagnostic and therapeutic targets for IDD. Meanwhile, this study can provide new insights into the functional characteristics and mechanism of mitophagy in the development of IDD.

Involvement of DJ-1 in the pathogenesis of intervertebral disc degeneration via hexokinase 2-mediated mitophagy

Intervertebral disc degeneration (IDD) is an important pathological basis for degenerative spinal diseases and is involved in mitophagy dysfunction. However, the molecular mechanisms underlying mitophagy regulation in IDD remain unclear. This study aimed to clarify the role of DJ-1 in regulating mitophagy during IDD pathogenesis. Here, we showed that the mitochondrial localization of DJ-1 in nucleus pulposus cells (NPCs) first increased and then decreased in response to oxidative stress. Subsequently, loss- and gain-of-function experiments revealed that overexpression of DJ-1 in NPCs inhibited oxidative stress-induced mitochondrial dysfunction and mitochondria-dependent apoptosis, whereas knockdown of DJ-1 had the opposite effect. Mechanistically, mitochondrial translocation of DJ-1 promoted the recruitment of hexokinase 2 (HK2) to damaged mitochondria by activating Akt and subsequently Parkin-dependent mitophagy to inhibit oxidative stress-induced apoptosis in NPCs. However, silencing Parkin, reducing mitochondrial recruitment of HK2, or inhibiting Akt activation suppressed DJ-1-mediated mitophagy. Furthermore, overexpression of DJ-1 ameliorated IDD in rats through HK2-mediated mitophagy. Taken together, these findings indicate that DJ-1 promotes HK2-mediated mitophagy under oxidative stress conditions to inhibit mitochondria-dependent apoptosis in NPCs and could be a therapeutic target for IDD.

Exploring the Impact of TLR-2 Signaling on miRNA Dysregulation in Intervertebral Disc Degeneration.

Toll-like receptors (TLRs) are key mediators of inflammation in intervertebral disc (IVD) degeneration. TLR-2 activation contributes to the degenerative process by increasing the expression of extracellular matrix-degrading enzymes, pro-inflammatory cytokines, and neurotrophins. As potent post-transcriptional regulators, microRNAs can modulate intracellular mechanisms, and their dysregulation is known to contribute to numerous pathologies. This study aims to investigate the impact of TLR-2 signaling on miRNA dysregulation in the context of IVD degeneration. Small-RNA sequencing of degenerated IVD cells shows the dysregulation of ten miRNAs following TLR-2 activation by PAM2CSK4. The miR-155-5p is most significantly upregulated in degenerated and non-degenerated annulus fibrosus and nucleus pulposus cells. Sequence-based target and pathway prediction shows the involvement of miR-155-5p in inflammation- and cell fate-related pathways and TLR-2-induced miR-155-5p expression leads to the downregulation of its target c-FOS. Furthermore, changes specific to the activation of TLR-2 through fragmented fibronectin are seen in miR-484 and miR-487. Lastly, miR-100-3p, miR-320b, and miR-181a-3p expression exhibit degeneration-dependent changes. These results show that TLR-2 signaling leads to the dysregulation of miRNAs in IVD cells as well as their possible downstream effects on inflammation and degeneration. The identified miRNAs provide important opportunities as potential therapeutic targets for IVD degeneration and low back pain.

Leptin Induces MMP-1 Expression Through the RhoA/ERK1/2/NF-κB Axis in Human Intervertebral Disc Cartilage Endplate-Derived Stem Cells.

