Apoptosis Of Human Nucleus Pulposus Cells Research Articles

The Role of the Bone Morphogenetic Protein Antagonist Noggin in Nucleus Pulposus Intervertebral Disc Cells

Low back pain (LBP) is a significant global health issue, contributing to disability and socioeconomic burdens worldwide. The degeneration of the human intervertebral disc (IVD) is a critical factor in the pathogenesis of LBP. Recent studies have emphasized the significance of a specific set of genes and extracellular matrix (ECM) in IVD health. In particular, Noggin has emerged as a critical gene due to its high expression levels in healthy nucleus pulposus cells (NPCs) observed in our previous research. In this study, it was hypothesized that decreased Noggin expression in NPCs is associated with IVD degeneration and contributes to LBP development. A lentivirus-mediated RNAi was applied to knock down Noggin expression in primary NPCs from six human donors. The NPCs after transduction were evaluated through cell viability analysis, XTT assay, and cell apoptosis analyses. After two weeks, a colony formation assay was used to examine the anchor-independent growth ability of transduced cells. At the transcript level, anabolic and catabolic markers were quantified using RT-qPCR. The results demonstrated that lentivirus-mediated downregulation of Noggin significantly inhibited cell proliferation, reduced cell viability, and suppressed colony formation, while inducing apoptosis in human NPCs in vitro. Notably, it disrupted cellular anabolic processes and promoted catabolic activity in human NPCs post-transduction. Our findings indicated that the degeneration of human IVD is possibly related to decreased Noggin expression in NPCs. This research provides valuable insights into the role of Noggin in IVD homeostasis and its implications in LBP treatment.

Panax notoginseng saponins inhibits oxidative stress- induced human nucleus pulposus cell apoptosis and delays disc degeneration in vivo and in vitro

Ethnopharmacological relevancePanax notoginseng (Burk) F. H. Chen has been a popular traditional Chinese medicine with a long history of treating low back pain. Its main active ingredient, Panax notoginseng saponins (PNS), can be found in several Chinese patent medicines that are frequently used to treat blood stasis type low back pain. Intervertebral disc degeneration (IDD) is the most common cause of back pain, and the injection of PNS has been used to relieve IDD-induced back pain in clinical practice. Despite its effectiveness, the exact mechanisms of action for PNS injections remain unclear. Aim of the studyIDD as a consequence of aging involves apoptosis of nucleus pulposus (NP) cells and imbalanced degradation of extracellular matrix (ECM) induced by several factors including oxidative stress. We hypothesized that PNS may have a therapeutic effect on IDD via inhibiting apoptosis of NP cells and degradation of ECM under oxidative stress. Materials and methodsIn this study, network pharmacology was initially employed to predict the targets of PNS against IDD. Subsequently, commercial PNS was analyzed by high-performance liquid chromatography to confirm the ingredients for in vitro and in vivo experiments. In vitro experiments were conducted on human nucleus pulposus (HNP) cells, including CCK-8, RT-PCR, Western blot, immunofluorescence staining, autophagic flux detection, and TUNEL assay. In vivo experiments were also performed on rats with IDD of tail discs induced by annular fibrosus needle puncture, which involved MRI, HE staining, and immunohistochemistry. ResultsOur study demonstrated the theoretical targets of PNS against IDD, including Caspase 3, MMP13, Akt, and autophagy, based on network pharmacology. Subsequently, in vitro experiments revealed that PNS attenuated cellular apoptosis of NP by suppressing the expression of cleaved-caspase 3 and the ratio of Bax/Bcl-2 under H2O2 stimulation. Autophagy was also inhibited by PNS treatment, and the protective effect was abolished with rapamycin, an autophagy inducer, indicating that autophagy inhibition was involved in the protective effect of PNS on IDD. Furthermore, Akt/mTOR pathway activation was observed in HNP cells responding to H2O2 with PNS treatment, which played a role in autophagy downregulation. PNS was also shown to promote the expression of anabolic genes such as COL2A1 and ACAN while inhibiting the expression of catabolic gene MMP13 in HNP cells. In addition, the in vivo study revealed that PNS treatment could ameliorate IDD in a puncture-induced rat tail model. The development of IDD was significantly reduced, and there was decreased MMP13 expression, as well as increased COL2A1 protein expression in NP tissues. ConclusionOur study showed that PNS could protect HNP cells against apoptosis via autophagy inhibition and ameliorate disc degeneration in vivo, indicating its potential to be a therapeutic agent for IDD.

