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Targeting oxidative stress with amobarbital to prevent intervertebral disc degeneration: Part I. in vitro and ex vivo
Dongrim Seol1, Mitchell C Coleman2, James A Martin3
1Departments of Orthopedics and Rehabilitation, University of Iowa, Iowa City, IA 52242, USA.
Background:
Mounting evidence that oxidative stress contributes to the pathogenesis of intervertebral disc (IVD) degeneration (IDD) suggests that therapies targeting oxidative stress may slow or prevent disease progression.
Purpose:
The objective of this study was to investigate the inhibitory effects of amobarbital (Amo) on the mitochondria of nucleus pulposus (NP) cells under tert-butyl hydrogen peroxide (tBHP)-induced oxidative stress or in NP tissues under oxidative stress from tissue injury as a means of identifying therapeutic targets for IDD.
Study Design/Setting:
We tested the effects inhibiting mitochondria, a major source of oxidants, with Amo in NP cells subjected to two different forms of insult: exposure to tBHP, and physical injury induced by disc transection. N-acetylcysteine (NAC), an antioxidant known to protect NP cells, was compared to the complex I inhibitor, Amo.
Methods:
NP cells were pre-treated for 2 hours with Amo, NAC, or both, and then exposed to tBHP for 1 hour. Apoptosis, necrosis, and reactive oxygen species (ROS) production were assessed using confocal microscopy and fluorescent probes (Annexin V, propidium iodide, and MitoSox Red, respectively). The activation of mitogen-activated protein kinases (MAPKs) involved in oxidative stress responses were interrogated by confocal imaging of immunofluorescence stains using phospho-specific antibodies to extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38. Mitochondrial function was assessed by imaging JC-1 staining, a probe for membrane potential.
Results:
Amo was modestly more protective than NAC by some measures, while both agents improved mitochondrial function and lowered tBHP-induced apoptosis, necrosis, and ROS production. Activation of MAPK by tBHP was significantly suppressed by both drugs. Physically injured IVDs were treated immediately after transection with Amo or NAC for 24 hours, and then stained with dihydroethidium (DHE), a fluorescent probe for ROS production. Immunofluorescence was used to track the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor that induces the expression of antioxidant genes. Amo and NAC significantly reduced ROS production and increased Nrf2 expression.
Conclusion:
These findings suggest that the progression of IDD may be forestalled by Amo via protection of NP cells from oxidative stress following IVD injury.
Clinical Significance:
This study will define the extent to which a novel, minimally invasive procedure targeting oxidative stress in NP cells can augment surgical interventions intended to retard IVD degeneration.
Insights
Amobarbital (Amo) protects nucleus pulposus cells from oxidative stress, potentially slowing intervertebral disc degeneration (IDD). This study investigated Amo
Area of Science:
- Biomedical Sciences
- Cell Biology
- Biochemistry
Background:
- Oxidative stress is a key factor in intervertebral disc degeneration (IDD).
- Targeting oxidative stress pathways offers a potential therapeutic strategy for IDD.
- Nucleus pulposus (NP) cells are central to disc health and vulnerable to oxidative damage.
Purpose of the Study:
- To investigate the protective effects of amobarbital (Amo) on NP cells against oxidative stress.
- To evaluate Amo's efficacy in mitigating oxidative stress in NP cells induced by tert-butyl hydrogen peroxide (tBHP) and physical injury.
- To identify amobarbital as a potential therapeutic target for intervertebral disc degeneration.
Main Methods:
- NP cells were exposed to tBHP or physical injury (disc transection).
- Cells were treated with amobarbital (Amo) or N-acetylcysteine (NAC) and assessed for apoptosis, necrosis, reactive oxygen species (ROS), and mitochondrial function.
- MAPK activation and Nrf2 expression were analyzed using immunofluorescence.
Main Results:
- Amobarbital demonstrated protective effects comparable to or exceeding NAC in reducing ROS and apoptosis.
- Both Amo and NAC improved mitochondrial function and suppressed MAPK activation.
- Treatment with Amo and NAC reduced ROS production and increased Nrf2 expression in injured IVD tissues.
Conclusions:
- Amobarbital protects NP cells from oxidative stress, suggesting its potential to slow IDD progression.
- Targeting mitochondrial function with amobarbital presents a novel therapeutic approach for IDD.
- Further research will explore minimally invasive procedures involving amobarbital to augment surgical interventions for IDD.
