Rat Nucleus Pulposus Cells Research Articles

Transcription factor 7-like 2 controls matrix degradation through nuclear factor κB signaling and is repressed by microRNA-155 in nucleus pulposus cells.

TCF7L2, a key transcription factor in the canonical Wnt pathway, plays a vital role in the matrix degradation of chondrocytes. However, it is unknown whether TCF7L2 is important in the matrix metabolism of inner gel-like nucleus pulposus (NP) cells; thus, the aim of this study was to explore the effect and mechanism of TCF7L2 in this process. Western blotting and immunofluorescence analyses were used to observe TCF7L2 expression in rat and human NP tissues. Real-time PCR and western blotting were performed to detect the expression of TCF7L2 stimulated by inflammatory cytokines. Dual luciferase reporter assay, real-time PCR, western blotting and knockdown experiments were performed to demonstrate the role of NF-κB signaling in matrix regulation by TCF7L2 and the regulation of TCF7L2 by miR-155 in intervertebral disc degeneration. TCF7L2 is present in rat and human NP tissues and is expressed in the nucleus of NP cells. TCF7L2 is refractory to stimulation of rat and human NP cells with the inflammatory cytokines TNF-α and IL-1β, in contrast to the results in other cell types. Loss-of-function experiments using TCF7L2 siRNA or lentiviral shTCF7L2 showed that TCF7L2 knockdown suppresses matrix degradation through p65/NF-κB signaling in the absence and presence of TNF-α. In addition, TCF7L2 expression is repressed by miR-155 overexpression and promoted by miR-155 inhibition. Overall, these results demonstrate that the suppression of TCF7L2, which is modulated by miR-155, inhibits matrix degradation through p65/NF-κB signaling. TCF7L2 suppression may have therapeutic potential in intervertebral disc degeneration.

Thursday, September 27, 2018 2:05 PM–3:05 PM Section on Biologics and Basic Research Abstract Presentations: 124. NF-κB dependent unfolded protein response in the survival and proliferation of nucleus pulposus cells under TNF-α stimulation

BACKGROUND CONTEXT Intervertebral disc (IVD) degeneration is a degenerative disease closely related to inflammation of nucleus pulposus (NP) cells. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine expressed by NP cells (NPCs) of degenerate IVD, which induces NPCs apoptosis and extracellular matrix (ECM) degradation, and accelerate IVD degeneration (IDD). The endoplasmic reticulum serves several important cell functions, which are essential for normal cell function and survival. Nuclear factor-kappaB (NF-κB) is important for genes involved in cell survival, adhesion, differentiation, and proliferation. However, the roles of ER stress and NF-κB in IDD remain to be elucidated. PURPOSE This study aims to clarify the roles of NF-κB and ER stress related unfolded protein response (UPR) in TNF-α-induced biological changes in rat NPCs and IVD degeneration. METHODS In vivo, different doses of exogenous TNF-α were injected into rat tail discs. Magnetic resonance imaging (MRI) yielding T2-weighted images were performed. Then, the discs were stained by Hematoxylin and Eosin, Masson's trichrome to visualize the histocytological changes, immunohistochemical staining was performed to evaluate the expression of ECM including aggrecan and collagen II. In vitro, we cultured rat NPCs with different concentrations of TNF-α, with or without UPR and NF-κB pathway small interfering RNA (siRNA). The mRNA expression and protein levels of UPR markers (XBP1s and p-eIF2α) and p-p65 were measured by immunofluorescence staining, qRT-PCR and western blot analysis respectively and were used to monitor UPR and NF-κB. Cell proliferation was evaluated by CCK-8 assay, cell-cycle analysis and cyclin D1 expression. Apoptosis was detected by flowcytometry and Western blot analyses. All the data were expressed as mean ± SD and from multiple independent experiments. The results were compared among different groups by using unpaired student-test. P-values RESULTS In vivo, after one month, a significant decrease in T2 value calculated from T2-mapping was observed in TNF-α injection groups along with clearly decrease in disc height and nucleus volumes in comparison to the control discs. Histological analysis showed degenerative changes with NPCs and ECM loss, while no degenerative changes were observed in control groups. In vitro, TNF-α induced the apoptosis of some NPCs in the early stage and then accelerated the proliferation of surviving cells. In addition, TNF-α stimulus up-regulated XBP1s, p-eIF2α and p-p65 at the protein level, which indicated that TNF-α activated UPR and NF-κB signals in rat NP cells. However, these effects could be reversed by UPR and NF-κB siRNA, and UPR interference decreased the expression of p-p65 notably. In parallel, both UPR and NF-κB interference reduced cell proliferation and enhanced apoptosis. CONCLUSIONS Our study demonstrated that TNF-α is an important early pathogenetic factor in disc degeneration. UPR reinforces the survival and proliferation of NPCs in TNF-α stimulus by activating NF-κB signaling, which could be an important therapeutic target in the future. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Resveratrol enhances matrix biosynthesis of nucleus pulposus cells through activating autophagy via the PI3K/Akt pathway under oxidative damage.

