Degenerated Disc Tissue Research Articles

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Astragaloside Ⅳ in treating intervertebral disc degeneration

This study aims to identify potential targets and regulatory mechanisms of Astragaloside Ⅳ (AS-Ⅳ) in treating intervertebral disc degeneration (IDD) through network pharmacology analysis with experimental validation. Lumbar spine instability (LSI) mouse models were first established and treated with AS-Ⅳ. Micro-CT, safranin O-fast green staining, IDD score, RT-PCR and immunohistochemistry staining were employed to demonstrate the effect of AS-Ⅳ. Network pharmacology was used to predict the signaling pathways and potential targets of AS-Ⅳ in treating IDD. RT-PCR and immunohistochemistry staining were used to elucidate and validate the mechanism of AS-Ⅳ in vivo. Animal experiments showed that AS-Ⅳ maintained disc height and volume, improved matrix metabolism in LSI mice, and restored Col2α1, ADAMTS-5, Aggrecan, and MMP-13 expression in degenerated discs. Network pharmacology analysis identified 32 cross-targets between AS-Ⅳ and IDD, and PPI network analysis filtered out 11 core genes, including ALB, MAPK1, MAPK14 (p38 MAPK), EGFR, TGFBR1, MAPK8, MMP3, ANXA5, ESR1, CASP3, and IGF1. Enrichment analysis revealed that 7 of the 11 core target genes enriched in the MAPK signaling pathway, and AS-Ⅳ exhibited stable binding to them according to molecular docking results. Experimental validation indicated that AS-Ⅳ reversed mRNA levels of 7 core targets in degenerated disc tissues in LSI mice. Immunohistochemistry staining further revealed that AS-Ⅳ treatment mainly depressed IDD-elevated protein levels of EGFR, p38 MAPK and CASP3 in the annulus fibrosus. This study elucidates that AS-Ⅳ alleviates lumbar spine instability-induced IDD in mice, suggesting the mechanism may involve inhibition of the EGFR/MAPK signaling pathway.

Oxidative Stress, inflammation and metallomatrix Proteinase-Regulated hydrogels for intervertebral disc regeneration

Intervertebral disc degeneration (IVDD) is still a challenge and the efficient treatment approaches are still lacking, due to the high oxidative stress and inflammation and matrix degradation. The utilization of bioactive hydrogels as carriers for therapeutic factors offers a powerful means to enhance IVDD treatment. However, the therapeutic efficacy of existing hydrogels heavily relies on the loaded therapeutic drugs, resulting in reduced treatment efficiency. Herein, we report a dual-component polymer network hydrogel (FPFG) based on gelatin methacrylate (GelMA) and poly (ferulic acid)-F127- poly (ferulic acid) (FPF). The FPFG hydrogel exhibited injectability, antioxidative properties, good biocompatibility, and biodegradability. FPFG hydrogel could efficiently suppress the intracellular reactive oxygen species (ROS) and metallomatrix proteinase in nucleus pulposus (NP) cells, inhibit the expression of inflammatory factors in macrophages and enhance the extracellular matrix formation. The rat puncture model revealed that the FPFG hydrogel effectively restored disc height, maintained disc hydration, and preserved tissue structure. By exerting anti-inflammatory and antioxidant activities, the FPFG hydrogel successfully ameliorated the hostile microenvironment commonly found in degenerated disc tissue. These findings highlight the potential application of FPFG hydrogel in enhancing IVDD treatment.

Mesenchymal stem cell exosomes inhibit nucleus pulposus cell apoptosis via the miR-125b-5p/TRAF6/NF-κB pathway axis.