Intervertebral disc (IVD) degeneration, associated with aging, may cause low back pain and disability, with obesity as a significant risk factor. In a prior study, we found a positive correlation between IVD degeneration and levels of matrix metalloproteinase-1 (MMP-1) and leptin. Yet, the interaction between MMP-1 and leptin in IVD degeneration is unclear. Our research seeks to explore leptin's influence on MMP-1 expression and the underlying mechanisms in human intervertebral disc cartilage endplate-derived stem cells, specifically SV40 cells. The mRNA and protein expression in leptin-stimulated SV40 cells were assessed using RT-real-time PCR and Western blotting or ELISA, respectively. We examined leptin-mediated RhoA activation through a GTP-bound RhoA pull-down assay. Furthermore, the phosphorylation levels of mitogen-activated protein kinases and AKT in leptin-stimulated SV40 cells were analyzed using Western blotting. The activation of NF-κB by leptin was investigated by assessing phosphorylation of IKKα/β, IκBα, and NF-κB p65, along with the nuclear translocation of NF-κB p65. To understand the underlying mechanism behind leptin-mediated MMP-1 expression, we employed specific inhibitors. Leptin triggered the mRNA and protein expression of MMP-1 in SV40 cells. In-depth mechanistic investigations uncovered that leptin heightened RhoA activity, promoted ERK1/2 phosphorylation, and increased NF-κB activity. However, leptin did not induce phosphorylation of JNK1/2, p38, or AKT. When we inhibited RhoA, ERK1/2, and NF-κB, it resulted in a decrease in MMP-1 expression. Conversely, inhibition of reactive oxygen species and NADPH oxidase did not yield the same outcome. Additionally, inhibiting RhoA or ERK1/2 led to a reduction in leptin-induced NF-κB activation. Moreover, inhibiting RhoA also decreased leptin-mediated ERK1/2 phosphorylation. These results indicated that leptin induced MMP-1 expression in SV40 cells through the RhoA/ERK1/2/NF-κB axis. This study provided the pathogenic role of leptin and suggested the potential therapeutic target for IVD degeneration.

CD206+ M2-like macrophages protect against intervertebral disc degeneration partially by targeting R-spondin-2

This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD). Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TUNEL assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD. Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD. Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD.

The involvement of DDX3X in compression-induced nucleus pulposus pyroptosis.

A study has been conducted to investigate the relationship between DDX3X and nucleus pulposus (NP) pyroptosis. DDX3X and pyroptosis-related proteins (Caspase-1, Full-length GSDMD, Cleaved GSDMD) were measured in compression-induced human NP cells and tissue. DDX3X was overexpressed or knocked down by gene transfection. The expressions of NLRP3, ASC, and pyroptosis-related proteins were detected by Western blot assay. IL-1β and IL-18 were detected by ELISA. HE staining and immunohistochemistry were used to observe the expression of DDX3X, NLRP3, and Caspase-1 in the rat model of compression-induced disc degeneration. DDX3X, NLRP3, and Caspase-1 were highly expressed in degenerated NP tissue. Overexpression of DDX3X induced pyroptosis in NP cells and increased levels of NLRP3, IL-1β, IL-18, and pyroptosis-related proteins. Knockdown of DDX3X showed an opposite trend to overexpression of DDX3X. The NLRP3 inhibitor CY-09 effectively prevented the up-regulation of the expression of IL-1β, IL-18, ASC, Pro-caspase-1, Full-length GSDMD, and Cleaved GSDMD. Increased expression of DDX3X, NLRP3, and Caspase-1 was observed in the rat model of compression-induced disc degeneration. Our study showed that DDX3X mediates pyroptosis of NP cells by upregulating NLRP3 expression, which ultimately leads to intervertebral disc degeneration (IDD). This discovery deepens the understanding of IDD pathogenesis and provides a promising and novel therapeutic target for IDD.

Identification of mitochondria-related key gene and association with immune cells infiltration in intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration and its inflammatory microenvironment can result in discogenic pain, which has been shown to stem from the nucleus pulposus (NP). Increasing evidence suggests that mitochondrial related genes are strictly connected to cell functionality and, importantly, it can regulate cell immune activity in response to damaged associated signals. Therefore, identification of mitochondria related genes might offer new diagnostic markers and therapeutic targets for IVD degeneration. In this study, we identified key genes involved in NP tissue immune cell infiltration during IVD degeneration by bioinformatic analysis. The key modules were screened by weighted gene co-expression network analysis (WCGNA). Characteristic genes were identified by random forest analysis. Then gene set enrichment analysis (GSEA) was used to explore the signaling pathways associated with the signature genes. Subsequently, CIBERSORT was used to classify the infiltration of immune cells. Function of the hub gene was confirmed by PCR, Western blotting and ELISA. Finally, we identified MFN2 as a crucial molecule in the process of NP cell pyroptosis and NLRP3 inflammasome activation. We speculate that the increased MFN2 expression in NP tissue along with the infiltration of CD8+ T cells, NK cell and neutrophils play important roles in the pathogenesis of IVD degeneration.

