Synthesis of 1,4-Dialkoxynaphthalene-Based Imidazolium Salts and Their Cytotoxicity in Cancer Cell Lines.

In this study, we designed and synthesized novel 1,4-dialkoxynaphthalene-2-alkyl imidazolium salt (IMS) derivatives containing both 1,4-dialkoxynaphthalene and imidazole, which are well known as pharmacophores. The cytotoxicities of these newly synthesized IMS derivatives were investigated in order to explore the possibility of using them to develop anticancer drugs. It was found that some of the new IMS derivatives showed good cytotoxic activities. In addition, an initial, qualitative structure-activity relationship is presented on the basis of observations of activity changes corresponding to structural changes.

Fine particulate matter (PM2.5) inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and skin keratinocytes.

Exposure to fine particulate matter (PM) with diameter <2.5 µm (PM2.5) causes epithelium injury and endothelial dysfunction. Primary cilia are sensory organelles that transmit extracellular signals into intracellular biochemical responses and have roles in physiology. To date, there have been no studies investigating whether PM2.5 affects primary cilia in skin. We addressed this in the present study using normal human epidermal keratinocytes (NHEKs) and retinal pigment epithelium (RPE) cells. We found that formation of primary cilium is increased in differentiated NHEKs. However, treatment with PM2.5 blocked increased ciliogenesis in NHEKs and RPE cells. Furthermore, PM2.5 transcriptionally upregulated small proline rich protein 3 (SPRR3) expression by activating c-Jun, and ectopic expression of SPRR3 inhibits suppressed the ciliogenesis. Accordingly, treatment with c-Jun activator (anisomycin) induced SPRR3 expression, whereas the inhibitor (SP600125) recovered the ciliated cells and cilium length in PM2.5-treated cells. Moreover, c-Jun inhibitor suppressed upregulation of SPRR3 in PM2.5-treated cells. Taken together, our finding suggested that PM2.5 inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and keratinocytes.

Salmonella­induced miR­155 enhances necroptotic death in macrophage cells via targeting RIP1/3.

Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella), a virulent pathogen, is known to induce host­cell death. Using reverse transcription­quantitative polymerase chain reaction, a 28­fold increase of microRNA (miR)­155 expression in RAW 264.7 macrophages was observed following infection with Salmonella for 24 h. This miR­155 upregulation increased macrophage cell death by up to 40% in 48 h following infection. Western blot analysis revealed that receptor interacting protein 1 (RIP1) and 3 (RIP3) were increased at 18 h following miR­155 transfection to macrophages, similar to Salmonella infection. In addition, inhibition of RIP1 by pre­incubating macrophages with necrostatin­1, a RIP1 specific inhibitor, increased the viability of Salmonella­infected cells and miR­155­transfected cells by up to 20%. The cleavage of poly (adenosine diphosphate­ribose) polymerase­1 (PARP­1) was also enhanced by miR­155 induction upon Salmonella infection. Therefore, it was suggested that RIP1/3­induced necroptosis and PARP­1­mediated necrosis caused by miR­155 induction may represent distinct routes of programmed necrotic cell death of Salmonella­infected macrophages.

Cynandione A inhibits lipopolysaccharide-induced cell adhesion via suppression of the protein expression of VCAM­1 in human endothelial cells.