Intervertebral disc degeneration (IVDD) is the pathological basis of a range of degenerative spinal diseases and is the primary cause of lower back pain. Mesenchymal stem cell (MSC) transplantation inhibits IVDD progression. However, the specific mechanisms that underlie these effects remain unclear. In this study, candidate microRNAs (miRNAs) are screened using bioinformatics and high-throughput sequencing. TNF-α is used to induce nucleus pulposus cell (NPC) degeneration. MSC-derived exosomes (MSC-exosomes) are obtained using high-speed centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis. Cell viability is determined by CCK-8 assay. Flow cytometry and TUNEL assays are used to detect cell apoptosis. The expression levels of miR-125b-5p are detected by RT-qPCR, and a dual-luciferase gene reporter assay confirms the downstream target genes of miR-125b-5p. Protein expression is determined by western blot analysis. Rat models are used to validate the function of miR-125b-5p in MSC-exosomes. The results show that miR-125b-5p is expressed at low levels in degenerated disc tissues compared with that in normal disc tissues; however, it is highly expressed in MSC-exosomes. Furthermore, MSC-exosomes are efficiently taken up by NPCs while miR-125b-5p is delivered into NPCs; thus, MSC-exosomes act as inhibitors of apoptosis in NPCs. Overexpression of miR-125b-5p downregulates TRAF6 expression and inhibits NF-κB activation. However, TRAF6 overexpression reverses these effects of miR-125b-5p. We demonstrate that MSC-exosomes attenuate IVDD in vivo by delivering miR-125b-5p. MSC-exosomes can deliver miR-125b-5p to target TRAF6, inhibit NF-κB activation, and attenuate the progression of IVDD.

Pro‑differentiating compounds for human intervertebral disc cells are present in Violina pumpkin leaf extracts.

Intervertebral disc (IVD) degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype, which current therapies are unable to reverse. In the present study, the effects of acetone extract from Violina pumpkin (Cucurbitamoschata) leaves on degenerated IVD cells were investigated. IVD cells were isolated from the degenerated disc tissue of patients undergoing spinal surgery and were exposed to acetone extract and three major thin layer chromatography subfractions. The results revealed that, in particular, the cells benefited from exposure to subfraction Fr7, which consisted almost entirely of p‑Coumaric acid. Western blot and immunocytochemical analysis showed that Fr7 induced a significant increase in discogenic transcription factors (SOX9 and tricho‑rhino‑phalangeal syndrome typeI protein, zinc finger protein), extracellular matrix components (aggrecan, collagen typeII), cellular homeostasis and stress response regulators, such as FOXO3a, nuclear factor erythroid2‑related factor 2, superoxide dismutase 2 and sirtuin 1. Two important markers related to the presence and activity of stem cells, migratory capacity and OCT4 expression, were assessed by scratch assay and western blotting, respectively, and were significantly increased in Fr7‑treated cells. Moreover, Fr7 counteracted H2O2‑triggered cell damage, preventing increases in the pro‑inflammatory and anti‑chondrogenic microRNA (miR), miR‑221. These findings strengthen the hypothesis that adequate stimuli can support resident cells to repopulate the degenerated IVD and restart the anabolic machinery. Taken together, these data contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the use of part of a plant, the pumpkin leaves, which is usually considered a waste product in the Western world, indicated that it contains substances with potential beneficial effects on human health.

Role of the mechanosensitive piezo1 channel in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a multifactorial skeletal disease involving mechanical, genetic, systemic, and biological factors, and it is characterized by apoptosis of the nucleus pulposus cells and breakdown of the extracellular matrix (ECM), which will impair the structure and function of the intervertebral disc (IVD), and cause low back pain. Recently, the piezo1 is recognized as a critical mechanically activated ion channel of IDD. Numerous studies have reported that the piezo1 ion channel was aberrantly activated in the degenerated disc tissues and deeply participated in the pathogenesis of IDD. Inactivating or interfering with the piezo1 channel could effectively prevent the progression of IDD under the experimental conditions. It may be a promising target for the prevention and treatment of the disabling disease. Therefore, we have to make a comprehensive investigation and understanding of the mechanisms and functions of the piezo1 in the biomechanics of the spine. This study mainly elucidates the role of the piezo1 channel in IDD, which may facilitate the development of therapeutic targets for this disease.

