Yang-Hyun Chun

Cytoprotective and anti-inflammatory effects of melatonin in hydrogen peroxide-stimulated CHON-001 human chondrocyte cell line and rabbit model of osteoarthritis via the SIRT1 pathway.

Abstract:  Melatonin has potent antioxidant, analgesic, and antinociceptive properties. However, the effects of melatonin against oxidative stress‐induced cytotoxicity and inflammatory mediators in human chondrocytes remain poorly understood. This study examined the effects and underlying mechanism of melatonin in hydrogen peroxide (H2O2)‐stimulated human chondrocytes and rabbit osteoarthritis (OA) model. Melatonin markedly inhibited hydrogen peroxide (H2O2)‐stimulated cytotoxicity, iNOS, and COX‐2 protein and mRNA expression, as well as the downstream products, NO and PGE2. Incubation of cells with melatonin decreased H2O2‐induced Sirtuin 1 (SIRT1) mRNA and protein expression. SIRT1 inhibition by sirtinol or Sirt1 siRNA reversed the effects of melatonin on H2O2‐mediated induction of pro‐inflammatory cytokines (NO, PGE2, TNF‐α, IL‐1β, and IL‐8) and the expression of iNOS, COX‐2, and cartilage destruction molecules. Melatonin blocked H2O2‐induced phosphorylation of PI3K/Akt, p38, ERK, JNK, and MAPK, as well as activation of NF‐κB, which was reversed by sirtinol and SIRT1 siRNA. In rabbit with OA, intra‐articular injection of melatonin significantly reduced cartilage degradation, which was reversed by sirtinol. Taken together, this study shows that melatonin exerts cytoprotective and anti‐inflammatory effects in an oxidative stress‐stimulated chondrocyte model and rabbit OA model, and that the SIRT1 pathway is strongly involved in this effect.

Hypertonic saline‐induced muscle nociception and c‐fos activation are partially mediated by peripheral NMDA receptors

In this study, the animal model of hypertonic saline (HS) infusion protocol was developed and utilized to test the hypothesis that HS causes peripheral release of glutamate, and that blockade of peripheral NMDA receptors significantly reduces HS‐induced nocifensive behavior and central neuronal activation. Nocifensive behavior and c‐fos immunoreactivity, as a marker of central neuronal activation, were assessed from the animals that received intramuscular HS infusion with and without the NMDA receptor antagonist, MK‐801. HS infusion (20μl/min for 10min) in the rat masseter produced prolonged nocifensive hindpaw shaking responses that peaked in the first minute and gradually diminished over the infusion period. The HS induced nocifensive behavior was dose‐dependently attenuated by MK‐801 pretreatments (0.3mg/kg and 0.1mg/kg), but not by vehicle pretreatment (isotonic saline; ISO), in the masseter muscle. HS infusion produced a significant number of Fos positive neurons in the ispsilateral subnucleus caudalis (Vc). Subsequent immunohistochemical studies showed that peripheral MK‐801 pretreatment effectively reduced the HS induced neuronal activation in the Vc. These results provide compelling evidence that HS‐induced muscle nociception is mediated, in part, by peripheral release of glutamate, and that blockade of peripheral glutamate receptors may provide effective means of preventing central neuronal activation.

Peripheral AMPA receptors contribute to muscle nociception and c-fos activation

In this study, involvement of peripheral AMPA receptors in mediating craniofacial muscle pain was investigated. AMPA receptor subunits, GluR1 and GluR2, were predominantly expressed in small to medium size neurons but more GluR2 positive labeling were encountered in trigeminal ganglia (TG) of male Sprague Dawley rats. A greater prevalence of GluR2 is reflected by the significantly higher percentage of GluR2 than GluR1 positive masseter afferents. Nocifensive behavior and c-fos immunoreactivity were assessed from the same animals that received intramuscular mustard oil (MO) with or without NBQX, a potent AMPA/KA receptor antagonist. Masseteric MO produced nocifensive hindpaw shaking responses that peaked in the first 30s and gradually diminished over a few minutes. There was a significant difference in both peak and overall MO-induced nocifensive responses between NBQX and vehicle pre-treated rats. Subsequent Fos studies also showed that peripheral NBQX pre-treatment effectively reduced the MO-induced neuronal activation in the subnucleus caudalis of the trigeminal nerve (Vc). These combined results provide compelling evidence that acute muscle nociception is mediated, in part, by peripherally located AMPA/KA receptors, and that blockade of multiple peripheral glutamate receptor subtypes may provide a more effective means of reducing muscular pain and central neuronal activation.