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

Intervertebral disc degeneration (IDD) is a complex chronic disease involving nucleus pulposus cells (NPCs) senescence, apoptosis, autophagy and extracellular matrix (ECM) degradation. In this study, we aimed to investigate the role of human adipose tissue stem cells (hASCs)-derived exosomal miR-155-5p targeting TGFβR2 in IDD and the mechanisms involved. Then miRNA sequencing was performed, and hASCs-derived Exo (hASCs-Exo) was extracted and characterized. First, NPCs were treated with different concentrations of LPS. Then miRNA sequencing was performed, and hASCs-Exo was extracted and characterized. NPCs were treated with PBS or autophagy inhibitor 3-MA. NPCs were transfected with miR-155-5p mimic, si-TGFβR2 and negative control. Cell viability, apoptosis, ROS, caspase-1+PI, pyroptosis markers, inflammatory cytokines, autophagy markers, Aggrecan, MMP13, and Akt/mTOR pathway-related factors were measured. Bioinformatics prediction and dual-luciferase were performed to verify the binding sites of miR-155-5p to TGFβR2. Finally, we validated the role of hASCs-derived exosomal miR-155-5p on IDD in vivo. LPS promoted pyroptosis of NPCs, and inhibited autophagy and ECM synthesis. MiR-155-5p was characterized as an inflammation-related miRNA in NPCs. HASCs-derived exosomal miR-155-5p inhibited pyroptosis of NPCs and promoted autophagy and ECM synthesis. After bioinformatics prediction and verification, it was found that miR-155-5p targeted TGFβR2. Moreover, miR-155-5p targeted TGFβR2 to promote autophagy and inhibit pyroptosis in NPCs. In vivo experiments revealed that hASCs-derived exosomal miR-155-5p alleviated IDD in rats. HASCs-derived exosomal miR-155-5p alleviated IDD by targeting TGFβR2 to promote autophagy and reduce pyroptosis. Our study may provide a new therapeutic target for IDD. HASCs-derived exosomal miR-155-5p is expected to be a biomarker for clinical treatment of IDD. Our study may provide a new therapeutic target for IDD.

Development of a Novel Inflammatory-Associated Gene Signature and Immune Infiltration Patterns in Intervertebral Disc Degeneration.

Background Both inflammatory factors and immune response play important roles in the pathogenesis of intervertebral disc degeneration (IDD). However, a comprehensive analysis of interaction between inflammatory response-associated genes (IRGs) and immune microenvironment in patients with IDD remains lacking. Hence, the current research is aimed at investigating the correlations between IRG signatures and immune cells in the progression of IDD. Methods The expression profiles (GSE27494 and GSE41883) and IRGs were downloaded from the Gene Expression Omnibus (GEO) database and Molecular Signature Database (MSigDB), respectively. Weighted gene coexpression network analysis (WGCNA) and differential expression analysis were used to identify the pivotal modules and common differentially expressed genes (DEGs) associated with IDD. Subsequently, we retrieved differentially expressed IRGs (DE-IRGs) by intersecting IRGs and DEGs for enrichment analysis. Next, LASSO regression analyses were performed to screen optimal marker genes for IDD prediction. Additionally, we validated differences DE-IRGs between IDD patients and controls in GSE150408. Finally, the infiltration alteration of immune cells was evaluated by the CIBERSORT, and the correlation between diagnostic markers and infiltrating immune cells was analyzed. Results A total of 10 upregulated differentially expressed inflammatory genes were identified that were obviously related to progression of IDD. Functional analysis results revealed that DE-IRGs were mainly enriched in signaling pathways TNF, IL-17, NOD-like receptor, and NF-kappa B pathway. A five-gene signature that consisted of IL-1β, LIF, LYN, NAMPT, and SLC7A2 was constructed by the LASSO Cox regression. IL1B, LYN, and NAMPT were further validated as optimal candidate genes in the pathophysiology of IDD. In addition, there was a remarkable immune cell infiltration difference between the healthy and IDD groups. The proportions for dendritic cells activated, mast cells activated, and neutrophils in the IDD group were significantly higher than those in the normal group, while the proportion of some cells was lower than that of the normal group, such as T cell CD4 memory resting, NK cells activated, and macrophage M0. Furthermore, correlation analysis indicated IL-1β, LYN, and NAMPT were closely implicated with immune cell infiltration in IDD development. Conclusions We explored an association between inflammatory response-associated signature and immune infiltration in IDD and validated that IL-1β, LYN, and NAMPT might serve as biomarkers and therapeutic targets for IDD in the future.

