Rat Annulus Fibrosus Cells Research Articles

Inhibition of PP2A ameliorates intervertebral disc degeneration by reducing annulus fibrosus cells apoptosis via p38/MAPK signal pathway

BackgroundIntervertebral disc degeneration (IVDD) is considered one of the main reasons for low back pain (LBP). To date, the specific pathology of IVDD remains unclear. The annulus fibrosus (AF) is an important part of the intervertebral disc, and AF cell oxidative stress, apoptosis plays a vital role in disc degeneration. Protein phosphatase 2 A (PP2A), a serine/threonine phosphatase, has regulatory functions in various processes, including apoptosis and autophagy. However, thus far, the effect of PP2A on IVDD is not clear. MethodsAF cells derived from caudal intervertebral discs in SD rats were used to analyze the levels of oxidative stress, apoptosis and degeneration as well as PP2A expression. A PP2A agonist (FTY720), inhibitor (microcystin-LR) and siRNA (si-PPP2CA) were employed in IVDD induced by H2O2 to investigate the levels of apoptosis and degeneration. The p38/MAPK signal pathways were evaluated, and a p38 inhibitor (SB203580) and ERK inhibitor (U0126) were added for verification. Finally, FTY720 and microcystin-LR were administered to IVDD rats to assess the effects on levels of apoptosis and degeneration and the relief of IVDD. ResultsThe expression of PP2A was increased in rat AF cells after H2O2 intervention.The levels of apoptosis and degeneration were higher with upregulation of PP2A but were significantly reduced after inhibition of PP2A. The PP2A inhibitor relieved cell apoptosis and degeneration by downregulating the p38/MAPK pathway. In vivo, the knockdown of PP2A resulted in a more complete morphology of discs and less apoptotic and degenerative expression. ConclusionsThis study suggests that the downregulation of PP2 A could reduce AF cell apoptosis and degeneration via the p38/MAPK pathway. It also revealed that the inhibition of PP2 A is expected to be a therapeutic target for IVDD.

Smad7 Is Highly Expressed in Human Degenerative Discs and Participates in IL-1β-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway.

Background Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1β was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1β stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1β-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Conclusion Smad7 is highly expressed in human degenerative discs and participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.

Investigation into the anti-inflammatory properties of metformin in intervertebral disc cells.

IntroductionIntervertebral disc degeneration (IDD) is closely related to heightened inflammation in the annulus fibrosis (AF) and nucleus pulposus (NP) cells in the intervertebral disc. An imbalanced matrix homeostasis has been shown to contribute to disc degeneration and associated discogenic low back pain. Metformin, a diabetes medication, has been noted to exhibit anti‐inflammatory properties through upregulation of the AMPK pathway, leading to various anti‐inflammatory‐related responses in hepatocytes. However, it is still unclear how metformin influences disc cellular response to inflammatory stress and the corresponding mechanism. Hence, the objective of this study is to elucidate the effects of metformin on expression of key pro‐inflammatory, catabolic, and anabolic factors within rat AF cells in response to inflammatory stimulation and mechanical tensile stress.MethodsFive Fischer 344 rats were sacrificed and their spines isolated. AF cells were cultured and plated in flexible silicone membrane‐based six‐well plates. Wells were split into eight groups and subjected to metformin, IL‐1β, mechanical stretch, and combined treatments. Relative gene expressions of MMP‐13, COX‐2, iNOS, AGC, and Col1 were assessed with quantitative real‐time polymerase chain reaction (qRT‐PCR), and downstream prostaglandin E2 (PGE2) production was quantified with enzyme‐linked immunosorbent assay (ELISA). NF‐kB nuclear translocation was also quantified.ResultsMetformin in the presence of the combined stress treatments (M + IL/S) significantly increased Col1, COX‐2, and MMP‐13 gene expression, decreased PGE2 production compared to IL/S conditions alone. Metformin treatment of cultured rat annulus fibrosus cells significantly reduced the nuclear translocation of NF‐κB after 4 h of IL‐1β treatment from 43.1% in case of IL‐1β treatment down to 26.2% in the case of metformin + IL‐1β treatment.DiscussionThe lack of metformin‐mediated suppression of inflammatory response in the nonstretch groups indicates that metformin may be enacting its effects through a stretch‐dependent manner. These results suggest a foundation for pursuing further research into metformin's potential role as an anti‐inflammatory agent for curtailing intervertebral disc degeneration.