Lactotransferrin promotes intervertebral disc degeneration by regulating Fas and inhibiting human nucleus pulposus cell apoptosis.

Background: In recent years, intervertebral disc (IVD) degeneration (IDD) has increased in age. There is still a lack of effective treatment in clinics, which cannot improve the condition of IDD at the level of etiology.Objective: To explore IDD pathogenesis at the cellular and gene levels and investigate lactotransferrin (LTF) expression in IDD patients and its possible mechanism.Methods: We downloaded the IDD data set from the Gene Expression Omnibus (GEO) database, screened the differentially expressed genes (DEGs) and hub genes and performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to construct a protein–protein interaction (PPI) network. Subsequently, we verified LTF's regulatory mechanism through cell experiments. IL-1β was used to intervene in nucleus pulposus cells (NPCs) to construct the IDD cell model, and LTF and Fas expression was detected by qRT–PCR. LTF inhibitor, Fas inhibitor, LTF mimic, and Fas mimic were used to intervene in each group. Western blotting was used to detect Fas, Caspase-3, Bax, and Bcl-2 expression.Results: A total of 131 DEGs and 10 hub genes were screened. LTF mRNA in the IDD model was significantly higher than that in the control group, while Fas' mRNA was significantly lower. When LTF was upregulated or downregulated in NPCs, apoptosis marker expression showed the opposite trend. The rescue test showed that LTF and Fas' overexpression greatly enhanced NPC apoptosis.Conclusion: LTF promotes IDD progression by regulating Fas in NPCs, and it may be an effective gene therapy target.

Role of MiR-27a-3p in Intervertebral Disc Degeneration through Targeting RASSF5 via MST1/LATS1 and RAS/RAC1 Signaling Pathway.

Background The apoptosis of nucleus pulposus (NP) cells reduces the number of nucleus pulposus cells in intervertebral disc tissue, resulting in intervertebral disc degeneration (IDD). MicroRNAs (miRNAs) play an important regulatory role in abnormal cell proliferation and apoptosis. Methods The miR-27a-3p expressions in degenerative NP tissue and cells were measured via qPCR. The impacts of miR-27a-3p on the proliferation and apoptosis of human NP cells were evaluated by flow cytometry assays, MTT assays, and western blot analyses. In addition, target scan and luciferase reporter assay were applied to confirm that RASSF5 was directly binding to miR-27a-3p. Western blot was applied to assess the relationship between miR-27a-3p, RASSF5 and MST1/LATS1, and RAS/RAC1 signaling pathway. Results MiR-27a-3p was downregulated in degenerative NP tissues and cells by comparison with the control group. MiR-27a-3p overexpression enhanced cell proliferation and suppressed apoptosis of NP cells, while the above factors showed an opposite tendency after in the miR-27a-3p inhibitor group. The western blot experiment similarly suggested mir-27a-3p apparently downregulated apoptosis-related proteins (Bax and caspase-3) and upregulated antiapoptotic proteins (Bcl-2). In addition, RASSF5 was confirmed to be directly regulated by miR-27a-3p using the luciferase reporter assay. Overexpressed RASSF5 could reverse the effects caused by miR-27a-3p mimic. Finally, miR-27a-3p could downregulate RASSF5 and affected the MST1/LATS1 and RAS/RAC1 pathway. Conclusion MiR-27a-3p may target RASSF5 and enhance cell proliferation and imped cell apoptosis of the nucleus pulposus cells via the MST1/LATS1 and RAS/RAC1 pathway, lessening the degeneration of intervertebral discs.

Bergenin alleviates H2 O2 -induced oxidative stress and apoptosis in nucleus pulposus cells: Involvement of the PPAR-γ/NF-κB pathway.

