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It has been reported that microRNA (miRNA/miR)‑25 is downregulated in patients with intervertebral disc degeneration (IVDD). However, the potential role of miR‑25 in IVDD remains unclear. Therefore, the present study aimed to investigate the effects of miR‑25 on human intervertebral disc nucleus pulposus cells (NPCs). The expression levels of miR‑25 and those of small ubiquitin‑related modifier 2 (SUMO2) were determined in human nucleus pulposus (NP) tissues by reverse transcription‑quantitative PCR (RT‑qPCR) and western blot analyses. Subsequently, the potential interaction between miR‑25 and SUMO2 was validated via dual‑luciferase reporter assay and RNA pull‑down assay with biotinylated miRNA. The effects of miR‑25 on NPC proliferation and apoptosis were evaluated using Cell Counting Kit‑8 assay, 5‑ethynyl‑2'‑deoxyuridine incorporation assay, and flow cytometry. The results showed that miR‑25 was downregulated in patients with IVDD. In addition, miR‑25 increased the proliferation of NPCs and inhibited their apoptosis. Furthermore, the current study verified that miR‑25 could directly target SUMO2 and regulate its expression via the p53 signaling pathway. Additionally, the effects of miR‑25 on NPCs were abrogated following SUMO2 overexpression. Overall, the results of the present study demonstrated that miR‑25 could promote the proliferation and inhibit the apoptosis of NPCs via targeting SUMO2, suggesting that miR‑25 may be a potential target in the treatment of IVDD.

Apoptosis of nucleus pulposus (NP) cells has an important role in the process of intervertebral disc degeneration (IDD), and the search for novel compounds to prevent apoptosis from occurring is urgently required. In the present study, syringic acid (SyrA) was found to exhibit no cytotoxicity on NP cells, and was able to reverse the cytotoxicity, as well as the abnormal expression of Bcl‑2 and caspase‑3, that were induced by lipopolysaccharide (LPS). The transcriptomes of each group were then analyzed using RNA‑Seq. A total of 65 differentially expressed genes (DEGs) were identified in LPS‑stimulated groups (LPS group vs. control group), 819 DEGs were identified in the SyrA‑reversed groups (SyrA plus LPS group vs. LPS group), and a further 25 DEGs were identified in the SyrA plus LPS group compared with the control group. Reverse transcription‑quantitative PCR validation indicated that the alterations in expression of uroplakin 3B‑like 1 (UPK3BL1), voltage‑dependent calcium channel subunit α‑2/δ‑1 (CACNA2D1) and polo‑like kinase 4 (PLK4) were consistent with the corresponding results of RNA‑Seq, and that these genes were involved in both LPS‑stimulation and SyrA‑reversion processes. Kyoto Encyclopedia of Genes and Genomes analyses indicated that the DEGs in SyrA‑reversed groups were involved in, amongst other pathways, 'Autophagy‑other' and 'Apoptosis‑multiple species'. In conclusion, the addition of SyrA to the NP cells co‑incubated with LPS appeared to help prevent the abnormal expression of mRNAs and apoptosis that had been identified in NP cells incubated with LPS alone. The potential mechanism underlying the reversion of SyrA might be attributed to the regulation of CACNA2D1 and PLK4.

