scholarly journals Hypoxia Helps Maintain Nucleus Pulposus Homeostasis by Balancing Autophagy and Apoptosis

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Han-Jun Kim ◽  
Hye-Rim Lee ◽  
Hyosung Kim ◽  
Sun Hee Do
Keyword(s):  
Nucleus Pulposus ◽  
Ex Vivo ◽  
Lumbar Disc ◽  
Serial Passage ◽  
Autophagic Flux ◽  
Nucleus Pulposus Cells ◽  
Gene Expressions ◽  
Lower Back ◽  
Transmission Electron ◽  
Effects Of Aging

Intervertebral disc degeneration (IVDD) is a common cause of lower back pain. Programmed cell death (PCD) including apoptosis and autophagy is known to play key mechanistic roles in the development of IVDD. We hypothesized that the nucleus pulposus cells that make up the center of the IVD can be affected by aging and environmental oxygen concentration, thus affecting the development of IVDD. Here, we evaluated the phenotype changes and PCD signaling in nucleus pulposus cells in two different oxygen percentages (5% (hypoxia) and 20% (normoxia)) up to serial passage 20. NP cells were isolated from the lumbar discs of rats, and the chondrogenic, autophagic, and apoptotic gene expressions were analyzed during cell culture up to serial passage 20. Hypoxia significantly increased the number of autophagosomes, as determined by monodansylcadaverine staining and transmission electron microscopy. Furthermore, hypoxia triggered the activation of autophagic flux (beclin-1, LC3-II/LC3-I ratio, and SIRT1) with a concomitant decrease in the expression of apoptotic proteins (Bax and caspase-3). Despite injury and age differences, no significant differences were observed between the ex vivo lumbar disc cultures of groups incubated in the hypoxic chamber. Our study provides a better understanding of autophagy- and apoptosis-related senescence in NP cells. These results also provide insight into the effects of aging on NP cells and their PCD levels during aging.

scholarly journals Ligustrazine Prevents Intervertebral Disc Degeneration via Suppression of Aberrant TGFβ Activation in Nucleus Pulposus Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Shufen Liu ◽  
Yuhao Cheng ◽  
Yuqi Tan ◽  
Jingcheng Dong ◽  
Qin Bian
Keyword(s):  
Growth Factor ◽  
Mouse Model ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Ex Vivo ◽  
Tissue Growth ◽  
Nucleus Pulposus Cells

Objectives. Aberrant transforming growth factor β (TGFβ) activation is detrimental to both nucleus pulposus (NP) cells and cartilage endplates (CEPs), which can lead to intervertebral disc degeneration (IDD). Ligustrazine (LIG) reduces the expression of inflammatory factors and TGFβ1 in hypertrophic CEP to prevent IDD. In this study, we investigate the effects of LIG on NP cells and the TGFβ signaling. Design. LIG was injected to the lumbar spinal instability (LSI) mouse model. The effect of LIG was evaluated by intervertebral disc (IVD) score in the LSI mouse model. The expression of activated TGFβ was examined using immunostaining with pSmad2/3 antibody. The upright posture (UP) rat model was also treated and evaluated in the same manner to assess the effect of LIG. In ex vivo study, IVDs from four-week old mice were isolated and treated with 10−5, 10−6, and 10−7 M of LIG. We used western blot to detect activated TGFβ expression. TGFβ-treated human nucleus pulposus cells (HNPCs) were cotreated with optimized dose of LIG in vitro. Immunofluorescence staining was performed to determine pSmad2/3, connective tissue growth factor (CCN2), and aggrecan (ACAN) expression levels. Results. IVD score and the percentage of pSmad2/3+ NP cells were low in LIG-treated LSI mice in comparison with LSI mice, but close to the levels in the Sham group. Similarly, LIG reduced the overexpression of TGFβ1 in NP cells. The inhibitory effect of LIG was dose dependent. A dose of 10−5 M LIG not only strongly attenuated Smad2/3 phosphorylation in TGFβ-treated IVD ex vivo but also suppressed pSmad2/3, CCN2, and ACAN expression in TGFβ-treated NP cells in vitro. Conclusions. LIG prevents IDD via suppression of TGFβ overactivation in NP cells.

scholarly journals SIRT1 Inhibits Apoptosis by Promoting Autophagic Flux in Human Nucleus Pulposus Cells in the Key Stage of Degeneration via ERK Signal Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Fei He ◽  
Qingshu Li ◽  
Bo Sheng ◽  
Haitao Yang ◽  
Wei Jiang
Keyword(s):  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Early Stage ◽  
Signalling Pathway ◽  
Autophagic Flux ◽  
Nucleus Pulposus Cells ◽  
Normal Height ◽  
Definition Of ◽  
Pfirrmann Grade ◽  
Erk Signal Pathway