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 H2O2 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 H2O2), 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.

Nucleus pulposus cell senescence is alleviated by resveratrol through regulating the ROS/NF-κB pathway under high-magnitude compression.

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.

Parathyroid hormone 1‑34 inhibits senescence in rat nucleus pulposus cells by activating autophagy via the m‑TOR pathway.

Osteoporosis is closely associated with intervertebral disc degeneration. While parathyroid hormone (PTH) 1–34, which is an established drug used to treatosteoporosis, is thought to inhibit the disc degeneration associated with osteoporosis, the precise mechanism involved remains unclear. In the present study, primary Sprague-Dawley rat nucleus pulposus cells (NPCs) were cultured, phenotyped and then treated with dexamethasone (DXM) for 48 h. Cell area analysis and β-galactosidase staining were used to investigate the effect of DXM on the senescence of NPCs. In addition, the protein levels of LC3-II, Beclin-1, P62, p-mTOR and p-p70S6k were determined by western blotting and analyzing the regulatory effect of PTH upon autophagy and the mTOR signaling pathway in cells treated with DXM. Following autophagic inhibition induced by ATG5 siRNA transfection, the regulatory effect of PTH on senescence in NPCs were investigated in addition to the potential role of autophagy. As the concentration of DXM increased, the size of the NPCs was significantly enlarged and the proportion of cells with positive β-galactosidase staining increased significantly (P<0.05). In terms of protein expression, PTH treatment led to an increase in LC3-II and Beclin-1 proteins, a reduction in P62 protein, and inhibited p-mTOR and p-p70S6k protein expression in DXM-treated NPCs (P<0.05). PTH attenuated the effect of DXM according to the cell size and percentage of β-galactosidase-positive cells. However, the inhibition of autophagy via ATG5 siRNA transfection reversed the protective effect of PTH on cell senescence (P<0.05). Collectively, the present findings suggest that PTH may inhibit the senescence of NPCs induced by DXM by activating autophagy via the mTOR pathway.

MicroRNA-223 inhibits lipopolysaccharide-induced inflammatory response by directly targeting Irak1 in the nucleus pulposus cells of intervertebral disc.

This study was aimed to research the effect of miR-223 on the inflammatory responses induced by lipopolysaccharide (LPS) in nucleus pulposus (NP) cells of rat intervertebral disc. Isolated rat NP cells were induced by LPS. Reverse transcriptase quantitative real-time polymerase chain reaction was used to detect gene expression. To detect protein expression, Western blot and enzyme-linked immunosorbent assay experiments were applied. The putative targeting relationship between miR-223 and Irak1 was determined using dual-luciferase reporter gene assay. We found that miR-223 was downregulated in LPS-induced NP cells. MiR-223 upregulated the expression of extracellular matrix-related genes (Aggrecan and Collagen II). Matrix degrading enzymes (ADAMTS4, ADAMTS5, MMP3 and MMP13), NO reaction-associated proteins (PGE2, COX-2 and INOS) and the expression of nuclear factor (NF)-κB signaling-related proteins were downregulated after miR-233 overexpression. In addition, luciferase reporter assays demonstrated that miR-223 directly targeted Irak1. MiR-223 overexpression could inhibit NF-κB signaling by targeting Irak1, and finally suppress the LPS-induced inflammation in NP cells. © 2018 IUBMB Life, 70(6):479-490, 2018.

