Human Intervertebral Disc Degeneration Research Articles

The mitochondrial UPR induced by ATF5 attenuates intervertebral disc degeneration via cooperating with mitophagy.

Intervertebral disc degeneration (IVDD) is an aging disease that results in a low quality of life and heavy socioeconomic burden. The mitochondrial unfolded protein response (UPRmt) take part in various aging-related diseases. Our research intents to explore the role and underlying mechanism of UPRmt in IVDD. Nucleus pulposus (NP) cells were exposed to IL-1β and nicotinamide riboside (NR) served as UPRmt inducer to treat NP cells. Detection of ATP, NAD + and NADH were used to determine the function of mitochondria. MRI, Safranin O-fast green and Immunohistochemical examination were used to determine the degree of IVDD in vivo. In this study, we discovered that UPRmt was increased markedly in the NP cells of human IVDD tissues than in healthy controls. In vitro, UPRmt and mitophagy levels were promoted in NP cells treated with IL-1β. Upregulation of UPRmt by NR and Atf5 overexpression inhibited NP cell apoptosis and further improved mitophagy. Silencing of Pink1 reversed the protective effects of NR and inhibited mitophagy induced by the UPRmt. In vivo, NR might attenuate the degree of IDD by activating the UPRmt in rats. In summary, the UPRmt was involved in IVDD by regulating Pink1-induced mitophagy. Mitophagy induced by the UPRmt might be a latent treated target for IVDD.

Conditioned Medium of Intervertebral Disc Cells Inhibits Osteo-Genesis on Autologous Bone-Marrow-Derived Mesenchymal Stromal Cells and Osteoblasts.

Low back pain (LBP) is associated with the degeneration of human intervertebral discs (IVDs). Despite progress in the treatment of LBP through spinal fusion, some cases still end in non-fusion after the removal of the affected IVD tissue. In this study, we investigated the hypothesis that the remaining IVD cells secrete BMP inhibitors that are sufficient to inhibit osteogenesis in autologous osteoblasts (OBs) and bone marrow mesenchymal stem cells (MSCs). A conditioned medium (CM) from primary human IVD cells in 3D alginate culture was co-cultured with seven donor-matched OB and MSCs. After ten days, osteogenesis was quantified at the transcript level using qPCR to measure the expression of bone-related genes and BMP antagonists, and at the protein level by alkaline phosphatase (ALP) activity. Additionally, cells were evaluated histologically using alizarin red (ALZR) staining on Day 21. For judging ALP activity and osteogenesis, the Noggin expression in samples was investigated to uncover the potential causes. The results after culture with the CM showed significantly decreased ALP activity and the inhibition of the calcium deposit formation in alizarin red staining. Interestingly, no significant changes were found among most bone-related genes and BMP antagonists in OBs and MSCs. Noteworthy, Noggin was relatively expressed higher in human IVD cells than in autologous OBs or MSCs (relative to autologous OB, the average fold change was in 6.9, 10.0, and 6.3 in AFC, CEPC, and NPC, respectively; and relative to autologous MSC, the average fold change was 2.3, 3.4, and 3.2, in AFC, CEPC, and NPC, respectively). The upregulation of Noggin in residual human IVDs could potentially inhibit the osteogenesis of autologous OB and MSC, thus inhibiting the postoperative spinal fusion after discectomy surgery.