Single-cell atlas unveils cellular heterogeneity and novel markers in human neonatal and adult intervertebral discs

SummaryThe origin, composition, distribution, and function of cells in the human intervertebral disc (IVD) have not been fully understood. Here, cell atlases of both human neonatal and adult IVDs have been generated and further assessed by gene ontology pathway enrichment, pseudo-time trajectory, histology, and immunofluorescence. Comparison of cell atlases revealed the presence of two subpopulations of notochordal cells (NCs) and their associated markers in both the neonatal and adult IVDs. Developmental trajectories predicted 7 different cell states that describe the developmental process from neonatal to adult cells in IVD and analyzed the NC’s role in the IVD development. A high heterogeneity and gradual transition of annulus fibrosus cells (AFCs) in the neonatal IVD was detected and their potential relevance in IVD development assessed. Collectively, comparing single-cell atlases between neonatal and adult IVDs delineates the landscape of IVD cell biology and may help discover novel therapeutic targets for IVD degeneration.

Discovery and Application of Postnatal Nucleus Pulposus Progenitors Essential for Intervertebral Disc Homeostasis and Degeneration.

Intervertebral disc degeneration (IDD) results from the dysfunction of nucleus pulposus (NP) cells and the exhaustion of NP progenitors (ProNPs). The cellular applications of NP cells during IDD are currently limited due to the lack of in vivo studies showing whether NP cells are heterogeneous and contain ProNPs throughout postnatal stages. In this study, single‐cell RNA sequencing of purified NP cells is used to map four molecularly defined populations and urotensin II receptor (UTS2R)‐expressing postnatal ProNPs is identified, which are markedly exhausted during IDD, in mouse and human specimens. The lineage tracing shows that UTS2R+ ProNPs preferentially resides in the NP periphery with its niche factor tenascin‐C and give rise to functional NP cells. It is also demonstrated that transplanting UTS2R+ ProNPs with tenascin‐C into injured intervertebral discs attenuate the progression of IDD. The study provides a novel NP cell atlas, identified resident ProNPs with regenerative potential, and revealed promising diagnostic and therapeutic targets for IDD.

The Roles of circRNAs in Intervertebral Disc Degeneration: Inflammation, Extracellular Matrix Metabolism, and Apoptosis.

Low back pain (LBP) is seriously harmful to human health and produces heavy economic burden. And most scholars hold that intervertebral disc degeneration (IDD) is the primary cause of LBP. With the study of IDD, aberrant expression of gene has become an important pathogenic factor of IDD. Circular RNAs (circRNAs), as a kind of noncoding RNA (ncRNA), participate in the regulation of genetic transcription and translation and further affect the expression of inflammatory cytokine, metabolism of extracellular matrix (ECM), the proliferation and apoptosis of cells, etc. Therefore, maybe it will become a new therapeutic target for IDD. At present, our understanding of the mechanism of circRNAs in IDD is limited. The purpose of this review is to summarize the mechanism and related signaling pathways of circRNAs in IDD reported in the past. Particularly, the roles of circRNAs in inflammation, ECM metabolism, and apoptosis are emphasized.

MiR-98 Protects Nucleus Pulposus Cells against Apoptosis by Targeting TRAIL in Cervical Intervertebral Disc Degeneration.

The excessive apoptosis of nucleus pulposus (NP) cells is a major risk factor in the progress of cervical intervertebral disc degeneration (IVDD). In this study, we investigated the impact of miR-98 on apoptosis of NP cells and the potential molecular mechanisms. Lipopolysaccharide (LPS) was used to establish an NP cell IVDD model. The sponging effect of miR-98 on TRAIL 3′UTR was predicted by ENCORI and assessed by the dual-luciferase reporter gene system. The expression levels of miR-98, TRAIL, and TRAIL pathway-related genes were tested by qRT-PCR, Western blot, and immunofluorescence analysis. Cell apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. Cell viability was analyzed by MTT assay. It was found that the expression level of miR-98 was downregulated, while the level of TRAIL was upregulated in IVDD tissues, and their levels were negatively and positively associated with the clinical MRI grade, respectively. The LPS treatment resulted in a significant decrease of the miR-98 expression level and an increase of the TRAIL expression level in NP cells. miR-98 reduced NP cell apoptosis under LPS treatment in vitro. miR-98 directly targeted TRAIL. Moreover, the mRNA and protein levels of DR5, FADD, cleaved caspase8, cleaved caspase3, and cleaved PARP were downregulated by miR-98 overexpression. Overexpression of TRAIL partially reversed the suppressive roles of miR-98 on cell apoptosis and activation of the TRAIL pathway. We concluded that miR-98 inhibited apoptosis of NP cells by inactivating the TRAIL pathway via targeting TRAIL in IVDD NP cells. These results indicated that miR-98 might be a therapeutic target for IVDD.

