Protects Nucleus Pulposus Cells Research Articles

MiR-486-5p Inhibits Inflammatory Response, Matrix Degradation and Apoptosis of Nucleus Pulposus Cells through Directly Targeting FOXO1 in Intervertebral Disc Degeneration.

microRNA-486-5p (miR-486-5p) and forkhead box protein O1 (FOXO1) play an important role in the development of intervertebral disc degeneration (IDD). However, their molecular mechanisms in IDD remain unknown. qRT-PCR assay was used to identify miR-486-5p expression in nucleus pulposus (NP) cells. In-vitro transfection, CCK-8, flow cytometry and luciferase reporter assay were used to validate the role and relationship of miR-486-5p and FOXO1 in lipopolysaccharides (LPS)-stimulated NP cells. qRT-PCR and Western blot were used to measure the expression levels of inflammatory cytokines, matrix degrading enzymes, and extracellular matrix (ECM)-related genes. miR-486-5p expression was significantly down-regulated, while FOXO1 expression was up-regulated in LPS-treated NP cells (P<0.001). miR-486-5p over-expression repressed LPS-induced expressions of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and matrix degrading enzymes (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5), and promoted the expressions of LPS-inhibited ECM-related genes (Aggrecan and Collagen II) (all P<0.001). In addition, miR-486-5p over-expression protected NP cells against LPS-induced apoptosis. However, inhibition of miR-486-5p led to the opposite effects. Mechanically, FOXO1 was a direct target gene of miR-486-5p. Over-expressed FOXO1 aggravated LPS-induced injury, and antagonized protection effects of miR-486-5p. miR-486-5p can inhibit inflammatory response, ECM degradation and apoptosis in NP cells by directly targeting FOXO1, which may contribute to the biological therapy of IDD.

β‑ecdysterone protects against apoptosis by promoting autophagy in nucleus pulposus cells and ameliorates disc degeneration.

Increasing cell apoptosis is one of the major causes of intervertebral disc degeneration (IDD). β-ecdysterone has been demonstrated to protect PC12 cells against neurotoxicity. A previous study revealed that β‑ecdysterone may be involved in the regulation of autophagy in osteoblasts. Therefore, we hypothesized that β‑ecdysterone may possess therapeutic effects on IDD via autophagy stimulation. The effect of β‑ecdysterone on IDD was explored by invitro experiments. The results demonstrated that β‑ecdysterone attenuated the apoptosis induced by tert‑butyl hydroperoxide via promoting autophagy in nucleus pulposus cells. Beclin‑1, an indispensable protein for the stimulation of autophagy, is upregulated and stabilized by β‑ecdysterone in a dose‑ and time‑dependent manner in nucleus pulposus cells. Inhibition of autophagy with 3‑methyladenine partially abrogated the protective function of β‑ecdysterone against apoptosis of nucleus pulposus cells, indicating that autophagy participated in the protective effect of β‑ecdysterone on IDD. Additionally, β‑ecdysterone promoted the expression of anabolic genes while inhibiting the expression of catabolic genes in nucleus pulposus cells. Collectively, the present study demonstrated that β‑ecdysterone may protect nucleus pulposus cells against apoptosis by autophagy stimulation and ameliorate disc degeneration, which indicates that β‑ecdysterone may be a potential therapeutic agent for IDD.

Melatonin ameliorates intervertebral disc degeneration via the potential mechanisms of mitophagy induction and apoptosis inhibition.