A Single Injection of NTG-101 Reduces the Expression of Pain-Related Neurotrophins in a Canine Model of Degenerative Disc Disease

Background: Tissue sources of pain emanating from degenerative discs remains incompletely understood. Canine intervertebral discs (IVDs) were needle puncture injured, 4-weeks later injected with either phosphate-buffered saline (PBS) or NTG-101, harvested after an additional fourteen weeks and then histologically evaluated for the expression of NGFr, BDNF, TrkB and CALCRL proteins. Quantification was performed using the HALO automated cell-counting scoring platform. Immunohistochemical analysis was also performed on human IVD tissue samples obtained from spinal surgery. Immunohistochemical analysis and quantification of neurotrophins and neuropeptides was performed using an in vivo canine model of degenerative disc disease and human degenerative disc tissue sections. Discs injected with NTG-101 showed significantly lower levels of Nerve Growth Factor receptor (NGFr/TrkA, p = 0.0001), BDNF (p = 0.009), TrkB (p = 0.002) and CALCRL (p = 0.008) relative to PBS injections. Human IVD tissue obtained from spinal surgery due to painful DDD show robust expression of NGFr, BDNF, TrkB and CALCRL proteins. A single intradiscal injection of NTG-101 significantly inhibits the expression of NGFr, BDNF, TrkB and CALCRL proteins in degenerative canine IVDs. These results strongly suggest that NTG-101 inhibits the development of neurotrophins that are strongly associated with painful degenerative disc disease and may have profound effects upon the management of patients living with discogenic pain.

Inhibitory activity of CXCR4 attenuates intervertebral disc degeneration by regulating TXNIP/NLRP3 expression

Purpose: To explore the inhibitory effect and mechanism of action of CXCR4 on intervertebral disc degeneration.Methods: The expression of CXCR4 was assessed in different degrees of degenerate disc tissues. Nucleus pulposus (NP) cells-induced degeneration were obtained using interleukin 1 beta (IL-1β) stimulation. CXCR4 inhibitor, AMD 3100, was employed to resist the function of CXCR4, in order to determine whether CXCR4 inhibition acted by targeting TXNIP/NLRP3 axis.Results: Expression of CXCR4 increased in severely degenerated disc tissues. IL-1β activated and promoted the progression of intervertebral disc degeneration, but AMD3100 treatment reversed the effects of IL-1β. Moreover, TXNIP and NLRP3 were significantly suppressed by AMD3100 stimuli.Conclusion: The results demonstrate that inhibition of CXCR4 prevents NP cells from degradation by regulating TXNIP/NLRP3 signaling pathway, thus indicating that this approach could be effective in the management of intervertebral disc degeneration.

IL-17 in intervertebral disc degeneration: Mechanistic insights and therapeutic implications.

Intervertebral disc degeneration (IDD) serves as an independent risk factor for lower back pain and is closely associated with spinal musculoskeletal disorders, including lumbar disc herniation, radiculopathy, and myelopathy. Interleukin-17 (IL-17), also named IL-17A, is a critical signature cytokine of T-helper 17 cells. Upon binding to the IL-17 receptor A/C heterodimeric complex, IL-17 can trigger multiple signal transduction pathways to stimulate gene transcription and increase messenger RNA stability. IL-17 expression is significantly increased in degenerative disc tissue and shows a positive correlation with disease severity. IL-17 has been shown to accelerate the development of IDD by promoting extracellular matrix degradation, enhancing inflammatory response, inducing neoangiogenesis, and inhibiting nucleus pulposus cell autophagy and proliferation. Targeting IL-17 represents a novel and promising approach for the therapeutic intervention of IDD. In this review, we summarized the recent progression about the role of IL-17 in IDD and highlighted its therapeutic implications.

Tissue Renin-Angiotensin System (tRAS) Induce Intervertebral Disc Degeneration by Activating Oxidative Stress and Inflammatory Reaction.