Naringin Inhibits Apoptosis Induced by Cyclic Stretch in Rat Annular Cells and Partially Attenuates Disc Degeneration by Inhibiting the ROS/NF-κB Pathway.

Oxidative stress and apoptosis play important roles in the pathogenesis of various degenerative diseases. Previous studies have shown that naringin can exert therapeutic effects in multiple degenerative diseases by resisting oxidative stress and inhibiting apoptosis. Although naringin is effective in treating degenerative disc disease, the underlying mechanism remains unclear. This study is aimed at investigating the effects of naringin on oxidative stress, apoptosis, and intervertebral disc degeneration (IVDD) induced by cyclic stretch and the underlying mechanisms in vitro and in vivo. Abnormal cyclic stretch was applied to rat annulus fibrosus cells, which were then treated with naringin, to observe the effects of naringin on apoptosis, oxidative stress, mitochondrial function, and the nuclear factor- (NF-) κB signaling pathway. Subsequently, a rat model of IVDD induced by dynamic and static imbalance was established to evaluate the effects of naringin on the degree of degeneration (using imaging and histology), apoptosis, and oxidative stress in the serum and the intervertebral disc. Naringin inhibited the cyclic stretch-induced apoptosis of annulus fibrosus cells, reduced oxidative stress, improved mitochondrial function, enhanced the antioxidant capacity, and suppressed the activation of the NF-κB signaling pathway. Additionally, it reduced the degree of IVDD (evaluated using magnetic resonance imaging) and the level of oxidative stress and inhibited apoptosis and p-P65 expression in the intervertebral discs of rats. Thus, naringin can inhibit cyclic stretch-induced apoptosis and delay IVDD, and the underlying mechanism may be related to the inhibition of oxidative stress and activation of the NF-κB signaling pathway. Naringin may be an effective drug for treating degenerative disc disease.

Mechanical Stretch Induces Annulus Fibrosus Cell Senescence through Activation of the RhoA/ROCK Pathway.

Background Intervertebral disc is responsible for absorbing and transmitting mechanical compression. Under physiological conditions, the peripheral annulus fibrosus (AF) cells are subjected to different magnitudes of transverse mechanical stretch depending on the swelling of the central nucleus pulposus tissue. However, the biological behavior of AF cells under mechanical stretch is not well studied. Objective This study was performed to study the effects of mechanical tension on AF cell senescence and the potential signaling transduction pathway. Methods Rat AF cells were made to experience different magnitudes of mechanical stretch (2% elongation and 20% elongation for 4 hours every day at 1 Hz) in a 10-day experiment period. The inhibitor RKI-1447 of the Rho-associated coiled-coil–containing protein kinases (ROCK) was added along with culture medium to investigate its role. Cell proliferation, cell cycle, telomerase activity, and expression of senescence markers (p16 and p53) were analyzed. Results We found that 20% elongation significantly decreased cell proliferation, promoted G0/G1 cell cycle arrest, decreased telomerase activity, and upregulated mRNA/protein expression of p16 and p53. Moreover, the inhibitor RKI-1447 partly resisted effects of 20% elongation on these parameters of cell senescence. Conclusion High mechanical stretch obviously induces AF cell senescence through the RhoA/ROCK pathway. This study provides us a deeper understanding on the AF cell's behavior under mechanical stretch.