Injectable hydrogel induces regeneration of naturally degenerate human intervertebral discs in a loaded organ culture model

Low back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc (IVD). This study investigates the ability of an injectable hydrogel (NPgel), to inhibit catabolic protein expression and promote matrix expression in human nucleus pulposus (NP) cells within a tissue explant culture model isolated from degenerate discs. Furthermore, the injection capacity of NPgel into naturally degenerate whole human discs, effects on mechanical function, and resistance to extrusion during loading were investigated. Finally, the induction of potential regenerative effects in a physiologically loaded human organ culture system was investigated following injection of NPgel with or without bone marrow progenitor cells. Injection of NPgel into naturally degenerate human IVDs increased disc height and Young's modulus, and was retained during extrusion testing. Injection into cadaveric discs followed by culture under physiological loading increased MRI signal intensity, restored natural biomechanical properties and showed evidence of increased anabolism and decreased catabolism with tissue integration observed. These results provide essential proof of concept data supporting the use of NPgel as an injectable therapy for disc regeneration. Statement of significanceLow back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc. This study investigated the potential regenerative properties of an injectable hydrogel system (NPgel) within human tissue samples. To mimic the human in vivo conditions and the unique IVD niche, a dynamically loaded intact human disc culture system was utilised. NPgel improved the biomechanical properties, increased MRI intensity and decreased degree of degeneration. Furthermore, NPgel induced matrix production and decreased catabolic factors by the native cells of the disc. This manuscript provides evidence for the potential use of NPgel as a regenerative biomaterial for intervertebral disc degeneration.

Intervertebral disc human nucleus pulposus cells associated with back pain trigger neurite outgrowth in vitro and pain behaviors in rats.

Low back pain (LBP) is often associated with the degeneration of human intervertebral discs (IVDs). However, the pain-inducing mechanism in degenerating discs remains to be elucidated. Here, we identified a subtype of locally residing human nucleus pulposus cells (NPCs), generated by certain conditions in degenerating discs, that was associated with the onset of discogenic back pain. Single-cell transcriptomic analysis of human tissues showed a strong correlation between a specific cell subtype and the pain condition associated with the human degenerated disc, suggesting that they are pain-triggering. The application of IVD degeneration-associated exogenous stimuli to healthy NPCs in vitro recreated a pain-associated phenotype. These stimulated NPCs activated functional human iPSC-derived sensory neuron responses in an in vitro organ-chip model. Injection of stimulated NPCs into the healthy rat IVD induced local inflammatory responses and increased cold sensitivity and mechanical hypersensitivity. Our findings reveal a previously uncharacterized pain-inducing mechanism mediated by NPCs in degenerating IVDs. These findings could aid in the development of NPC-targeted therapeutic strategies for the clinically unmet need to attenuate discogenic LBP.

Retracted] Role of microRNA‑210 in human intervertebral disc degeneration.

[This retracts the article DOI: 10.3892/etm.2016.3176.].

Matrix stiffness induces Drp1-mediated mitochondrial fission through Piezo1 mechanotransduction in human intervertebral disc degeneration

BackgroundExtracellular matrix stiffness is emerging as a crucial mechanical cue that drives the progression of various diseases, such as cancer, fibrosis, and inflammation. The matrix stiffness of the nucleus pulposus (NP) tissues increase gradually during intervertebral disc degeneration (IDD), while the mechanism through which NP cells sense and react to matrix stiffness remains unclear. In addition, mitochondrial dynamics play a key role in various cellular functions. An in-depth investigation of the pathogenesis of IDD can provide new insights for the development of effective therapies. In this study, we aim to investigate the effects of matrix stiffness on mitochondrial dynamics in IDD.MethodsTo build the gradient stiffness model, NP cells were cultured on polystyrene plates with different stiffness. Western blot analysis, and immunofluorescence staining were used to detect the expression of mitochondrial dynamics-related proteins. Flow cytometry was used to detect the mitochondrial membrane potential and intracellular Ca2+ levels. Apoptosis related proteins, ROS level, and TUNEL staining were performed to assess the effect of substrate stiffness on NP cells.ResultsStiff substrate increased phosphorylation of dynamin-related protein 1 (Drp1) at Ser616 by activating extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which promoted mitochondrial fission and apoptosis in NP cells. Furthermore, Piezo1 activation was involved in the regulation of the post-translational modifications of Drp1 and mitochondrial fission caused by matrix stiffness. Inhibition of Piezo1 and ERK1/2 can effectively reduce stiffness-induced ROS elevation and apoptosis in NP cells.ConclusionsOur results revealed that stiff substrate causes Piezo1 activation and Ca2+ influx, results in ERK1/2 activation and phosphorylation of Drp1 at S616, and finally leads to mitochondrial fission and apoptosis in NP cells. These findings reveal a new mechanism of mechanotransduction in NP cells, providing novel insights into the development of therapies for treating IDD.