Bergenin is a C-glucoside of 4-O-methyl gallic acid with a variety of biological activities, such as antioxidant and anti-inflammatory. Herein, we investigated the involvement of bergenin in the protective effect against H2 O2 -induced oxidative stress and apoptosis in human nucleus pulposus cells (HNPCs) and the underlying mechanisms. HNPCs were cotreated with various concentrations of bergenin and 200 μM H2 O2 for 24 h. Cell viability was detected by Cell Counting Kit-8 and lactate dehydrogenase release assays. Reactive oxygen species (ROS) was evaluated utilizing 2',7'-dichlorofluorescein-diacetate. Superoxide dismutase (SOD) and catalase (CAT) activities and glutathione (GSH) levels were measured to assess oxidative stress. Apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase-3/7 activity assays. Expression of protein was determined by western blotting. Results indicated that treatment with bergenin significantly alleviated H2 O2 -induced viability reduction and ROS overproduction in HNPCs in a dose-dependent manner. Bergenin alleviated H2 O2 -induced oxidative stress in HNPCs by increased activity of superoxide dismutase and level of glutathione peroxidase. H2 O2 -induced apoptosis and activity of caspase-3/7 were also suppressed by bergenin treatment in HNPCs. Western blotting showed that H2 O2 -induced decrease in expression of peroxisome proliferator-activated receptor γ (PPAR-γ) and increase in nuclear factor κB (NF-κB) were inhibited by bergenin. However, the inhibitory effect of bergenin on H2 O2 -induced viability reduction, oxidative stress and apoptosis were noticeably abrogated in PPAR-γ knockdown HNPCs. In conclusion, our results indicated that bergenin alleviates H2 O2 -induced oxidative stress and apoptosis in HNPCs by activating PPAR-γ and suppressing NF-κB pathway.

Higenamine mitigates interleukin-1β-induced human nucleus pulposus cell apoptosis by ROS-mediated PI3K/Akt signaling.

Intervertebral disc degeneration (IDD) is a natural problem linked to the inflammation. Higenamine exerts multiple pharmacological properties in inflammation-related disorders. Our study aimed to explore the function of higenamine on interleukin (IL)-1β-caused apoptosis of human nucleus pulposus cells (HNPCs). Cell apoptosis was investigated by TUNEL and flow cytometry. Apoptosis-related biomarkers were determined by qRT-PCR or Western blotting. The protein in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling was measured by Western blotting. We found that higenamine showed little effect on cell apoptosis, but mitigated IL-1β-caused apoptosis in a dose-dependent pattern. Higenamine attenuated IL-1β-induced decrease of Bcl-2 and increase of Bax and cleaved caspase-3. Higenamine did not affect the reactive oxygen species (ROS) level and the PI3K/Akt signaling, but attenuated IL-1β-induced ROS production and inhibition of the PI3K/Akt signaling. IL-1β repressed the activation of the PI3K/Akt pathway, but ROS inhibition using N-acetylcysteine (NAC) rescued this pathway. The PI3K/Akt signaling suppression using LY294002 reversed the inhibitive effect of higenamine on IL-1β-caused apoptosis, and this effect was weakened by ROS inhibition. In conclusion, higenamine attenuates IL-1β-caused apoptosis of HNPCs via ROS-mediated PI3K/Akt pathway.

Bromodomain-containing protein 7 regulates matrix metabolism and apoptosis in human nucleus pulposus cells through the BRD7-PI3K-YAP1 signaling axis

Intervertebral disc degeneration (IDD) results from dysregulated metabolism of the extracellular matrix of the nucleus pulposus (NP) and involves the participation of inflammatory factors such as TNF-α. Bromodomain-containing protein 7 (BRD7) shows considerable potential for anti-inflammatory applications. Herein, we investigated the role of BRD7 in IDD. The immunohistochemistry results demonstrated decreased BRD7 expression in severely degenerated human NP tissues compared to those showing mild degeneration. Lentiviruses and adenoviruses were used to knock down or overexpress BRD7 and YAP1, respectively. Our results revealed that BRD7 knockdown promoted matrix degradation and suppressed PI3K and YAP1 expression, while BRD7 overexpression alleviated matrix degradation and promoted YAP1 and PI3K expression. In addition, PI3K inhibition augmented matrix degradation, enhanced apoptosis, and reduced YAP1 expression, whereas YAP1 overexpression promoted matrix synthesis, suppressed apoptosis and promoted PI3K expression. Besides, BRD7 overexpression reversed the reductions in sulfated glycosaminoglycan levels induced by TNF-α, but this effect was blocked by PI3K or YAP1 inhibitors. Moreover, YAP1 and PI3K were shown to interact through coimmunoprecipitation analysis. In summary, our results demonstrate that BRD7 can regulate matrix metabolism and apoptosis in human NP cells through the BRD7-PI3K-YAP1 signaling axis. This study might provide new insights into the prevention and treatment of IDD.

