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Excessive reactive oxygen species are therapeutic targets for intervertebral disc degeneration.

Suzuki S, Fujita N, Hosogane N, Watanabe K, Ishii K, Toyama Y, Takubo K, Horiuchi K, Miyamoto T, Nakamura M, Matsumoto M - Arthritis Res. Ther. (2015)

Bottom Line: A high frequency of nitrotyrosine-positive cells was observed in rat and human degenerative discs. mRNA expression of catabolic factors such as tumor necrosis factor-alpha (TNF-alpha), matrix metalloprotease-3 (MMP-3), and cyclooxygenase-2 (COX-2) was significantly induced by treatment with H2O2 or buthionine sulfoximine, whereas that of aggrecan, an important chondrogenic proteoglycan, was reduced in a dose-dependent manner.Treatment with mitogen-activated protein kinase (MAPK) inhibitors blocked the inductive effect of excessive ROS on COX-2 mRNA expression.Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated the catabolic effect of excessive ROS and TNF-alpha in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. ssatosea@yahoo.co.jp.

ABSTRACT

Introduction: Oxidative stress has been reported to be involved in numerous human diseases, including musculoskeletal disorders such as osteoarthritis. However, the interaction between intervertebral disc (IVD) degeneration and oxidative stress is not well understood. The purpose of the present study was to elucidate the contribution of oxidative stress to IVD degeneration and the efficacy of antioxidant treatment for degenerative discs.

Methods: The expression level of an oxidative stress marker, nitrotyrosine, was assessed by immunohistochemistry and Western blotting. For evaluating intracellular reactive oxygen species (ROS) levels and oxidative stress in rat annulus fibrosus (AF) cells, flow cytometry and luciferase assay with an OKD48 construct were performed. The grade of IVD degeneration was assessed by magnetic resonance imaging and histological analysis.

Results: A high frequency of nitrotyrosine-positive cells was observed in rat and human degenerative discs. mRNA expression of catabolic factors such as tumor necrosis factor-alpha (TNF-alpha), matrix metalloprotease-3 (MMP-3), and cyclooxygenase-2 (COX-2) was significantly induced by treatment with H2O2 or buthionine sulfoximine, whereas that of aggrecan, an important chondrogenic proteoglycan, was reduced in a dose-dependent manner. Treatment with mitogen-activated protein kinase (MAPK) inhibitors blocked the inductive effect of excessive ROS on COX-2 mRNA expression. Western blotting confirmed the phosphorylation of MAPKs in H2O2 and BSO-treated AF cells. Conversely, we showed that TNF-α induced oxidative stress with increased intracellular ROS levels in AF cells. Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated the catabolic effect of excessive ROS and TNF-alpha in vitro. Finally, we showed that oral administration of NAC prevented IVD degeneration in rat degenerative model.

Conclusions: A positive feedback loop was formed between excessive ROS and TNF-alpha in AF cells. Thus, oxidative stress contributes to the progression of IVD degeneration and NAC can be a therapeutic option for IVD degeneration.

No MeSH data available.


Related in: MedlinePlus

Schematic of relationship between oxidative stress and disc degeneration. Schematic showing positive catabolic feedback loop between excessive ROS and catabolic factors in AF cells. Excessive ROS induced expression of catabolic factors, which are upregulated in the degenerative state, and reduced cartilage ECM aggrecan via the signaling pathways of p38 in AF cells. Conversely, TNFα increased intracellular ROS levels in AF cells through p38, JNK, ERK, and p65. These pathways were neutralized by NAC. COX cyclooxygenase, ERK extracellular signal-regulated kinase, JNK c-Jun N-terminal kinase, MAPK mitogen-activated protein kinase, MMP matrix metalloprotease, NAC N-acetyl cysteine, NF nuclear factor, TNF tumor necrosis factor
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Fig8: Schematic of relationship between oxidative stress and disc degeneration. Schematic showing positive catabolic feedback loop between excessive ROS and catabolic factors in AF cells. Excessive ROS induced expression of catabolic factors, which are upregulated in the degenerative state, and reduced cartilage ECM aggrecan via the signaling pathways of p38 in AF cells. Conversely, TNFα increased intracellular ROS levels in AF cells through p38, JNK, ERK, and p65. These pathways were neutralized by NAC. COX cyclooxygenase, ERK extracellular signal-regulated kinase, JNK c-Jun N-terminal kinase, MAPK mitogen-activated protein kinase, MMP matrix metalloprotease, NAC N-acetyl cysteine, NF nuclear factor, TNF tumor necrosis factor

