Photobiomodulation transiently increases the spontaneous firing in the superficial layer of the rat spinal dorsal horn.

The therapeutic benefits of photobiomodulation (PBM) in pain management, although well documented, are accompanied by concerns about potential risks, including pain, particularly at higher laser intensities. This study investigated the effects of laser intensity on pain perception using behavioral and electrophysiological evaluations in rats. Our results show that direct laser irradiation of 1000 mW/cm2 to the sciatic nerve transiently increases the frequency of spontaneous firing in the superficial layer without affecting the deep layer of the spinal dorsal horn, and this effect reverses to pre-irradiation levels after irradiation. Interestingly, laser irradiation at 1000 mW/cm2, which led to an increase in spontaneous firing, did not prompt escape behavior. Furthermore, a significant reduction in the time to initiate escape behavior was observed only at 9500 mW/cm2 compared to 15, 510, 1000, and 4300 mW/cm2. This suggests that 1000 mW/cm2, the laser intensity at which an increase in spontaneous firing was observed, corresponds to a stimulus that did not cause pain. It is expected that a detailed understanding of the risks and mechanisms of PBM from a neurophysiological perspective will lead to safer and more effective use of PBM.

Photobiomodulation inhibits neuronal firing in the superficial but not deep layer of a rat spinal dorsal horn.

Photobiomodulation (PBM) has attracted attention as a treatment for chronic pain. Previous studies have reported that PBM of the sciatic nerve inhibits neuronal firing in the superficial layers (lamina I-II) of the spinal dorsal horn of rats, which is evoked by mechanical stimulation that corresponds to noxious stimuli. However, the effects of PBM on the deep layers (lamina III-IV) of the spinal dorsal horn, which receive inputs from innocuous stimuli, remain poorly understood. In this study, we examined the effect of PBM of the sciatic nerve on firing in the deep layers of the spinal dorsal horn evoked by mechanical stimulation. Before and after PBM, mechanical stimulation was administered to the cutaneous receptive field using 0.6-26.0 g von Frey filaments (vFFs), and vFF-evoked firing in the deep layers of the spinal dorsal horn was recorded. The vFF-evoked firing frequencies were not altered after the PBM for any of the vFFs. The inhibition rate for 26.0 g vFF-evoked firing was approximately 13 % in the deep layers and 70 % in the superficial layers. This suggests that PBM selectively inhibits the transmission of pain information without affecting the sense of touch. PBM has the potential to alleviate pain while preserving the sense of touch.

Combined Experiments with in Vivo Fiber Photometry and Behavior Tests Can Facilitate the Measurement of Neuronal Activity in the Primary Somatosensory Cortex and Hyperalgesia in an Inflammatory Pain Mice Model.

The pain matrix, which includes several brain regions that respond to pain sensation, contribute to the development of chronic pain. Thus, it is essential to understand the mechanism of causing chronic pain in the pain matrix such as anterior cingulate (ACC), or primary somatosensory (S1) cortex. Recently, combined experiment with the behavior tests and in vivo calcium imaging using fiber photometry revealed the interaction between the neuronal function in deep brain regions of the pain matrix including ACC and the phenotype of chronic pain. However, it remains unclear whether this combined experiment can identify the interaction between neuronal activity in S1, which receive pain sensation, and pain behaviors such as hyperalgesia or allodynia. In this study, to examine whether the interaction between change of neuronal activity in S1 and hyperalgesia in hind paw before and after causing inflammatory pain was detected from same animal, the combined experiment of in vivo fiber photometry system and von Frey hairs test was applied. This combined experiment detected that amplitude of calcium responses in S1 neurons increased and the mechanical threshold of hind paw decreased from same animals which have an inflammatory pain. Moreover, we found that the values between amplitude of calcium responses and mechanical thresholds were shifted to negative correlation after causing inflammatory pain. Thus, the combined experiment with fiber photometry and the behavior tests has a possibility that can simultaneously consider the interaction between neuronal activity in pain matrix and pain induced behaviors and the effects of analgesics or pain treatments.

Chronic Treatment with Aß42 with a Toxic Conformer and LPS Induces Inflammatory Responses in BV-2 Microglia with Dysregulation of Hypoxia-Inducible Factor Expression.