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.

Cynarin alleviates intervertebral disc degeneration via protecting nucleus pulposus cells from ferroptosis

Intervertebral disc degeneration (IVDD) leads to a series of degenerative spine diseases. Clinical treatment of IVDD is mainly surgery, lacking effective drugs to alleviate intervertebral disc degeneration. In this study, we analysed the mRNA sequencing dataset of human degenerative intervertebral disc tissues and revealed the participation of ferroptosis in IVDD. Furthermore, we confirmed that TNF-α, an important cytokine in IVDD, induces ferroptosis in nucleus pulposus cells. Subsequently, a ferroptosis inhibitors screening strategy using multiple ferroptosis indicators was developed. Through the screen of various natural compounds, cynarin, a natural product enriched in Artichoke, was discovered to inhibit ferroptosis of nucleus pulposus cells. Cynarin can dose-dependently inhibit the catabolism of nucleus pulposus cells, increase the expression of key ferroptosis-inhibiting genes (GPX4 and NRF2), inhibit the increment of cellular Fe2+, lipid peroxides, and reactive oxygen species. It can also prevent mitochondria shrinkage, reduce mitochondria cristae density in ferroptosis, and prevent IVDD in the rat model. In conclusion, cynarin is a potential candidate for the drug development for IVDD.

Single-cell transcriptomics reveals heterogeneity and intercellular crosstalk in human intervertebral disc degeneration

The complexity of the human intervertebral disc (IVD) has hindered the elucidation of the microenvironment and mechanisms underlying IVD degeneration (IVDD). Here we determined the landscapes of nucleus pulposus (NP), annulus fibrosus (AF), and immunocytes in human IVD by scRNA-seq. Six NP subclusters and seven AF subclusters were identified, whose functional differences and distribution during different stages of degeneration (Pfirrmann I-V) were investigated. We found MCAM+ progenitor in AF, as well as CD24+ progenitor and MKI67+ progenitor in NP, forming a lineage trajectory from CD24+/MKI67+ progenitors to EffectorNP_⅓ during IVDD. There is a significant increase in monocyte/macrophage (Mφ) in degenerated IVDs (p= 0.044), with Mφ-SPP1 exclusively found in IVDD but not healthy IVDs. Further analyses of the intercellular crosstalk network revealed interactions between major subpopulations and changes in the microenvironment during IVDD. Our results elucidated the unique characteristics of IVDD, thereby shedding light on therapeutic strategies.

Ex vivo intervertebral disc cultures: degeneration-induction methods and their implications for clinical translation

Because low back pain is frequently a result of intervertebral disc degeneration (IVDD), strategies to regenerate or repair the IVD are currently being investigated. Often, ex vivo disc cultures of non-human IVD organs or tissue explants are used that usually do not exhibit natural IVDD. Therefore, degenerative changes mimicking those reported in human IVDD need to be induced. To support researchers in selecting ex vivo disc cultures, a systematic search was performed for them and their potential use for studying human IVDD reviewed. Five degeneration induction categories (proinflammatory cytokines, injury/damage, degenerative loading, enzyme, and other) were identified in 129 studies across 7 species. Methods to induce degeneration are diverse and can induce mild to severe degenerative changes that progress over time, as described for human IVDD. The induced degenerative changes are model-specific and there is no "one-fits-all" IVDD induction method. Nevertheless, specific aspects of human IVDD can be well mimicked. Currently, spontaneously degenerated disc cultures from large animals capture human IVDD in most aspects. Combinatorial approaches of several induction methods using discs derived from large animals are promising to recapitulate pathological changes on several levels, such as cellular behaviour, extracellular matrix composition, and biomechanical function, and therefore better mimic human IVDD. Future disc culture setups might increase in complexity, and mimic human IVDD even better. As ex vivo disc cultures have the potential to reduce and even replace animal trials, especially during preclinical development, advancement of such models is highly relevant for more efficient and cost-effective clinical translation from bench-to-bedside.