Icariin Attenuates Serum Deprived-Induced Premature Senescence in Rat Annulus Fibrosus Cells and Ameliorates Disc Degeneration In Vivo

Icariin Attenuates Serum Deprived-Induced Premature Senescence in Rat Annulus Fibrosus Cells and Ameliorates Disc Degeneration In Vivo

TGF-\u03b21 reduces the oxidative stress-induced autophagy and apoptosis in rat annulus fibrosus cells through the ERK signaling pathway

BackgroundThe aim of this study is to explore the effects of TGF-β1 on autophagy and apoptosis induced by exogenous hydrogen peroxide (H2O2) in annulus fibrosus (AF) cells and possible signal pathways involved in this process.MethodsAF cells were isolated from rat lumbar discs and subjected to different concentrations of exogenous H2O2 (50, 100, 200 μmol/L) for different time periods (0.5, 1, 2, and 4 h). Cell viability was determined by CCK-8 assay, and the levels of autophagy and apoptosis were evaluated by Western blotting and caspase 3, 8, 9 activity assay. By administration with different concentrations of TGF-β1 (5, 10, 20 ng/mL), the effects of TGF-β1 on autophagy and apoptosis induced by H2O2 were observed, and the possible signaling pathways were also investigated by using various apoptosis inhibitors or an autophagy inhibitor Bafilomycin A (Baf A) in AF cells.ResultsH2O2 significantly impaired cell viability in a dose- and time-dependent manner. H2O2 also induced a sudden and the highest level of autophagy at 1 h, and gradually increased apoptosis through ERK pathway. The mitochondrial pathway was involved in H2O2-induced apoptosis in AF cells. TGF-β1 reduced the expression of p-ERK and downregulated the expressions of Beclin-1, LC3 II/I, and mitochondrial-related apoptotic proteins (Bax/Bcl-2, caspase-9). Meanwhile, TGF-β1 downregulated the level of intracellular H2O2 through upregulating the expression level of glutathione peroxidase-1 (GPx-1).ConclusionsTGF-β1 reduced autophagy and apoptosis induced by exogenous H2O2 through downregulating the expression of ERK in AF cells. TGF-β1 could downregulate the level of ERK and intracellular H2O2 by upregulating GPx-1.

High glucose promotes annulus fibrosus cell apoptosis through activating the JNK and p38 MAPK pathways.

Diabetes mellitus (DM) is an important risk factor of intervertebral disc degeneration. A high glucose niche-mediated disc cell apoptosis is an implicate causative factor for the spine degenerative diseases related with DM. However, the effects of a high glucose niche on disc annulus fibrosus (AF) cell apoptosis and the potential signaling transduction pathway is unclear. The present study is to investigate the effects of high glucose on disc AF cell apoptosis and the role of two potential signaling pathways in this process. Rat AF cells were cultured in baseline medium or medium with different concentrations (0.1 and 0.2 M) of glucose for 3 days. Flow cytometry was used to assess the degree of apoptosis. Activity of caspase 3/9 was evaluated by chemical kit. Expression of pro-apoptotic and anti-apoptotic molecules was analyzed by real-time polymerase chain reaction and Western blot. In addition, activity of the C-Jun NH2-terminal kinases (JNK) pathway and p38 mitogen-activated protein kinase (MAPK) pathway was evaluated by Western blot. Compared with the control group, high glucose culture increased cell apoptosis ratio and caspase-3/9 activity, up-regulated expression of bax, caspase-3, cleaved caspase-3 and cleaved PARP, and down-regulated expression of bcl-2 in a glucose concentration-dependent manner. Additionally, high glucose culture increased expression of the p-JNK and p-p38 MAPK in a concentration-dependent manner. Further results showed that inhibition of the JNK or p38 MAPK pathway attenuated the effects of high glucose on AF cell apoptosis. Together, high glucose promoted disc AF cell apoptosis through regulating the JNK pathway and p38 MAPK pathway in a glucose concentration-dependent manner.

Secreted Factors From Intervertebral Disc Cells and Infiltrating Macrophages Promote Degenerated Intervertebral Disc Catabolism.