Amyloid ß (Aß) plays a key role in the pathology of Alzheimer's disease (AD) and is toxic owing to its ability to aggregate into oligomers and fibrils. Aß has high aggregative ability and potent toxicity due to the "toxic turn" at positions 22 and 23. Furthermore, APP knock-in mice producing E22P-Aß with the toxic turn exhibited AD-related phenotypes such as cognitive impairment, Aß plaque accumulation, and tau hyperphosphorylation. In these mice, it is suggested that the activation of neuroinflammation and dysregulation of hypoxia-inducible factor (HIF) expression in the hippocampus contribute to the pathogenesis of AD-related phenotype. However, it remains unclear which cells are responsible for the dysregulation of HIF expression and the neuroinflammation which was induced by E22P-Aß with the toxic turn. Here, we investigated the effects of chronic treatment with E22P-Aß42 and lipopolysaccharides (LPS) on the inflammatory response in BV-2 microglia. Chronic treatment with E22P-Aß42 and LPS increased nitric oxide production and the expression of interleukin-6 (IL-6), whereas it reduced the expression of HIF-1α and HIF-3α in BV-2 microglia. The reduction of HIF-1α caused by E22P-Aß42 and LPS was milder than that caused by LPS. Furthermore, chronic treatment with E22P-Aß42 and LPS increased the nuclear translocation of nuclear factor-kappaB (NF-κB). E22P-Aß42 could enhance the inflammatory response of microglia with abnormal HIF signaling and contribute to the progression of AD pathology.

Isolation and Purification of Harpagogenin as an Nrf2-ARE Activator from the Tubers of Chinese Artichoke (Stachys sieboldii).

Chinese artichoke tuber (Stachys sieboldii Miq.) is used as an herbal medicine as well as edible food. This study examined the effect of the Chinese artichoke extracts on the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway that induces the expression of antioxidant enzymes to explore its novel characteristics. Hot water extracts exhibited relatively high ARE activity. ARE activity was observed in two fractions when the hot water extracts were separated in the presence of trifluoroacetic acid using HPLC. Conversely, the highly active fraction disappeared when the hot water extracts were separated in the absence of trifluoroacetic acid. These results indicate that acidic degradation produces active ingredients. The structural analysis of the two active fractions identified harpagide, which is an iridoid glucoside, and harpagogenin. In vitro experiments revealed that harpagide was converted into harpagogenin under acidic conditions and that harpagogenin, but not harpagide, had potent ARE activity. Therefore, this study identified harpagogenin, which is an acid hydrolysate of harpagide, as an ARE activator and suggests that Nrf2-ARE pathway activation by Chinese artichoke contributes to the antioxidative effect.

Near-Infrared Photobiomodulation of the Peripheral Nerve Inhibits the Neuronal Firing in a Rat Spinal Dorsal Horn Evoked by Mechanical Stimulation.

Photobiomodulation has analgesic effects via inhibition of nerve activity, but few reports have examined the effects on the spinal dorsal horn, the entry point for nociceptive information in the central nervous system. In this study, we evaluated the effects of laser irradiation of peripheral nerve axons, which are conduction pathways for nociceptive stimuli, on the neuronal firing in lamina II of the spinal dorsal horn of a rat evoked by mechanical stimulation with von Frey filaments (vFF). In order to record neuronal firing, electrodes were inserted into lamina II of the exposed rat spinal dorsal horn. The exposed sciatic nerve axons were irradiated with an 808 nm laser. The 26.0 g vFF-evoked firing frequency was inhibited from 5 min after laser irradiation and persisted for 3 h. Sham irradiation did not alter the firing frequency. Laser irradiation selectively inhibited 15.0 and 26.0 g vFF-evoked firing, which corresponded to nociceptive stimuli. Histopathological evaluation revealed no damage to the sciatic nerve due to laser irradiation. These results indicate that neuronal firing is inhibited in lamina II of the spinal dorsal horn, suggesting that laser irradiation inhibits Aδ and/or C fibers that conduct nociceptive stimuli.

Structure Optimization of the Toxic Conformation Model of Amyloid ß42 by Intramolecular Disulfide Bond Formation.