Lumbar intervertebral disc degeneration (IDD) has been the major contributor to low back pain (LBP). IDD is an chronic inflammation process, with the activation of plentiful inflammation-related cytokines and ECM degradation-related enzymes. In the past few years, hypertension has been reported to correlate with LBP. In addition, the local tissue renin-angiotensin system (tRAS) has been identified in multiple tissues, including the spinal cord, skin, kidney, heart, and bone. Recently, tRAS has also been established in both bovine and human intervertebral disc tissues, especially in the degenerated disc tissue. However, the exact of tRAS and IDD remains unknown. In this present study, proteomic analysis, molecular biology analysis, and animal model were all used. Firstly, we revealed that tRAS was excessively activated in the human degenerated intervertebral disc tissue via proteomic analysis and molecular biology analysis. Then, in vitro experiment suggested that Ang II could decrease the cell viability of human NP cells and promote NP cell apoptosis, senescence, oxidative stress, and NLRP3 activation in human NP cells. In addition, Ang II could also trigger degeneration and fibrosis phenotype in human NP cells. Finally, the animal model demonstrated that the local activated ACE/Ang II axis in the NP tissue could accelerate IDD in aging spontaneously hypertensive rats (SHR). Collectively, the degenerated intervertebral disc tissue showed excessively activated tRAS, and local activated tRAS could induce NP cell senescence, apoptosis, oxidative stress, and inflammatory reaction to promote IDD. These biological effects of Ang II on human NP cells may provide novel insight into further treatment of IDD.

TNFAIP3 ameliorates the degeneration of inflammatory human nucleus pulposus cells by inhibiting mTOR signaling and promoting autophagy.

Autophagy is involved in degenerative diseases such as osteoarthritis and disc degeneration. Although, tumor necrosis factor α-induced protein 3 (TNFAIP3) is well-known as a key regulator of inflammation and autophagy, it is still not clear whether TNFAIP3 regulates autophagy to protect from human disc cells degeneration. We hypothesize that TNFAIP3 may also regulate autophagy to inhibit pro-inflammatory cytokines expression in human nucleus pulposus cells (NPCs). In this study, TNFAIP3 expression was increased in degenerative disc tissue as well as LPS-stimulated human NPCs, and the effect of TNFAIP3 in LPS-induced NPCs was further explored. The results demonstrated that pro-inflammatory cytokines expression in TNFAIP3-His cells was decreased, while it was increased in TNFAIP3-siRNA cells. Further molecular mechanism research showed that TNFAIP3-siRNA cells enhanced the phosphorylation of mammalian target of rapamycin (mTOR) and inhibited autophagy. Meanwhile, after treatment of TNFAIP3-siRNA cells with the mTOR inhibitor Torin1, the level of autophagy increased and the decrease of extracellular matrix was reversed. In summary, overexpressed TNFAIP3 can promote autophagy and reduce inflammation in LPS-induced human NPCs. Moreover, autophagy triggered by TNFAIP3 can ameliorate the degeneration of inflammatory human NPCs, providing a potential and an attractive therapeutic strategy for degenerative disease.

SF1670 inhibits apoptosis and inflammation via the PTEN/Akt pathway and thus protects intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is associated with the apoptosis of nucleus pulposus (NP) cells. Previous studies have shown that PTEN plays crucial roles in cell survival and apoptosis. The effect of PTEN inhibitors on cell survival following IVDD has been rarely examined. In this study, we investigated the protective effect of SF1670, as a specific PTEN inhibitor, on an in vitro NP cells degenerated model. We collected human disc samples from IVDD patients and detected PTEN expression in them with different degenerated degrees. NP cells were isolated from the samples and exposed to IL-1β with or without SF1670. Then, cells viability was determined by CCK-8 assay. We also measured the levels of collagen II, p16, p53, PTEN, Akt, aggrecan, caspase 3/9, Bax, Bcl-2, and several inflammatory factors in NP cells. We found that the expression of PTEN markedly increased in severely degenerated disc tissues. The data showed that IL-1β upregulated the expressions of p16, p53, PTEN, caspase 3/9, and Bax, but decreased the expressions of collagen II, Akt, aggrecan, and Bcl-2. Surprisingly, the treatment with SF1670 could significantly reverse the regulatory effects of IL-1β. Moreover, relative levels of IL-6, IL-8, TNF-α, and MMP3/9/13 were significantly suppressed by SF1670 stimuli compared with IL-1β group. Overall, these results demonstrated that SF1670 prevented NP degradation via suppressing apoptosis and inflammation through inhibition of PTEN and activation of Akt. SF1670 may become a novel target for the therapy of IVDD in the future.