Expression of GADD45G and CAPRIN1 in Human Nucleus Pulposus: Implications for Intervertebral Disc Degeneration.

Marked cellular changes occur in human intervertebral disc (IVD) degeneration during disc degeneration with biochemical changes. Genome-wide analysis of the DNA methylation profile has identified 220 differentially methylated loci associated with human IVD degeneration. Among these, two cell-cycle-associated genes, growth arrest and DNA damage 45 gamma (GADD45G) and cytoplasmic activation/proliferation-associated protein-1 (CAPRIN1), were focused on. The expression of GADD45G and CAPRIN1 in human IVDs remains unknown. We aimed to examine the expression of GADD45G and CAPRIN1 in human nucleus pulposus (NP) cells and evaluate those in human NP tissues in the early and advanced stages of degeneration according to Pfirrmann magnetic resonance imaging (MRI) and histological classifications. Human NP cells were cultured as monolayers after isolation from NP tissues by sequential enzyme digestion. Total RNA was isolated, and the mRNA expression of GADD45G and CAPRIN1 was quantified using real-time polymerase chain reaction. To examine the effects of pro-inflammatory cytokines on mRNA expression, human NP cells were cultured in the presence of IL-1β. Protein expression was evaluated using Western blotting and immunohistochemistry. GADD45G and CAPRIN1 expression was identified in human NP cells at both mRNA and protein levels. The percentage of cells immunopositive for GADD45G and CAPRIN1 significantly increased according to the Pfirrmann grade. A significant correlation between the histological degeneration score and the percentage of GADD45G-immunopositive cells was identified, but not with that of CAPRIN1-immunopositive cells. The expression of cell-cycle-associated proteins (GADD45G and CAPRIN1) was enhanced in human NP cells at an advanced stage of degeneration, suggesting that it may be regulated during the progression of IVD degeneration to maintain the integrity of human NP tissues by controlling cell proliferation and apoptosis under epigenetic alteration.

Evaluation of the concentration of selected elements in serum patients with intervertebral disc degeneration

Quantitative analysis of the trace element content of human intervertebral discs (IVDs) is essential because it can identify specific enzymes or metabolites that may be related to human intervertebral disc degeneration (IVDD). The goal of this study was to assess the concentrations of copper (Cu), iron (Fe), manganese (Mn), lead (Pb), zinc (Zn), sodium (Na), magnesium (Mg), potassium (K), phosphorus (P), and calcium (Ca) in serum samples obtained from patients with IVDD in comparison to healthy volunteers (a control group). The study group consisted of 113 Caucasian patients qualified by a specialist neurosurgeon for microdiscectomy. The control group consisted of 113 healthy volunteers who met the eligibility criteria for blood donors. The examined clinical material was the serum samples obtained from both groups.Based on the quantitative analysis of selected elements, there were statistically significantly (p 0.05) higher concentrations of Cu (1180 μg/L±800 μg/L vs. 1230 μg/L±750 μg/L), Zn (790 μg/L±300 μg/L vs. 850 μg/L±200 μg/L), and Mg (21730 μg/L±4360 μg/L vs. 23820 μg/L±4990 μg/L) in the serum of healthy volunteers compared to those in the study group. In addition, statistically significant changes were not detected in the concentrations of any elements among either sex in either the study or control group or in their body mass index (BMI) values (p > 0.05). In the serum samples from the study group, the strongest relationships were noted between the concentrations of Zn and Pb (r = 0.61), Zn and P (r = 0.69), Zn and Ca (r = 0.84), Zn and Cu (r = 0.83), Mg and Ca (r = 0.74), and Ca and P (r = 0.98).It has been indicated that, above all, the concentrations of Cu, Zn, Ca, and Mg depend on the advancement of radiological changes, according to the Pfirrmann scale. However, no influence on pain intensity was found, depending on the concentration of the assessed elements.The analysis indicates that the determination of serum Cu, Zn, Ca, and Mg concentrations may have diagnostic significance in predicting the onset of lumbosacral IVDD. The predictive evaluation of changes in the concentrations of selected elements in patients with degenerative lumbar IVD lesions appears to be a promising, cost-effective strategy.