Critical contribution of RIPK1 mediated mitochondrial dysfunction and oxidative stress to compression-induced rat nucleus pulposus cells necroptosis and apoptosis.

The aim of this study was to investigate whether RIPK1 mediated mitochondrial dysfunction and oxidative stress contributed to compression-induced nucleus pulposus (NP) cells necroptosis and apoptosis, together with the interplay relationship between necroptosis and apoptosis in vitro. Rat NP cells underwent various periods of 1.0MPa compression. To determine whether compression affected mitochondrial function, we evaluated the mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP), mitochondrial ultrastructure and ATP content. Oxidative stress-related indicators reactive oxygen species, superoxide dismutase and malondialdehyde were also assessed. To verify the relevance between oxidative stress and necroptosis together with apoptosis, RIPK1 inhibitor necrostatin-1(Nec-1), mPTP inhibitor cyclosporine A (CsA), antioxidants and small interfering RNA technology were utilized. The results established that compression elicited a time-dependent mitochondrial dysfunction and elevated oxidative stress. Nec-1 and CsA restored mitochondrial function and reduced oxidative stress, which corresponded to decreased necroptosis and apoptosis. CsA down-regulated mitochondrial cyclophilin D expression, but had little effects on RIPK1 expression and pRIPK1 activation. Additionally, we found that Nec-1 largely blocked apoptosis; whereas, the apoptosis inhibitor Z-VAD-FMK increased RIPK1 expression and pRIPK1 activation, and coordinated regulation of necroptosis and apoptosis enabled NP cells survival more efficiently. In contrast to Nec-1, SiRIPK1 exacerbated mitochondrial dysfunction and oxidative stress. In summary, RIPK1-mediated mitochondrial dysfunction and oxidative stress play a crucial role in NP cells necroptosis and apoptosis during compression injury. The synergistic regulation of necroptosis and apoptosis may exert more beneficial effects on NP cells survival, and ultimately delaying or even retarding intervertebral disc degeneration.

Resveratrol attenuates high glucose-induced nucleus pulposus cell apoptosis and senescence through activating the ROS-mediated PI3K/Akt pathway.

Background: Diabetes mellitus is closely correlated with disc degeneration. Nucleus pulposus (NP) cell apoptosis and senescence are typical cellular features within the degenerative disc. Resveratrol is a newly identified phytoalexin that has protective effects on cartilaginous tissue.Objective: To investigate the whether resveratrol can protect against high glucose-induced NP cell apoptosis and senescence, and the potential mechanism in this process.Methods: Rat NP cells were cultured in either 10% FBS culture medium (control group) or 10% FBS with a high glucose concentration (0.2 M, experiment group) for 3 days. Resveratrol or the combination of resveratrol and LY294002 was added into the culture medium of experiment group to investigate the effects of resveratrol and the PI3K/Akt pathway.Results: High glucose significantly promoted NP cell apoptosis and NP cell senescence compared with the control group. Resveratrol exhibited protective effects against high glucose-induced NP cell apoptosis and senescence. Further analysis showed that resveratrol suppressed reactive oxygen species (ROS) generation and increased the activity of the PI3K/Akt pathway under the high glucose condition. However, the LY294002 had no significant effects on ROS content in the resveratrol-treated high glucose group.Conclusion: Resveratrol can attenuate high glucose-induced NP cell apoptosis and senescence, and the activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration.