Cynandione A (CA) is one of the most active compounds in the roots of Cynanchum wilfordii, the extracts of which have been used extensively in East Asia to treat various diseases including anti­ischemic stroke. In the present study, the anti­adherent activity of CA in lipopolysaccharide (LPS)­stimulated human umbilical vascular endothelial cells (HUVECs) was investigated. CA markedly reduced the expression of vascular adhesion molecule­1 (VCAM­1) by LPS in HUVECs. The results also demonstrated that CA significantly reduced the expression of pro­inflammatory and chemoattractant cytokines, including interleukin (IL)­1ß, IL­6, IL­8, monocyte chemoattractant protein­1 and tumor necrosis factor­α, in LPS­activated human endothelial cells. CA inhibited the phosphorylation of mitogen­activated protein kinases, including the extracellular signal­regulated kinase 1/2 and p38 kinases. It was found that CA decreased the IKK/IκB­α phosphorylation of inhibitor of nuclear factor (NF)­κB kinase/inhibitor of NF­κB­α, suppressed translocation of the NF­κB p65 subunit into the nucleus and inhibited the transcriptional activity of NF­κB. CA also decreased human monocyte cell adhesion to endothelial cells in LPS­stimulated conditions. These results demonstrated that CA inhibited the protein expression of VCAM­1 and pro­inflammatory cytokines by suppressing the transcriptional activity of NF­κB. The results also suggested that CA may be important in the development of anti­inflammatory drugs by inhibiting the expression of cell adhesion molecules.

Inhibitory effect of 5,6-dihydroergosteol-glucoside on atopic dermatitis-like skin lesions via suppression of NF-κB and STAT activation.

BACKGROUND: Atopic dermatitis (AD) is a Th2-type disease. Keratinocytes, a major type in the skin, produce Th2 chemokines such as thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22, which play pivotal roles in the development of Th2-dominant inflammatory skin diseases. Recently, it was reported that 5,6-dihydroergosterol-glucoside (DHE-Glc) was synthesized and exhibited strong anti-inflammatory activity. OBJECTIVE: We aimed to investigate the effects of DHE-Glc, a synthetic molecule derived from ergosterol, on AD-like skin lesions induced by 2,4-dinitrochlorobenzene (DNCB) in mice and to elucidate the effects of DHE-Glc on TNF-α/IFN-γ-induced production of CCL17 and CCL22 in human keratinocytes (HaCaTs) and DNCB induced skin inflammation mice model. METHOD: Mice were sensitized and challenged on the skin of their backs with DNCB. At 30-60 days after sensitization, mice were treated with cutaneous administration of DHE-Glc by skin smear. HaCaT cells were used to evaluate the effects of DHE-Glc on production of CCL17 and CCL22 and investigate mechanisms of action by RT-PCR, ELISA, Western blot, and reporter assays. RESULT: Topical administration of DHE-Glc attenuated AD-like skin inflammatory symptoms. DHE-Glc decreased infiltration of epidermal eosinophils and mast cells, and reduced levels of IgE, histamine, and mRNA expression and protein levels of CCL17/CCL22 in the plasma of DNCB-treated animals. In addition, DHE-Glc suppressed TNF-α/IFN-γ-induced expression of the Th2 chemokines CCL17 and CCL22 by inhibiting NF-κB and STAT activation in TNF-α/IFN-γ-induced HaCaT cells. CONCLUSION: DHE-Glc improved AD-like skin inflammatory symptoms on the backs of DNCB-induced mice, partly by suppressing production of Th2 chemokines, CCL17 and CCL22 in inflamed skin. Therefore, DHE-Glc is a potential therapeutic agent for skin inflammatory diseases such as AD.

Tetramethylpyrazine, a natural alkaloid, attenuates pro-inflammatory mediators induced by amyloid ß and interferon-γ in rat brain microglia.