Background. The application of biomolecular interventions in the early stage of intervertebral disc degeneration (IVDD) is considered an ideal method for the treatment of IVDD. However, the precise definition of the “early stage” of IVDD is unclear. Silent information regulation 2 homologue-1 (SIRT1) can protect human degenerative nucleus pulposus (NP) cells from apoptosis by activating autophagy. However, the mechanism of this effect is still unclear. This study tried to confirm the “early stage” of IVDD and the role of NP cell autophagy during IVDD as well as to determine the mechanism by which SIRT1 protects NP cells. Methods. The characteristics of the NP in various stages of degeneration were assessed to confirm the “early stage” of IVDD. Then, autophagy and apoptosis were detected in NP cells after SIRT1 upregulation/downregulation. Finally, LY294002 and PD98059 were used to inhibit the AKT/ERK pathway to determine the mechanism by which SIRT1 regulates autophagy in NP cells. Results. Our data showed that mildly degenerative (Pfirrmann grade III with normal height of intervertebral disc) NP cells may be the key target for biomolecular interventions in IVDD and that SIRT1 protects human mildly degenerative NP cells from apoptosis by activating autophagy via the ERK signalling pathway. Conclusion. Our data showed that SIRT1 inhibits apoptosis by promoting the autophagic flux in NP cells via the ERK signalling pathway during the key stage of degeneration. These findings will assist in the development of novel therapeutic approaches for IVDD treatment.

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

2019 ◽  
Vol 27 (2) ◽  
pp. 347-357 ◽  
Author(s):  
Gang Zheng ◽  
Zongyou Pan ◽  
Yu Zhan ◽  
Qian Tang ◽  
Fanghong Zheng ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Autophagic Flux ◽  
Nucleus Pulposus Cells

Optimizing Oxygenation and Serum Concentration in Ex Vivo Culture of Mouse Nucleus Pulposus Cells Derived from the Notochord

2012 ◽  
Vol 2 (1_suppl) ◽  
pp. s-0032-1319943-s-0032-1319943
Author(s):  
D. Nakajima ◽  
D. Sakai ◽  
M. Tanaka ◽  
K. Yokoyama ◽  
A. Hiyama ◽  
...  
Keyword(s):  
Serum Concentration ◽  
Nucleus Pulposus ◽  
Ex Vivo ◽  
Nucleus Pulposus Cells ◽  
Ex Vivo Culture

scholarly journals Promoting Nrf2/Sirt3-Dependent Mitophagy Suppresses Apoptosis in Nucleus Pulposus Cells and Protects against Intervertebral Disc Degeneration

2021 ◽  
Vol 2021 ◽  
pp. 1-20
Author(s):  
Sunli Hu ◽  
Chenxi Zhang ◽  
Tianchen Qian ◽  
Yue Bai ◽  
Liang Chen ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Protein Docking ◽  
Tunel Staining ◽  
Autophagic Flux ◽  
Potential Candidate ◽  
Nucleus Pulposus Cells

One of the causes of intervertebral disc degeneration (IVDD) is nucleus pulposus cell (NPC) death, possibly apoptosis. In this study, we explored the role of the Nrf2/Sirt3 pathway and tert-butylhydroquinone (t-BHQ) in IVDD and elucidated the potential working mechanism. Reactive oxygen species (ROS) assay kits and malondialdehyde (MDA) assay kits were used to assess oxidative stress. Western blot and TUNEL staining were used to examine apoptosis. After siRNA against Nrf2 or lentivirus against Sirt3 was transfected into NPCs, the mechanism of the effect of the Nrf2/Sirt3 pathway on NPCs was assessed. The interaction between t-BHQ and its potential interacting protein NRF2 was further investigated through protein docking analysis. ChIP examined the binding affinity between Nrf2 and Sirt3 promoter. In vivo experiments, X-ray, hematoxylin-eosin (HE) staining, Safranin O staining, and immunohistochemistry were used to evaluate IVDD grades. The results demonstrated that activation of the Nrf2/Sirt3 pathway inhibited tert-butyl hydroperoxide- (TBHP-) induced apoptosis and mitochondrial dysfunction in vitro. In addition to apoptosis, upregulation of the Nrf2/Sirt3 pathway induced by t-BHQ restored TBHP-induced autophagic flux disturbances. However, its protective effect was reversed by chloroquine and Si-ATG5. Furthermore, t-BHQ ameliorated IVDD development in a rat model. In conclusion, our findings indicate that the Nrf2/Sirt3 pathway and its agonist represent a potential candidate for treating IVDD.

Large Cytoplasmic Vacuoles within Notochordal Nucleus Pulposus Cells: A Possible Regulator of Intracellular Pressure That Shapes the Cytoskeleton and Controls Proliferation

2018 ◽  
Vol 206 (1-2) ◽  
pp. 9-15 ◽  
Author(s):  
Xin Hong ◽  
Cong Zhang ◽  
Feng Wang ◽  
Xiao-Tao Wu
Keyword(s):  
Nucleus Pulposus ◽  
Mechanical Loading ◽  
Biological Function ◽  
Biological Behavior ◽  
Nucleus Pulposus Cells ◽  
Lower Back ◽  
Cytoplasmic Vacuoles ◽  
Motor Deficiency ◽  
Vacuolated Cells ◽  
Intracellular Pressure

