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Background: During disc degeneration, inflammatory cytokine tumor necrosis factor (TNF)-α is correlated with nucleus pulposus (NP) cell apoptosis. Transforming growth factor (TGF)-β1 has the potential to regenerate degenerative disc., Objective: To investigate the protective role of TGF-β1 against TNF-α-mediated NP cell apoptosis and the underlying mechanism., Methods: Rat NP cells were treated with TNF-α (100 ng/ml) for 48 h. TGF-β1 was added into the culture medium to investigate its protective effects against TNF-α-induced NP cell apoptosis. Exogenous FasL was used to investigate the potential role of the Fas/FasL pathway in this process. Flow cytometry assay was used to analyze NP cell apoptosis. Real-time PCR and Western blotting were used to analyze gene and protein expression of apoptosis-related molecules., Results: In TNF-α-treated NP cells, TGF-β1 significantly decreased NP cell apoptosis, declined caspase-3 and -8 activity, and decreased expression of Bax and caspase-3 (cleaved-caspase-3) but increased expression of Bcl-2. However, exogenous FasL partly reversed these effects of TGF-β1 in NP cells treated with TNF-α. Additionally, expression of Fas and FasL in TNF-α-treated NP cells partly decreased by TGF-β1, whereas exogenous FasL increased expression of Fas and FasL in NP cells treated with TGF-β1 and TNF-α., Conclusion: TGF-β1 helps to inhibit TNF-α-induced NP cell apoptosis and the Fas/FasL pathway may be involved in this process. The present study suggests that TGF-β1 may be effective to retard inflammation-mediated disc degeneration., (© 2020 The Author(s).)

The present study was designed to investigate the protective effect of moracin on primary culture of nucleus pulposus cells in intervertebral disc and explore the underlying mechanism. Moracin treatment significantly inhibited the LPS-induced inflammatory cytokine accumulation (IL-1β, IL-6 and TNF-α) in nucleus pulposus cells. And moracin also dramatically decreased MDA activity, and increased the levels of SOD and CAT induced by LPS challenge. Moreover, the expressions of Nrf-2 and HO-1 were decreased and the protein levels of p-NF-κBp65, p-IκBα, p-smad-3 and TGF-β were increased by LPS challenge, which were significantly reversed after moracin treatments. Moracin treatments also decreased the levels of matrix degradation enzymes (MMP-3, MMP-13) as indicated by RT-PCR analysis. However, Nrf-2 knockdown abolished these protective effects of moracin. Together, our results demonstrated the ability of moracin to antagonize LPS-mediated inflammation in primary culture of nucleus pulposus in intervertebral disc by partly regulating the Nrf2/HO-1 and NF-κB/TGF-β pathway in nucleus pulposus cells., (© 2019 The Author(s).)

Nucleus pulposus (NP) cell senescence is involved in disc degeneration. The in situ osmolarity within the NP region is an important regulator of disc cell's biology. However, its effects on NP cell senescence remain unclear. The present study was aimed to investigate the effects and mechanism of hyper-osmolarity on NP cell senescence. Rat NP cells were cultured in the in situ -osmolarity medium and hyper-osmolarity medium. The reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) was added along with the medium to investigate the role of oxidative injury. Cell cycle, cell proliferation, senescence associated β-galactosidase (SA-β-Gal) activity, telomerase activity, expression of senescence markers (p16 and p53) and matrix molecules (aggrecan and collagen II) were tested to assess NP cell senescence. Compared with the in situ -osmolarity culture, hyper-osmolarity culture significantly decreased cell proliferation and telomerase activity, increased SA-β-Gal activity and cell fraction in the G 0 /G 1 phase, up-regulated expression of senescence markers (p16 and p53) and down-regulated expression of matrix molecules (aggrecan and collagen II), and increased intracellular ROS accumulation. However, addition of NAC partly reversed these effects of hyper-osmolarity culture on cellular senescence and decreased ROS content in NP cells. In conclusion, a hyper-osmolarity culture promotes NP cell senescence through inducing oxidative stress injury. The present study provides new knowledge on NP cell senescence and helps us to better understand the mechanism of disc degeneration., (© 2019 The Author(s).)