Neuroinflammation has been consistently reported as a pathological hallmark of Alzheimer׳s disease and other neurodegenerative diseases. Microglial cells are activated by diverse pathological stimuli and play key roles in development of neuroinflammation. Amyloid ß peptide (Aß), the major constituent of amyloid plaques in Alzheimer׳s brain, is known to activate cultured microglial cells to produce increased amounts of proinflammatory and neurotoxic factors. Tetramethylpyrazine (TMP) is the main bioactive alkaloid isolated from Ligusticum chuanxiong. TMP has multiple pharmacological activities, including anti-oxidant, anti-inflammatory, and anti-cancer effects. Neuroprotective potential of TMP has been demonstrated in animal models of neuropathologies. However, the efficacy of this compound for controlling Aß-related neuropathology has not been explored yet. We examined the efficacy of TMP in the repression of inflammatory response in cultured microglial cells stimulated with Aß25-35 in the presence of interferon (IFN)-γ. TMP significantly inhibited the Aß25-35 and IFN-γ-stimulated productions of nitric oxide, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, monocyte chemoattractant protein-1, and intracellular reactive oxygen species from primary microglial cells. TMP also effectively reduced Aß25-35 and IFN-γ-elicited NF-κB activation. In organotypic hippocampal slice cultures (OHSCs), TMP significantly blocked Aß25-35-induced reactive oxygen species generation and phosphorylation of Akt. Furthermore, TMP also inhibited Aß1-42-induced TNF-α and IL-1ß production in primary microglial cells and neuronal death in OHSCs. These results suggest that TMP provide a possible therapeutic approach for alleviating the inflammatory progression of Alzheimer׳s disease.

Down-regulation of mortalin exacerbates Aß-mediated mitochondrial fragmentation and dysfunction.

Mitochondrial dynamics greatly influence the biogenesis and morphology of mitochondria. Mitochondria are particularly important in neurons, which have a high demand for energy. Therefore, mitochondrial dysfunction is strongly associated with neurodegenerative diseases. Until now various post-translational modifications for mitochondrial dynamic proteins and several regulatory proteins have explained complex mitochondrial dynamics. However, the precise mechanism that coordinates these complex processes remains unclear. To further understand the regulatory machinery of mitochondrial dynamics, we screened a mitochondrial siRNA library and identified mortalin as a potential regulatory protein. Both genetic and chemical inhibition of mortalin strongly induced mitochondrial fragmentation and synergistically increased Aß-mediated cytotoxicity as well as mitochondrial dysfunction. Importantly we determined that the expression of mortalin in Alzheimer disease (AD) patients and in the triple transgenic-AD mouse model was considerably decreased. In contrast, overexpression of mortalin significantly suppressed Aß-mediated mitochondrial fragmentation and cell death. Taken together, our results suggest that down-regulation of mortalin may potentiate Aß-mediated mitochondrial fragmentation and dysfunction in AD.

ARP101 inhibits α-MSH-stimulated melanogenesis by regulation of autophagy in melanocytes.

Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular organelles. Although autophagy regulates the turnover of cellular components, its role in melanogenesis is not clearly established. Previously, we reported that ARP101 induces autophagy in various cancer cells. Here, we show that ARP101 inhibits melanogenesis by regulation of autophagy. ARP101 inhibited α-MSH-stimulated melanin synthesis and suppressed the expression of tyrosinase and TRP1 in immortalized mouse melanocytes. ARP101 also induced autophagy in melanocytes. Knockdown of ATG5 reduced both anti-melanogenic activity and autophagy mediated by ARP101 in α-MSH treated melanocytes. Electron microscopy analysis further revealed that autophagosomes engulf melanin or melanosome in α-MSH and ARP101-treated cells. Collectively, our results suggest that ARP101 inhibits α-MSH-stimulated melanogenesis through the activation of autophagy in melanocytes.

Paeoniflorin, a monoterpene glycoside, attenuates lipopolysaccharide-induced neuronal injury and brain microglial inflammatory response.

Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Paeoniflorin (PF), a water-soluble monoterpene glycoside found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, such as anti-oxidant, anti-inflammatory, and anti-cancer effects. Neuroprotective potential of PF has also been demonstrated in animal models of neuropathologies. Here, we have examined the efficacy of PF in the repression of inflammation-induced neurotoxicity and microglial inflammatory response. In organotypic hippocampal slice cultures, PF significantly blocked lipopolysaccharide (LPS)-induced hippocampal cell death and productions of nitric oxide (NO) and interleukin (IL)-1ß. PF also inhibited the LPS-stimulated productions of NO, tumor necrosis factor-α, and IL-1ß from primary microglial cells. These results suggest that PF possesses neuroprotective activity by reducing the production of proinflammatory factors from activated microglial cells.