Mentions: IVD degeneration is characterized by increases in levels of the proinflammatory cytokines TNFα, IL-1α, IL-1β, IL-6, and IL-17, secreted by IVD cells [12, 13, 44, 45]. In particular, it is known that TNFα and IL-1β play pivotal roles in the progression of these degenerative changes [12, 13]. Cytokines have been shown to upregulate chemokines and various catabolic mediators, including ADAMTS-4, ADAMTS-5, MMP-1, MMP-2, MMP-3, MMP-13, and syndecan-4, and to suppress the expression of important ECM genes through NF-κB and MAPK signaling pathways in degenerative disc cells [14–17, 46, 47]. In a previous study, Yoshimura et al. [48] demonstrated that IL-1β induced cell death in rat primary chondrocytes and mouse chondrocytic ATDC5 cells via mitochondrial dysfunction in a ROS-dependent manner. Our flow cytometry analysis clearly showed that intracellular ROS levels were significantly induced by treatment with TNFα in AF cells. Furthermore, to evaluate oxidative stress conditions in TNFα-treated AF cells, a luciferase assay was carried out using OKD48 construct. Under oxidative stress conditions, Nrf2, a basic region leucine zipper transcription factor, is known to be stabilized at the posttranscriptional level [49]. OKD48 consists of an oxidative stress-inducible promoter, luciferase, and an Nrf2 fragment that contributes to stress-dependent stabilization [36]. This analysis clearly showed that oxidative stress was significantly induced by TNFα in AF cells. Vice versa, our results showed that the expression of catabolic factors, including TNFα, was induced by treatment with H2O2 or BSO in AF cells, which indicated that there was a reciprocal interaction between excessive ROS and TNFα in inflammatory AF cells (Fig. 8). We previously reported that ROS activated the ERK and p38 MAPK pathways but not the JNK pathway in ATDC5 cells [50]. On the other hand, the present study showed that JNK signaling was also activated by treatment of AF cells with ROS, suggesting that the downstream pathway of ROS is cell or tissue specific.Fig. 8


Excessive reactive oxygen species are therapeutic targets for intervertebral disc degeneration.

Suzuki S, Fujita N, Hosogane N, Watanabe K, Ishii K, Toyama Y, Takubo K, Horiuchi K, Miyamoto T, Nakamura M, Matsumoto M - Arthritis Res. Ther. (2015)

Schematic of relationship between oxidative stress and disc degeneration. Schematic showing positive catabolic feedback loop between excessive ROS and catabolic factors in AF cells. Excessive ROS induced expression of catabolic factors, which are upregulated in the degenerative state, and reduced cartilage ECM aggrecan via the signaling pathways of p38 in AF cells. Conversely, TNFα increased intracellular ROS levels in AF cells through p38, JNK, ERK, and p65. These pathways were neutralized by NAC. COX cyclooxygenase, ERK extracellular signal-regulated kinase, JNK c-Jun N-terminal kinase, MAPK mitogen-activated protein kinase, MMP matrix metalloprotease, NAC N-acetyl cysteine, NF nuclear factor, TNF tumor necrosis factor
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4635526&req=5