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert‐butyl hydroperoxide (TBHP)‐treated nucleus pulposus cells of SD rats in vitro as well as in a puncture‐induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen‐II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts‐5. Additionally, autophagy blockade using 3‐MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine‐treated rats had an accentuated T2‐weighted signal and a diminished histological degenerative grade than vehicle‐treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.

Transcriptome and alternative splicing analysis of nucleus pulposus cells in response to high oxygen tension: Involvement of high oxygen tension in the pathogenesis of intervertebral disc degeneration

High oxygen tension caused by neovascularization in the microenvironment of intervertebral discs (IVDs) is associated with the pathogenesis of IVD degeneration (IDD). Pre-mRNAs undergo alternative splicing (AS) to produce structurally and functionally diverse mRNA and proteins. However, the precise role of high oxygen tension in IDD and the relationship between AS and high oxygen tension in disc cells remain unknown. To investigate the effect of high oxygen tension on disc cells, Affymetrix Rat Transcriptome Array 1.0 was used to determine differentially expressed genes (DEGs) and alternative splicing genes (ASGs) in rat nucleus pulposus (NP) cells treated with 20% O2. NP cells at 1% O2 served as the control. PCR was used for validation. GO and KEGG pathway analysis was performed. Furthermore, the reactive oxygen species (ROS) production, growth, cell cycle and matrix metabolism of NP cells were also investigated. In total, 2499 DEGs and 8451 ASGs were identified. Various GO terms and KEGG pathways were potently associated with IDD, including autophagy, mTOR signaling pathway and angiogenesis. Especially, high oxygen tension increased ROS production in NP cells. It also accelerated the matrix metabolism of NP cells and induced NP cell cycle arrest to retard cell growth. This study, for the first time, analyzes the transcriptome and AS of NP cells in response to high oxygen tension, indicating that high oxygen tension is involved in the establishment and progression of IDD through its wide effects on the viability and function of disc cells.

Hydrogen sulfide is expressed in the human and the rat cultured nucleus pulposus cells and suppresses apoptosis induced by hypoxia.

Apoptosis plays pivotal role in the pathogenesis of degenerative disc diseases, which is the primary contributor to low back pain. Although the role of hydrogen sulfide (H2S) in cell apoptosis is well appreciated, the effects and mechanism that H2S regulates the program death of intervertebral disc cell are not yet elucidated. In this study, we utilized the nucleus pulposus (NP) from patients with lumbar disc herniation to investigate the relationship between endogenous H2S and NP cells apoptosis in human. Furthermore, we analyzed primary rat NP cells to study the effects of exogenous H2S on hypoxia induced cell apoptosis. Human NP samples were obtained from patients with lumbar disc herniation and were divided into uncontained and contained herniation groups. Using immunohistochemistry staining and sulphur-sensitive electrode, we detected the expression of cystathionine-β-synthase (CBS) and cystathionine γ-lyase (CSE), as well as the production of endogenous H2S in human NP. Tunel staining showed increased apoptosis in NP from herniated disc; and there was significant correlation between H2S generation and apoptosis in human NP. CoCl2 was then used to induce hypoxia in cultured primary rat NP cells. Annexin V staining indicated that exogenous NaHS attenuated hypoxia induced apoptosis in rat NP cells. Furthermore, hypoxia significantly increased the levels of multiple apoptosis associated proteins (Fas, Cytochromes C, Caspase 9 and cleaved-Caspase-3) in cells, which were eliminated by NaHS.Our study demonstrates the presence of endogenous H2S in human intervertebral disc; and the endogenous H2S generation rate is associated with NP apoptosis in herniated disc. In vitro study showes exogenous H2S donor attenuates hypoxia induced apoptosis in primary rat NP cells. Thus, our work provides insights that H2S may have beneficial effects in treating degenerative disc diseases.