IL-38 alleviates the inflammatory response and the degeneration of nucleus pulposus cells via inhibition of the NF-κB signaling pathway in vitro

Previous studies have suggested that the inflammatory response contributes to the onset of intervertebral disc degeneration (IVDD). Interleukin (IL)-38, a newly discovered cytokine of the IL-1 family, has been demonstrated to play an anti-inflammatory role in autoimmune diseases, such as Crohn’s disease, rheumatoid arthritis and psoriasis. However, whether IL-38 participates in the pathogenesis of IVDD remains unknown. In this study, human disc tissues from IVDD patients and rat disc tissues from an IVDD model were collected to measure the expression of IL-38 in the IVDD groups and the control groups by western blot and immunohistochemical staining. To further determine the role of IL-38 in IVDD, human nucleus pulposus cells (HNPCs) were stimulated with TNF-α to generate an in vitro model of inflammation to mimic the local inflammatory environment of the lumbar disc. The inflammatory response and HNPC degeneration markers were measured after stimulation with TNF-α and IL-38. IL-38 was upregulated in both the human and rat degenerated disc tissues compared with the control tissues. In vitro, IL-38 significantly decreased the TNF-α-induced expression of IL-1β, IL-6, COX-2, MMP-13 and ADAMTS-5 in the HNPCs, and IL-38 also alleviated the TNF-α-induced reductions in type II collagen and aggrecan. Moreover, IL-38 inhibited the activation of the NF-κB signaling pathway in the HNPC-based model of inflammation by reducing the expression level of the NF-κB P-P65 protein. In conclusion, IL-38 could alleviate the inflammatory response and HNPC degeneration in vitro via the inhibition of the NF-κB signaling pathway. These results suggest that IL-38 may be a new strategy for the treatment of IVDD.

Circulating miR-155-5p as a Novel Biomarker of Lumbar Degenerative Disc Disease.

Case-control study measuring differential gene expression of circulating microRNA (miRNA) in patients with degenerative disc disease (DDD). To identify miRNA dysregulation in serum samples of patients with DDD compared to healthy controls (HC). Early DDD can be a difficult diagnosis to make clinically, with lack of positive and specific findings on physical exam or advanced imaging. miRNAs are a class of molecules that act as gene regulators and have been shown to be dysregulated in local degenerative disc tissue. However, to date no studies have identified dysregulation of serum miRNA in patients with DDD. Whole blood samples were obtained from 69 patients with DDD and 16 HC. Patient-reported outcomes were collected preoperatively and degree of DDD was classified using Pfirrmann grade on preoperative imaging. Differential gene expression analysis using a screening assay for several hundred miRNAs and further characterization for five specific miRNAs (miR-16-5p, miR-21-5p, miR-142-3p, miR-146a-5p, and miR-155-5p) was performed. In addition, a pro-inflammatory cytokine multiplex assay and bioinformatics analysis were done. The initial screening assay showed 13 miRNA molecules that were significantly dysregulated in DDD patients, with miR-155-5p showing significant downregulation (p = 0.027) and direct interactions with the pro-inflammatory cytokine IL-1β, and the tumor suppressor genes p53 and BRAF. Analyzing the whole cohort, miR-155 showed an almost four-fold downregulation in DDD patients (-3.94-fold, P < 0.001) and was the sole miRNA that accurately predicted the presence of disc degeneration (P = 0.006). Downregulation of miR-155 also correlated with increased leg pain (P = 0.018), DDD (P = 0.006), and higher Pfirrmann grade (P = 0.039). On cytokine analysis, TNF-α (0.025) and IL-6 (P < 0.001) were significantly higher in DDD patients. Serum miR-155-5p is significantly downregulated in patients with DDD and may be a diagnostic marker for degenerative spinal disease. N/A.

Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades.

Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor β1 (TGFβ1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFβ1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.

The effects of age, pathology, and fusion on cervical neural foramen area.

Cervical radiculopathy is a relatively common neurological disorder, often resulting from mechanical compression of the nerve root within the neural foramen. Anterior cervical discectomy and fusion (ACDF) is a common treatment for radicular symptoms that do not resolve after conservative treatment. One mechanism by which ACDF is believed to resolve symptoms is by replacing degenerated disc tissue with bone graft to increase the neural foramen area, however in vivo evidence demonstrating this is lacking. The aim of this study was to evaluate the effects of age, pathology, and fusion on bony neural foramen area. Participants included 30 young adult controls (<35 years old), 23 middle-aged controls (36 to 60 years old), and 36 cervical arthrodesis patients tested before and after ACDF surgery. Participants' cervical spines were imaged in the neutral, full flexion, and full extension positions while seated within a biplane radiography system. A validated model-based tracking technique determined three-dimensional vertebral position and orientation and automated software identified the neural foramen area in each head position. The neural foramen area decreased throughout the entire sub-axial cervical spine with age and pathology, however, no changes in neural foramen area were observed due solely to replacing degenerated disc tissue with bone graft. The neural foramen area was not associated with disc height in young adult controls, but moderate to strong associations were observed in middle-aged controls. The results provide evidence to inform the debate regarding localized versus systemic spinal degeneration and provide novel insight into the mechanism of pain relief after ACDF.

Necrosulfonamide (NSA) protects intervertebral disc degeneration via necroptosis and apoptosis inhibition.

Previous studies have shown that nucleus pulposus (NP) cell death plays an extremely important role in the progress of intervertebral disc degeneration (IVDD). This research aimed to investigate the protective effect of the MLKL inhibitor necrosulfonamide (NSA) on human NP cells. We collected human NP tissues from the patients undergoing disc herniation operations and isolated NP cell from the samples. IL-1β (10 ng/ml) was used to establish a NP cells degenerated model. We analyzed the expression of caspase 3, caspase 8, RIPK1, RIPK 3, and MLKL in different degree of degenerate disc tissues. Cell viability was analyzed by the Cell Counting Kit-8 (CCK-8) assay. The expression levels of collagen Ⅱ, β-galactosidase (β-gal), caspase 3, caspase 8, RIPK1, RIPK 3, and MLKL, several inflammatory and anti-oxidant enzymes of different NP cell treat groups were detected by Western blotting, immunofluorescence staining, or RT-PCR. Flow cytometry was used to measure the ROS level and cell apoptosis. The data showed that expression of caspase 3, caspase 8, RIPK1, RIPK 3, and MLKL markedly increased in severely degenerated disc tissues. IL-1β promoted the cell death of NP cells, while NSA could reverse the effects of IL-1β. We found that NAS increased the antioxidant SOD1, SOD2, CAT, and GPX3 expression and suppressed oxidative stress in the disc. Moreover, MMP3, MMP10, IL-6, and TNF-α were significantly suppressed by the NSA. These results suggest that NSA prevented NP degradation via inhibiting apoptosis and necroptosis of NP cells. Besides, the protective function of antagonizing cell death may owe to the inflammation and oxidative stress suppression.

