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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

Downstream signaling of ROS in AF cells. a, b Western blot analysis showed that mitogen-activated protein kinases (MAPKs), including p38, ERK, and JNK, were maximally phosphorylated 10 minutes after treatment with H2O2a and BSO b. c, d Real-time RT-PCR analysis. Treatment of AF cells with MAPK signaling inhibitors, including p38 inhibitor (SB), JNK inhibitor (SP), and ERK inhibitor (PD), significantly abolished H2O2-mediated c or BSO-mediated d induction of COX-2 mRNA expression. Data presented as mean ± SD of three independent experiments performed in triplicate (n = 3); *p <0.05. BSO buthionine sulfoximine, COX cyclooxygenase, ERK extracellular signal-regulated kinase, H2O2 hydrogen peroxide, HPRT hypoxanthine phosphoribosyl transferase, JNK c-Jun N-terminal kinase
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Fig3: Downstream signaling of ROS in AF cells. a, b Western blot analysis showed that mitogen-activated protein kinases (MAPKs), including p38, ERK, and JNK, were maximally phosphorylated 10 minutes after treatment with H2O2a and BSO b. c, d Real-time RT-PCR analysis. Treatment of AF cells with MAPK signaling inhibitors, including p38 inhibitor (SB), JNK inhibitor (SP), and ERK inhibitor (PD), significantly abolished H2O2-mediated c or BSO-mediated d induction of COX-2 mRNA expression. Data presented as mean ± SD of three independent experiments performed in triplicate (n = 3); *p <0.05. BSO buthionine sulfoximine, COX cyclooxygenase, ERK extracellular signal-regulated kinase, H2O2 hydrogen peroxide, HPRT hypoxanthine phosphoribosyl transferase, JNK c-Jun N-terminal kinase

Mentions: To clarify the pathophysiological role of intracellular ROS, we examined the phenotype of the AF cells treated with H2O2 and BSO, which is a glutathione synthesis inhibitor that activates oxidative stress. Flow cytometry confirmed that the intracellular level of ROS was significantly increased by treatment with both H2O2 and BSO in AF cells (Fig. 2a). Next, we treated rat cultured AF cells with H2O2 and evaluated the expression of catabolic and anabolic factors of IVD degeneration by real-time RT-PCR analysis. We found that the mRNA expression of TNFα, MMP-3, and COX-2 was significantly induced, whereas that of aggrecan was reduced in a dose-dependent manner (Fig. 2b). Expectedly, real-time RT-PCR showed similar results with BSO treatment (Fig. 2c). To investigate the downstream signaling of ROS in AF cells, we evaluated the phosphorylation of MAPKs, including p38, ERK, and JNK, as well as p65 by western blotting. This analysis showed that the three signaling pathways of MAPK were maximally phosphorylated 10 minutes after treatment with H2O2 and BSO (Fig. 3a, b). On the other hand, the phosphorylation of p65 was not activated by their treatments (Fig. 3a, b). Next, to investigate further the involvement of MAPKs, we cultured H2O2-treated or BSO-treated AF cells with MAPK signaling inhibitors, including p38 inhibitor (SB203580), JNK inhibitor (SP600125), and ERK inhibitor (PD98059), and assessed the mRNA expression of COX-2, TNFα, and MMP-3 by real time RT-PCR analysis. Figure 3c, d shows that all inhibitors significantly abolished H2O2-mediated or BSO-mediated induction of COX-2 mRNA expression. These results suggest that the catabolic effect of excessive ROS is mediated through the signaling pathways of p38, ERK, and JNK in AF cells. However, the results of TNFα and MMP-3 did not clearly show the effect of these inhibitors compared with COX-2 (Additional file 2: Figure S2). The gene regulatory network of TNFα and MMP3 can be more complicated than that of COX-2. The experiment concerned with the mechanisms of the difference is in progress.Fig. 2


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)

Downstream signaling of ROS in AF cells. a, b Western blot analysis showed that mitogen-activated protein kinases (MAPKs), including p38, ERK, and JNK, were maximally phosphorylated 10 minutes after treatment with H2O2a and BSO b. c, d Real-time RT-PCR analysis. Treatment of AF cells with MAPK signaling inhibitors, including p38 inhibitor (SB), JNK inhibitor (SP), and ERK inhibitor (PD), significantly abolished H2O2-mediated c or BSO-mediated d induction of COX-2 mRNA expression. Data presented as mean ± SD of three independent experiments performed in triplicate (n = 3); *p <0.05. BSO buthionine sulfoximine, COX cyclooxygenase, ERK extracellular signal-regulated kinase, H2O2 hydrogen peroxide, HPRT hypoxanthine phosphoribosyl transferase, JNK c-Jun N-terminal kinase
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Fig3: Downstream signaling of ROS in AF cells. a, b Western blot analysis showed that mitogen-activated protein kinases (MAPKs), including p38, ERK, and JNK, were maximally phosphorylated 10 minutes after treatment with H2O2a and BSO b. c, d Real-time RT-PCR analysis. Treatment of AF cells with MAPK signaling inhibitors, including p38 inhibitor (SB), JNK inhibitor (SP), and ERK inhibitor (PD), significantly abolished H2O2-mediated c or BSO-mediated d induction of COX-2 mRNA expression. Data presented as mean ± SD of three independent experiments performed in triplicate (n = 3); *p <0.05. BSO buthionine sulfoximine, COX cyclooxygenase, ERK extracellular signal-regulated kinase, H2O2 hydrogen peroxide, HPRT hypoxanthine phosphoribosyl transferase, JNK c-Jun N-terminal kinase
Mentions: To clarify the pathophysiological role of intracellular ROS, we examined the phenotype of the AF cells treated with H2O2 and BSO, which is a glutathione synthesis inhibitor that activates oxidative stress. Flow cytometry confirmed that the intracellular level of ROS was significantly increased by treatment with both H2O2 and BSO in AF cells (Fig. 2a). Next, we treated rat cultured AF cells with H2O2 and evaluated the expression of catabolic and anabolic factors of IVD degeneration by real-time RT-PCR analysis. We found that the mRNA expression of TNFα, MMP-3, and COX-2 was significantly induced, whereas that of aggrecan was reduced in a dose-dependent manner (Fig. 2b). Expectedly, real-time RT-PCR showed similar results with BSO treatment (Fig. 2c). To investigate the downstream signaling of ROS in AF cells, we evaluated the phosphorylation of MAPKs, including p38, ERK, and JNK, as well as p65 by western blotting. This analysis showed that the three signaling pathways of MAPK were maximally phosphorylated 10 minutes after treatment with H2O2 and BSO (Fig. 3a, b). On the other hand, the phosphorylation of p65 was not activated by their treatments (Fig. 3a, b). Next, to investigate further the involvement of MAPKs, we cultured H2O2-treated or BSO-treated AF cells with MAPK signaling inhibitors, including p38 inhibitor (SB203580), JNK inhibitor (SP600125), and ERK inhibitor (PD98059), and assessed the mRNA expression of COX-2, TNFα, and MMP-3 by real time RT-PCR analysis. Figure 3c, d shows that all inhibitors significantly abolished H2O2-mediated or BSO-mediated induction of COX-2 mRNA expression. These results suggest that the catabolic effect of excessive ROS is mediated through the signaling pathways of p38, ERK, and JNK in AF cells. However, the results of TNFα and MMP-3 did not clearly show the effect of these inhibitors compared with COX-2 (Additional file 2: Figure S2). The gene regulatory network of TNFα and MMP3 can be more complicated than that of COX-2. The experiment concerned with the mechanisms of the difference is in progress.Fig. 2

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