Fig8: Schematic of relationship between oxidative stress and disc degeneration. Schematic showing positive catabolic feedback loop between excessive ROS and catabolic factors in AF cells. Excessive ROS induced expression of catabolic factors, which are upregulated in the degenerative state, and reduced cartilage ECM aggrecan via the signaling pathways of p38 in AF cells. Conversely, TNFα increased intracellular ROS levels in AF cells through p38, JNK, ERK, and p65. These pathways were neutralized by NAC. COX cyclooxygenase, ERK extracellular signal-regulated kinase, JNK c-Jun N-terminal kinase, MAPK mitogen-activated protein kinase, MMP matrix metalloprotease, NAC N-acetyl cysteine, NF nuclear factor, TNF tumor necrosis factor
Mentions: IVD degeneration is characterized by increases in levels of the proinflammatory cytokines TNFα, IL-1α, IL-1β, IL-6, and IL-17, secreted by IVD cells [12, 13, 44, 45]. In particular, it is known that TNFα and IL-1β play pivotal roles in the progression of these degenerative changes [12, 13]. Cytokines have been shown to upregulate chemokines and various catabolic mediators, including ADAMTS-4, ADAMTS-5, MMP-1, MMP-2, MMP-3, MMP-13, and syndecan-4, and to suppress the expression of important ECM genes through NF-κB and MAPK signaling pathways in degenerative disc cells [14–17, 46, 47]. In a previous study, Yoshimura et al. [48] demonstrated that IL-1β induced cell death in rat primary chondrocytes and mouse chondrocytic ATDC5 cells via mitochondrial dysfunction in a ROS-dependent manner. Our flow cytometry analysis clearly showed that intracellular ROS levels were significantly induced by treatment with TNFα in AF cells. Furthermore, to evaluate oxidative stress conditions in TNFα-treated AF cells, a luciferase assay was carried out using OKD48 construct. Under oxidative stress conditions, Nrf2, a basic region leucine zipper transcription factor, is known to be stabilized at the posttranscriptional level [49]. OKD48 consists of an oxidative stress-inducible promoter, luciferase, and an Nrf2 fragment that contributes to stress-dependent stabilization [36]. This analysis clearly showed that oxidative stress was significantly induced by TNFα in AF cells. Vice versa, our results showed that the expression of catabolic factors, including TNFα, was induced by treatment with H2O2 or BSO in AF cells, which indicated that there was a reciprocal interaction between excessive ROS and TNFα in inflammatory AF cells (Fig. 8). We previously reported that ROS activated the ERK and p38 MAPK pathways but not the JNK pathway in ATDC5 cells [50]. On the other hand, the present study showed that JNK signaling was also activated by treatment of AF cells with ROS, suggesting that the downstream pathway of ROS is cell or tissue specific.Fig. 8

Bottom Line: A high frequency of nitrotyrosine-positive cells was observed in rat and human degenerative discs. mRNA expression of catabolic factors such as tumor necrosis factor-alpha (TNF-alpha), matrix metalloprotease-3 (MMP-3), and cyclooxygenase-2 (COX-2) was significantly induced by treatment with H2O2 or buthionine sulfoximine, whereas that of aggrecan, an important chondrogenic proteoglycan, was reduced in a dose-dependent manner.Treatment with mitogen-activated protein kinase (MAPK) inhibitors blocked the inductive effect of excessive ROS on COX-2 mRNA expression.Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated the catabolic effect of excessive ROS and TNF-alpha in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo, 160-8582, Japan. ssatosea@yahoo.co.jp.

ABSTRACT

Introduction: Oxidative stress has been reported to be involved in numerous human diseases, including musculoskeletal disorders such as osteoarthritis. However, the interaction between intervertebral disc (IVD) degeneration and oxidative stress is not well understood. The purpose of the present study was to elucidate the contribution of oxidative stress to IVD degeneration and the efficacy of antioxidant treatment for degenerative discs.

Methods: The expression level of an oxidative stress marker, nitrotyrosine, was assessed by immunohistochemistry and Western blotting. For evaluating intracellular reactive oxygen species (ROS) levels and oxidative stress in rat annulus fibrosus (AF) cells, flow cytometry and luciferase assay with an OKD48 construct were performed. The grade of IVD degeneration was assessed by magnetic resonance imaging and histological analysis.

Results: A high frequency of nitrotyrosine-positive cells was observed in rat and human degenerative discs. mRNA expression of catabolic factors such as tumor necrosis factor-alpha (TNF-alpha), matrix metalloprotease-3 (MMP-3), and cyclooxygenase-2 (COX-2) was significantly induced by treatment with H2O2 or buthionine sulfoximine, whereas that of aggrecan, an important chondrogenic proteoglycan, was reduced in a dose-dependent manner. Treatment with mitogen-activated protein kinase (MAPK) inhibitors blocked the inductive effect of excessive ROS on COX-2 mRNA expression. Western blotting confirmed the phosphorylation of MAPKs in H2O2 and BSO-treated AF cells. Conversely, we showed that TNF-α induced oxidative stress with increased intracellular ROS levels in AF cells. Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated the catabolic effect of excessive ROS and TNF-alpha in vitro. Finally, we showed that oral administration of NAC prevented IVD degeneration in rat degenerative model.

Conclusions: A positive feedback loop was formed between excessive ROS and TNF-alpha in AF cells. Thus, oxidative stress contributes to the progression of IVD degeneration and NAC can be a therapeutic option for IVD degeneration.

No MeSH data available.


Related in: MedlinePlus