Intervertebral disc degeneration (IDD) is a complicated disease in patients. The pathogenesis of IDD encompasses cellular oxidative stress, mitochondrion dysfunction and apoptosis. Melatonin eliminates oxygen free radicals, regulates mitochondrial homoeostasis and function, stimulates mitophagy and protects against cellular apoptosis. Therefore, we hypothesize that melatonin has beneficial effect on IDD by mitophagy stimulation and inhibition of apoptosis. The effects of melatonin on IDD were investigated in vitro and in vivo. For the former, melatonin diminished cellular apoptosis caused by tert‐butyl hydroperoxide in nucleus pulposus (NP) cells. Mitophagy, as well as its upstream regulator Parkin, was activated by melatonin in both a dose and time‐dependent manner. Mitophagy inhibition by cyclosporine A (CsA) partially eliminated the protective effects of melatonin against NP cell apoptosis, suggesting that mitophagy is involved in the protective effect of melatonin on IDD. In addition, melatonin was demonstrated to preserve the extracellular matrix (ECM) content of Collagen II, Aggrecan and Sox‐9, while inhibiting the expression of matrix degeneration enzymes, including MMP‐13 and ADAMTS‐5. In vivo, our results demonstrated that melatonin treatment ameliorated IDD in a puncture‐induced rat model. To conclude, our results suggested that melatonin protected NP cells against apoptosis via mitophagy induction and ameliorated disc degeneration, providing the potential therapy for IDD.

TFEB protects nucleus pulposus cells against apoptosis and senescence via restoring autophagic flux

Excessive apoptosis and senescence of nucleus pulposus (NP) cells are major pathological changes in intervertebral disc degeneration (IVDD) development; previous studies demonstrated pharmacologically or genetically stimulation of autophagy may inhibit apoptosis and senescence in NP cells. Transcription factor EB (TFEB) is a master regulator of autophagic flux via initiating autophagy-related genes and lysosomal biogenesis. This study was performed to confirm whether TFEB was involved in IVDD development and its mechanism. TFEB activity was detected in NP tissues in puncture-induced rat IVDD model by immunofluorescence as well as in tert-Butyl hydroperoxide (TBHP), the reactive oxygen species (ROS) donor to induce oxidative stress, treated NP cells by western blot. After TFEB overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. In invivo study, the lentivirus-normal control (LV-NC) or lentivirus-TFEB (LV-TFEB) were injected into the center space of the NP tissue, after 4 or 8 weeks, Magnetic resonance imaging (MRI), X ray, Hematoxylin-Eosin (HE) and Safranin O staining were used to evaluate IVDD grades. The nuclear localization of TFEB declined in degenerated rat NP tissue as well as in TBHP treated NP cells. Applying lentivirus to transfect NP cells, TFEB overexpression restored the TBHP-induced autophagic flux blockage and protected NP cells against apoptosis and senescence; these protections of TFEB are diminished by chloroquine-medicated autophagy inhibition. Furthermore, TFEB overexpression ameliorates the puncture-induced IVDD development in rats. Experimental IVDD inhibited the TFEB activity. TFEB overexpression suppressed TBHP-induced apoptosis and senescence via autophagic flux stimulation in NP cell and alleviates puncture-induced IVDD development invivo.

17β-estradiol protects nucleus pulposus cells from serum deprivation-induced apoptosis and regulates expression of MMP-3 and MMP-13 through promotion of autophagy

Serum deprivation is a likely contributor to intervertebral disc (IVD) degeneration (IVDD).17β-estradiol (E2) have been noted to protect nucleus pulposus cells (NPCs) against apoptosis. Autophagy and apoptosis play a paramount role in maintaining the homeostasis of IVD. So far, little research has been published on whether autophagy plays a role for the E2 mediated protection of NPCs. The aim of this study is to understand whether autophagy is involved in the protective effect of E2 against serum deprivation-induced cell apoptosis and expression of matrix metalloproteinase (MMP)-3 and MMP-13. mCherry-GFP-LC3-adenovirus transfection is used to monitor autophagy detection. The expression levels of autophagy-related proteins were measured by Western blotting, Apoptosis and MMPs were detected by flow cytometry and Western blotting. Accordingly, Autophagy and apoptosis was detected in NP cells under serum deprivation conditions, the autophagy incidence began to reached a peak value at 48 h, the apoptosis and MMPs incidence began reached a minimum value treat with E2 (10−7 M). Whereas the combined use of E2 and 3-MA led to a dramatic decrease in autophagy, while aberrantly elevated expression levels of apoptotic and MMPs. These data suggest that serum deprivation-induced apoptosis and MMP-3, MMP-13, which was efficiently suppressed by the E2 through promoting autophagy in rat NPCs.

Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration.

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert‐butyl hydroperoxide (TBHP)‐treated nucleus pulposus cells of SD rats in vitro as well as in a puncture‐induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen‐II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts‐5. Additionally, autophagy blockade using 3‐MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine‐treated rats had an accentuated T2‐weighted signal and a diminished histological degenerative grade than vehicle‐treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.

Therapeutic Potential of Naringin for Intervertebral Disc Degeneration: Involvement of Autophagy Against Oxidative Stress-Induced Apoptosis in Nucleus Pulposus Cells.

Intervertebral disc degeneration (IDD) is a major cause of lower back pain, but few efficacious medicines have been developed for IDD. Increased nucleus pulposus cells apoptosis is a dominant pathogenesis of IDD and is considered a therapeutic target. Previously, our group proved that autophagy may protect nucleus pulposus cells against apoptosis. As one of the major bioflavonoids of citrus, naringin activates autophagy. Therefore, we hypothesize that naringin may have therapeutic potential for IDD by activating autophagy in nucleus pulposus cells. In this study, we evaluated the effects of naringin on TBHP-induced oxidative stress in nucleus pulposus cells in vitro as well as in puncture-induced rat IDD model in vivo. Our results showed that naringin could reduce the incidence of oxidative stress-induced apoptosis in nucleus pulposus cells and promoted the expression of autophagy markers LC3-II/I and beclin-1. Meanwhile, inhibition of autophagy by 3-MA may partially reverse the anti-apoptotic effect of naringin, indicating that autophagy was involved in the protective effect of naringin in nucleus pulposus cells. Further study showed that autophagy regulation of naringin may be related to AMPK signaling. Also, we found that naringin treatment can regulate the expression of collagen II, aggrecan and Mmp13 to sustain the extracellular matrix. Furthermore, our in vivo study showed that naringin can ameliorate IDD in puncture-induced rat model. In conclusion, our study suggests that naringin can protect nucleus pulposus cells against apoptosis and ameliorate IDD in vivo, the mechanism may relate to its autophagy regulation.

Tauroursodeoxycholic Acid Protects Nucleus Pulposus Cells from Compression-Induced Apoptosis and Necroptosis via Inhibiting Endoplasmic Reticulum Stress.

Tauroursodeoxycholic acid (TUDCA) is a kind of hydrophilic bile acid, which could protect cells from death via inhibiting endoplasmic reticulum (ER) stress. However, the role of TUDCA in compression-induced intervertebral disc degeneration (IVDD) has not been elucidated. Here, we used a previously described device to mimic in vivo compression conditions. NP cells treated with DMSO or TUDCA were exposed to compression. Then, cell viability, morphology, and apoptosis were detected. Furthermore, apoptosis-related proteins and necroptosis markers were detected too. To investigate the specific cytoprotective mechanisms of TUDCA in IVDD, we detected the ER morphology by electron microscopy. In addition, the ER stress of nucleus pulposus (NP) cells was quantitatively evaluated by analyzing the level of ER-stress-associated proteins. Our results revealed that TUDCA could protect NP cells from excessive compression-induced death by reducing the apoptosis and necroptosis. In addition, ER stress is involved in pathogenesis of IVDD induced by excessive compression and plays a detrimental role. TUDCA exerts its protective functions by inhibiting ER stress. In conclusion, TUDCA could protect NP cells from compression-induced death, which suggested that treatment by TUDCA may be a potential method to retard IVDD.