Muramyl dipeptide activates human beta defensin 2 and pro-inflammatory mediators through Toll-like receptors and NLRP3 inflammasomes in human dental pulp cells.

PurposeThe expression levels of intracellular pyrin domain-containing 3 (NLRP3) and microbial pattern-recognition receptors, such as nucleotide-binding oligomerization domain 2 (NOD2), have been reported in human dental pulp cells (HDPCs) and inflamed dental pulp tissue, but the role of NLRP3 and Toll-like receptors (TLRs) in the production of human beta defensin 2 (hBD2) and inflammatory cytokines against invading pathogens remains poorly defined. The aim of this study was to determine whether the NOD2 ligand muramyl dipeptide (MDP) upregulates hBD2 and inflammatory cytokines and whether this response is dependent on TLRs and NLRP inflammasomes in HDPCs.MethodologyThe effects of MDP on the expression of hBD2, TLRs, inflammasomes, and pro-inflammatory mediators in HDPCs were examined using Western blotting and reverse transcription–polymerase chain reaction. Levels of pro-inflammatory cytokines, such as nitric oxide (NO) and prostaglandin E2 (PGE2), were determined by enzyme-linked immunosorbent assay.ResultsMDP upregulated hBD2, TLR2, and TLR4 mRNAs and protein levels in a dose- and time-dependent manner. TLR2 and TLR4 neutralizing blocking antibodies and NOD2- and hBD2-specific small interfering RNAs (siRNAs) attenuated the MDP-induced production of NO, PGE2, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-8 and upregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) in HDPCs. Additionally, MDP activated inflammasome-related genes, such as NLRP3, caspase 1, apoptotic speck protein containing a caspase recruitment domain, and IL-1β. Furthermore, silencing of the NLRP3 gene using a siRNA significantly decreased the MDP-induced expression of hBD2 and cytokines, such as iNOS-derived NO, COX2, PGE2, TNF-α, IL-6, and IL-8.ConclusionThese results suggest that NOD2 activates the TLR2, TLR4, and NLRP3 inflammasome-signaling pathways in HDPCs to induce the production of multiple inflammatory mediators and antimicrobial peptides, which in turn promote pulp immune defense against microbial challenge.Clinical relevanceThe TLR and NLRP3 inflammasome pathways may represent an important modulatory mechanism of immune defense responses during the progression of pulpitis. Our results suggest that local inhibition of NLRP3 and TLRs may reduce the impact of cytokine-mediated host destructive processes in pulpitis.

Orofacial complex regional pain syndrome: pathophysiologic mechanisms and functional MRI

Complex regional pain syndrome (CRPS) is one of the most challenging chronic pain conditions and is characterized by burning pain, allodynia, hyperalgesia, autonomic changes, trophic changes, edema, and functional loss involving mainly the extremities. Until recently, very few reports have been published concerning CRPS involving the orofacial area. We report on a 50-year-old female patient who presented with unbearable pain in all of her teeth and hypersensitivity of the facial skin. She also reported intractable pain in both extremities accompanied by temperature changes and orofacial pain that increased when the other pains were aggravated. In the case of CRPS with trigeminal neuropathic pain, protocols for proper diagnosis and prompt treatment have yet to be established in academia or in the clinical field. We performed functional magnetic resonance imaging for a thorough analysis of the cortical representation of the affected orofacial area immediately before and immediately after isolated light stimulus of the affected hand and foot and concluded that CRPS can be correlated with trigeminal neuropathy in the orofacial area. Furthermore, the patient was treated with carbamazepine administration and stellate ganglion block, which can result in a rapid improvement of pain in the trigeminal region.