scholarly journals Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway

2018 ◽  
Vol 39 (6) ◽  
pp. 912-922 ◽  
Author(s):  
Zong-chao Liu ◽  
Zhen-long Wang ◽  
Chen-yi Huang ◽  
Zhi-jiang Fu ◽  
Yong Liu ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Matrix Degradation ◽  
Nucleus Pulposus Cells

scholarly journals Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture

2018 ◽  
Vol 46 (2) ◽  
pp. 482-491 ◽  
Author(s):  
Yichun Xu ◽  
Hui Yao ◽  
Pei Li ◽  
Wenbin Xu ◽  
Junbin Zhang ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Dynamic Compression ◽  
Akt Pathway ◽  
Bioreactor Culture ◽  
Nucleus Pulposus Cells ◽  
Matrix Synthesis ◽  
Static Compression ◽  
Matrix Deposition ◽  
The Matrix

Background/Aims: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Methods: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Results: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Conclusion: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.

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 Caveolin-1 regulates oxidative stress-induced senescence in nucleus pulposus cells primarily via the p53/p21 signaling pathway in vitro

2017 ◽  
Vol 16 (6) ◽  
pp. 9521-9527 ◽  
Author(s):  
Lei Ding ◽  
Qingmin Zeng ◽  
Jingping Wu ◽  
Defang Li ◽  
Houlei Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signaling Pathway ◽  
Nucleus Pulposus ◽  
Nucleus Pulposus Cells ◽  
Caveolin 1

scholarly journals Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in vitro and in vivo study

2021 ◽  
Vol 41 ◽  
pp. 90-107
Author(s):  
S Tang ◽  
◽  
A Salazar-Puerta ◽  
J Richards ◽  
S Khan ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Viral Vectors ◽  
Extracellular Vesicle ◽  
Proteoglycan Synthesis ◽  
Nucleus Pulposus Cells ◽  
Keratin 19 ◽  
Altered Gene ◽  
Ivd Degeneration

Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration. Bulk electroporation was used to investigate FOXF1 effects on human NP cells during a 4-week culture in 3D agarose constructs. Engineered EV delivery of FOXF1 into human IVD cells in monolayer was determined, with subsequent in vivo validation in a pilot mouse IVD puncture model. FOXF1 transfection significantly altered gene expression by upregulating healthy NP markers [FOXF1, keratin 19 (KRT19)], decreasing inflammatory cytokines [interleukin (IL)-1β, -6], catabolic enzymes [metalloproteinase 13 (MMP13)] and nerve growth factor (NGF), with significant increases in glycosaminoglycan accumulation in human NP cells. Engineered EVs loaded with FOXF1 demonstrated successful encapsulation of FOXF1 cargo and effective uptake by human NP cells cultured in monolayer. Injection of FOXF1-loaded EVs into the mouse IVD in vivo resulted in a significant upregulation of FOXF1 and Brachyury, compared to controls at 7 d post-injection, with no evidence of cytotoxicity. This is the first study to demonstrate non-viral delivery of FOXF1 and reprogramming of human NP cells in vitro and mouse IVD cells in vivo. This strategy represents a non-addictive approach for treating IVD degeneration and associated back pain.

scholarly journals LncRNA GAS5 as miR-26a-5p Sponge Regulates the PTEN/PI3K/Akt Axis and Affects Extracellular Matrix Synthesis in Degenerative Nucleus Pulposus Cells in vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Liang Tan ◽  
Yifang Xie ◽  
Ye Yuan ◽  
Kai Hu
Keyword(s):  
Extracellular Matrix ◽  
Nucleus Pulposus ◽  
Intervertebral Disc Degeneration ◽  
Nucleus Pulposus Cell ◽  
Nucleus Pulposus Cells ◽  
Akt Inhibitor ◽  
Matrix Synthesis ◽  
Lncrna Gas5

The role of lncRNA growth arrest specific 5 (GAS5) in degenerative nucleus pulposus cell (NPC) apoptosis has been reported, but the mechanism of GAS5 in extracellular matrix (ECM) synthesis in intervertebral disc degeneration (IDD) remains unknown. We aimed to investigate the mechanism of GAS5 in ECM synthesis in degenerative NPCs. GAS5 expression was measured in degenerative NPCs (CP-H170) and normal NPCs (CP-H097). siRNA-mediated GAS5 knockdown was transfected to NPCs to detect cell viability and the expression of ECM-related genes (Collagen II, aggrecan, Collagen I, and MMP-3). Subcellular localization of GAS5 was analyzed. The downstream gene and pathway of GAS5 in degenerative NPCs were explored. As our results indicated, lncRNA GAS5 was upregulated in degenerative NPCs. Silencing GAS5 improved the viability of degenerative NPCs and increased ECM synthesis. GAS5 was mainly located in the cytoplasm of NPCs. LncRNA GAS5 sponged miR-26a-5p to regulate PTEN. Overexpression of miR-26a-5p promoted ECM synthesis in degenerative NPCs. Akt inhibitor LY294002 reversed the promotion of silencing GAS5 on ECM synthesis of degenerative NPCs. In conclusion, lncRNA GAS5 sponged miR-26a-5p to upregulate PTEN and inhibit the PI3K/Akt pathway, thus inhibiting ECM synthesis of degenerative NPCs.