The incidence of intervertebral disc degeneration (IDD) is increasing, especially among elderly individuals. The present study aimed to investigate the effects of the NF‑κB/p53 signaling pathway on IDD and its regulatory effect on associated cytokines. In the present study, human nucleus pulposus cells were isolated from patients with thoracic‑lumbar fractures and patients with IDD to observe cellular morphology and detect phosphorylated (p)‑p65/p53 expression levels. The locality and expression levels of p65 in interleukin (IL)‑1β‑stimulated nucleus pulposus cells, with or without the addition of ammonium pyrrolidinedithiocarbamate (PDTC; a NF‑κB signaling pathway‑specific blocker), were measured. Furthermore, the effects of IL‑1β stimulation on the protein and gene expression levels of IDD‑related cytokines were determined following p53 knockdown and inhibition of the NF‑κB signaling pathway. The results suggested that p‑p65 and p53 expression was significantly increased in IDD cells compared with normal nucleus pulposus cells. Moreover, nucleus pulposus cells isolated from patients with IDD contained less cytoplasm compared with normal nucleus pulposus cells, and p65 expression levels were higher in the cytoplasm than the nucleus of IL‑1β‑stimulated PDTC‑treated healthy nucleus pulposus cells. Moreover, the p53 expression levels were significantly decreased following transfection with sip53. PDTC treatment and p53 knockdown significantly decreased matrix metallopeptidase (MMP)‑3, MMP‑13, metallopeptidases with thrombospondin type 1 motif (ADAMTS)‑4 and ADAMTS‑5 expression levels, and increased aggrecan and collagen type II expression levels in IL‑1β‑stimulated cells. The present study indicated that activation of the NF‑κB/p53 signaling pathway might be related to the occurrence of IDD; therefore, the NF‑κB/p53 signaling pathway may serve as a therapeutic target for IDD.

Intervertebral disc degeneration (IVDD) is the main pathological basis of spinal degenerative diseases, and aberrant apoptosis of nucleus pulposus cells (NPCs) is the main cellular process that causes IVDD. In our previous studies, 17β‑estradiol (E2) was demonstrated to protect rat NPCs from interleukin‑1β (IL‑1β)‑induced apoptosis via the PI3K/Akt signaling pathway. However, the downstream signaling pathway of PI3K/Akt is currently unclear. The present study aimed to explore the signaling pathways that are downstream of the PI3K/Akt pathway, including mTOR, NF‑κB and glycogen synthase kinase‑3β (GSK‑3β). Annexin V/propidium iodide double staining was used to determine the incidence of apoptosis. Cell Counting kit‑8 and MTS assays were used to determine the proliferation and viability of NPCs, respectively. Cellular binding was evaluated using a cell‑collagen binding assay. Western blotting was used to determine the protein expression levels of mTOR, NF‑κB and GSK‑3β, and their phosphorylation levels, as well as the expression levels of active caspase‑3. The results revealed that IL‑1β induced NPC apoptosis and increased the early apoptotic rate of NPCs. However, E2 reduced the early apoptosis of NPCs induced by IL‑1β. In addition, E2 suppressed the decrease in cell viability and binding ability caused by IL‑1β cytotoxicity. Western blotting revealed that E2 also reduced the expression of activated caspase‑3, and increased the expression of activated mTOR. As a specific inhibitor of mTOR, rapamycin effectively attenuated the effects of E2. These findings indicated that E2 protected NPCs against apoptosis via activation of the mTOR/caspase‑3 pathway.

It has been reported that bupivacaine, the most widely used local anesthetic to relieve discogenic back pain in clinical settings, is cytotoxic to intervertebral disc (IVD) cells in vitro; however, the precise mechanisms of cytotoxicity induced by bupivacaine remain unclear. Autophagy is an intracellular lysosomal degradation process that is important for cellular survival. The present study investigated the role of autophagy in the survival of IVD cells subjected to bupivacaine treatment. Human nucleus pulposus (NP) cells isolated from IVD cells were exposed to various concentrations of bupivacaine for 2, 6 and 12 h, and analyzed for cellular viability using MTT assay and western blotting. Additionally, autophagosome formation and autophagy‑associated biomarkers were evaluated by electron microscopy and western blotting to determine the autophagic activity and signaling alterations in NP cells under bupivacaine treatment. Furthermore, autophagic activity was inhibited in vitro using 3‑methyladenine to further analyze the association between autophagy and apoptosis in bupivacaine‑treated NP cells. Bupivacaine exhibited time‑ and dose‑dependent cytotoxic effects on human IVD cells at clinically relevant concentrations. Bupivacaine increased autophagic activity by promoting autophagosome formation, and LC3‑II and Beclin‑1 production. Additionally, bupivacaine inhibited protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/S6 kinase (S6K) signaling, which is a negative regulator of autophagic activity. Of note, pharmacological inhibition of autophagy alleviated bupivacaine‑induced cytotoxicity of IVD cells. The findings indicated that application of clinically relevant concentrations of bupivacaine upregulated autophagic activity via inhibition of Akt/mTOR/S6K signaling. In addition, the inhibition of autophagic activation served as a protective mechanism against bupivacaine‑induced cytotoxicity. Collectively, these findings may provide novel insight into the mechanisms underlying cytotoxicity induced by bupivacaine when controlling spine‑associated pain.