Amyloid ß (Aß) oligomers play a critical role in the pathology of Alzheimer's disease. Recently, we reported that a conformation-restricted Aß42 with an intramolecular disulfide bond through cysteine residues at positions 17/28 formed stable oligomers with potent cytotoxicity. To further optimize this compound as a toxic conformer model, we synthesized three analogues with a combination of cysteine and homocysteine at positions 17/28. The analogues with Cys-Cys, Cys-homoCys, or homoCys-Cys, but not the homoCys-homoCys analogue, exhibited potent cytotoxicity against SH-SY5Y and THP-1 cells even at 10 nM. In contrast, the cytotoxicity of conformation-restricted analogues at positions 16/29 or 18/27 was significantly weaker than that of wild-type Aß42. Furthermore, thioflavin-T assay, non-denaturing gel electrophoresis, and morphological studies suggested that the majority of these conformation-restricted analogues exists in an oligomeric state in cell culture medium, indicating that the toxic conformation of Aß42, rather than the oligomeric state, is essential to induce cytotoxicity.

Histamine enhances ATP-induced itching and responsiveness to ATP in keratinocytes.

Mechanical stimulation of cultured keratinocytes and a living epidermis increases intracellular calcium ion concentrations ([Ca2+]i) in stimulated cells. This action propagates a Ca2+ wave to neighboring keratinocytes via ATP/P2Y2 receptors. Recent behavioral, pharmacological studies revealed that exogenous ATP induces itching via P2X3 receptors in mice. We previously showed that alloknesis occurs when an external stimulus is applied to the skin with increased epidermal histamine in the absence of spontaneous pruritus. Based on these results, we investigated the effects of histamine at a concentration that does not cause itching on ATP-induced itching. The mean number of scratching events induced by the mixture of ATP and histamine increased by 28% over the sum of that induced by histamine alone or ATP alone. A317491, a P2X3 receptor antagonist, suppressed the mixture-induced scratching more often than the ATP-induced scratching. Next, we examined the ATP-induced [Ca2+]i change before and after histamine stimulation using normal human epidermal keratinocytes. Some cells did not respond to ATP before histamine stimulation but responded to ATP afterward, the phenomenon suppressed by chlorpheniramine maleate. These findings suggest that histamine enhances ATP-induced itching and that a potential mechanism could involve increased responsiveness to ATP in keratinocytes.

TPNA10168, an Nrf-2 activator, attenuates inflammatory responses independently of Nrf2 in microglial BV-2 cells: Involvement of the extracellular-signal-regulated kinase pathway.

Some chemical Nrf2 inducers possess antioxidant and anti-inflammatory properties. TPNA10168, which was identified from a chemical library as a potential activator of the Keap1-Nrf2-ARE pathway, exhibits a neuroprotective effect against oxidative stress-induced injury. However, it has not been investigated as an anti-inflammatory agent. Here we examined the effect of TPNA10168 on interferon-γ-induced proinflammatory gene expression in mouse microglial BV-2 cells. TPNA10168 significantly reduced the transcription of inflammatory genes, including TNF-α, IL-1ß, IL-6, and iNOS; however, the inhibition of proinflammatory cytokine gene expression was not attenuated by inhibitors of Nrf2-regulated enzymes. Furthermore, TPNA10168 showed anti-inflammatory effects, even in Nrf2-deficient cells, and inhibited interferon-γ-induced phosphorylation of extracellular-signal-regulated kinase (ERK). Studies with an ERK pathway inhibitor demonstrated a role for ERK in the transcription of inflammatory genes. These results suggest that TPNA10168 attenuates microglial proinflammatory activation independently of Nrf2, at least in part, by suppressing interferon-γ-induced ERK signaling.

Light-Touch-Induced Afterdischarge Firing in the Superficial Spinal Dorsal Horn Neurons in Hairless Mice with Irritant Contact Dermatitis.

The skin is an important barrier that protects against invasion by foreign substances, including irritants and harmful microorganisms, and holds water in the body. Washing the skin with cleansers and shampoos containing anionic surfactants, for example sodium dodecyl sulfate (SDS), is important for maintaining skin homeostasis. However, surfactants can cause dermatitis, cutaneous hypersensitivity (e.g., alloknesis), and pruritus in humans. Our previous studies revealed an alloknesis response in the skin with SDS-induced dermatitis in C57BL/6 mice. In addition, we found that alloknesis responses and afterdischarge responses following stimulation with light touch are related because they are observed contemporaneously. In this study, we used Hos:HR-1 hairless mice to establish a mouse model to evaluate long-term drug application for alloknesis responses. Alloknesis was observed in HR-1 mice with SDS-induced dermatitis. The mean number of c-Fos (a marker of neural activity) immunopositive neurons was increased in the lamina 1-2 (L1-2) spinal dorsal horn, but not in L3-4, of SDS-treated HR-1 mice compared to vehicle-treated mice. We also discovered that afterdischarge responses were observed in neurons in L1-2. There was also a correlation between the intensity of the afterdischarge responses and depth of the recording site. Thus, the following were suggested: 1) neurons that mediate these afterdischarge responses are located on the superficial layer of the spinal cord; 2) afterdischarge responses can be an index of alloknesis responses, and 3) the mouse model of SDS-induced dermatitis is an appropriate alloknesis model.