Establishment of Ferroptosis-Related Key Gene Signature and Its Validation in Compression-Induced Intervertebral Disc Degeneration Rats.

Cell death and functional loss of nucleus pulposus cell play essential roles in intervertebral disc degeneration (IDD). Ferroptosis is a newly identified cell death type, and its role in IDD is still under investigation. Identifying the key genes of ferroptosis in IDD helps to identify the therapeutic targets of IDD. In this study, we downloaded the human IDD mRNA microarray data from the Gene Expression Omnibus and ferroptosis genes from FerrDb, then performed a series of analyses using strict bioinformatics algorithms. In general, we obtained 40 ferroptosis-related differential expression genes (FerrDEGs) and identified six ferroptosis key gene signatures, namely, ATF3, EIF2S1, AR, NQO1, TXNIP, and AKR1C3. In addition, enrichment analysis of the FerrDEGs was conducted, the protein-protein interaction network was constructed, the correlations between ferroptosis key genes and immune infiltrating cells were analyzed, and the lncRNA-miRNA-mRNA ceRNA network was constructed. In particular, ATF3 and EIF2S1 showed the strongest correlation with immune cell function, which might lead to the development of IDD. Finally, the expressions of ferroptosis key genes were verified in the rat compression-induced IDD. In conclusion, this preliminary study analyzed and verified the mechanism of ferroptosis in IDD, laid a foundation for the follow-up study of the mechanism of ferroptosis in IDD, and provided new targets for preventing and delaying IDD.

An update on animal models of intervertebral disc degeneration and low back pain: Exploring the potential of artificial intelligence to improve research analysis and development of prospective therapeutics.

Animal models have been invaluable in the identification of molecular events occurring in and contributing to intervertebral disc (IVD) degeneration and important therapeutic targets have been identified. Some outstanding animal models (murine, ovine, chondrodystrophoid canine) have been identified with their own strengths and weaknesses. The llama/alpaca, horse and kangaroo have emerged as new large species for IVD studies, and only time will tell if they will surpass the utility of existing models. The complexity of IVD degeneration poses difficulties in the selection of the most appropriate molecular target of many potential candidates, to focus on in the formulation of strategies to effect disc repair and regeneration. It may well be that many therapeutic objectives should be targeted simultaneously to effect a favorable outcome in human IVD degeneration. Use of animal models in isolation will not allow resolution of this complex issue and a paradigm shift and adoption of new methodologies is required to provide the next step forward in the determination of an effective repairative strategy for the IVD. AI has improved the accuracy and assessment of spinal imaging supporting clinical diagnostics and research efforts to better understand IVD degeneration and its treatment. Implementation of AI in the evaluation of histology data has improved the usefulness of a popular murine IVD model and could also be used in an ovine histopathological grading scheme that has been used to quantify degenerative IVD changes and stem cell mediated regeneration. These models are also attractive candidates for the evaluation of novel anti-oxidant compounds that counter inflammatory conditions in degenerate IVDs and promote IVD regeneration. Some of these compounds also have pain-relieving properties. AI has facilitated development of facial recognition pain assessment in animal IVD models offering the possibility of correlating the potential pain alleviating properties of some of these compounds with IVD regeneration.