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 in vitro 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.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine. The present study aimed to determine the expression and effects of LIF on nucleus pulposus generation. Degenerated nucleus pulposus samples were obtained from animal models and patients with lumbar intervertebral disc herniation. Degradation scores of intervertebral discs were evaluated via magnetic resonance imaging (MRI) and histology, and the protein expression levels of LIF were detected. Furthermore, cultured primary human degenerated nucleus pulposus cells (DNPCs) were stimulated with various concentrations of recombinant human LIF protein (rhLIF), and aggrecan and collagen type II α1 (COL2α1) protein expression levels were detected by western blotting. In addition, aggrecan expression was determined by toluidine blue staining. The effects of rhLIF on proliferation and apoptosis of DNPCs were evaluated by Cell Counting Kit‑8 and flow cytometry, respectively. The results revealed that the degradation scores of intervertebral discs were significantly associated with modeling time, as determined by MRI and histology. In addition, the protein expression levels of LIF were initially increased in patients with lumbar disc herniation and in rabbit models, particularly in the 2‑week modeling group; however, its expression decreased with the progression of disc degeneration. Notably, LIF expression in each modeling group was higher than that in the control and 0 week modeling group. The in vitro study revealed that the protein expression levels of aggrecan and COL2α1 were significantly increased in response to rhLIF, in a dose‑dependent manner, and statistical differences were identified between the treatment groups and control group. The results of toluidine blue staining were consistent with this finding. Although rhLIF had no effect on proliferation, it inhibited apoptosis of DNPCs in a concentration‑dependent manner. In conclusion, LIF was upregulated during the process of intervertebral disc degeneration, and may promote the expression of extracellular matrix components. It may also be hypothesized that LIF acts as a potential protective factor by inhibiting apoptosis of DNPCs without affecting cell proliferation.

The molecular mechanisms underlying protection and pathogenesis in spinal degenerative diseases remain unclear. Tumor necrosis factor-α (TNF-α) has been demonstrated to induce apoptosis of inte rvertebral disc (IVD) cells during IVD degeneration, and 17β‑estradiol (17β‑E2) has a protective effect against IVD cell apoptosis. However, the underlying molecular mechanism by which 17β‑E2 protects nucleus pulposus (NP) cells remains to be investigated. The aim of the present study was to evaluate whether 17β‑E2 modulates apoptosis of human NP cells induced by TNF‑α. In addition, the concentration‑response effect of 17β‑E2 on human NP cells was investigated. Human NP cells were cultured in complete medium, which was replaced every three days until the culture was ~80% confluent. Cells were treated with 100 ng/ml TNF‑α for 48 h, with or without pretreatment with various concentrations of 17β‑E2, and ICI 182,780, for 30 min. Morphologic alterations characteristic of apoptosis were observed by inverted phase‑contrast microscopy and Hoechst 33258 staining; the apoptosis rate was analyzed by flow cytometry. A Cell Counting kit‑8 assay was used to assess cell proliferation. Furthermore, caspase‑3 activity was determined and proteins associated with apoptosis were analyzed by western blotting. The level of apoptosis and caspase‑3 activity in human NP cells increased, whereas proliferation and the expression of poly ADP‑ribose polymerase decreased following TNF‑α treatment. These effects of TNF‑α were abolished by pretreatment with 17β‑E2 in a concentration‑dependent manner. The results of the present study indicated that 17β‑E2 serves a critical role in the survival of degenerative human NP cells.