scholarly journals Inverse Agonist of Retinoid-Related Orphan Receptor-Alpha Prevents Apoptosis and Degeneration in Nucleus Pulposus Cells via Upregulation of YAP

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Tongzhou Liang ◽  
Jincheng Qiu ◽  
Shaoguang Li ◽  
Zhihuai Deng ◽  
Xianjian Qiu ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Cell Metabolism ◽  
Type Ii Collagen ◽  
Inverse Agonist ◽  
Orphan Receptor ◽  
Nucleus Pulposus Cells ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
A Disintegrin And Metalloproteinase

Intervertebral disc degenerative disease (IDD) is the most common degenerative spine disease, which leads to chronic low back pain and symptoms in the lower extremities. In this study, we found that RORα, a member of the retinoid-related orphan receptor family, is significantly elevated in nucleus pulposus tissue in IDD patients. The elevation of RORα is associated with increased apoptosis of nucleus pulposus (NP) cells. Therefore, we applicated a well-established inverse agonist of RORα, SR3335, to investigate its role in regulating NP cell metabolism and apoptosis. To further investigate the mechanism that SR3335 regulates the pathogenesis of IDD in vitro, tumor necrosis factor alpha (TNF-α) stimulation was used in human NP cells to mimic the hostile environment that leads to degeneration. We found that SR3335 treatment reversed the trend of increased apoptosis in NP cells induced by TNF-α treatment. Next, TNF-α treatment upregulated the expression of type II collagen and aggrecan and downregulated MMP13 (matrix-degrading enzyme matrix metalloproteinase 13) and ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4). However, these effects were reversed after SR3335 treatment. Furthermore, we find that SR3335 mediated the effect in NP cells by regulating the YAP signaling pathway, especially by affecting the phosphorylation state of YAP. In conclusion, the reduction of matrix degradation enzymes and apoptosis upon SR3335 treatment suggests that SR3335 is a promising drug in reversing the deleterious microenvironment in IDD patients.

scholarly journals Oxidative Stress Aggravates Apoptosis of Nucleus Pulposus Cells through m6A Modification of MAT2A Pre-mRNA by METTL16

2022 ◽  
Vol 2022 ◽  
pp. 1-15
Author(s):  
Peng-Bo Chen ◽  
Gui-Xun Shi ◽  
Tao Liu ◽  
Bo Li ◽  
Sheng-Dan Jiang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Animal Model ◽  
Pilot Study ◽  
Nucleus Pulposus ◽  
Intervertebral Disc Degeneration ◽  
Nucleus Pulposus Cell ◽  
Nucleus Pulposus Cells ◽  
Human Npcs

The process of intervertebral disc degeneration (IVDD) is complex, and its mechanism is considered multifactorial. Apoptosis of oxidative stressed nucleus pulposus cells (NPCs) should be a fundamental element in the pathogenesis of IVDD. In our pilot study, we found that the expression of MAT2A decreased, and METTL16 increased in the degenerative nucleus pulposus tissues. Previous studies have shown that the balance of splicing, maturation, and degradation of MAT2A pre-mRNA is regulated by METTL16 m6A modification. In the current study, we aimed to figure out whether this mechanism was involved in the aberrant apoptosis of NPCs and IVDD. Human NPCs were isolated and cultured under oxidative stress. An IVDD animal model was established. It showed that significantly higher METTL16 expression and lower MAT2A expression were seen in either the NPCs under oxidative stress or the degenerative discs of the animal model. MAT2A was inhibited with siRNA in vitro or cycloleucine in vivo. METTL16 was overexpressed with lentivirus in vitro or in vivo. Downregulation of MAT2A or upregulation of METTL16 aggravated nucleus pulposus cell apoptosis and disc disorganization. The balance of splicing, maturation, and degradation of MAT2A pre-mRNA was significantly inclined to degradation in the NPCs with the overexpression of METTL16. Increased apoptosis of NPCs under oxidative stress could be rescued by reducing the expression of METTL16 using siRNA with more maturation of MAT2A pre-mRNA. Collectively, oxidative stress aggravates apoptosis of NPCs through disrupting the balance of splicing, maturation, and degradation of MAT2A pre-mRNA, which is m6A modified by METTL16.

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