Intervertebral disc degeneration (IDD) is a natural progression of the aging process associated with inflammation. Higenamine, a plant-based alkaloid, has been identified to possess various pharmacological properties, including anti-inflammatory activity. In the present study, we aimed to evaluate the role of higenamine in interleukin (IL)-1β-induced inflammation in human nucleus pulposus cells (NPCs). The results showed that higenamine improved cell viability in IL-1β-induced NPCs. The IL-1β-dependent up-regulation of inflammatory molecules including inducible nitric oxide synthase (iNOS), nitric oxide (NO), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and IL-6 was attenuated by higenamine in NPCs. The increased productions of matrix metalloproteinases (MMP-3 and MMP-13), as well as a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-4 and ADAMTS-5) were significantly mitigated by higenamine treatment. Furthermore, we also found that higenamine suppressed the IL-1β-induced activation of NF-κB signaling pathway in NPCs. In conclusion, the present study proved that higenamine exhibited anti-inflammatory activity against IL-1β-induced inflammation in NPCs via inhibiting NF-κB signaling pathway. These results suggested that higenamine might be a therapeutic agent for the treatment of IDD., (© 2019 The Author(s).)

Mechanical overload is a risk factor of disc degeneration. It can induce disc degeneration through mediating cell apoptosis. Mechano growth factor (MGF) has been reported to inhibit mechanical overload-induced apoptosis of chondrocytes. The present study is aimed to investigate whether MGF can attenuate mechanical overload-induced nucleus pulposus (NP) cell apoptosis and the possible signaling transduction pathway. Rat NP cells were cultured and subjected to mechanical overload for 7 days. The control NP cells did not experience mechanical load. The exogenous MGF peptide was added into the culture medium to investigate its protective effects. NP cell apoptosis ratio, caspase-3 activity, gene expression of Bcl-2, Bax and caspase-3, protein expression of cleaved caspase-3, cleaved PARP, Bax and Bcl-2 were analyzed to evaluate NP cell apoptosis. In addition, activity of the p38 MAPK pathway was also detected. Compared with the control NP cells, mechanical overload significantly increased NP cell apoptosis and caspase-3 activity, up-regulated gene/protein expression of pro-apoptosis molecules (i.e. Bax, caspase-3, cleaved caspase-3 and cleaved PARP) whereas down-regulated gene/protein expression of anti-apoptosis molecule (i.e. Bcl-2). However, exogenous MGF partly reversed these effects of mechanical overload on NP cell apoptosis. Further results showed that activity of the p38 MAPK pathway of NP cells cultured under mechanical overload was decreased by addition of MGF peptide. In conclusion, MGF is able to attenuate mechanical overload-induced NP cell apoptosis, and the p38 MAPK signaling pathway may be involved in this process. The present study provides that MGF supplementation may be a promising strategy to retard mechanical overload-induced disc degeneration., (© 2019 The Author(s).)

Nucleus pulposus (NP) cell apoptosis is a classical cellular character during intervertebral disc degeneration (IDD). Previous studies have shown that inflammatory cytokine-induced NP cell apoptosis plays an important role in disc degeneration. The present study was aimed to investigate whether resveratrol can suppress IL-1β-mediated NP cell apoptosis and the potential signal transduction pathway. Experimental rat NP cells were treated with culture medium containing IL-1β (20 ng/ml) for 7 days. Control NP cells were cultured in the baseline medium. Resveratrol was added along with culture medium to investigate its effects. The inhibitor LY294002 was used to study the role of the PI3K/Akt pathway. NP cell apoptosis was reflected by the caspase-3 activity, cell apoptosis ratio, and expression of apoptosis-related molecules (Bcl-2, Bax, caspase-3, cleaved caspase-3, and cleaved PARP). Compared with the control NP cells, IL-1β significantly increased caspase-3 activity, NP cell apoptosis ratio and mRNA/protein expression of Bax, caspase-3, cleaved caspase-3 and cleaved PARP, but decreased mRNA expression of Bcl-2. However, resveratrol partly suppressed the effects of IL-1β on those cell apoptosis-related parameters. Further analysis showed that IL-1β significantly decreased activity of the PI3K/Akt pathway whereas resveratrol partly increased activity of the PI3K/Akt pathway in NP cells treated with IL-1β. Additionally, when the inhibitor LY294002 was added along with the resveratrol, its protective effects against IL-1β-induced NP cell apoptosis were attenuated. In conclusion, resveratrol suppresses IL-1β-mediated NP cell apoptosis through activating the PI3K/Akt pathway. Resveratrol may be an effective drug to attenuate inflammatory cytokine-induced disc degenerative changes., (© 2019 The Author(s).)