Degeneration of the intervertebral disc, which is closely associated with the loss of vacuolated notochordal nucleus pulposus cells (NNPC), remains a major cause of lower-back pain and motor deficiency. Being the most defining characteristic of NNPC, large cytoplasmic vacuoles not only modulate the cytoskeleton and shape cell morphology but they also respond to the disc microenvironment and regulate the biological behavior of vacuolated cells as a potent reporter of the histocytological changes that occur at the beginning of disc aging and degeneration. Here we hypothesize a model in which large cytoplasmic vacuoles primarily function to maintain a reasonable intracellular pressure (Pv) that facilitates NNPC in resisting the extracellular mechanical loading (Pe), part of which is absorbed by the extracellular matrix (Pm), forming the equation Pe = Pm + Pv. By mimicking a situation of contact-induced growth inhibition, the crowded cytoplasmic vacuoles slow down the proliferation of NNPC and restrain the generation of nonvacuolated chondrocytic nucleus pulposus cells (CNPC), whereas increased mechanical loading (↑Pe) alters cytoskeletons and breaches cytoplasmic vacuoles, which in turn weakens the vacuoles-mediated proliferation check, increases the generation of CNPC that accumulates fibrocartilaginous matrix, and rebalances the increased loading with elevated Pm (↑Pm) and lowered Pv (↓Pv), equating to ↑Pe = ↑Pm + ↓Pv. By depicting the biological function and the disappearance of the cytoplasmic vacuoles, our model highlights a mechanical exhaustion of the notochordal cell resources, which might help to elucidate the histocytological changes that initiate disc aging and degeneration.

Transplantation of Activated Nucleus Pulposus Cells after Cryopreservation: Efficacy Study in Canine Disc Degeneration Model

2014 ◽  
Vol 4 (1_suppl) ◽  
pp. s-0034-1376573-s-0034-1376573
Author(s):  
T. Nukaga ◽  
D. Sakai ◽  
A. Hiyama ◽  
T. Ishii ◽  
T. Nakai ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Efficacy Study ◽  
Nucleus Pulposus Cells

Sirtuin 1 Maitains Survival via PKB Signaling in Degenerative Human Disc Nucleus Pulposus Cells*

2012 ◽  
Vol 39 (6) ◽  
pp. 563-573
Author(s):  
Da-Wu WANG ◽  
Zhen-Ming HU ◽  
Jie HAO ◽  
Bin HE ◽  
Qiang GAN ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Sirtuin 1 ◽  
Nucleus Pulposus Cells

TNF-α Suppresses Autophagic Flux in Acinar Cells in IgG4-Related Sialadenitis

2019 ◽  
Vol 98 (12) ◽  
pp. 1386-1396 ◽  
Author(s):  
X. Hong ◽  
S.N. Min ◽  
Y.Y. Zhang ◽  
Y.T. Lin ◽  
F. Wang ◽  
...  
Keyword(s):  
Acinar Cell ◽  
Cell Injury ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Autophagic Flux ◽  
C6 Cells ◽  
Tnf Α ◽  
Α Level

IgG4-related sialadenitis (IgG4-RS) is a newly recognized immune-mediated systemic fibroinflammatory disease that affects salivary glands and leads to hyposalivation. Tumor necrosis factor–α (TNF-α) is a critical proinflammatory cytokine involved in several salivary gland disorders, but its role and mechanism regarding acinar cell injury in IgG4-RS are unknown. Here, we found that TNF-α level was significantly increased in serum and submandibular gland (SMG) of patients and that serum TNF-α level was negatively correlated with saliva flow rate. Ultrastructural observations of IgG4-RS SMGs revealed accumulation of large autophagic vacuoles, as well as dense fibrous bundles, decreased secretory granules, widened intercellular spaces, swollen mitochondria, and expanded endoplasmic reticulum. Expression levels of LC3 and p62 were both increased in patients’ SMGs. TNF-α treatment led to elevated levels of LC3II and p62 in both SMG-C6 cells and cultured human SMG tissues but did not further increase their levels when combined with bafilomycin A1 treatment. Moreover, transfection of Ad-mCherry-GFP-LC3B in SMG-C6 cells confirmed the suppression of autophagic flux after TNF-α treatment. Immunofluorescence imaging revealed that costaining of LC3 and the lysosomal marker LAMP2 was significantly decreased in patients, TNF-α–treated SMG-C6 cells, and cultured human SMGs, indicating a reduction in autophagosome-lysosome fusion. Furthermore, the ratio of pro/mature cathepsin D was elevated in vivo, ex vivo, and in vitro. TNF-α also appeared to induce abnormal acidification of lysosomes in acinar cells, as assessed by lysosomal pH and LysoTracker DND-26 fluorescence intensity. In addition, TNF-α treatment induced transcription factor EB (TFEB) redistribution in SMG-C6 cells, which was consistent with the changes observed in IgG4-RS patients. TNF-α increased the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2, and inhibition of ERK1/2 by U0126 reversed TNF-α–induced TFEB redistribution, lysosomal dysfunction, and autophagic flux suppression. These findings suggest that TNF-α is a key cytokine related to acinar cell injury in IgG4-RS through ERK1/2-mediated autophagic flux suppression.

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