Resveratrol protects against sodium nitroprusside induced nucleus pulposus cell apoptosis by scavenging ROS

Oxidative stress induced disc cell apoptosis plays an important role in intervertebral disc (IVD) degeneration. The present study aims to investigate effects of resveratrol (RV), a natural polyphenol compound, on sodium nitroprusside (SNP) induced nucleus pulposus (NP) cell apoptosis and related mechanism. Rat NP cells were pretreated with RV, N-acetyl cysteine (NAC) and carboxy-PTIO (PTIO) before SNP treatment. Cell Counting Kit-8 assay was carried out for cell viability evaluation. Annexin V/propidium iodide (PI), Hoechst 33258 and Actin-Tracker Green and Tubulin-Tracker Red staining were conducted to detect NP cell apoptosis and apoptotic structural changes. Mitochondrial membrane potential (ΔΨm) was analyzed with tetramethylrhodamine methyl ester staining. DCFH-DA and DAF-FM DA staining was used to determine intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels. An ex vivo experiment was also carried out followed by TUNEL assay of sections of discs. SNP induced NP cell apoptosis, excessive production of intracellular ROS and NO, reduction of ΔΨm as well as disruption of cytoskeletal and morphological structure. Meanwhile, organ culture results showed that SNP induced NP cell apoptosis ex vivo. RV and NAC siginificantly inhibited SNP induced NP cell apoptosis, production of intracellular ROS, deline of ΔΨm as well as disruption of cytoskeletal and morphological structure, while RV did not suppress NO production. RV and NAC could also suppress SNP induced NP cell apoptosis ex vivo. However, PTIO did not prevent SNP induced NP cell apoptosis, though it scavenged NO significantly. In conclusion, RV protects against SNP induced NP cell apoptosis by scavenging ROS but not NO, suggesting a promising prospect of RV in IVD degeneration retardation.

Endoplasmic Reticulum Stress Facilitates the Survival and Proliferation of Nucleus Pulposus Cells in TNF-α Stimulus by Activating Unfolded Protein Response.

Intervertebral disc (IVD) degeneration is closely related to inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α). The endoplasmic reticulum (ER) serves several important cell functions, which are essential for normal cell metabolism and survival. This study aims to clarify the role of ER stress and unfolded protein response (UPR) in TNF-α-induced biological changes in rat nucleus pulposus cells (NPCs) and IVD degeneration. In our research, rat NPCs were cultured with different concentrations of TNF-α in the presence or absence of ER stress inhibitors. Related genes and proteins were measured by immunofluorescence staining, quantitative real-time PCR, and Western blot analyses to monitor ER stress. Cell proliferation was evaluated by CCK-8 assay and cyclin D1 expression. Apoptosis was detected by flow cytometry and Western blot analyses. Our results showed that TNF-α induced the apoptosis of some NPCs in the early stage and then accelerated the proliferation of surviving cells. In addition, TNF-α stimulus upregulated ER stress markers and initiated UPR. However, these effects could be reversed by inhibitors, thereby reducing cell proliferation and enhancing apoptosis. In conclusion, ER stress reinforces the survival and proliferation of NPCs in TNF-α stimulus by activating UPR signaling, which could be an important therapeutic target in the future.

Aquaporin-3 Attenuates Oxidative Stress-Induced Nucleus Pulposus Cell Apoptosis Through Regulating the P38 MAPK Pathway

Background/Aims: Previous studies have shown that oxidative damage is a main contributor to disc nucleus pulposus (NP) cell apoptosis. Aquaporin-3 (AQP-3) facilitates reactive oxygen species (ROS) scavenging and thus alleviates oxidative injury in other cells. This study aims to investigate the role and mechanism of AQP-3 in regulating NP cell apoptosis under oxidative damage. Methods: Rat NP cells were treated with H₂O₂ for 48 hours, while control NP cells were free of H₂O₂. Recombinant AQP-3 lentiviral vectors were used to investigate the effect of enhanced AQP-3 expression levels in NP cells. NP cell apoptosis was assessed by flow cytometry, caspase-3 activity, gene expression of apoptosis-related molecules (Bax, Bcl-2 and caspase-3), and protein expression of cellular apoptosis markers (cleaved PARP and cleaved caspase-3). Additionally, intracellular ROS content and activity of the p38 MAPK pathway were evaluated. Results: Compared with the control NP cells, oxidative damage in the treatment cells significantly increased cell apoptosis ratios and caspase-3 activity, upregulated gene expression of Bax and caspase-3, downregulated gene expression of Bcl-2, and increased protein expression of cleaved PARP and cleaved caspase-3, as well as increased intracellular ROS content and activity of the p38 MAPK pathway. However, AQP-3 overexpression partly alleviated cell apoptosis, decreased intracellular ROS content, and inhibited the p38 MAPK pathway in NP cells under oxidative damage. Conclusion: Oxidative damage can significantly downregulate AQP-3 expression. Enhancing AQP-3 expression in NP cells partly attenuates cellular apoptosis through regulating the p38 MAPK pathway under oxidative damage.