MiR-502 Suppresses TNF-α-Induced Nucleus Pulposus Cell Apoptosis by Targeting TARF2.

Intervertebral disc degeneration (IVDD) is a common cause of low back pain. This study is aimed at investigating the role of microRNAs (miRNAs) in regulating human nucleus pulposus (NP) cell injury induced by tumor necrosis factor- (TNF-) α in IVDD. In this study, we induced NP cells with 20 ng/mL TNF-α in vitro, which promoted the obvious apoptosis of NP cells and the activation of nuclear transcription factor (NF)-κB. In contrast, using the specific NF-κB inhibitor BAY 11-7082 to treat cells greatly impaired the activation of NF-κB and increased the sensitivity of NP cells to TNF-α-induced apoptosis. Moreover, both TNF-α and BAY 11-7082 treatments were associated with marked miRNA dysregulation, with miR-502 being upregulated by TNF-α treatment and downregulated by BAY 11-7082 treatment, respectively. And the overexpression of miR-502 enhanced NF-κB activation and suppressed apoptosis of human NP cells induced by TNF-α, whereas the opposite was observed following miR-502 inhibition. Last, through bioinformatic analyses and luciferase reporter gene experiments, we identified TRAF2, an important activator of NF-κB, as a miR-502 target gene. Similarly, siRNA-mediated knockdown of the TRAF2 expression also suppressed TNF-α-induced apoptosis and enhanced NF-κB activation. Our findings provide evidence indicating that miR-502 is a key regulator of apoptosis of human NP cells induced by TNF-α by targeting TRAF2 and activating NF-κB.

Roles of miR-182 in Intervertebral Disc Degeneration and its Potential Prognostic Value

MicroRNAs were reported to be involved in the progression of intervertebral disc degeneration (IDD). This study focused on the potential prognostic value and the underlying mechanism of miR-182 in IDD. The expression level of miR-182 in plasma samples from 60 IDD patients and 60 healthy controls were examined in the present study. Then, the relationship between miR-182 expression and clinical features of IDD patients was analyzed. Moreover, the nucleus pulposus (NP) cells were cultured and transfected with either miR-182 inhibitor, mimics, or NC to explore the effects of miR-182 on cell proliferation and apoptosis. Furthermore, expression levels of proliferation and apoptosis-related proteins Bcl-2, Bax, and Caspase-3 were also evaluated. The expression level of miR-182 was dramatically increased in plasma samples of IDD patients compared with the controls. Moreover, ROC analysis indicated that miR-182 was a feasible diagnostic indicator for the diagnosis of IDD. According to the Japanese Orthopaedic Association (JOA) score, the prognosis of patients with the lower expression levels of miR-182 was better than for those with the higher expression levels of miR-182 in IDD. Furthermore, the miR-182 inhibitor significantly increased the proliferation, decreased the apoptosis of human NP cells, and altered the expression of proliferation and apoptosis-related proteins Bcl-2, Bax, and Caspase-3. On the contrary, miR-182 mimics notably inhibited proliferation but promoted apoptosis of human NP cells and increased Bax and Caspase-3 expressions while reducing the Bcl-2 level. miR-182 was negatively correlated with the prognosis of the IDD patients and affected the proliferation and apoptosis of NP cells in vitro.

Long Noncoding RNA ANPODRT Overexpression Protects Nucleus Pulposus Cells from Oxidative Stress and Apoptosis by Activating Keap1-Nrf2 Signaling.

Oxidative stress and subsequent nucleus pulposus (NP) cell apoptosis are important contributors to the development of intervertebral disc degeneration (IDD). Emerging evidences show that long noncoding RNAs (lncRNAs) play a role in the pathogenesis of IDD. In this study, we investigated the role of lncRNA ANPODRT (anti-NP cell oxidative damage-related transcript) in oxidative stress and apoptosis in human NP cells. We found that ANPODRT was downregulated in degenerative NP tissues and in NP cells treated with tert-butyl hydroperoxide (TBHP, the oxidative stress inducer). ANPODRT overexpression alleviated oxidative stress and apoptosis in NP cells exposed to TBHP, while ANPODRT knockdown exerted opposing effects. Mechanistically, ANPODRT facilitated nuclear factor E2-related factor 2 (Nrf2) accumulation and nuclear translocation and activated its target genes by disrupting the kelch-like ECH-associated protein 1- (Keap1-) Nrf2 association in NP cells. Nrf2 knockdown abolished the antioxidative stress and antiapoptotic effects of ANPODRT in NP cells treated with TBHP. Collectively, our findings suggest that ANPODRT protects NP cells from oxidative stress and apoptosis, at least partially, by activating Nrf2 signaling, implying that ANPODRT may be a potential therapeutic target for IDD.