The CRL4DCAF6 E3 ligase ubiquitinates CtBP1/2 to induce apoptotic signalling and promote intervertebral disc degeneration.

Inflammation and apoptosis are two important pathological causes of intervertebral disc degeneration (IDD). The crosstalk between these two biological processes during IDD pathogenesis remains elusive. Herein, we discovered that chronic inflammation induced apoptosis through a cullin-RING E3 ligase (CRL)-dependent mechanism. Two cullin proteins, CUL4A and 4B, recruited DNA damage-binding protein 1 (DDB1), RING-box protein 1 (RBX1) and DDB1- and CUL4-associated factor 6 (DCAF6) to assemble a CRL4DCAF6 E3 ligase in intervertebral discs (IVDs) derived from IDD patients. The CRL4DCAF6 E3 ligase ubiquitinated and degraded C-terminal-binding protein 1 and 2 (CtBP1/2), two homologues of transcriptional corepressors. The degradation of CtBP1/2 disassociated from the p300-forkhead box O3a (FOXO3a) complex, inducing the expression of B-cell lymphoma 2 (Bcl2)-binding component 3 (BBC3) and causing BBC3-dependent apoptosis. TSC01131, a small molecule that specifically targets CUL4-DDB1 interaction, could inhibit the ubiquitination of CtBP1/2 in vitro and in vivo, thereby decreasing the BBC3 expression level and preventing apoptosis signalling. Using a mouse chronic inflammation model, we found that chronic inflammation could accelerate the IDD process through a conserved CRL4DCAF6-mediated mechanism. The administration of TSC01131 to mice could significantly improve the outcome of IDD. Collectively, our results revealed that inflammation-dependent CRL4DCAF6 E3 ligase triggered apoptosis through the removal of CtBP-mediated transrepression. The blockage of the CRL4DCAF6 E3 ligase by TSC01131 may represent a new therapeutic strategy for IDD treatment. KEY MESSAGES: CUL4A and CUL4B recruited DDB1, RBX1 and DCAF6 to assemble a CRL4DCAF6 E3 ligase in human IDD biopsies. The CRL4DCAF6 E3 ligase ubiquitinated and degraded CtBP1/2, causing BBC3-dependent apoptosis. A small molecule TSC01131 that specifically targets CUL4-DDB1 interaction could inhibit the ubiquitination of CtBP1/2, improving the outcome of IDD in a mouse model.

Injectable biomaterial induces regeneration of the intervertebral disc in a caprine loaded disc culture model.

Back pain is the leading cause of disability with half of cases attributed to intervertebral disc (IVD) degeneration, yet currently no therapies target this cause. We previously reported an ex vivo caprine loaded disc culture system (LDCS) that accurately represents the cellular phenotype and biomechanical environment of human IVD degeneration. Here, the efficacy of an injectable hydrogel system (LAPONITE® crosslinked pNIPAM-co-DMAc, (NPgel)) to halt or reverse the catabolic processes of IVD degeneration was investigated within the LDCS. Following enzymatic induction of degeneration using 1 mg mL-1 collagenase and 2 U mL-1 chondroitinase ABC within the LDCS for 7 days, IVDs were injected with NPgel alone or with encapsulated human bone marrow progenitor cells (BMPCs). Un-injected caprine discs served as degenerate controls. IVDs were cultured for a further 21 days within the LDCS. Tissues were then processed for histology and immunohistochemistry. No extrusion of NPgel was observed during culture. A significant decrease in histological grade of degeneration was seen in both IVDs injected with NPgel alone and NPgel seeded with BMPCs, compared to un-injected controls. Fissures within degenerate tissue were filled by NPgel and there was evidence of native cell migration into injected NPgel. The expression of healthy NP matrix markers (collagen type II and aggrecan) was increased, whereas the expression of catabolic proteins (MMP3, ADAMTS4, IL-1β and IL-8) was decreased in NPgel (±BMPCs) injected discs, compared to degenerate controls. This demonstrates that NPgel promotes new matrix production at the same time as halting the degenerative cascade within a physiologically relevant testing platform. This highlights the potential of NPgel as a future therapy for IVD degeneration.