Rat nucleus pulposus (NP) cells or annulus fibrosus (AF) cells were stimulated with conditioned media of RAW 264.7 macrophages and vice versa under healthy culture conditions and in the presence of pro-inflammatory mediators. The gene expression of pro-inflammatory mediators, extracellular matrix (ECM)-modifying enzymes, and chemokines, which play important roles in intervertebral disc degeneration (IDD), was determined. To test whether the interaction between native disc cells and infiltrating macrophages accelerates inflammation state, disrupts matrix homeostasis, and promotes inflammatory cells infiltration. With macrophages infiltration, the disc resident cells would be inevitably exposed to macrophages. Macrophages have been shown to play pro-inflammatory role in the cellular interactions with disc cells under healthy culture conditions. However, the biologic interactions between macrophages and disc cells under degenerated disc inflammatory environment remain unknown. Murine Macrophages RAW 264.7 were cultured in the conditioned media of Rat AF or NP cells culture in the presence or absence of IL-1β stimulation. Similarly, Rat AF or NP cells were also cultured in the conditioned media of Murine Macrophages RAW 264.7 culture in the presence or absence of IFN-γ stimulation. The mRNA levels difference of pro-inflammatory genes, catabolic genes and chemokines genes for AF cells, NP cells and Macrophages RAW 264.7 were analyzed by qRT-PCR, respectively. Compared with serum-free media exposure, RAW 264.7 macrophages exposed to AF or NP cells conditioned media selectively modestly upregulated mRNA levels of the aforementioned cytokines. Exposure of RAW 264.7 macrophages to conditioned media from AF or NP cells with IL-1β stimulation dramatically increased mRNA levels of all the investigated cytokines. Similarly, compared with serum-free media exposure, AF or NP cells exposed to RAW 264.7 macrophages conditioned media selectively modestly upregulated mRNA levels of the aforementioned cytokines. Exposure of AF or NP cells to conditioned media from RAW 264.7 macrophages with IFN-γ stimulation dramatically increased mRNA levels of all the investigated cytokines. The biologic interactions between infiltrating macrophages and native disc cells under degenerated disc inflammatory environment lead to an increasingly severe inflammatory conditions, which may be a self-stimulated process from the macrophages infiltration occurrenceLevel of Evidence: 5.

Extensive mechanical tension promotes annulus fibrosus cell senescence through suppressing cellular autophagy.

Background: Mechanical load contributes a lot to the initiation and progression of disc degeneration. Annulus fibrosus (AF) cell biology under mechanical tension remains largely unclear.Objective: The present study was aimed to investigate AF cell senescence under mechanical tension and the potential role of autophagy.Methods: Rat AF cells were cultured and experienced different magnitudes (5% elongation and 20% elongation) of mechanical tension for 12 days. Control AF cells were kept static. Cell proliferation, telomerase activity, cell cycle fraction, and expression of senescence-related molecules (p16 and p53) and matrix macromolecules (aggrecan and collagen I) were analyzed to evaluate cell senescence. In addition, expression of Beclin-1 and LC3, and the ratio of LC3-II to LC3-I were analyzed to investigate cell autophagy.Results: Compared with the control group and 5% tension group, 20% tension group significantly decreased cell proliferation potency and telomerase activity, increased G1/G0 phase fraction, and up-regulated gene/protein expression of p16 and p53, whereas down-regulated gene/protein expression of aggrecan and collagen I. In addition, autophagy-related parameters such as gene/protein expression of Beclin-1 and LC3, and the ratio of LC3-II to LC3-I, were obviously suppressed in the 20% tension group.Conclusion: High mechanical tension promotes AF cell senescence though suppressing cellular autophagy. The present study will help us to better understand AF cell biology under mechanical tension and mechanical load-related disc degeneration.

Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells

Background: There is an increasing local application of methylene blue (MB) in the treatment of discogenic low back pain (LBP) and percutaneous transforaminal endoscopic discectomy (PTED) procedures. MB could generate DNA damage and induce apoptosis in different cell types; however, the effects of MB on intervertebral disc (IVD) annulus fibrosus (AF) cells are not clearly understood. Objective: The objective of this study was to investigate the effects of different concentrations of MB on rat AF cells in vitro. Study Design: This study used an experimental design. Setting: This research was conducted at the Orthopaedic Institute of the Clinical Medical College of Yangzhou University. Methods: AF cells were isolated and cultured with different concentrations of MB (0, 2, 20, and 200 μg/mL) and assessed to determine the possible cytotoxic effects of MB. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells, and flow cytometry was used to measure the incidence of cell apoptosis. The mRNA and protein expression levels of apoptosis-associated genes (caspase-3, Bcl-2, and Bax) and other related genes (collagen type I, transforming growth factor β1 [TGF-β1], fibroblast growth factor [bFGF], and tissue inhibitor of metalloproteinase-1 [TIMP-1]) were analyzed by quantitative real-time PCR (RT-PCR) and Western blotting. Results: Our results indicated that MB reduced cell viability in a concentration- and timedependent manner. MB also induced marked AF cell apoptosis in a concentration-dependent manner observed by inverted phase-contrast microscopy, flow cytometry, and indicated by the increased expression of caspase-3. Both RT-PCR and Western blotting revealed significant upregulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl2 in a concentration-dependent manner. Moreover, collagen type I, TGF-β1, bFGF, and TIMP-1 mRNA and protein levels were also found to be decreased by MB in a concentration-dependent manner. Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of MB on human IVD cells. Conclusions: Our results indicate that a high concentration of MB can not only inhibit proliferation and paracrine function of AF cells, but can also induce cell apoptosis in a concentration-dependent manner, suggesting that it is necessary to choose low concentrations of MB in practical application and limit the use of MB in the treatment of discogenic LBP to research protocols. Key words: Methylene blue, annulus fibrosus cell, proliferation, apoptosis, paracrine

Potential involvement of semaphorin 3A in maintaining intervertebral disc tissue homeostasis.

Intervertebral discs (IVDs) are avascular; however, ingrowth of blood vessels into their outer regions has been noted during the progression of degeneration. The mechanisms underlying vascularization in IVD degeneration are not completely understood. Semaphorin 3A (Sema3A), originally characterized as a chemorepulsive factor for growing axons in the developing nervous system, inhibits angiogenesis. This study aimed to elucidate the potential involvement of Sema3A in maintaining tissue homeostasis within the avascular IVD. We demonstrated that the mRNA expression of Sema3A was higher in rat annulus fibrosus (AF) than in nucleus pulposus (NP) and that its expression level decreased with age. Both mRNA and protein expression level of Sema3A was also markedly suppressed in AF tissues of a rat IVD degeneration model. Both real-time RT-PCR and Western blot clearly indicated that Sema3A expression significantly reduced by treating inflammatory cytokines in rat AF cells. In a gain- and loss-of-function study, we observed that Sema3A reduced the catabolic shift in rat AF cells. In addition, our results indicated that Sema3A potentially inhibited the IL-6/JAK/STAT pathway. Finally, BrdU assay and tube formation assay revealed that treatment of recombinant Sema3A significantly blocks both proliferation and tube formation of HUVEC. Our results indicate that Sema3A may help maintain IVD tissue homeostasis. Thus, although further studies are needed, Sema3A may be a potential molecular target for suppressing IVD degeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Responses of apoptosis and matrix metabolism of annulus fibrosus cells to different magnitudes of mechanical tension in vitro.

Background: Annulus fibrosus (AF) is important to confine disc nucleus pulposus (NP) tissue during mechanical load experience. However, the knowledge on AF cell biology under mechanical load is much limited compared with disc NP. Objective: The present study aimed to investigate responses of apoptosis and matrix metabolism of AF cells to different magnitudes of mechanical tension in vitro. Methods: Rat AF cells were subjected to different magnitudes (5, 10, and 20% elongations at a frequency of 1.0 Hz for 6 h per day) of mechanical tension for 7 days. Control AF cells were cultured without mechanical tension. Cell apoptosis ratio, caspase-3 activity, gene/protein expression of apoptosis-related molecules (Bcl-2, Bax, caspase-3/cleaved caspase-3 and cleaved PARP), matrix macromolecules (aggrecan and collagen I) and matrix metabolism-related enzymes (TIMP-1, TIMP-3, MMP-3, and ADAMTS-4) were analyzed. Results: Compared with 5% tension group and control group, 10 and 20% tension groups significantly increased apoptosis ratio, caspase-3 activity, up-regulated gene/protein expression of Bax, caspase-3/cleaved caspase-3, cleaved PARP, MMP-3, and ADAMTS-4, whereas down-regulated gene/protein expression of Bcl-2, aggrecan, collagen I, TIMP-1, and TIMP-3. No significant difference was found in these parameters apart from Bcl-2 expression between the control group and 5% tension group. Conclusion: High mechanical tension promotes AF cell apoptosis and suppresses AF matrix synthesis compared with low mechanical tension. The present study indirectly indicates how mechanical overload induces disc degeneration through affecting AF biology.