Mechanosensitive Ion Channel Piezo1 Activated by Matrix Stiffness Regulates Oxidative Stress-Induced Senescence and Apoptosis in Human Intervertebral Disc Degeneration.

Mechanical stimulation plays a crucial part in the development of intervertebral disc degeneration (IDD). Extracellular matrix (ECM) stiffness, which is a crucial mechanical microenvironment of the nucleus pulposus (NP) tissue, contributes to the pathogenesis of IDD. The mechanosensitive ion channel Piezo1 mediates mechanical transduction. This study purposed to investigate the function of Piezo1 in human NP cells under ECM stiffness. The expression of Piezo1 and the ECM elasticity modulus increased in degenerative NP tissues. Stiff ECM activated the Piezo1 channel and increased intracellular Ca2+ levels. Moreover, the activation of Piezo1 increased intracellular reactive oxygen species (ROS) levels and the expression of GRP78 and CHOP, which contribute to oxidative stress and endoplasmic reticulum (ER) stress. Furthermore, stiff ECM aggravated oxidative stress-induced senescence and apoptosis in human NP cells. Piezo1 inhibition alleviated oxidative stress-induced senescence and apoptosis, caused by the increase in ECM stiffness. Finally, Piezo1 silencing ameliorated IDD in an in vivo rat model and decreased the elasticity modulus of rat NP tissues. In conclusion, we identified the mechanosensitive ion channel Piezo1 in human NP cells as a mechanical transduction mediator for stiff ECM stimulation. Our results provide novel insights into the mechanism of mechanical transduction in NP cells, with potential for treating IDD.

The REDD1/TXNIP Complex Accelerates Oxidative Stress‐Induced Apoptosis of Nucleus Pulposus Cells through the Mitochondrial Pathway

The death of nucleus pulposus (NP) cells is an important cause of intervertebral disc (IVD) degeneration. Redox disturbance caused by dysfunctional mitochondria has been considered as a vital risk for NP cell survival. It is valuable to identify key proteins maintaining mitochondrial function in NP cells. A previous study found that regulated in development and DNA damage response 1 (REDD1) are upregulated during intervertebral disc degeneration and that REDD1 can cause NP cell apoptosis. Thus, the present study further explores the effect of REDD1 on IVD degeneration. Our results showed that REDD1 promotes NP cell apoptosis via the mitochondrial pathway. Importantly, REDD1 formed a complex with TXNIP to strengthen its own action, and the combination was consolidated under H2O2-induced oxidative stress. The combined inhibition of the REDD1/TXNIP complex was better than that of REDD1 or TXNIP alone in restoring cell proliferation and accelerating apoptosis. Moreover, p53 acts as the transcription factor of REDD1 to regulate the REDD1/TXNIP complex under oxidative stress. Altogether, our results demonstrated that the REDD1/TXNIP complex mediated H2O2-induced human NP cell apoptosis and IVD degeneration through the mitochondrial pathway. Interferences on these sites to achieve mitochondrial redox homeostasis may be a novel therapeutic strategy for oxidative stress-associated IVD degeneration.

Allicin Attenuated Advanced Oxidation Protein Product-Induced Oxidative Stress and Mitochondrial Apoptosis in Human Nucleus Pulposus Cells.