Prevention of inflammation-mediated neurotoxicity by butylidenephthalide and its role in microglial activation.

Microglial cells are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). During pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory molecules and neurotoxins. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. The rhizome of Ligusticum chuanxiong Hort. (Ligusticum wallichii Franch) has been widely used for the treatment of vascular diseases in traditional oriental medicine. Butylidenephthalide (BP), a major bioactive component from L. chuanxiong, has been reported to have a variety of pharmacological activities, including vasorelaxant, anti-anginal, anti-platelet and anti-cancer effects. The aim of this study was to examine whether BP represses microglial activation. In rat brain microglia, BP significantly inhibited the lipopolysaccharide (LPS)-induced production of nitric oxide (NO), tumour necrosis factor-α and interleukin-1ß. In organotypic hippocampal slice cultures, BP clearly blocked the effect of LPS on hippocampal cell death and inhibited LPS-induced NO production in culture medium. These results newly suggest that BP provide neuroprotection by reducing the release of various proinflammatory molecules from activated microglia.

Suppression of autophagy exacerbates Mefloquine-mediated cell death.

Mefloquine is an effective treatment drug for malaria. However, it can cause several adverse side effects, and the precise mechanism associated with the adverse neurological effects of Mefloquine is not clearly understood. In this study, we investigated the effect of Mefloquine on autophagy in neuroblastoma cells. Mefloquine treatment highly induced the formation of autophagosomes and the conversion of LC3I into LC3II. Moreover, Mefloquine-induced autophagy was efficiently suppressed by an autophagy inhibitor and by down regulation of ATG6. The autophagy was also completely blocked in ATG5 deficient mouse embryonic fibroblast cells. Moreover, suppression of autophagy significantly intensified Mefloquine-mediated cytotoxicity in SH-SY5Y cells. Our findings suggest that suppression of autophagy may exacerbate Mefloquine toxicity in neuroblastoma cells.

Neuroprotective effects of INM-176 against lipopolysaccharide-induced neuronal injury.

Neuroinflammation plays a critical role in the etiology of chronic neurodegenerative diseases such as Alzheimer's disease. INM-176 is a standardized ethanolic extract of Angelica gigas, which has been traditionally used as a tonic to treat anemia. In the present study, we investigated whether INM-176 exhibits neuroprotective activities against lipopolysaccharide (LPS)-induced neuronal damage in vitro and in vivo. In primary microglial cells, INM-176 significantly inhibited LPS-induced nitric oxide release and expression of tumor necrosis factor-α and interleukin-1ß. The expression levels of inducible nitric oxide synthase and cylcooxygenase-2 in BV2 microglial cells were markedly upregulated by LPS, but this increased expression was counteracted by INM-176. LPS-mediated neuronal damage in an organotypic hippocampal slice culture was also attenuated by the administration of INM-176. In addition, LPS (1 µg/2 µl, i.c.v.)-induced cognitive dysfunction in mice, as determined by passive avoidance and Y-maze tasks, was significantly attenuated by the administration of INM-176. Furthermore, the activation of microglia or astrocytes by LPS in the hippocampal regions of mice was suppressed by INM-176. These results suggest that the neuroprotective and cognition ameliorating effects of INM-176 against LPS-induced damage are mediated, in part, by its anti-inflammatory activities.

Indirubin-3'-oxime inhibits inflammatory activation of rat brain microglia.