Evaluation of the expression and proliferation of degenerative markers in primary cell cultures obtained from human intervertebral disc tissue

Aim: A major cause of low back pain is disc degeneration. Nevertheless, no specific and reliable markers of the degeneration of the nucleus pulposus (NP) are available. This presented study aimed to examine changes in the expressions of genes in primary cell cultures isolated from intact and degenerated tissues to give insights into the biopathogenesis of intervertebral disc (IVD) tissue.Material and Methods: Tissues of eight patients (n = 8; average age: 41.74 ± 9.86 years) were resected through microdiscectomy, and primary cell cultures were prepared using degenerated disc tissue. The cultured degenerated tissues served as the study group. The samples in the control group comprised the intact tissues of patients (n = 8; average age: 38.68 ± 7.91 years) resected following a trauma. Morphology of the cell surface were evaluated using an inverted light/fluorescent microscopy at 0 and 24 h on days 10 and 21. The expressions of the chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1 beta (IL-1 beta), and matrix metalloproteinase (MMP)-7 and MMP-19 genes were evaluated using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The data obtained were statistically analyzed.Results: The four genes investigated, except COMP (P > 0.05), changed significantly in primary cell cultures isolated from degenerative IVD tissues. This result was statistically significant (P 0.05). The gene expressions in the samples derived from intact IVD tissues changed markedly and these changes were associated with proliferation (P 0.05).Conclusion: Analyzing the changes in gene expression levels associated with IVD should contribute to future studies on the prevention and treatment of such pathologies. The data obtained from the present study will shed light on cellular-based personal targeted therapies through which genetic information can be transmitted to cells.

FBW7 inhibits nucleus pulposus cells proliferation by downregulation of cyclin E in the intervertebral disc degeneration.

TF-box and WD repeat domain-containing 7 (FBW7), a component of SCF ubiquitin ligase complex, usually acts as a tumor suppressor because it has an ability in the inhibition of cell proliferation. Nevertheless, the role of FBW7 in intervertebral disc degeneration (IDD) is not quite understood. The total protein and RNA were isolated from patients' disc tissues. WB was carried out to analyze the collagen II and FBW7 protein levels of different Pfirrmann grades disc degeneration. Reverse transcription-polymerase chain reaction (RT-PCR) was used to test the collagen II and FBW7 mRNA expression in these disc samples. NP cells were transfected with siRNA-FWB7 to downregulate the FBW7 expression. SiRNA-NC was used as the sham group. Cyclin E, E2F1, and E2F2 were analyzed with WB and RT-PCR. In this study, different kinds of degenerated disc tissues were analyzed, and it was found that FBW7 was overexpressed in much severe degeneration condition, which was also proved by the IL-1β stimuli nucleus pulposus (NP) cells degeneration model in vitro. Interestingly, the results showed that FBW7 suppression could reverse the degeneration of NP cells. Furthermore, we found that FBW7 induced NP cell cycle arrest in the G1 phase of and inhibited cell proliferation by upregulating p27 expression in vitro. The overexpression of p27 resulted in the inhibition of cyclin E, which promotes cell proliferation. Taken together, our study uncovered that FBW7 played an essential inhibitory role in NP cells proliferation, providing new insights that FBW7 may be a potential strategy for IDD treatments.

Coblation annuloplasty in cervical discogenic pain without radiculopathy.