Intervertebral disc degeneration (IDD) is one of the most common chronic degenerative musculoskeletal disorders. Oxidative stress-induced apoptosis of the nucleus pulposus (NP) cells plays a key role during IDD progression. Advanced oxidation protein products (AOPP), novel biomarkers of oxidative stress, have been reported to function in various diseases due to their potential for disrupting the redox balance. The current study is aimed at investigating the function of AOPP in the oxidative stress-induced apoptosis of human NP cells and the alleviative effects of allicin during this process which was known for its antioxidant properties. AOPP were demonstrated to hamper the viability and proliferation of NP cells in a time- and concentration-dependent manner and cause cell apoptosis markedly. High levels of reactive oxygen species (ROS) and lipid peroxidation product malondialdehyde (MDA) were detected in NP cells after AOPP stimulation, which resulted in depolarized mitochondrial transmembrane potential (MTP). Correspondingly, higher levels of AOPP were discovered in the human degenerative intervertebral discs (IVD). It was also found that allicin could protect NP cells against AOPP-mediated oxidative stress and mitochondrial dysfunction via suppressing the p38-MAPK pathway. These results disclosed a significant role of AOPP in the oxidative stress-induced apoptosis of NP cells, which could be involved in the primary pathogenesis of IDD. It was also revealed that allicin could be a promising therapeutic approach against AOPP-mediated oxidative stress during IDD progression.

Silencing of Long Non-coding RNA NEAT1 Upregulates miR-195a to Attenuate Intervertebral Disk Degeneration via the BAX/BAK Pathway.

Background/AimsAn increasing body of evidence has demonstrated that long non-coding RNAs (lncRNAs) play a vital regulatory role in intervertebral disk degeneration (IVDD). Nucleus enriched abundant transcript 1 (NEAT1), a novel cancer-related lncRNA, is associated with many malignancies, including ovarian cancer, and esophageal squamous cell carcinoma. Nevertheless, the role of NEAT1 in the progression of IVDD remains to be studied. Here, we explored the effect of NEAT1 on the progression of IVDD and the mechanisms involved.MethodsAn IVDD model was constructed in SD rats in vivo, and degeneration was induced by advanced glycation end product (AGE) in human nucleus pulposus cells (HNPC) in vitro. Quantitative real-time PCR was performed to detect the relative NEAT1 and miR-195a expressions and further confirmed the relationship between NEAT1 and miR-195a. Cell apoptosis was evaluated by TUNEL assay. The related mechanisms were explored by Western blot assay.ResultsThe relative NEAT1 expression was significantly upregulated in the IVDD rat model and the denatured HNPC. Silencing of NEAT1 expression in HNPC significantly promoted the Collagen II and TIMP-1 expression induced by AGE while greatly suppressing the expressions of MMP-3 and cleaved caspase-3. Besides, downregulation of NEAT1 obviously reversed the AGE-induced apoptosis in HNPC. More interestingly, these effects of NEAT1 knockout on HNPC were largely reversed by silencing of miR-195a or overexpression of BAX under the AGE treatment. Mechanically, the direct combination of NEAT1 with miR-195a resulted in upregulation of MMP-3, cleaved caspase-3, BAX, and BAK, as well as downregulation of Collagen II and TIMP-1, which are associated with EMT and apoptosis. We also demonstrated similar results in the in vivo experiments.ConclusionNEAT1 played its role in IVDD progression via partly by mediating the miR-195 expression and might be used as a potential target for IVDD therapy.

Marein protects human nucleus pulposus cells against high glucose-induced injury and extracellular matrix degradation at least partly by inhibition of ROS/NF-κB pathway

Intervertebral disc degeneration (IDD), a major cause of discogenic low back pain, is a musculoskeletal disorder involving the apoptosis of nucleus pulposus cells (NPCs) and extracellular matrix (ECM) degradation. Marein is a major active flavonoid ingredient extracted from the hypoglycemic plant Coreopsis tinctoria with several beneficial biological activities including anti-diabetic effects. Nevertheless, there are no reports concerning the effects of marein on IDD. Our study aimed to evaluate the effects of marein on high glucose (HG)-induced injury and ECM degradation in human NPCs (HNPCs). CCK-8 assay was applied to evaluate cell viability. Flow cytometry analysis, a cell death detection ELISA, and caspase-3 activity assay were used to assess apoptosis. The mRNA expression of ECM-related proteins matrix metalloproteinase (MMP)-3, MMP-13, Collagen II, and aggrecan were determined by qRT-PCR. The changes of the nuclear factor-kappa B (NF-κB) pathway were examined by western blot. Stimulation with HG significantly reduced cell viability and induced apoptosis in HNPCs. Moreover, HG exposure increased MMP-3 and MMP-13 expression and decreased Collagen II and aggrecan expression in HNPCs. Notably, marein effectively alleviated HG-induced viability reduction, apoptosis and ECM degradation in HNPCs. We also found that marein inhibited HG-induced ROS generation and NF-κB activation in HNPCs. Inhibition of NF-κB pathway reinforced HG-induced injury and ECM degradation in HNPCs. In summary, marein protected HNPCs against HG-induced injury and ECM degradation at least partly by inhibiting the ROS/NF-κB pathway.