Periodic Mechanical Stress Induces Extracellular Matrix Expression and Migration of Rat Nucleus Pulposus Cells Through Src-GIT1-ERK1/2 Signaling Pathway

Background/Aims: Periodic mechanical stress has been shown to promote extracellular matrix (ECM) synthesis and cell migration of nucleus pulposus (NP) cells, however, the mechanisms need to be fully elucidated. The present study aimed to investigate the signal transduction pathway in the regulation of NP cells under periodic mechanical stress. Methods: Primary rat NP cells were isolated and seeded on glass slides, and then treated in our self-developed periodic stress field culture system. To further explore the mechanisms, data were analyzed by scratch-healing assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, and co-immunoprecipitation assay. Results: Under periodic mechanical stress, the mRNA expression of ECM collagen 2A1 (Col2A1) and aggrecan, and migration of NP cells were significantly increased (P < 0.05 for each), associating with increases in the phosphorylation of Src, GIT1, and ERK1/2 (P < 0.05 for each). Pretreatment with the Src inhibitor PP2 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each), while the phosphorylation of GIT1 and ERK1/2 were inhibited. ECM synthesis, cell migration, and phosphorylation of ERK1/2 were inhibited after pretreatment with the small interfering RNA for GIT1 in NP cells under periodic mechanical stress (P < 0.05 for each), whereas the phosphorylation of Src was not affected. Pretreatment with the ERK1/2 inhibitor PD98059 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each). Co-immunoprecipitation assay showed that there was a direct interaction between Src and GIT1 and between GIT1 and ERK1/2. Conclusion: In conclusion, periodic mechanical stress induced ECM expression and migration of NP cells via Src-GIT1-ERK1/2 signaling pathway, playing an important role in regulation of NP cells.

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

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.

Hydrogen peroxide induces programmed necrosis in rat nucleus pulposus cells through the RIP1/RIP3-PARP-AIF pathway.

This study aimed to systematically investigate whether programmed necrosis contributes to H2 O2 -induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H2 O2 for various time periods. The cell viability was measured using a cell counting kit-8, and the death rate was detected by Hoechst 33258/propidium iodide (PI) staining. The programmed necrosis-related molecules receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), poly (ADP-ribose) polymerase (PARP), and apoptosis inducing factor (AIF) were determined by real-time polymerase chain reaction and Western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy (TEM). In addition, the necroptosis inhibitor Necrostatin-1 (Nec-1), the PARP inhibitor diphenyl-benzoquinone (DPQ) and small interfering RNA (siRNA) technology were used to indirectly evaluate programmed necrosis. Our results indicated that H2 O2 induced necrotic morphologic and ultrastructural changes and an elevated PI positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec-1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3-mediated programmed necrosis, executed through the PARP-AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1269-1282, 2018.

Reverse Reconstruction and Bioprinting of Bacterial Cellulose-Based Functional Total Intervertebral Disc for Therapeutic Implantation.

