Abstract and Introduction
Study Design. An in vitro study to investigate the anti-inflammatory effects of fullerol on mouse dorsal root ganglia (DRG) under tumor necrosis factor (TNF)-α induction.
Objective. To evaluate the potential of a free radical scavenger, fullerol nanoparticles, to prevent DRG tissue and neuron inflammatory responses under TNF-α induction in vitro.
Summary of Background Data. Low back pain is one of the most common reasons for clinician visits in Western societies. Symptomatic intervertebral disc degeneration is strongly implicated as a cause of low back pain, as it results in DRG inflammation. Increased production of reactive oxygen species (ROS) is associated with DRG inflammation.
Methods. With or without fullerol treatment, DRG tissue and DRG neurons isolated from wild-type C3H/HeNCrl (Charles River Laboratories, Wilmington, MA) mice were cultured under TNF-α induction. The amount of intracellular ROS was measured with H2DCFDA (Life Technologies Corporation, Grand Island, NY) fluorescence staining. Cellular apoptosis was detected via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The expression of inflammatory as well as antioxidative enzyme genes in neurons was analyzed by real-time polymerase chain reaction. In addition, inflammatory cytokine expression in DRG tissue was determined by immunofluorescence staining and enzyme-linked immunosorbent assay.
Results. Fluorescence staining results indicated that TNF-α markedly increased the production of intracellular ROS and the number of apoptotic cells. Under fullerol treatment, cellular apoptosis was reduced along with concomitant suppression of ROS. The expression of inflammatory cytokines interleukin 1 β, interleukin 6, cyclooxygenase-2, and prostaglandin E2, was also inhibited by fullerol in a dose-dependent manner. Furthermore, fullerol-treated cells exhibited upregulation of antioxidative enzyme genes superoxide dismutase 2 and catalase.
Conclusion. The results obtained from this study clearly suggest that fullerol treatment suppresses the inflammatory responses of DRG and neurons, as well as cellular apoptosis by decreasing the level of ROS and potentially enhancing antioxidative enzyme gene expression. Therefore, fullerol has potential to serve as a novel therapeutic agent for low back pain treatment.
Low back pain is one of the most common pathophysiological conditions for clinical visits, with a lifetime prevalence of 60% to 90%.[1,2] Ingrowth of nociceptive neural fiber into deeper parts of the degenerative intervertebral disc (IVD) is considered as one of the most widely accepted pathomechanisms related to chronic discogenic pain. In normal disc, the outer layers of the annulus fibrosus are innervated by sensory nerve endings from the dorsal root ganglia (DRG). As the disc degeneration proceeds, disc inflammation may promote axonal growth of afferent fibers innervating the disc by secreting proinflammatory mediators, such as tumor necrosis factor (TNF) and interleukin 6 (IL-6). The pain signal could be triggered as the neurons of the DRG transmit the inflammatory signal through the spinal cord to the pain centers of the brain. Thus, relieving the inflammatory tension of the DRG would be of great significance to treat low back pain caused by IVD degeneration.
Clinically, epidural injection of steroids, such as corticosteroids that decrease inflammation through inhibition of prostaglandins, has been well accepted practice and a rational approach to treat low back pain. However, some absolute contraindications for this strategy exist: infection, history of severe allergic reaction to any of the injected materials, and local malignancy. In addition, steroids have no tissue specificity and affect not only inflammation, but also development, homeostasis, metabolism, and cognition. Administration of inhibitors for inflammatory mediators has also been investigated to treat low back pain. The cytokine TNF-α is a mediator of chronic pain produced by the onset of inflammation or sensory nerve damage. The levels of TNF-α are shown to be elevated in animal models of inflammatory response or neuropathic pain around the spinal cord and at the site of injury. Several clinical trials showed that application of the TNF-α inhibitor etancercept onto the spinal nerve produced pain relief.[10–12] However, it was noted that etancercept has serious potential toxicity and widespread adoption of this method should await further studies. Therefore, discovering alternative novel agents to treat low back pain is of utmost importance.
Reactive oxygen species (ROS), at a moderate level, are recognized to be physically involved in cell signaling and required for biochemical energetics of life. However, when produced in excess, ROS can be associated with several pathological conditions, including cellular inflammatory responses.[13,14] Under potentially pathological conditions, a cellular oxidative burst, or respiratory burst occurs, resulting in synthesis of an array of soluble factors, such as ROS, TNF-α, and IL-1 α/β, which further induce neuronal dysfunction and degeneration. Therefore, attenuation of oxidative tension by quenching ROS may be a novel way to rescue DRG inflammation caused by IVD degeneration, toward low back pain treatment.
Recently, fullerene (C60) and its derivatives have drawn a great deal of attention in biomedical research fields due to their antioxidative features. Fullerene is considered to function as a "free radical sponge" and is shown to quench varieties of free radicals more efficiently than conventional antioxidants. The antioxidative properties of fullerene are attributed to a highly delocalized π double bond system forming the carbon cage. Gharbi et al demonstrated that fullerene has strong protective effects on rodent liver against free radical damage due to its antioxidative features.
In this study, we investigated the anti-inflammatory effects of fullerol, a polyhydroxylated derivative of fullerene, which possesses good biocompatibility and excellent efficiency in eliminating reactive free radicals. It was hypothesized that fullerol nanoparticles protect DRG tissue and neurons from TNF-α–induced inflammatory responses by decreasing oxidative tension as well as other pathways. Our results demonstrated the anti-inflammatory effects of fullerol on TNF-α–induced mouse DRG tissue and neurons. This study is of clinical significance for relieving low back pain caused by IVD degeneration.
Spine. 2013;38(17):1443-1451. © 2013 Lippincott Williams & Wilkins