Pterostilbene inhibits inflammation by promoting the nuclear translocation of Nrf2 in the rat nucleus pulposus

Pterostilbene (PTE), a natural plant extract, has an anti-inflammatory effect; however, whether PTE could protect nucleus pulposus cells (NPCs) in the intervertebral disk from inflammation remains unclear. Primary NPCs isolated from Sprague-Dawley (SD) rats were cultured, and Cell Counting Kit-8 (CCK-8) analysis was used to test the cytotoxicity of PTE. The effect of PTE on interleukin-1β (IL-1β)-induced inflammation was analyzed using an enzyme-linked immunosorbent assay, real-time polymerase chain reaction (PCR), and a Griess test. Western blotting, immunofluorescence, and a nuclear factor erythroid 2-related factor 2 (Nrf2) small interfering RNA (siRNA) transfection were used to assess the involvement of Nrf2 in the anti-inflammatory mechanism of PTE on NPCs. The results of the CCK-8 analysis showed that PTE produced no cytotoxicity in NPCs at 20 μM for 24 h. PTE suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) and inhibited the messenger RNA (mRNA) expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induced by IL-1β. PTE could promote the nuclear translocation of Nrf2 in NPCs. In addition, Nrf2 silence reversed the inhibitory effect of PTE on the production of NO and PGE2 and the expression of COX-2 and iNOS. These results indicate that PTE inhibits inflammation in the rat nucleus pulposus by promoting the nuclear translocation of Nrf2.

Nicotinamide Phosphoribosyltransferase Inhibitor APO866 Prevents IL-1β-Induced Human Nucleus Pulposus Cell Degeneration via Autophagy

Background/Aims: Intervertebral discs consist of an extracellular matrix (ECM) with a central gelatinous nucleus pulposus (NP) enclosed in an outer layer known as the annulus fibrosus. ECM metabolic disorders result in loss of boundary between the annulus fibrosus and NP, which can lead to intervertebral disc degeneration (IDD). Proinflammatory cytokines, such as interleukin (IL)-1β, mediate the progression of IDD. Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD) and is known to be induced by IL-1β. APO866 is an inhibitor of NAD biosynthesis and is involved in autophagy. LC3 (microtubule-associated protein 1 light chain 3) is a key regulator of autophagy and is used as an indicator of increased autophagy. Herein, we investigate the role of APO866 in regulating autophagy in NP cells and IL-1β mediated NP cell degeneration and apoptosis. Methods: NP cells were extracted from IDD tissues and cultured in DMEM/F12 medium. Nampt was induced by different concentrations of IL-1β (0, 0.5, 1, 5, 10 ng/mL) for 24 h or NP cells were treated with 10 ng/mL IL-1β for 0, 6, 12, 48 h. QRT-PCR and western blots were used to detect Nampt and ECM-related protein expression in NP tissue of patients with IDD and in NP cells. Confocal analysis was used to detect membrane-bound LC3, Aggrecan, and Collagen II. Results: Nampt is expressed in NP tissue at higher levels in severe grades of IDD (Grade IV and V) compared with low grades (Grade II and III). In NP cells, 10 ng/mL IL-1β induced Nampt expression for 48 h, increased expression of the degradative-associated proteins, ADAMTS4/5 and MMP-3/13, and decreased expression of ECM-related proteins, Aggrecan and Collagen II. However, the Nampt inhibitor APO866 blocked IL-1β induction, and the knockdown of Nampt expression increased the expression of ECM proteins that were inhibited by IL-1β. Moreover, evidence provided by the autophagic markers LC3 and Beclin-1 indicated that APO866 induced NP cell autophagy. Furthermore, although APO866 inhibited the downregulated expression of ECM-related proteins by IL-1β, this function was blocked by autophagy inhibitor, 3-methyladenine. Conclusion: APO866 protects NP cells and induces autophagy by inhibiting IL-1β-induced NP cell degeneration and apoptosis, which may have therapeutic potential in IDD.