APP Knock-In Mice Produce E22P-Aß Exhibiting an Alzheimer's Disease-like Phenotype with Dysregulation of Hypoxia-Inducible Factor Expression.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that requires further pathological elucidation to establish effective treatment strategies. We previously showed that amyloid ß (Aß) toxic conformer with a turn at positions 22-23 is essential for forming highly toxic oligomers. In the present study, we evaluated phenotypic changes with aging in AD model AppNL-P-F/NL-P-F (NL-P-F) mice with Swedish mutation (NL), Iberian mutation (F), and mutation (P) overproducing E22P-Aß, a mimic of toxic conformer utilizing the knock-in technique. Furthermore, the role of the toxic conformer in AD pathology was investigated. NL-P-F mice produced soluble toxic conformers from an early age. They showed impaired synaptic plasticity, glial cell activation, and cognitive decline, followed by the accumulation of Aß plaques and tau hyperphosphorylation. In addition, the protein expression of hypoxia-inducible factor (HIF)-1α was increased, and gene expression of HIF-3α was decreased in NL-P-F mice. HIF dysregulation due to the production of soluble toxic conformers may be involved in AD pathology in NL-P-F mice. This study could reveal the role of a highly toxic Aß on AD pathogenesis, thereby contributing to the development of a novel therapeutic strategy targeting the toxic conformer.

Inhibitory effect of panaxytriol on BV-2 microglial cell activation.

Activated microglia induce brain inflammation and neuronal death. Panaxytriol, ((3R,9R,10R)-Heptadec-1-en-4,6-diyne-3,9,10-triol), is a component of Panax ginseng C. A. Meyer extracts and activates the Nrf2-ARE signaling pathway. However, little is known about its effects on activated microglia in the brain. In this study, we investigated the effect of panaxytriol on lipopolysaccharide (LPS)-induced activated microglia in BV-2 cells. Panaxytriol suppressed LPS-induced NO production and inhibited the increase in iNOS protein expression in BV-2 cells. Besides, panaxytriol inhibited the mRNA expression of proinflammatory cytokines such as TNF-α, IL-1ß, and IL-6. The inhibitory effect of panaxytriol on microglia activation did not affect the Nrf2-ARE pathway and the MAPK pathway. However, panaxytriol suppressed LPS-induced NF-κB nuclear translocation. These results suggest that panaxytriol inhibits the LPS-induced activation of microglia via the inhibition of NF-κB signaling pathway.

Establishing a high throughput drug screening system for cerebral ischemia using zebrafish larvae.

We previously generated an ischemic stroke in a zebrafish model using N2 gas perfusion; however, this model was an unsuitable drug screening system due to low throughput. In this study, we examined a zebrafish ischemic stroke model using an oxygen absorber to assess drug effects. Hypoxic exposure more than 2 h using the oxygen absorber significantly induced cell death in the brain and damage to the neuronal cells. To confirm the utility of the ischemic model induced by the oxygen absorber, we treated zebrafish with neuroprotective agents. MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist, significantly suppressed cell death in the brain, and edaravone, a free radical scavenger, significantly reduced the number of dead cells. These results suggest that the activation of NMDA receptors and the production of reactive oxygen species induce neuronal cell damage in accordance with previous mammalian reports. We demonstrate the suitability of an ischemic stroke model in zebrafish larvae using the oxygen absorber, enabling a high throughput drug screening.

Panaxytriol Inhibits Lipopolysaccharide-Induced Microglia Activation in Brain Inflammation in Vivo.

Brain inflammation is a pathological characteristic of neurodegenerative diseases. In this condition, excessively activated microglia elevate proinflammatory mediator levels. We previously reported that panaxytriol inhibited lipopolysaccharide (LPS)-induced microglia activation in vitro. However, the effects of panaxytriol on microglia activation in vivo require confirmation. In the present study, we found that panaxytriol suppressed both microglia and astrocyte activation by injected LPS intracerebrally to mice with LPS-induced brain inflammation. Panaxytriol was more effective on microglia than astrocytes. Moreover, panaxytriol tended to reduce LPS-induced spontaneous motor activity dysfunction. These results suggested that panaxytriol could improve brain health by suppressing microglia activation in neurodegenerative diseases.