Potential Involvement of the IL-6/JAK/STAT3 Pathway in the Pathogenesis of Intervertebral Disc Degeneration.

Laboratory study. To elucidate the potential involvement of the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducers and activator of transcription (STAT3) pathway in the development of intervertebral disc (IVD) degeneration. IL-6 plays a crucial role in IVD degeneration; however, the downstream intracellular signaling of IL-6 in the IVD is not fully understood. The expression levels of IL-6 and suppressors of cytokine signaling 3 (SOCS3), a target gene of the IL-6/JAK/STAT3 pathway, were evaluated in rat and human degenerated IVD samples. The effects of IL-6 on primary rat annulus fibrosus (AF) cells were analyzed using quantitative PCR, immunocytochemistry, and Western blotting. The potential efficacy of a JAK inhibitor, CP690,550, in neutralizing the effect of IL-6 was evaluated in vitro. A high expression of IL-6 and SOCS3 was observed in both rat and human degenerated IVD samples. In rat AF cells, IL-6 markedly induced the phosphorylation of STAT3 and the expression of cyclooxygenase-2 and matrix metalloprotease-13. CP690,550 significantly suppressed the phosphorylation of STAT3 and offset the catabolic effect of IL-6 in rat AF cells. Our results suggest that the IL-6/JAK/STAT3 pathway is involved in the pathogenesis of IVD degeneration and that CP690,550 suppresses the catabolic effect of the IL-6 in the IVD. N/A.

17β-Estradiol Protects Rat Annulus Fibrosus Cells Against Apoptosis via α1 Integrin-Mediated Adhesion to Type I Collagen: An In-vitro Study.

Background17β-Estradiol (E2) has been reported to protect annulus fibrosus (AF) cells in vitro against interleukin-1β (IL-1β)-induced apoptosis in a concentration-dependent manner. However, its time-response effect remains unexplored. In addition, integrin α2/collagen II interaction has been reported to influence the apoptosis of nucleus pulposus cells in vitro. Thus, we hypothesized that integrin α1/collagen II might play a role in exerting the anti-apoptosis effect by E2. The aim of the current study was to further investigate the anti-apoptotic effect of E2 and determine the role of integrin α1/collagen II interaction.Material/MethodsRat AF cells were primary cultured and used for the following experiments. AF cells were identified by immunocytochemistry of type I collagen. Cell apoptosis was detected by fluorescence-activated cell sorter (FACS) analysis. The activity of active caspase-3 was determined by use of a caspase-3 detection kit. AF cell adhesion to type I collagen was determined by cell adhesion assay. Protein level of integrin subunit α1 was quantified by Western blot and mRNA expression was determined by real-time qPCR.ResultsThe immunocytochemistry of type I collagen revealed that cell purity was eligible for the following experiments with 98% of purity. FACS analysis indicated time-dependent anti-apoptosis effect of E2 at time points of 6 h, 12 h, and 24 h, which was confirmed by Caspase-3 activity. Furthermore, cell adhesion assay showed that E2 significantly increased cell binding to 95% of control, and qPCR and Western blot analysis showed that E2 effectively upregulated integrin α1. However, estrogen receptor antagonist ICI182780 prohibited the effect of E2.ConclusionsThis study shows that E2 protects against apoptosis in a time-dependent manner, and α1 integrin-mediated adhesion to collagen II is essential for estrogen-dependent anti-apoptosis in rat annulus fibrosus cells in vitro.