Edaravone ameliorates compression-induced damage in rat nucleus pulposus cells

AimsEdaravone is a strong free radical scavenger most used for treating acute ischemic stroke. In this study we investigated the protective effects and underlying mechanisms of edaravone on compression-induced damage in rat nucleus pulposus (NP) cells. Materials and methodsCell viability was determined using MTT assay methods. NP cell apoptosis was measured by Hoechst 33,258 staining and Annexin V/PI double staining. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and intracellular calcium ([Ca2+]i) were determined by fluorescent probes DCFH-DA, JC-1 and Fluo-3/AM, respectively. Apoptosis-related proteins (cleaved caspase-3, cytosolic cytochrome c, Bax and Bcl-2) and extracellular matrix proteins (aggrecan and collagen II) were analyzed by western blot. Key findingsEdaravone attenuated the compression-induced decrease in viability of NP cells in a dose-dependent manner. 33,258 and Annexin V/PI double staining showed that edaravone protected NP cells from compression-induced apoptosis. Further studies confirmed that edaravone protected NP cells against compression-induced mitochondrial pathway of apoptosis by inhibiting overproduction of ROS, collapse of MMP and overload of [Ca2+]i. In addition, edaravone promoted the expression of aggrecan and collagen II in compression-treated NP cells. SignificanceThese results strongly indicate that edaravone ameliorates compression-induced damage in rat nucleus pulposus cells. Edaravone could be a potential new drug for treatment of IDD.

Chitosan oligosaccharides protect nucleus pulposus cells from hydrogen peroxide-induced apoptosis in a rat experimental model

Chitosan has been investigated for its protective effect on nucleus pulposus (NP) cells against intervertebral disc degeneration(IDD), but its high molecular weight prohibits its clinical application. The low molecular derivatives, chitosan oligosaccharides (COS) are easier to absorb; however, the protective effect of COS against IDD remains unclear. In this study, we investigated the effects of COS on NP degeneration. NP cells derived from rats were treated with H2O2 to induce an IDD-like transition. Then, COS was used to pre-treat cells before administering H2O2 and changes occurring in the cells were observed. As shown by the result of a cell counting kit-8(CCK-8) assay, COS protected the viability of the cells. The induced apoptosis rate fell when cells were pre-treated with COS, revealed by annexin-V FITC/PI double staining analysis and Hoechst 33342 staining. COS administration also protected ECM synthesis and prevented its degradation, as shown by western blotting(WB) and polymerase chain reaction(PCR). We analyzed the activity of the PI3K/Akt pathway in H2O2 treated NP cells by WB and the result showed that COS could enhance activity of the pathway. To investigate the relationship between the PI3K/Akt pathway and the protective effects of COS on NP cells, the PI3K/Akt pathway was inhibited by wortmannin, and we subsequently found that this abolished the protective effects. These results support the hypothesis that COS exerts its protective effect on NP cells against H2O2-induced apoptosis via the PI3K/Akt pathway.

Mesenchymal Stem Cells Protect Nucleus Pulposus Cells from Compression-Induced Apoptosis by Inhibiting the Mitochondrial Pathway.

Objective Excessive apoptosis of nucleus pulposus cells (NPCs) induced by various stresses, including compression, contributes to the development of intervertebral disc degeneration (IVDD). Mesenchymal stem cells (MSCs) can benefit the regeneration of NPCs and delay IVDD, but the underlying molecular mechanism is poorly understood. This study aimed to evaluate the antiapoptosis effects of bone marrow-derived MSC (BMSC) on rat NPCs exposed to compression and investigate whether the mitochondrial pathway was involved. Methods BMSCs and NPCs were cocultured in the compression apparatus at 1.0 MPa for 36 h. Cell viability, apoptosis, mitochondrial function, and the expression of apoptosis-related proteins were evaluated. Results The results showed that coculturing with BMSCs increased the cell viability and reduced apoptosis of NPCs exposed to compression. Meanwhile, BMSCs could relieve the compression-induced mitochondrial damage of NPCs by decreasing reactive oxygen species level and maintaining mitochondrial membrane potential as well as mitochondrial integrity. Furthermore, coculturing with BMSCs suppressed the activated caspase-3 and activated caspase-9, decreased the expressions of cytosolic cytochrome c and Bax, and increased the expression of Bcl-2. Conclusions Our results suggest that BMSCs can protect against compression-induced apoptosis of NPCs by inhibiting the mitochondrial pathway and thus enhance our understanding on the MSC-based therapy for IVDD.