Protective Effect of 2',3'-Dihydroxy-4',6'-dimethoxychalcone on Glutamate-Induced Neurotoxicity in Primary Cortical Cultures.

We have previously isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla leaves as the activator of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. This study aims to evaluate the effects of DDC against glutamate neurotoxicity using rat primary cortical cultures. Treatment of cultures with DDC for 24 h before glutamate exposure significantly inhibited glutamate neurotoxicity in a concentration-dependent manner. The involvement of hemeoxygenase-1 (HO-1) and reduced glutathione (GSH) in the protective effects of DDC on cortical cultures was also evaluated. While an HO-1 inhibitor did not have a significant effect on DDC-induced neuroprotection, a γ-glutamylcystein synthetase (γ-GCS) inhibitor significantly suppressed the protective effect of DDC. In an astrocyte culture, DDC induced a marked increase in the levels of intracellular reduced GSH. These results suggest that DDC mainly activates the Nrf2-ARE pathway of astrocytes, resulting in the increased extracellular release of reduced GSH, protecting neurons from glutamate neurotoxicity.

Evaluation of Toxic Amyloid 42 Oligomers in Rat Primary Cerebral Cortex Cells and Human iPS-derived Neurons Treated with 10-Me-Aplog-1, a New PKC Activator.

Amyloid ß42 (Aß42), a causative agent of Alzheimer's disease (AD), is derived extracellularly from Aß precursor protein (APP) following the latter's cleavage by ß-secretase, but not α-secretase. Protein kinase Cα (PKCα) activation is known to increase α-secretase activity, thereby suppressing Aß production. Since Aß42 oligomer formation causes potent neurotoxicity, APP modulation by PKC ligands is a promising strategy for AD treatment. Although bryostatin-1 (bryo-1) is a leading compound for this strategy, its limited natural availability and the difficulty of its total synthesis impedes further research. To address this limitation, Irie and colleagues have developed a new PKC activator with few side effects, 10-Me-Aplog-1, (1), which decreased Aß42 in the conditioned medium of rat primary cerebral cortex cells. These results are associated with increased α-secretase but not PKCε-dependent Aß-degrading enzyme. The amount of neuronal embryonic lethal abnormal vision (nELAV), a known ß-secretase stabilizer, was reduced by treatment with 1. Notably, 1 prevented the formation of intracellular toxic oligomers. Furthermore, 1 suppressed toxic oligomerization within human iPS-derived neurons such as bryo-1. Given that 1 was not neurotoxic toward either cell line, these findings suggest that 1 is a potential drug lead for AD therapy.

Increased CCL6 expression in astrocytes and neuronal protection from neuron-astrocyte interactions.

Astrocytes have been reported to exhibit neuroprotective action via various chemokines. Reports of the chemokine CCL6 in central nervous system cells show expression in cultured microglia, but many unexplained effects on neurons and astrocytes remain. In this study, cultured cerebral cortical neurons, astrocytes, and a mixed culture system were constructed, and expression levels of CCL6 and its effects on glutamate neurotoxicity were examined. When neuron cultures and neuron-astrocyte mixed cultures were treated with glutamate, neuronal cell death was observed in both, but was induced by lower concentrations of glutamate in monocultured neurons. In addition, pretreatment of neuron cultures with conditioned media from neuron-astrocyte mixed cultures inhibited glutamate neurotoxicity. CCL6 expression was not observed in fluorescence activated cell sorting analyses of neuron and astrocyte cultures, but was observed in astrocytes from cocultures of neurons and astrocytes. Higher CCL6 concentrations were found in media from cocultures of neurons and astrocytes than in culture media from neuron cultures. Pretreatment of neuron cell cultures with CCL6 for 24 h also protected against glutamate neurotoxicity. This protective effect was suppressed by an antagonist of the chemokine receptor CCR1. Furthermore, glutamate neurotoxicity in mixed neuron and astrocyte cultures was enhanced by pretreatments with the CCR1 antagonist. Finally, cotreatments with the phosphatidylinositol-3 kinase (PI3K) inhibitor and CCL6 abolished the neuroprotective effects of CCL6. These data suggest that astrocytes protect neurons by activating CCR1 in neurons. Moreover, this neuroprotective action of astrocyte CCL6 is mediated by CCR1, and downstream by PI3K.