Restoration of Autophagic Flux Rescues Oxidative Damage and Mitochondrial Dysfunction to Protect against Intervertebral Disc Degeneration.

Oxidative stress-induced mitochondrial dysfunction and nucleus pulposus (NP) cell apoptosis play crucial roles in the development of intervertebral disc degeneration (IDD). Increasing studies have shown that interventions targeting impaired autophagic flux can maintain cellular homeostasis by relieving oxidative damage. Here, we investigated the effect of curcumin (CUR), a known autophagy activator, on IDD in vitro and in vivo. CUR suppressed tert-butyl hydroperoxide- (TBHP-) induced oxidative stress and mitochondrial dysfunction and thereby inhibited human NP cell apoptosis, senescence, and ECM degradation. CUR treatment induced autophagy and enhanced autophagic flux in an AMPK/mTOR/ULK1-dependent manner. Notably, CUR alleviated TBHP-induced interruption of autophagosome-lysosome fusion and impairment of lysosomal function and thus contributed to the restoration of blocked autophagic clearance. These protective effects of CUR in TBHP-stimulated human NP cells resembled the effects produced by the autophagy inducer rapamycin, but the effects were partially eliminated by 3-methyladenine- and compound C-mediated inhibition of autophagy initiation or chloroquine-mediated obstruction of autophagic flux. Lastly, CUR also exerted a protective effect against puncture-induced IDD progression in vivo. Our results showed that suppression of excessive ROS production and mitochondrial dysfunction through enhancement of autophagy coupled with restoration of autophagic flux ameliorated TBHP-induced human NP cell apoptosis, senescence, and ECM degradation. Thus, maintenance of the proper functioning of autophagy represents a promising therapeutic strategy for IDD, and CUR might serve as an effective therapeutic agent for IDD.

Liraglutide inhibits the apoptosis of human nucleus pulposus cells induced by high glucose through PI3K/Akt/caspase-3 signaling pathway.

Diabetes mellitus (DM) is a potential etiology of disc degeneration. Glucagon-like peptide-1 (GLP-1) is currently regarded as a powerful treatment option for type 2 diabetes. Apart from the beneficial effects on glycaemic control, GLP-1 has been reported to exert functions in a variety of tissues on modulation of cell proliferation, differentiation, and apoptosis. However, little is known regarding the effects of GLP-1 on nucleus pulposus cells (NPCs). In the present study, we investigated the effects of liraglutide (LIR), a long-lasting GLP-1 analogue, on apoptosis of human NPCs and the underlying mechanisms involved. We confirmed the presence of GLP-1 receptor (GLP-1R) in NPCs. Our data demonstrated that liraglutide inhibited the apoptosis of NPCs induced by high glucose (HG), as detected by Annexin V/Propidium Iodide (PI) and ELISA assays. Moreover, liraglutide down-regulated caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further analysis suggested that liraglutide suppressed reactive oxygen species (ROS) generation and stimulated the phosphorylation of Akt under HG condition. Pretreatment of cells with the Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY) and small interfering RNAs (siRNAs) GLP-1R abrogated the liraglutide-induced activation of Akt and the protective effects on NPCs’ apoptosis. In conclusion, liraglutide could directly protect NPCs against HG-induced apoptosis by inhibiting oxidative stress and activate the PI3K/Akt/caspase-3 signaling pathway via GLP-1R.

TNF-α induces apoptosis of human nucleus pulposus cells via activating the TRIM14/NF-κB signalling pathway