IntroductionThe nerve fibers innervating the annulus fibrosus are the major origin of degeneration-associated discogenic pain. Coblation is a tissue-dissociating technique in which the nerve fibers in the degenerative disc tissue are ablated. We hypothesized that coblation annuloplasty would be an effective maneuver for cervical discogenic pain without radiculopathy.AimTo observe the therapeutic efficacy of coblation annuloplasty in patients with cervical discogenic pain without radiculopathy.Material and methodsForty patients diagnosed with cervical discogenic pain without radiculopathy were screened for coblation annuloplasty therapy. The patient-rated visual analog scale (VAS) score for pain, significant pain relief rate, and Modified MacNab pain-relieving effect were adopted to evaluate the therapeutic effect within a 1-year follow-up period.ResultsThirty-three patients eventually completed the study. The average pain duration was 4.6 ±1.6 years (range: 0.5–8 years). The mean VAS pain score decreased from preoperative 6.8 ±0.9 to postoperative 2.5 ±1.3 (p < 0.01). For all participants, the immediate pain relief rate was 78.7% (26/33), which continued to postoperative 6 months. One year later, 22 (66.6%) subjects reported that their pain was significantly alleviated. According to the Modified MacNab criteria, 63.6–82.1% considered the effect of surgery for their pain therapy as “excellent” during the 1-year follow-up period. No significant complications such as hemorrhage, paresthesia, or infection were observed.ConclusionsThis study is the first to demonstrate that coblation annuloplasty is an effective intervention providing significant alleviation of neck pain from cervical discogenic injury without radiculopathy.

139. Serum MicroRNA miR-155-5p as a potential biomarker of degenerative disc disease in patients with low back pain

BACKGROUND CONTEXT MicroRNAs (miRNAs) are a class of stable, noncoding RNA molecules that are dysregulated in various disease states and are readily detected in the serum, making them useful biomarkers. Prior studies have found down regulation of local levels of miR-155-5p in degenerative disc tissue, directly increasing Fas-mediated apoptosis of nucleus pulposus cells. However, no studies are currently assessing whether these changes are detectable in the serum. PURPOSE To determine the serum concentration of miR-155-5p in patients with degenerative disc disease (DDD) undergoing lumbar fusion and compare with young, healthy controls. STUDY DESIGN/SETTING Prospective cohort study. PATIENT SAMPLE Sixty-three patients undergoing a single-level lumbar fusion with a Pfirrmann grade of 3, 4 or 5 on MRI T2 sequence were prospectively enrolled at a high-volume academic center. Patients with a history of previous surgery, active infection or systemic chronic illnesses such as diabetes, autoimmune diseases, or malignancy were excluded. Samples from 15 healthy controls were obtained for comparison. OUTCOME MEASURES Fold change of miR-155-5p expression level in patients with DDD compared to healthy controls. Preoperative ODI, SF-12 PCS and MCS, VAS Back and VAS Leg scores. METHODS After informed consent was obtained in the preoperative area, a venous whole blood sample was collected and transported to the laboratory for isolation of serum and storage. miRNA was extracted from the serum using a commercially available kit. Real-time RT-PCR analysis was completed for confirmation of gene expression using TaqMan™ primers for human miR-155-5p. Bioinformatics analysis was completed using Ingenuity Pathway Analysis (Qiagen, Germany). RESULTS Average age for patients in the DDD group was 57.5 ± 13.6 compared to 29.5 ± 10.2 in the control group. In the DDD group, 11.1% of patients were Pfirrmann grade 3, 54.0% were grade 4, 34.9% were grade 5. Average preoperative patient-reported outcomes were: ODI 41.9 ± 18.4, SF-12 PCS 32.2 ± 8.6, SF-12 MCS 46.4 ± 11.3, VAS Back 6.43 ± 2.94, and VAS Leg 6.13 ± 3.05. Serum levels of miR-155-5p were significantly decreased in the DDD group (Fold change −8.523 [-11.585, −5.461]) compared to the healthy control group. Bioinformatics analysis showed involvement of miR-155-5p in mostly cellular development and cellular growth and proliferation pathways. It also showed significant interactions with cell cycle and apoptosis pathways (P38/MAPk, Akt) as well as with pro-inflammatory cytokines. CONCLUSIONS Local levels of miR-155-5p have been shown to be down regulated in DDD by increasing Fas-mediated apoptosis in nucleus pulposus cells. However, this is the first study to detect down regulation of miR-155-5p in the serum of patients with DDD, correlating with local tissue levels and potentially providing a useful biomarker for detecting early degenerative change. Further studies are needed to identify other miRNAs involved in the regulation of intervertebral disc degeneration to create a robust gene profile. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.