The degradation of intervertebral discs (IVD), a typical hierarchical structured tissue, causes serious neck and back pain. The current methods cannot fully reconstitute the unique structure and function of native IVD. In this study, by reverse reconstruction of the structure of native IVD and bioprinting bacterial cellulose (BC) nanofibers with a high-throughput optimized micropattern screening microchip, a total IVD is created that contained type II collagen-based nucleus pulposus (NP) and hierarchically organized and micropatterned BC-based annulus fibrosus (AF), mimicking native IVD tissue. The artificial NP contains rat NP cells, whereas the AF contains concentrically arranged BC layers with aligned micropatterns and attached AF cells in +/-30° alternate directions between adjacent layers. Long-term (3 months) implantation experiments on rats demonstrate the excellent structural (shape maintenance, hydration, tissue integration) and functional (mechanical support and flexibility) performance of the artificial IVD. This study provides a novel strategy for creating highly sophisticated artificial tissues.

TIGAR mediates the inhibitory role of hypoxia on ROS production and apoptosis in rat nucleus pulposus cells.

Hypoxia has been shown to inhibit reactive oxygen species (ROS) production in nucleus pulposus (NP) cells. The TP53-induced glycolysis and apoptosis regulator (TIGAR) has been reported to suppress oxidative stress. We sought to explore the role of TIGAR in the effect of hypoxia on ROS production and apoptosis. An intervertebral disc degeneration (IDD) model of Sprague-Dawley (SD) rat caudal spine was established by puncturing the Co6-7 disc. TIGAR expression was detected by immunohistochemistry and western blotting in human and SD rat NP tissues of degenerated discs. Rat primary NP cells treated with hypoxia and cobalt chloride (CoCl2) were analyzed by western blotting for TIGAR expression. After TIGAR silence with TIGAR siRNA transfection, apoptosis percentage, mitochondrial and total intracellular ROS levels were measured. H2O2 was used to further check the effects of TIGAR on oxidative stress. Finally, NADPH/NADP+ and GSH/GSSH ratio were examined after TIGAR silencing under hypoxic conditions and after H2O2 treatment. A degree-dependent increase in TIGAR expression was observed in human and rat degenerated NP tissues. Hypoxia and hypoxia-inducer CoCl2 enhanced TIGAR and P53 expressions in rat NP cells. TIGAR silence reversed the inhibitory effects of hypoxia on intracellular and mitochondrial ROS production, as well as apoptosis percentage. However, TIGAR silence aggravated H2O2-induced ROS production. In addition, TIGAR increased NADPH/NADP+ and GSH/GSSH ratio in NP cells. These results suggested that TIGAR appears to mediate the protective role of hypoxia on ROS production and apoptosis percentage by enhancing NADPH/NADP+ and GSH/GSSH ratio.

Edaravone ameliorates compression-induced damage in rat nucleus pulposus cells

AimsEdaravone is a strong free radical scavenger most used for treating acute ischemic stroke. In this study we investigated the protective effects and underlying mechanisms of edaravone on compression-induced damage in rat nucleus pulposus (NP) cells. Materials and methodsCell viability was determined using MTT assay methods. NP cell apoptosis was measured by Hoechst 33,258 staining and Annexin V/PI double staining. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and intracellular calcium ([Ca2+]i) were determined by fluorescent probes DCFH-DA, JC-1 and Fluo-3/AM, respectively. Apoptosis-related proteins (cleaved caspase-3, cytosolic cytochrome c, Bax and Bcl-2) and extracellular matrix proteins (aggrecan and collagen II) were analyzed by western blot. Key findingsEdaravone attenuated the compression-induced decrease in viability of NP cells in a dose-dependent manner. 33,258 and Annexin V/PI double staining showed that edaravone protected NP cells from compression-induced apoptosis. Further studies confirmed that edaravone protected NP cells against compression-induced mitochondrial pathway of apoptosis by inhibiting overproduction of ROS, collapse of MMP and overload of [Ca2+]i. In addition, edaravone promoted the expression of aggrecan and collagen II in compression-treated NP cells. SignificanceThese results strongly indicate that edaravone ameliorates compression-induced damage in rat nucleus pulposus cells. Edaravone could be a potential new drug for treatment of IDD.