Animal models of chronic pain increase spontaneous glutamatergic transmission in adult rat spinal dorsal horn in vitro and in vivo.

The ability to detect noxious stimulation is essential to an organism's survival and wellbeing. Chronic pain is characterized by abnormal sensitivity to normal stimulation coupled with a feeling of unpleasantness. This condition afflicts people worldwide and severely impacts their quality of life and has become an escalating health problem. The spinal cord dorsal horn is critically involved in nociception and chronic pain. Especially, the substantia gelatinosa (SG) neurons of lamina II, which receives nociceptive inputs from primary afferents. Two major models are used to study chronic pain in animals, including nerve injury and the injection of a complete Freund's adjuvant (CFA) into the hind paw. However, how these models induce glutamatergic synaptic plasticity in the spinal cord is not fully understood. Here, we studied synaptic plasticity on excitatory transmissions in the adult rat SG neurons. Using in vitro and in vivo whole-cell patch-clamp recording methods, we analyzed spontaneous excitatory postsynaptic currents (sEPSCs) 2 weeks following nerve injury and 1 week following CFA injection. In the spinal slice preparation, these models increased both the frequency and amplitude of sEPSCs in SG neurons. The frequency and amplitude of sEPSCs in the nerve injury and the CFA group were reduced by the presence of tetrodotoxin (TTX). By contrast, TTX did not reduce the sEPSCs compared with miniature EPSCs in naïve rats. Next, we analyzed the active electrophysiological properties of neurons, which included; resting membrane potentials (RMPs) and the generation of action potentials (APs) in vitro. Interestingly, about 20% of recorded SG neurons in this group elicited spontaneous APs (sAPs) without changing the RMPs. Furthermore, we performed in vivo whole-cell patch-clamp recording in SG neurons to analyze active electrophysiological properties under physiological conditions. Importantly, in vivo SG neurons generated sAPs without affecting RMP in the nerve injury and the CFA group. Our study describes how animal models of chronic pain influence both passive and active electrophysiological properties of spinal SG neurons.

Effects of 2'-3'-dihydroxy-4',6'-dimethoxychalcone derived from green perilla on auricle thickness in chronic contact dermatitis model mice.

Oxidative stress has been implicated in the pathogenesis of allergic contact dermatitis. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, an in vivo antioxidant system, induces antioxidant enzymes. In our previous studies, we isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla and identified it as a novel activator of the Nrf2-ARE pathway. We also discovered that it exerted cytoprotective effects against oxidative stress in PC12 cells. However, its effects on skin disease model animals in vivo remain unclear. In the present study, auricular thickness time-dependently increased with the repeated application of picryl chloride, and significant increases were observed from Day 2 in chronic contact hypersensitivity (cCHS) model mice. Histological changes, such as higher numbers of cells in the epidermis, were observed with increases in auricular thickness. The administration of DDC every two days from Day 6 suppressed the increases in auricular thickness and the number of scratching events in a dose-dependent manner. The expression levels of antioxidant enzymes increased in the mouse auricle 24 h after the administration of DDC. These results presume that DDC inhibits increases in auricular thickness in cCHS mice by up-regulating the expression of antioxidative enzymes through the activation of the Nrf2-ARE pathway.

Protective Effect of Green Perilla-Derived Chalcone Derivative DDC on Amyloid ß Protein-Induced Neurotoxicity in Primary Cortical Neurons.

Amyloid ß protein (Aß) causes neurotoxicity and cognitive impairment in Alzheimer's disease (AD). Oxidative stress is closely related to the pathogenesis of AD. We have previously reported that 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC), a component of green perilla, enhances cellular resistance to oxidative damage through the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. Here, we investigated the effects of DDC on cortical neuronal death induced by Aß. When Aß and DDC had been preincubated for 3 h, the aggregation of Aß was significantly suppressed. In this condition, we found that DDC provided a neuroprotective action on Aß-induced cytotoxicity. Treatment with DDC for 24 h increased the expression of heme oxygenase-1 (HO-1), and this was controlled by the activation of the Nrf2-ARE pathway. However, DDC did not affect Aß-induced neuronal death under any of these conditions. These results suggest that DDC prevents the aggregation of Aß and inhibits neuronal death induced by Aß, and although it activates the Nrf2-ARE pathway, this mechanism is less involved its neuroprotective effect.