Ji Tu

Inhibition of microRNA-34a prevents IL-1β-induced extracellular matrix degradation in nucleus pulposus by increasing GDF5 expression

Accumulating evidence indicates that miRNAs, a class of small non-coding RNAs, are implicated in the pathogenesis of various diseases such as cancer and intervertebral disc degeneration. The aim of this study was to investigate the expression and the biological function of microRNA-34a in intervertebral disc degeneration. In this study, microRNA-34a expression was assessed in nucleus pulposus specimens and in IL-1β-stimulated nucleus pulposus cells by real-time polymerase chain reaction. microRNA-34a functions were investigated by using gain and loss of function experiments in nucleus pulposus cells and a dual luciferase reporter assay in 293T cells. microRNA-34a was dramatically up-regulated in degenerative nucleus pulposus tissues and in IL-1β-stimulated nucleus pulposus cells when compared with controls. Furthermore, growth differentiation factor 5 was identified as a target of microRNA-34a. Aberrant expression of microRNA-34a inhibited growth differentiation factor 5 expression by direct binding to its 3′-untranslated region. This inhibition was abolished by mutation of the microRNA-34a binding sites. In addition, microRNA-34a silencing reversed IL-1β-induced decrease in type II collagen and aggrecan expression in nucleus pulposus cells. This effect was substantially suppressed by growth differentiation factor 5 silencing. Our results suggested that microRNA-34a inhibition prevents IL-1β-induced extracellular matrix degradation in human nucleus pulposus by increasing growth differentiation factor 5 expression. microRNA-34a inhibition may be a novel molecular target for intervertebral disc degeneration treatment through the prevention of nucleus pulposus extracellular matrix degradation.

MicroRNA-494 promotes apoptosis and extracellular matrix degradation in degenerative human nucleus pulposus cells

Purpose This study investigated the expression and function of the microRNA-494 in intervertebral disc degeneration (IDD). Results MicroRNA-494 expression was upregulated during IDD progression; its overexpression increased the expression of ECM catabolic factors such as matrix metalloproteinase and A disintegrin and metalloproteinase with thrombospondin motif in NP cells while decreasing that of anabolic genes such as type II collagen and aggrecan; it also induced the apoptosis of NP cells, as determined by flow cytometry. These effects were reversed by microRNA-494 inhibitor treatment. SOX9 was identified as a target of negative regulation by microRNA-494. Promoter hypomethylation and NF-κB activation were associated with microRNA-494 upregulation in IDD. Materials and Methods MicroRNA-494 expression in degenerative nucleus pulposus (NP) tissue was assessed by quantitative real-time PCR. The effect of microRNA-494 on extracellular matrix (ECM) metabolism and NP cell apoptosis was evaluated by transfection of microRNA-494 mimic or inhibitor. The regulation of SRY-related high mobility group box (SOX)9 expression by microRNA-494 was assessed with the luciferase reporter assay, and the methylation status of the microRNA-494 promoter was evaluated by methylation-specific PCR and bisulfite sequencing PCR. The role of activated nuclear factor (NF)-κB in the regulation of microRNA-494 expression was evaluated using specific inhibitors. Conclusions MicroRNA-494 promotes ECM degradation and apoptosis of degenerative human NP cells by directly targeting SOX9.

Simvastatin Inhibits IL-1β-Induced Apoptosis and Extracellular Matrix Degradation by Suppressing the NF-kB and MAPK Pathways in Nucleus Pulposus Cells

Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway. Some studies have shown that statins may have the potential to inhibit intervertebral disk (IVD) degeneration (IDD). Interleukin (IL)-1β, a catabolic cytokine, is a key regulator of IDD. This study aimed to investigate the mechanism underlying the effect of simvastatin on IDD. The viability of nucleus pulposus (NP) cells was determined by the methyl-thiazolyl-tetrazolium (MTT) assay. The apoptosis of NP cells was measured by flow cytometric analysis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blotting of relevant apoptotic proteins. The protein levels of catabolic factors and anabolic factors were determined by western blotting. The cells were stimulated with IL-1β in the absence or presence of simvastatin to investigate the effects on matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, type II collagen, and aggrecan expression. Our findings indicate that simvastatin considerably inhibited IL-1β-induced apoptosis in NP cells. We also found that simvastatin attenuated IL-1β-induced expression and MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 activities and also reduced the decrease in type II collagen and aggrecan expression. In addition, simvastatin considerably suppressed the nuclear translocation and activation of nuclear factor-kappa B (NF-KB) by inhibiting p65 phosphorylation and translocation and blocking inhibitor kB-α degradation. It also inhibited MAPK pathway activation by blocking c-Jun N-terminal kinase (JNK), p38, and ERK phosphorylation. The results of our study revealed that simvastatin is a potential agent for IDD prevention and treatment.

Icariin Attenuates Interleukin-1β-Induced Inflammatory Response in Human Nucleus Pulposus Cells

BACKGROUND Low back pain is a common problem, mainly caused by intervertebral disc degeneration (IDD). An important pathophysiological characteristic of IDD is the loss of homeostatic balance of the extracellular matrix metabolism. Interleukin-1β (IL-1β) is one of the inflammatory mediators stimulating the degradation of extracellular matrix in the nucleus pulposus (NP) and contributing to IDD pathogenesis. Icariin, which is isolated from Epimedium brevicornum, acts as an anti-inflammatory drug. OBJECTIVE This study aimed to explore the pharmacological effects of icariin in IDD by simulating NP inflammation in vitro. METHOD Human NP cells were isolated and cultured in vitro. NP cells were pretreated with icariin (0.1, 1 and 10 µM) and stimulated by IL-1β (10 ng/ml). The concentration of Prostaglandin E2 was determined by enzymelinked immunosorbent assay. Nitric oxide was determined by Griess reagent assay. The expression of cyclooxygenase- 2 (COX-2), inducible nitric oxide synthase (iNOS), degrading enzymes, collagen II, aggrecan, mitogenactivated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB)-related signaling molecules was assessed via western blotting. RESULTS IL-1β induced pronounced expression of COX-2 and iNOS, and stimulated production of prostaglandin E2 and nitric oxide. Icariin exhibited significant anti-inflammatory effect, inhibiting IL-1β-induced production of degrading enzymes, as well as extracellular matrix reduction. Finally, icariin suppressed IL-1β-induced activation of MAPK- and NF-κB-related signaling pathways. CONCLUSION The present findings suggest that icariin may have a protective effect on NP cells. The antiinflammatory effect may contribute to the therapeutic action of icariin in IDD.

Dysregulated miR-127-5p contributes to type II collagen degradation by targeting matrix metalloproteinase-13 in human intervertebral disc degeneration.

BACKGROUND Intervertebral disc degeneration (IDD) is a chronic disease associated with the degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMP)-13 is a major enzyme that mediates the degradation of ECM components. MMP-13 has been predicted to be a potential target of miR-127-5p. However, the exact function of miR-127-5p in IDD is still unclear. OBJECTIVE We designed this study to evaluate the correlation between miR-127-5p level and the degeneration of human intervertebral discs and explore the potential mechanisms. METHODS miR-127-5p levels and MMP-13 mRNA levels were detected by quantitative real-time polymerase chain reaction (qPCR). To determine whether MMP-13 is a target of miR-127-5p, dual luciferase reporter assays were performed. miR-127-5p mimic and miR-127-5p inhibitor were used to overexpress or downregulate miR-127-5p expression in human NP cells, respectively. Small interfering RNA (siRNA) was used to knock down MMP-13 expression in human NP cells. Type II collagen expression in human NP cells was detected by qPCR, western blotting, and immunofluorescence staining. RESULTS We confirmed that miR-127-5p was significantly downregulated in nucleus pulposus (NP) tissue of degenerative discs and its expression was inversely correlated with MMP-13 mRNA levels. We reveal that MMP-13 may act as a target of miR-127-5p. Expression of miR-127-5p was inversely correlated with type II collagen expression in human NP cells. Moreover, suppression of MMP-13 expression by siRNA blocked downstream signaling and increased type II collagen expression. CONCLUSION Dysregulated miR-127-5p contributed to the degradation of type II collagen by targeting MMP-13 in human IDD. Our findings highlight that miR-127-5p may serve as a new therapeutic target in IDD.