The role of the miR-4306/PAK6 axis in degenerative nucleus pulposus cells in human intervertebral disc degeneration

Intervertebral disc degeneration (IDD), characterized by degenerative changes that occur in intervertebral discs due to aging or structural injury, is thought to be the most common cause of lower back pain. Recent studies have shown that microRNAs (miRNAs) have a critical role in the etiopathogenesis of IDD. In the current study, we aimed to determine the role of miRNAs in mediating the underlying mechanisms associated with IDD. First, differentially expressed miRNAs (DEmiRNAs) were identified using the GEO database, and subsequently confirmed by RT-qPCR and in situ hybridization. We found that miR-4306 expression was significantly decreased in human nucleus pulposus (NP) tissues compared with healthy controls, and was negatively correlated with the patients' Pfirrmann grade. To determine the mechanism by which miR-4306 was involved in IDD pathogenesis, we examined the effects of overexpressing or silencing miR-4306 on extracellular matrix (ECM) synthesis/degradation, proliferation, autophagy and apoptosis of human degenerated NP cells isolated from IDD patients. Next, we used dual-luciferase reporter assays to demonstrate that miR-4306 interacted with the 3′-untranslated regions of p21-activated kinase 6 (PAK6) mRNA, resulting in significant suppression of PAK6 expression. This effect was abolished by miR-4306 binding site mutations. Using miR-4306/PAK6 gain-of-function and loss-of-function studies in human degenerated NP cells, we demonstrated that miR-4306 promoted NP cell proliferation, ECM synthesis and autophagy, while inhibiting apoptosis and ECM degradation via PAK6. Thus, our findings indicate that miR-4306, acting via PAK6, has an important role in IDD and can be used as a promising therapeutic target for the treatment of patients with IDD.

Vascular anatomy-based localization of intervertebral discs assisting needle puncture for constructing a mouse model of mechanical injury-induced lumbar intervertebral disc degeneration

Intervertebral disc degeneration (IDD) may be the primary cause of low back pain. Potential therapeutics for IDD must be validated in animal models, and their effectiveness quantified using functional metrics. Needle puncture of intervertebral discs (IVDs) has been used to induce IDD in mice and rats. Due to operational challenges, most animal IDD models are constructed using needle puncture of the caudal IVDs in mice, or by using larger animals, such as rats and rabbits. However, mouse IDD models involving lumbar IVD puncture are preferable because mice are genetically similar to humans and are the most commonly used transgenic animals, and because human IDD commonly affects the lumbar spine. We constructed a needle puncture-based mouse IDD model that relies on vascular anatomy to pinpoint lumbar IVDs. We evaluated the morphological and molecular changes in this model by using radiological, pathological, and immunostaining examinations. In our mechanical injury-induced IDD model, lumbar IVDs were accurately localized by injecting colored perfusates into the common iliac artery and vein, and right iliolumbar vein, which helped to visualize puncture positions, avoid neuromuscular injury, shorten the operation time, and decrease bleeding. Nucleus pulposus cells, defined by Krt19, and the disc height index gradually decreased after the surgery, and the degenerative effects peaked at 1 week. In conclusion, we established a mouse IDD model by performing precise puncture of lumbar IVDs via the ventral anterior approach assisted by vessel position. Our model effectively simulated the effects of IDD, and may serve as an efficient research tool.

MiR-328-5p Induces Human Intervertebral Disc Degeneration by Targeting WWP2.