Background: Allogeneic disc cell is the main cellular resource in tissue engineering (TE)-based strategy to retard disc degeneration. However, the accessible disc cells often exhibit senescent phenotype when they are subcultured in vitro Hence, alleviating senescence of human disc cells during cell subculture is important for TE-based strategy to regenerate degenerative disc tissue., Objective: The present study was aimed to investigate whether bone morphogenetic protein-7 (BMP-7) can alleviate subculture-induced senescence of human nucleus pulposus (NP) cells in vitro Methods: NP cells from human disc tissue were subcultured in vitro for six passages. Exogenous BMP-7 was added along with the culture medium to investigate its effects on senescence of NP cells. The inhibitor LY294002 was used to investigate the role of the PI3K/Akt pathway., Results: Compared with the human disc NP cells cultured in the baseline culture medium, addition of BMP-7 increased cell proliferation potency and telomerase activity, decreased senescence-associated β-galactosidase (SA-β-Gal) activity and G 0 /G 1 phase fraction, and down-regulated the expression of p16 and p53. Moreover, these positive effects of BMP-7 against senescence of human disc NP cells coincided with activation of the PI3K/Akt pathway. Further analysis showed that inhibitor LY294002 partly inhibited these protective effects of BMP-7 against senescence of human disc NP cells., Conclusion: BMP-7 alleviates subculture-induced senescence of human disc NP cells through activating the PI3K/Akt pathway. The present study provides new knowledge on allogeneic disc NP cell-based TE strategy to regenerate degenerative human disc tissue., (© 2019 The Author(s).)

Background: Nucleus pulposus (NP) cell senescence is an important cellular feature within the degenerative disc. It is known that a very acidic niche exists in the degenerative disc, which participates in regulating disc cell viability and matrix metabolism., Objective: The present study was aimed to investigate the role and potential signaling transduction pathway of an acidic pH in regulating NP cell senescence., Methods: Rat NP cells were cultured in an acidic pH of 7.2 close to that in a healthy disc (Control group) or in an acidic pH of 6.2 close to that in a severe degenerative disc (Experiment group) for 10 days. Additionally, the experimental NP cells were incubated along with the inhibitor SB203580 to analyze the role of p38 MAPK pathway in this process., Results: Compared with the control NP cells, experimental NP cells showed a suppressed cell proliferation potency, an increased G 0 /G 1 phase fraction whereas a decreased S-phase fraction and a declined telomerase activity, an up-regulated expression of senescence-related molecules (p16 and p53), and a down-regulated expression of matrix-related moleucles (aggrecan and collagen II). Further analysis showed that inhibition of the p38 MAPK pathway partly reversed effects of acidic pH of 6.2 on the experimental NP cells., Conclusion: The very acidic niche identified in a severe degenerative disc promotes NP cell senescence through regulating the p38 MAPK pathway. The present study provides a new mechanism that drives NP cell senescence during disc degeneration., (© 2018 The Author(s).)

Disc nucleus pulposus (NP) cell experiences periodic osmolarity alterations during daily activities, which has been proved to affect cell biology in vitro The present study was aimed to investigate the effects of cartilage-derived morphogenetic protein-2 (CDMP-2) on NP cell apoptosis under the hyperosmolarity culture and the potential mechanism. Isolated rat NP cells were cultured in the in situ -osmolarity medium or hyperosmolarity medium for 3 days. CDMP-2 was added into the hyperosmolarity medium to investigate its effects on NP cell apoptosis. Cell apoptosis rate, caspase-3 activity, gene expression of Bcl-2, Bax, and caspase-3, and protein expression of Bcl-2, Bax, and cleaved caspase-3 were analyzed to evaluate NP cell apoptosis. Additionally, the intracellular reactive oxygen species (ROS) and the total superoxide dismutase (SOD) activity were analyzed to investigate the potential role of oxidative damage in this process. In the hyperosmolarity culture, NP cells showed a significantly increased cell apoptosis rate and caspase-3 activity, an up-regulated expression of Bax and caspase-3/cleaved-caspase-3 and a down-regulated expression of Bcl-2. However, CDMP-2 partly inhibited these effects of hyperosmolarity culture on NP cells. Additionally, the hyperosmolarity culture significantly increased ROS content and decreased the total SOD activity compared with the in situ -osmolarity culture, whereas exogenous CDMP-2 partly decreased the ROS content and increased the total SOD activity in the hyperosmolarity culture. In conclusion, CDMP-2 is effective in attenuating hyperosmolarity environment-induced NP cell apoptosis, and this process may be mediated through inhibiting oxidative stress damage. The present study indicates that CDMP-2 may be helpful to retard hyperosmolarity niche-mediated disc degeneration., (© 2018 The Author(s).)