The role of angiopoietin-2 in nucleus pulposus cells during human intervertebral disc degeneration

Although evidence shows that intervertebral disc degeneration is generally characterized by angiogenesis, the role of angiopoietin has not been investigated. This study examined the presence of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) within the native intervertebral disc (IVD) and elucidated their functions in the regulation of nucleus pulposus (NP) cells. Initial investigation of uncultured NP tissue revealed that Ang-1 and Ang-2 were expressed by native NP cells. Ang-2 expression was significantly increased in infiltrated and degenerate samples relative to normal samples. The ratio of Ang-2/Ang-1 in tissues from patients increased markedly with increasing age and level of degeneration of the IVD. The ratio of both Ang-2/Ang-1 mRNA and protein increased over time when cells were subjected to constant pressure at 1 Mpa in vitro. Our findings indicate that Ang-2 plays a role in suppressing cell adhesion and viability, and promotes the apoptosis of NP cells and that Ang-2 can inhibit the pathways stimulated by Ang-1 and fibronectin. Ang-2 release during IVD degeneration causes higher ratio of Ang-2 to Ang-1, further inhibits NP cell viability and adhesion, promoting apoptosis by blocking PI3K/Akt signaling. The present study therefore provides new insights into the role of the angiopoietin-Tie system in the pathogenesis of IVD degeneration.

MicroRNA-15b silencing inhibits IL-1β-induced extracellular matrix degradation by targeting SMAD3 in human nucleus pulposus cells

ObjectivesTo determine the role of microRNA-15b (miR-15b) in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation in the nucleus pulposus (NP).ResultsMiR-15b was up-regulated in degenerative NP tissues and in IL-1β-stimulated NP cells, as compared to the levels in normal controls (normal tissue specimens from patients with idiopathic scoliosis). Bioinformatics and luciferase activity analyses showed that mothers against decapentaplegic homolog 3 (SMAD3), a key mediator of the transforming growth factor-β signaling pathway, was directly targeted by miR-15b. Functional analysis demonstrated that miR-15b overexpression aggravated IL-1β-induced ECM degradation in NP cells, while miR-15b inhibition had the opposite effects. Prevention of IL-1β-induced NP ECM degeneration by the miR-15b inhibitor was attenuated by small-interfering-RNA-mediated knockdown of SMAD3. In addition, activation of MAP kinase and nuclear factor-κB up-regulated miR-15b expression and down-regulated SMAD3 expression in IL-1β-stimulated NP cells.ConclusionsMiR-15b contributes to ECM degradation in intervertebral disc degeneration (IDD) via targeting of SMAD3, thus providing a novel therapeutic target for IDD treatment.

Sirtuin 3-dependent mitochondrial redox homeostasis protects against AGEs-induced intervertebral disc degeneration