Synergistic Effect of Nerve Growth Factor and Insulin-Like Growth Factor-1 on Anti-Apoptosis and Extracellular Matrix Synthesis of Rat Intervertebral Disc Cells

Introduction Decreased cellularity due to excessive apoptosis of disc cells is a major predisposing factor for disc degeneration. Therefore, the inhibition of apoptosis of disc cells might be a possible therapeutic approach for modulating the process of disc degeneration. The purpose of the current study was to investigate the effect of nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1) in preventing apoptosis and increasing extracellular matrix synthesis in rat intervertebral disc cells. Material and Methods Expression of tropomyosin-related kinase A (TrkA) receptor for NGF and IGF-1 receptor (IGF-1R) for IGF-1 was examined in rat annulus fibrosus cells. In addition, rat annulus fibrosus cells were isolated, cultured, and placed in either 10% (normal control) or 0% (apoptosis-promoting condition) fetal bovine serum (FBS). We identified and quantified the degree of expression of TrkA and IGF-1R and apoptosis of the cells. Finally, we analyzed the effect of NGF (100 ng/ml) and IGF-1 (500 ng/ml) in preventing apoptosis and increasing synthesis of extracellular matrix proteins, such as aggrecan, collagen-1 and -2, in the cells, in 0% FBS. Results In situ expression of TrkA and IGF-1R was identified in rat annulus fibrosus cells. In addition, the degree of expression of TrkA and IGF-1R was decreased in the cells treated with 0% FBS but some degree of expression was still maintained. Apoptosis of the cells was increased in 0% FBS compared with 10% FBS ( p < 0.001). Despite extreme survival condition (0% FBS), each application of NGF (100 ng/ml) and IGF-1 (500 ng/ml) reduced apoptosis of the cells by 2% and 5%, respectively, which led to subsequently increased synthesis of aggrecan, collagen-1 and -2 (all, p < 0.05). The combined application of NGF (100 ng/ml) and IGF-1 (500 ng/ml) more significantly decreased apoptosis of the cells by 9% and increased synthesis of aggrecan, collagen-1 and -2 in the cells (all, p < 0.01). Conclusion The current findings demonstrate that apoptosis of intervertebral disc cells can be attenuated by NGF and IGF-1, which lead to increased extracellular matrix synthesis. Combined application of NGF and IGF-1 has a synergistic effect than single use of NGF and IGF-1. Our result suggests that NGF and IGF-1 may play a therapeutic role in slowing disc degeneration, which is due to inappropriate or excessive apoptosis of intervertebral disc cells.

Compression Induced Stress Response of Nucleus Pulposus Cells in 3D Collagen Gel

Introduction The cause of degenerative disc disease is multi-factorial and is often associated with mechanical loading. However, mechanism of the cells' response to compressive loading is not fully known. It was demonstrated that in mechanical force can induce chondrocytes to upregulate expression of heat shock proteins (HSPs) as a form of cellular stress response.1 Similarly, stretching annulus fibrosus cells was found to upregulate endoplasmic reticulum stress markers.2 However, it is unclear that whether cells in the intervertebral disc (IVD) would experience compressive force as cellular stress in 3D environment. The objective of this study is to investigate the stress response of nucleus pulposus cells (NPCs) during compression in 3D culture environment. Materials and Methods To simulate the condition in the IVD, NPCs were isolated from bovine caudal disc and encapsulated in 3D collagen gel as described previously.3 The culture system was shown to be able to maintain NPCs' phenotype.4 The cell-encapsulated gels were cultured for 4 days and then compressed with different types of loading (static and dynamic loading), different strains (10% - 70%) and different durations (2h - 8h). Samples were retrieved to study the expressions of stress response genes in two groups: Heat Shock Response (HSP70, HSP27, HSP90 and HSF1) and Unfolded Protein Response (GRP78, GRP94, ATF4 and CHOP). Results Encapsulation in 3D collagen gel was found to upregulate HSP70 in NPCs. Compression further induced stress response of NPCs which depended on multiple factors. The upregulation of stress response genes was generally insignificant with increasing loading strain in short duration. However the upregulations increased with longer duration where increasing loading duration has a larger effect in the upregulation of HSP70 and HSP27. When high static strain was applied, the expressions of Heat Shock Response genes were continued to be upregulated with time after the load was removed while Unfolded Protein Response genes were downregulated with incubation duration. The changes in expression with incubation duration show that cellular stress response may play a role in NPC survival and protein homeostasis post-loading. Conclusion The NPCs demonstrated cellular stress response during compression in collagen gel. The expression of stress response genes were mainly affected by loading duration and incubation duration after loading. This study helps us to understand how the NPCs cope with mechanical stress. Acknowledgments This project was funded by TBRS (T12–708/12-N). References Kaarniranta K, Elo M, Sironen R, et al. Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci U S A 1998;95(5):2319–2324 Zhang YH, Zhao CQ, Jiang LS, Dai LY. Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production. Eur Spine J 2011;20(8):1233–1243 Chan BP, Hui TY, Yeung CW, Li J, Mo I, Chan GC. Self-assembled collagen-human mesenchymal stem cell microspheres for regenerative medicine. Biomaterials 2007;28(31):4652–4666 Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011;11(10):947–960