Metformin protects against apoptosis and senescence in nucleus pulposus cells and ameliorates disc degeneration in vivo.

Intervertebral disc degeneration (IDD) is a complicated process that involves both cellular apoptosis and senescence. Metformin has been reported to stimulate autophagy, whereas autophagy is shown to protect against apoptosis and senescence. Therefore, we hypothesize that metformin may have therapeutic effect on IDD through autophagy stimulation. The effect of metformin on IDD was investigated both in vitro and in vivo. Our study showed that metformin attenuated cellular apoptosis and senescence induced by tert-butyl hydroperoxide in nucleus pulposus cells. Autophagy, as well as its upstream regulator AMPK, was activated by metformin in nucleus pulposus cells in a dose- and time-dependent manner. Inhibition of autophagy by 3-MA partially abolished the protective effect of metformin against nucleus pulposus cells' apoptosis and senescence, indicating that autophagy was involved in the protective effect of metformin on IDD. In addition, metformin was shown to promote the expression of anabolic genes such as Col2a1 and Acan expression while inhibiting the expression of catabolic genes such as Mmp3 and Adamts5 in nucleus pulposus cells. In vivo study illustrated that metformin treatment could ameliorate IDD in a puncture-induced rat model. Thus, our study showed that metformin could protect nucleus pulposus cells against apoptosis and senescence via autophagy stimulation and ameliorate disc degeneration in vivo, revealing its potential to be a therapeutic agent for IDD.

MicroRNA-494 inhibition protects nucleus pulposus cells from TNF-α-induced apoptosis by targeting JunD.

Human nucleus pulposus cell (HNPC) apoptosis plays an important role in the development of intervertebral disc degeneration (IVDD). Our previous research revealed that among all of the dysregulated microRNAs in the degenerated nucleus pulposus tissues of patient with IVDD, miRNA-494 (miR-494) is the most significantly increased. However, the influence of miR-494 HNPC apoptosis has not been confirmed. This study was designed to evaluate the effect of miR-494 on the HNPC apoptosis induced by TNF-α and to explore the possible mechanism of this process. First, HNPCs were stimulated with TNF-α at different concentrations (0 ng/ml, 10 ng/ml, 50 ng/ml, or 100 ng/ml) for 0 h, 8 h, 16 h, or 24 h. Annexin V-PE/7-AAD assays and real-time quantitative PCR were used to detect the cell apoptosis rates and miR-494 expression. Second, we successfully knocked down endogenous miR-494 in HNPCs via lentiviral antigomiR-494 vector infection and then stimulated with TNF-α (100 ng/ml, 16 h). The rates of apoptosis and miR-494 expression were then detected again. Additionally, a dual-luciferase reporter assay and western blotting were used to determine whether JunD is a target of miR-494. Finally, western blotting was used to analyze the expression of cytochrome C. We found that the rate of apoptosis increased with concentration, time (p < 0.05) and miR-494 expression (p < 0.05). The rate of apoptosis in the 100 ng/ml, 16 h group appeared to be suitable. After transfection, the apoptosis rate and miR-494 expression were significantly decreased in the antigomiR-494+TNF-α group compared to the controls (p < 0.05). We also revealed that JunD is a target of miR-494. Western blotting analysis demonstrated that treatment with the lentiviral antigomiR-494 vector resulted in increased expression of JunD (p < 0.05) and decreased expression of cytochrome C (p < 0.05). These results indicated that miR-494 is a novel regulator of HNPC apoptosis induced by TNF-α. The knock-out of miR-494 expression protected the HNPCs from apoptosis via the up-regulation of JunD, which was possibly mediated via cytochrome C apoptotic signaling. These findings suggest that the miR-494/JunD signaling pathway might represent a novel therapeutic target for the prevention of IVDD.

Adipose-derived stromal cells protect intervertebral disc cells in compression: implications for stem cell regenerative disc therapy.