A potential target gene CD63 for different degrees of intervertebral disc degeneration

Understanding molecular mechanisms of intervertebral disc degeneration (IDD) and providing a novel target for the treatment of IDD have important implications. We sought to explore a new promising gene target for the treatment of IDD. This study integrated 19,678 genes of 38 IDD patients from two gene datasets. Differentially Expressed Genes (DEGs) of annulus fibrosus were analyzed in groups with mild disc degeneration (MDD) and severe disc degeneration (SDD). We screened the hub gene through biological information technology (bioinformatic) methods. Then, we further validated the hub gene using annulus fibrosus and nucleus pulposus tissues from 12 patients with qRT-PCR. In addition, we explored its underlying molecular mechanism with GO, KEGG and GSEA. Through multiple screening bioinformatics methods, the hub gene CD63 was identified. The qRT-PCR explored that CD63 decreased significantly in SDD group compared to that in MDD group (P < 0.001). The GO, KEGG and GSEA of CD63 explored significant enrichment of the molecular features (P < 0.001), including the cellular component (Extracellular matrix, P < 0.001), the molecular function (collagen binding, P < 0.001), the biological processes (protein targeting, collagen fibril organization and platelet degranulation, P < 0.001) and the signaling pathways. Our research explored and validated a new regulatory gene, CD63 for different degrees of IDD. A new novel form of therapeutic target for IDD may be developed.

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. Statement of significanceIntervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

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

Inflammatory responses of nucleus pulposus (NP) can induce imbalanced anabolism and catabolism of extracellular matrix, and the cytosolic dsDNA accumulation and STING–NF–κB pathway activation found in NP inflammation are considered as fairly important cause of intervertebral disc (IVD) degeneration. Herein, we constructed a siSTING delivery hydrogel of aldehyde hyaluronic acid (HA-CHO) and poly(amidoamine) PAMAM/siRNA complex to intervene the abnormal STING signal for IVD degeneration treatment, where the formation of dynamic Schiff base bonds in the system (siSTING@HPgel) was able to overcome the shortcomings such as low cellular uptake, short half-life, and rapid degradation of siRNA-based strategy. PAMAM not only formed complexes with siRNA to promote siRNA transfection, but also served as dynamic crosslinker to construct hydrogel, and the injectable and self-healing hydrogel efficiently and steadily silenced STING expression in NP cells. Finally, the siSTING@HPgel significantly eased IVD inflammation and slowed IVD degeneration by prolonging STING knockdown in puncture-induced IVD degeneration rat model, revealing that STING pathway was a therapeutic target for IVD degeneration and such novel hydrogel had great potential for being applied to many other diseases for gene delivery.

Down-Regulation of microRNA-30d Alleviates Intervertebral Disc Degeneration Through the Promotion of FOXO3 and Suppression of CXCL10.

MicroRNAs (miRNAs/miRs) are important biomarkers for the progression of intervertebral disc degeneration (IDD). We investigated the role of miR-30d in IDD progression through its interactions with forkhead box O3 (FOXO3) and C-X-C motif ligand 10 (CXCL10). We first measured the expression of miR-30d, FOXO3, and CXCL10 in NP cells cultured from IDD patients. RNA-immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were then employed to test the relationship among miR-30d, FOXO3, and CXCL10. Besides, gain- and loss-of function approaches were performed to assess the functional roles of miR-30d and FOXO3 in IDD in vitro and in vivo. We found high expression of miR-30d and CXCL10 and low expression of FOXO3 in IDD. We showed that miR-30d specifically targeted FOXO3, and that down-regulation of miR-30d promoted proliferation and inhibited apoptosis of NP cells in IDD by increasing the expression of FOXO3. Besides, FOXO3 inhibited apoptosis of NP cells by downregulation of CXCL10 expression. Moreover, inhibition of miR-30d promoted proliferation and inhibited apoptosis of NP cells in IDD by decreasing CXCL10. Furthermore, findings in the mouse IDD model confirmed the inhibitory role of decreased miR-30d in IDD progression. Thus, we show that downregulation of miR-30d could promote the proliferation of NP cells by increasing FOXO3 and decreasing CXCL10 expression, which may provide a novel therapeutic target for IDD.