Cervical spondylosis is a degenerative disease commonly found in older adults and characterized by progressive osteophyte formation and disc collapse. Apoptosis in nucleus pulposus (NP) cells which induced by TNF-α has been widely known to associate with the disc degeneration. However, the exactly underlying molecular mechanism was still unclear. The aim of our study was to investigate whether TRIM14/NF-κB signalling pathway is associated with the apoptosis of human NP cells (HNPC) induced by TNF-α. Our data demonstrated that TNF-α treatment obviously decreased the cell viability, induced apoptosis and increased TRIM14 expression and NF-κBp65 activity in HNPC in a dose-dependent manner. Down-regulation of TRIM14 or NF-κB inhibitor PDTC treatment significantly inhibited cell apoptosis, Bax/Bcl-2 ratio and NF-κBp65 activation induced by TNF-α in HNPC. Meanwhile, up-regulation of TRIM14 obviously increased cell apoptosis, Bax/Bcl-2 ratio and NF-κBp65 activation in HNPC. Then, we found that the protein PPM1A was identified as a binding partner of TRIM14 and ubiquitinated by TRIM14. These findings provide insights into the function of TRIM14 and NF-κB signalling and might, therefore, represent a novel therapeutic target for treatment of cervical spondylosis.

The effect of mechanical stretching stress on the proliferation, apoptosis of degenerative disc nucleus pulposus cells and expression of extracellular matrix

Objective To observe in vitro the effect of mechanical stress at different intensities on the proliferation, apoptosis and extracellular matrix of degenerative human nucleus pulposus cells. Methods The cells were isolated and cultured in vitro, and divided into a control group, a low-intensity group, a medium-intensity group and a high-intensity group. The low-, medium- and high-intensity groups were stretched mechanically by 1000 μ, 2000 μ or 4000 μ respectively for 6 hours using a four-point bending system, while the control group was not stressed. Flow cytometry was used to explore any changes in the cell cycle and the proliferation index (PI). The expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (BCL-2)/Bax, collagen II and aggrecan were measured using real-time quantitative polymerase chain reactions. Results The mechanical stretching significantly influenced proliferation, apoptosis and the extracellular matrices compared with the control group. The PI and PCNA expression increased at first and then decreased gradually with the exercise intensity. Compared with the control group, the mRNA expression level of Bcl-2/Bax increased significantly to 1.53 times that of the control group after 1000 μ stretching, but to only 0.71 times that of the control group at 2000 μ and 0.43 times at 4000 μ. The gene expression of collagen II increased significantly by 1.1 times and that of aggrecan by 1.3 times after 1000 μ stress stimulation compared with the control group (P≤0.05). However, the expression of collagen II and aggrecan was inhibited significantly at 2000 and 4000 μ , with the lowest levels at 4000 μ (P≤0.05). Conclusion Stretching at different intensities has different effects on the proliferation, apoptosis and extracellular matrix of human pulposus cells with degenerate nuclei. Key words: Mechanical stress; Stretching; Degenerate nuclei; Pulposus cells; Proliferation; Apoptosis; Extracellular matrices

Upregulation of miR-199 attenuates TNF-α-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5

MicroRNA-199 has been reported to play a potential role in the apoptosis of Human nucleus pulposus cells. However, the effect of miR-199 in regulating Human nucleus pulposus cell injury induced by TNF-α has not been previously illustrated. This study searched to probe the effect and the molecular mechanism of miR-199 on Human nucleus pulposus cell injury induced by TNF-α. Using the TNF-α model of Human nucleus pulposus cell in vitro, we found that miR-199 was extremely decreased in Human nucleus pulposus cells after TNF-α treatment. Knockdown the expression of miR-199 by recombinant adeno-associated viral vector infection markedly promoted the apoptosis of Human nucleus pulposus cells induced by TNF-α treatment, whereas miR-199 overexpression significantly decreased Human nucleus pulposus cell apoptosis. Both Dual-luciferase reporter and western blot assay proved that MAP3K5 was a direct target gene of miR-199, and miR-199 inhibited the expression of MAP3K5 via binding to its 3′-UTR. Furthermore, we proved that overexpression of miR-199 could inhibit the expression of MAP3K5 at the transcription and translation levels, whereas the inhibition of miR-199 could upregulate the expression of MAP3K5. Moreover, MAP3K5 was highly expressed in TNF-α treated Human nucleus pulposus cells and the apoptosis rate induced by TNF-α was associated with the increase in MAP3K5 expression. Importantly, knockdown the expression of MAP3K5 apparently abrogated the inhibitory effect of miR-199 mimics on TNF-α induced Human nucleus pulposus cell apoptosis. In conclusion, these results indicate that upregulation of miR-199 could inhibit Human nucleus pulposus cells injury through downregulation of MAP3K5 expression, providing an important molecular target mechanism for Human nucleus pulposus cells injury.