Introduction: Abnormal biomechanics plays a role in intervertebral disc degeneration. Adipose-derived stromal cells (ADSCs) have been implicated in disc integrity; however, their role in the setting of mechanical stimuli upon the disc's nucleus pulposus (NP) remains unknown. As such, the present study aimed to evaluate the influence of ADSCs upon NP cells in compressive load culture.Methods: Human NP cells were cultured in compressive load at 3.0MPa for 48 hours with or without ADSCs co-culture (the ratio was 50:50). We used flow cytometry, live/dead staining and scanning electron microscopy (SEM) to evaluate cell death, and determined the expression of specific apoptotic pathways by characterizing the expression of activated caspases-3, -8 and -9. We further used real-time (RT-) PCR and immunostaining to determine the expression of the extracellular matrix (ECM), mediators of matrix degradation (e.g. MMPs, TIMPs and ADAMTSs), pro-inflammatory factors and NP cell phenotype markers.Results: ADSCs inhibited human NP cell apoptosis via suppression of activated caspase-9 and caspase-3. Furthermore, ADSCs protected NP cells from the degradative effects of compressive load by significantly up-regulating the expression of ECM genes (SOX9, COL2A1 and ACAN), tissue inhibitors of metalloproteinases (TIMPs) genes (TIMP-1 and TIMP-2) and cytokeratin 8 (CK8) protein expression. Alternatively, ADSCs showed protective effect by inhibiting compressive load mediated increase of matrix metalloproteinases (MMPs; MMP-3 and MMP-13), disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs; ADAMTS-1 and 5), and pro-inflammatory factors (IL-1beta, IL-6, TGF-beta1 and TNF-alpha).Conclusions: Our study is the first in vitro study assessing the impact of ADSCs on NP cells in an un-physiological mechanical stimulation culture environment. Our study noted that ADSCs protect compressive load induced NP cell death and degradation by inhibition of activated caspase-9 and -3 activity; regulating ECM and modulator genes, suppressing pro-inflammatory factors and preserving CK8. Consequently, the protective impact of ADSCs found in this study provides an essential understanding and expands our knowledge as to the utility of ADSCs therapy for intervertebral disc regeneration.

Glucosamine protects nucleus pulposus cells and induces autophagy via the mTOR-dependent pathway.

Although glucosamine has been suggested to be effective in the treatment of osteoarthritis, its effect on disc degeneration remains unclear. We sought to explore whether glucosamine can activate autophagy in rat nucleus pulposus (NP) cells and protect cells treated with IL-1β or hydrogen peroxide (H2 O2 ). Autophagy in cells was examined by detecting for LC3, Beclin-1, m-TOR, and p70S6K, as well as by analyzing autophagosomes. To inhibit autophagy, 3-methyladenine (3-MA) was used. In the cells treated with IL-1β, the levels of Adamts-4, Mmp-13, aggrecan, and Col2a1 were analyzed by real-time PCR and immunofluorescence. Apoptosis was analyzed by TUNEL. Cell senescence under H2 O2 was revealed by SA-β-Gal staining. Glucosamine could activate autophagy in a dose-dependent manner within 24 h and inhibit the phosphorylation of m-TOR and p70S6K. Autophagy in IL-1β or H2 O2 -treated cells was increased by glucosamine. Glucosamine attenuated the decrease of aggrecan and prevented the apoptosis of the NP cells induced by IL-1β, whereas 3-MA partly reversed these effects. The percentage of SA-β-Gal-positive cells induced by H2 O2 treatment was decreased by glucosamine, accompanied by the decline of p70S6K phosphorylation. Glucosamine protects NP cells and up-regulates autophagy by inhibiting the m-TOR pathway, which might point a potential therapeutic agent for disc degeneration.

A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells

IntroductionIncreased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.MethodsCells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.ResultsTNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.ConclusionsHere, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.

Notochordal Cells Protect Nucleus Pulposus Cells from Degradation and Apoptosis: Implications for the Mechanisms of Intervertebral Disk Degeneration

IntroductionDegenerative disk disease (DDD) is an extremely common and costly health care condition which to date has no curative strategy which would be greatly aided by an ability to re-establish...