The decrease in nucleus pulposus (NP) matrix production is a classic feature during disc degeneration. Resveratrol (RSV) is reported to play protective effects under many pathological factors.The present study aims to study the effects of RSV on NP matrix homeostasis under oxidative damage and the potential mechanism. Rat NP cells were exposed to H 2 O 2 solution to create an oxidative damage. RSV and the 3-methyladenine (3-MA) were added along with the culture medium to respectively investigate the role of RSV and cellular autophagy. NP matrix synthesis was evaluated by the expression of macromolecules (aggrecan and collagen II) and glycosaminoglycan (GAG) content. Activation of cellular autophagy was assessed by the expression of several molecular markers. Additionally, activity of the PI3K/Akt pathway was also evaluated to study its potential role. Compared with the control group (NP cells treated with H 2 O 2 ), RSV significantly up-regulated expression of matrix macromolecules (aggrecan and collagen), promoted GAG production, and increased the expression of autophagy-related markers (Beclin-1 and LC-3). Further analysis showed that inhibition of autophagy by 3-MA partly attenuated NP matrix production. Additionally, RSV increased activity of the PI3K/Akt pathway compared with the control NP cells, but it was not affected by the addition of 3-MA. RSV plays a protective role in enhancing NP matrix synthesis under oxidative damage. Mechanistically, activation of the cellular autophagy via the PI3K/Akt pathway may participate in this process. RSV may be an effective drug to attenuate oxidative stress-induced disc degeneration., (© 2018 The Author(s).)

Mechanical overloading is a risk factor of disc degeneration. Studies have demonstrated that resveratrol helps to maintain the disc cell's healthy biology. The present study aims to investigate whether resveratrol can suppress mechanical overloading-induced nucleus pulposus (NP) cell senescence in vitro and the potential mechanism. The isolated rat NP cells were seeded in the decalcified bone matrix (DBM) and cultured under non-compression (control) and compression (20% deformation, 1.0 Hz, 6 h/day) for 5 days using the mechanically active bioreactor. The resveratrol (30 and 60 μM) was added into the culture medium of the compression group to investigate its protective effects against the NP cell senescence. NP cell senescence was evaluated by cell proliferation, cell cycle, senescence-associated β-galactosidase (SA-β-Gal) activity, telomerase (TE) activity, and gene expression of the senescence markers (p16 and p53). Additionally, the reactive oxygen species (ROS) content and activity of the NF-κB pathway were also analyzed. Compared with the non-compression group, the high-magnitude compression significantly promoted NP cell senescence, increased ROS generation and activity of the NF-κB pathway. However, resveratrol partly attenuated NP cell senescence, decreased ROS generation and activity of the NF-κB pathway in a concentration-dependent manner under mechanical compression. Resveratrol can alleviate mechanical overloading-induced NP cell senescence through regulating the ROS/NF-κB pathway. The present study provides that resveratrol may be a potential drug for retarding mechanical overloading-induced NP cell senescence., (© 2018 The Author(s).)

Disc nucleus pulposus (NP) matrix homeostasis is important for normal disc function. Mechanical overloading seriously decreases matrix synthesis and increases matrix degradation. The present study aims to investigate the effects of resveratrol on disc NP matrix homeostasis under a relatively high-magnitude mechanical compression and the potential mechanism underlying this process. Porcine discs were perfusion-cultured and subjected to a relatively high-magnitude mechanical compression (1.3 MPa at a frequency of 1.0 Hz for 2 h once per day) for 7 days in a mechanically active bioreactor. The non-compressed discs were used as controls. Resveratrol was added along with culture medium to observe the effects of resveratrol on NP matrix synthesis under mechanical load respectively. NP matrix synthesis was evaluated by histology, biochemical content (glycosaminoglycan (GAG) and hydroxyproline (HYP)), and expression of matrix macromolecules (aggrecan and collagen II). Results showed that this high-magnitude mechanical compression significantly decreased NP matrix content, indicated by the decreased staining intensity of Alcian Blue and biochemical content (GAG and HYP), and the down-regulated expression of NP matrix macromolecules (aggrecan and collagen II). Further analysis indicated that resveratrol partly stimulated NP matrix synthesis and increased activity of the PI3K/Akt pathway in a dose-dependent manner under mechanical compression. Together, resveratrol is beneficial for disc NP matrix synthesis under mechanical overloading, and the activation of the PI3K/Akt pathway may participate in this regulatory process. Resveratrol may be promising to regenerate mechanical overloading-induced disc degeneration., (© 2017 The Author(s).)