Effects of the NF‑κB/p53 signaling pathway on intervertebral disc nucleus pulposus degeneration.

The incidence of intervertebral disc degeneration (IDD) is increasing, especially among elderly individuals. The present study aimed to investigate the effects of the NF-κB/p53 signaling pathway on IDD and its regulatory effect on associated cytokines. In the present study, human nucleus pulposus cells were isolated from patients with thoracic-lumbar fractures and patients with IDD to observe cellular morphology and detect phosphorylated (p)-p65/p53 expression levels. The locality and expression levels of p65 in interleukin (IL)-1β-stimulated nucleus pulposus cells, with or without the addition of ammonium pyrrolidinedithiocarbamate (PDTC; a NF-κB signaling pathway-specific blocker), were measured. Furthermore, the effects of IL-1β stimulation on the protein and gene expression levels of IDD-related cytokines were determined following p53 knockdown and inhibition of the NF-κB signaling pathway. The results suggested that p-p65 and p53 expression was significantly increased in IDD cells compared with normal nucleus pulposus cells. Moreover, nucleus pulposus cells isolated from patients with IDD contained less cytoplasm compared with normal nucleus pulposus cells, and p65 expression levels were higher in the cytoplasm than the nucleus of IL-1β-stimulated PDTC-treated healthy nucleus pulposus cells. Moreover, the p53 expression levels were significantly decreased following transfection with sip53. PDTC treatment and p53 knockdown significantly decreased matrix metallopeptidase (MMP)-3, MMP-13, metallopeptidases with thrombospondin type 1 motif (ADAMTS)-4 and ADAMTS-5 expression levels, and increased aggrecan and collagen type II expression levels in IL-1β-stimulated cells. The present study indicated that activation of the NF-κB/p53 signaling pathway might be related to the occurrence of IDD; therefore, the NF-κB/p53 signaling pathway may serve as a therapeutic target for IDD.

LINC01121 induced intervertebral disc degeneration via modulating miR-150-5p/MMP16 axis.

Growing evidence indicates that Long noncoding RNAs contribute to cell differentiation, invasion, metabolism, proliferation and metastasis. However, the potential role of LINC01121 in progression of intervertebral disc degeneration (IDD) remains unclear. LINC01121, matrix metalloprotease (MMP)-16 and miR-150-5p expression was determined by a quantitative-reverse transcriptase-polymerase chain reaction assay. Inflammatory cytokines level was measured by an enzyme-linked immunosorbent assay and cell counting kit-8 analysis was used to assess cell proliferation. MMP-16-specific binding with miR-150-5p was verified with a luciferase reporter assay. We noted that interleukin (IL)-1β and tumor necrosis factor (TNF)-α treatment enhanced LINC01121 and MMP-16 expression in nucleus pulposus (NP) cells. LINC01121 was higher in IDD specimens compared to that in control specimens. Higher expression of LINC01121 was correlated with disc degeneration degree. Ectopic expression of LINC01121 enhanced cell proliferation and promoted ki-67, MMP-3 and ADAMTS5 expression and also suppressed collagen II expression in NP cells. We observed that overexpression of LINC01121 increased the secretion of three inflammatory cytokines, including IL-6, TNF-α and IL-1β. We found that ectopic expression of LINC01121 decreased the miR-150-5p level in NP cells. Luciferase reporter data confirmed that MMP-16 was one direct target of miR-150-5p. Overexpression of miR-150-5p inhibited MMP-16 level and elevated the expression of LINC01121 enhanced MMP-16 level. We also found that MMP-16 was up-regulated in IDD specimens compared to that in control specimens. Higher expression of MMP-16 was correlated with disc degeneration degree. Interestingly, MMP-16 expression was positively related to LINC01121 in IDD specimens. Finally, overexpression of LINC01121 regulated cell growth, extracellular matrix degradation and inflammatory cytokine secretion via modulating MMP-16. our data suggested LINC01121 may be a new therapeutic target for IDD.