Notochordal Cells Protect Nucleus Pulposus Cells from Degradation and Apoptosis: Implications for the Mechanisms of Intervertebral Disk Degeneration

IntroductionDegenerative disk disease (DDD) is an extremely common and costly health care condition which to date has no curative strategy which would be greatly aided by an ability to re-establish equilibrium between catabolic and anabolic tissue remodeling.1The nonchondrodystrophic (NCD) canine is unique amongst the canine subspecies in which this animal retains a high notochordal cell population throughout life, and is relatively resistant to the development of DDD and an emerging body of evidence indicates that notochordal cells confer anabolic capacity upon nucleus pulposus (NP) cells and that their absence is associated with susceptibility to degenerative changes.2–5Regulation of cellular turnover is vital to tissue homeostasis and is dependent upon a highly regulated form of programmed cell death (apoptosis). Some cells, classified as type I cells, function independently of the mitochondria and signal via Fas-induced apoptotic cell death involving the Caspase-8 pathway. Type II cells (such as NP cells) have a critical reliance upon the mitochondria whereby apoptosis is mediated via Caspase-9.8,9 Here, we demonstrate that notochordal cell-conditioned medium or “NCCM” is capable of protecting NP cells from matrix protein degradation and proinflammatory cytokine secretion induced by IL-1β and FasL. Furthermore, IL-1β and FasL-mediated cell death is inhibited by NCCM via the inhibition of activated caspases -9 and -3. Materials and MethodsWe obtained NP cells from bovine caudal disks as per our established methods to be used as the “target” cells in our experiments. We also developed bovine NP cell conditioned medium (BCCM) in exactly the same fashion as we obtained bovine NP “target” cells but in the case of BCCM cultured the bovine NP cells within alginate beads.We developed conditioned medium from the NPs of lower thoracic and entire lumbar IVDs from 11 nonchondrodystrophic canines and after overnight digestion seeded the cells within alginate beads for subsequent culture. Conditioned medium was generated from both nonchondrodystrophic canine notochordal cells (NCCM) and bovine NP cells (BCCM). We then determined the ability of NCCM or BCCM to protect bovine NP cells from cell death in the presence of IL-1β + Fas ligand (FasL) as well as the ability of NCCM to protect the expression of salient extracellular matrix genes and other molecules using qRT-PCR methods. We used flow cytometry and activated caspase assays to determine protection from cell death and to discern the apoptotic pathways involved. ResultsNCCM inhibits bovine NP cell apoptosis via suppression of activated caspase-9 and caspase-3/7. Furthermore, NCCM protects NP cells from the degradative effects of IL-1β and IL-1β + Fas-L by up-regulating the expression of anabolic/matrix protective genes (aggrecan, collagen typeII, CD44, link protein and TIMP-1) and downregulating matrix degrading genes such as MMP-3. Expression of ADAMTS-4 is increased. NCCM also protects against IL-1 + FasL-mediated downregulation of Ankexpression. Furthermore, NCCM downregulates the expression of IL-6by almost 50%. BCCM does not mediate cell death/apoptosis in target bovine NP cells. ConclusionWe have demonstrated in this study that NCCM protects against NP apoptosis via suppression of activated caspase-9 and -3/7. Possible mechanisms include stabilization of the mitochondrial membrane via inhibition of Bcl-2 activity, BID activation, or through the P53 growth factor-related pathway. It may be that components of NCCM suppress the formation of the apoptoses and in doing so they suppress the formation of activated caspase-3 thereby preventing apoptotic cell death. The novel finding of the activation of the Ankgene by NCCM under degenerative/death-inducing conditions suggests a possible role for this gene in the maintenance of NP cell phenotype. These areas are under examination by our group.The results of the present study provide evidence in vitro that notochordal cell-secreted soluble factors provide essential molecular signals that mediate apoptosis and degradation of NP cells induced by IL-1β + Fas-L. 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