Intervertebral disc (IVD) degeneration contributes largely to pathoanatomical and degenerative changes of spinal structure that increase the risk of low back pain. Apoptosis in nucleus pulposus (NP) can aggravate IVD degeneration, and increasing studies have shown that interventions targeting NP cell apoptosis can ameliorate IVD degeneration, exhibiting their potential for use as therapeutic strategies. Recent data have shown that advanced glycation end products (AGEs) accumulate in NP tissues in parallel with the progression of IVD degeneration and form a microenvironment of oxidative stress. This study examined whether AGEs accumulation aggravates NP cell apoptosis and IVD degeneration, and explored the mechanisms underlying these effects. We observed that the viability and proliferation of human NP cells were significantly suppressed by AGEs treatment, mainly due to apoptosis. Furthermore, activation of the mitochondrial apoptosis pathway was detected after AGEs treatment. In addition, the molecular data showed that AGEs could significantly aggravate the generation of mitochondrial reactive oxygen species and prolonged activation of the mitochondrial permeability transition pore, as well as the increased level of Bax protein and decreased level of Bcl-2 protein in mitochondria. These effects could be reduced by antioxidant (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) and Visomitin (SKQ1). Importantly, we identified that impairment of Sirtuin3 (SIRT3) function and the mitochondrial antioxidant network were vital mechanisms in AGEs-induced oxidative stress and secondary human NP cell apoptosis. Finally, based on findings that nicotinamide mononucleotide (NMN) could restore SIRT3 function and rescue human NP cell apoptosis through adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor-γ coactivator 1α (AMPK-PGC-1α) pathway in vitro, we confirmed its protective effect on AGEs-induced IVD degeneration in vivo. In conclusion, our data demonstrate that SIRT3 protects against AGEs-induced human NP cell apoptosis and IVD degeneration. Targeting SIRT3 to improve mitochondrial redox homeostasis may represent a potential therapeutic strategy for attenuating AGEs-associated IVD degeneration.

The involvement of regulated in development and DNA damage response 1 (REDD1) in the pathogenesis of intervertebral disc degeneration

Abstract Regulated in development and DNA damage response 1 (REDD1) is an evolutionarily conserved, ubiquitous protein that responds to various cell stresses. Studies have proved REDD1 is involved in many diseases, such as osteoarthritis and cancer. The present study aimed to investigate the potential role of REDD1 in the pathogenesis of intervertebral disc degeneration (IDD). Analysis of clinical tissue samples showed REDD1 expression was up‐regulated during IDD and was correlated with the grade of disc degeneration. Overexpression of REDD1 in normal human nucleus pulposus (NP) cells resulted in extracellular matrix (ECM) degeneration. Further, we investigated the function of REDD1 using a serum deprivation‐induced IDD vitro model and found that REDD1 was up‐regulated in a temporal manner. However, hypoxia abolished this increase through down‐regulation of NF‐&kgr;B. Knockdown of REDD1 or NF‐&kgr;B by si‐RNA significantly rescued ECM from degeneration both in normoxia and hypoxia. In addition, NF‐&kgr;B/REDD1 mediated the protection of hypoxia from serum deprivation‐induced apoptosis and autophagy in NP cells. These results suggest that REDD1 might play a pivotal role in IDD pathogenesis, thereby potentially providing a new therapeutic target for IDD treatment.

Autophagy attenuates compression-induced apoptosis of human nucleus pulposus cells via MEK/ERK/NRF1/Atg7 signaling pathways during intervertebral disc degeneration.

ABSTRACT Autophagy dysfunction has been observed in intervertebral disc degeneration (IVDD) cells, a main contributing factor to cell death, but the precise role of autophagy during IVDD is still controversial. This study aimed to investigate the role of autophagy involved in the pathogenesis of human IVDD and determine the signal transduction pathways responsible for compression‐induced autophagy in human nucleus pulposus (NP) cells. Autophagy, suppressing the induction of apoptosis, was activated in NP cells exposed to compression. Molecular analysis showed that compression promoted the activity of NRF1, a transcription regulator increasing Atg7 expression by binding to its promoter, through activating the Ras/MEK/ERK signaling in NP cells. Loss‐ and gain‐of‐function studies demonstrate that NRF1 induced autophagy and dampened the apoptotic response by promoting Atg7 expression in NP cells subjected to compression. This study confirmed that compression‐induced autophagy could be induced by Ras via MEK/ERK/NRF1/Atg7 signaling pathways, while inhibiting Ras/MEK/ERK/NRF1/Atg7 signaling pathways attenuated this autophagic process, implicating a promising therapeutic strategy for IVDD.