Rho-Associated Kinase Inhibitor Immortalizes Rat Nucleus Pulposus and Annulus Fibrosus Cells: Establishment of Intervertebral Disc Cell Lines With Novel Approaches.

Establishment of immortalized cell lines derived from rat intervertebral disc cells by Rho-associated kinase (ROCK) inhibitor, Y-27632. To determine whether rat nucleus pulposus (NP) and annulus fibrosus (AF) cells could be immortalized, retain their phenotype, and used as cell lines for in vitro cell biology. Intervertebral disc degeneration is a major factor for most low-back pain. However, the mechanism of the disease is not well understood by the limitation to obtain sufficient amounts of primary disc cells. Therefore, the establishment of disc cell lines will help in vitro molecular signaling studies to understand the mechanism of degenerative disc disease. Cells were isolated from the NP and AF tissues of lumbar discs of adult Sprague Dawley rat. Tissues were digested and cultured with DMEM/Ham's F-12 (1:1) and 20% FBS and antibiotics. From day 3, cells were grown in the presence of 10 μM Y-27632, a well-characterized inhibitor of the ROCK, and subcultured by trypsinization, passaging them 1:3 onto 100 mm culture dishes. Morphologic and genetic analyses were performed on the different passaged cells. ROCK inhibitor successfully immortalized rat NP and AF cells. They passaged for over 50 generations with sustained expression levels of several NP and AF cell markers. In addition, they retained phenotypic features similar to the primary parental NP and AF cells when the cells were challenged with different cytokines and growth factors. We established immortalized rat NP and AF cell lines using a method of treating cells with ROCK inhibitor Y-27632 and demonstrated that these immortalized cells retain the properties of primary cells and could serve as useful tools for in vitro signaling studies or drug screening studies to develop novel therapeutic strategies. N/A.

Beta1 integrin inhibits apoptosis induced by cyclic stretch in annulus fibrosus cells via ERK1/2 MAPK pathway.

Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.

17β-estradiol protects against apoptosis induced by interleukin-1β in rat nucleus pulposus cells by down-regulating MMP-3 and MMP-13.

In our previous study, 17β-estradiol was proved to protect rat annulus fibrosus cells against apoptosis induced by interleukin-1β (IL-1β). However, whether 17β-estradiol has protective effect on rat nucleus pulposus cells remains unclear. The purpose of this study was to further explore the effects of 17β-estradiol on rat nucleus pulposus cells based on IL-1β-induced apoptosis. TUNEL assay and Annexin V/PI double staining were used to detect apoptosis and revealed that IL-1β induced notable apoptosis, which was reversed by 17β-estradiol. Meanwhile, cell viability and binding ability were decreased by IL-1β, but activated caspase-3 was increased. However, all of the detected effects of IL-1β were eliminated by 17β-estradiol. Furthermore, real-time quantitative RT-PCR was used to further find that IL-1β downregulated expression level of type II collagen, aggrecan, tissue inhibitor of matrix metalloproteinase (TIMP)-1, while upregulated matrix metalloproteinase (MMP)-3, MMP-13 and Bcl-2, which was further confirmed by western blot. Finally, 17β-estradiol was proved to abolish the above negative effects of IL-1β. In summary, this work presented that IL-1β maybe induced apoptosis of rat nucleus pulposus cells, which was resisted by 17β-estradiol by down-regulating MMP-3 and MMP-13 via a mitochondrial pathway. This research provides a novel insight into the anti-apoptotic effect of 17β-estradiol on IL-1β-induced cytotoxicity, and may potentially lead to a better understanding of the clinical effects of 17β-estradiol, especially in terms of intervertebral disc degeneration.