Intervertebral disc degeneration (IDD) development is regulated by miRNA, including inflammatory reactions, cell apoptosis, and degradation of extracellular matrix. Nucleus pulposus cells apoptosis has a absolute influence in the development of IDD. This experiment explores the mechanism of miR-328-5p regulating IDD. Through the analysis of miRNA and mRNA microarray database, we screened the target genes miR-328-5p and WWP2. We verified the expression of miR-328-5p, WWP2, and related apoptotic genes in normal and degenerative nucleus pulposus tissues by qRT-PCR. The expressions of WWP2, Bcl-2, and Bax were detected by qRT-PCR and western blot after transfection to nucleus pulposus cell. The nucleus pulposus cell proliferation and apoptosis after transfection were confirmed by CCK8 and flow cytometry. Luciferase reporter assay and bioinformatics analyzed the targeting relationship between miR-328-5p and WWP2. Firstly, the qRT-PCR experiments confirmed the significant increase of miR-328-5p expression, while significant reduction of WWP2 in a degenerative tissues compared to the normal tissues. Surprisingly, miR-328-5p expression was positively, while that of WWP2 negatively correlated with the degeneration grade of IDD. And we also identified the high expression of Bax and Caspase3, while low expression of Bcl-2 in a degenerative tissues. After miR-328-5p mimic transfected into nucleus pulposus cell, qRT-PCR and western blot confirmed that WWP2 and Bcl-2 expressions were downregulated, while Bax and Caspase3 expressions were upregulated, and the same results were obtained by knocking down WWP2. CCK8 and flow cytometry confirmed that miR-328-5p inhibited the proliferation and induced apoptosis of nucleus pulposus cells. WWP2 is a target gene of miR-328-5p by bioinformatics and luciferase reporter assay. In summary, miR-328-5p targets WWP2 to regulate nucleus pulposus cells apoptosis and then participates in the development of IDD. Furthermore, this study may provide new references and ideas for IDD treatment.

Can transcription factors in the intervertebral disc of lopinavir/ritonavir prevent degeneration in the nucleus pulposus by mediating the regulation of inflammation through signaling pathways?

This study was conducted to examine whether lopinavir/ritonavir (Lop/r), an HIV protease inhibitor, can improve disc physiology and slow down intervertebral disc (IVD) degeneration through in vitro experimental methods, as well as whether it can suppress inflammation with interleukin-1 beta (IL-1β) and sex-determining region Y (SRY) protein-related high-mobility group box genes-9 (SOX9) through hypoxia-inducible factor 1-alpha (HIF-1α) and the nuclear factor kappa B (NF-κB) signaling pathway. The aim was to investigate whether Lop/r application is toxic to IVD cells and the microenvironment simultaneously. Human primary cell cultures were prepared using herniated IVD tissues obtained from patients with lumbar disc hernia who were unresponsive toconservative and medical treatment, and thereby, were operated on. The untreated culture samples served as control group, and the samples treated with Lop/r served as study group. Microscopic evaluations were performed simultaneously using fluorescent and supravital dyes in all groups. In addition to cell viability, toxicity, and proliferation analysis through a commercial kit, IL-1β, SOX9, HIF-1α, and NF-κB protein expressions were evaluated using Western blotting. In the statistical comparison of the obtained data, an alpha value less than 0.05 was considered significant. Cell proliferation decreased in the Lop/r group, but no cell death was observed (p < 0.05). Moreover, at the end of 72 hours after Lop/r application, IL-1β and NF-kB protein expressions decreased by 40% and 52%, respectively, while HIF-1α and SOX9 protein expressions increased by 4% and 59%, respectively (p< 0.05). Although these data were obtained from an in vitro experimental study, it is believed that these findings could make significant contributions to the pharmaco-regenerative treatment modalities of IVD degeneration. Lop/r suppresses the IL-1β and NF-κB and induces SOX9 and HIF-1α, since these signaling pathways may be related to human IVD degeneration.