Microglial cells play critical roles in the immune and inflammatory responses of the brain. Under pathological conditions, the activation of microglia helps to restore brain homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. As such, regulators of microglial activation have been considered as potential therapeutic candidates to reduce the risk of neurodegeneration associated with neurodegenerative diseases, including Alzheimer's and, Parkinson's diseases. Indirubin-3'-oxime, a potent inhibitor of cyclin-dependent kinases and glycogen synthase kinase-3ß, has been shown to have neuroprotective potential. The specific aim of this study was to examine the efficacy of indirubin-3'-oxime in the repression of microglial activation. Indirubin-3'-oxime was shown to effectively inhibit lipopolysaccharide (LPS)-induced nitric oxide release from cultured rat brain microglia. This compound reduced the LPS-stimulated productions of tumor necrosis factor-α, interleukin-1ß, prostaglandin E(2), and intracellular reactive oxygen species and also effectively reduced LPS-elicited NF-κB activation. In organotypic hippocampal slice cultures, indirubin-3'-oxime blocked LPS-related hippocampal cell death. These results suggest that indirubin-3'-oxime provides neuroprotection by reducing the productions of various neurotoxic molecules in activated microglia.

Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells.

Microglial cells play critical roles in the immune and inflammatory responses of the central nervous system (CNS). Under pathological conditions, the activation of microglia helps in restoring CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that observed in Alzheimer's and Parkinson's diseases. Crocin and crocetin, found in the fruits of gardenia and in the stigmas of saffron, have been considered for the treatment of various disorders in traditional oriental medicine. Crocin and crocetin have been reported to have diverse pharmacological functions, such as anti-hyperlipidemic, anti-atherosclerotic, and anti-cancer effects. Specifically, the neuroprotective potential of crocetin derivatives has previously been demonstrated. The specific aim of this study was to examine whether crocin or crocetin represses microglial activation. Crocin and crocetin were shown to be effective in the inhibition of LPS-induced nitric oxide (NO) release from cultured rat brain microglial cells. These compounds reduced the LPS-stimulated productions of tumor necrosis factor-α, interleukin-1ß, and intracellular reactive oxygen species. The compounds also effectively reduced LPS-elicited NF-κB activation. In addition, crocin reduced NO release from microglia stimulated with interferon-γ and amyloid-ß. In organotypic hippocampal slice cultures, both crocin and crocetin blocked the effect of LPS on hippocampal cell death. These results suggest that crocin and crocetin provide neuroprotection by reducing the production of various neurotoxic molecules from activated microglia.

Protective effects of Chunghyuldan against ROS-mediated neuronal cell death in models of Parkinson's disease.

Previous reports have suggested that the herbal medicine Chunghyuldan (CHD, Qingxue-dan in Chinese and Daio-Orengedokuto in Japanese) has wide-ranging biological effects, including anti-hyperlipidaemic, anti-ischaemic, anti-inflammatory and antioxidant activities. Reactive oxygen species (ROS)-mediated mitochondrial dysfunction is thought to be one of the major pathological mechanisms responsible for Parkinson's disease (PD) and may underlie the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) that is a hallmark of this disease. In this study, we examined the neuroprotective effects of CHD in PD models produced by treatment with neurotoxins that act via ROS-mediated mitochondrial dysfunction. In an in vitro PD model using 6-hydroxydopamine, CHD applied at concentrations of 10 and 100 µg/ml exhibited significant protective effects in PC12 cells by inhibiting intracellular ROS generation. CHD applied at 10 and 100 µg/ml also prevented 6-hydroxydopamine-induced mitochondrial depolarization and elevation of caspase-3 activity. At the same doses, CHD showed regulatory effects on the haem oxygenase-1 and gp91 phagocytic oxidase which have critical roles in generating ROS. In addition, CHD protected dopaminergic neurons in a primary mesencephalic culture against MPP+ neurotoxicity. In an in vivo PD model produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment (20 mg/kg, 4 times, i.p.), co-administration of CHD (50 mg/kg, 5 days, p.o.) ameliorated PD-like behavioural symptoms (bradykinesia) and reduced dopaminergic neuronal damage in the SNpc and striatum as measured by immunocytochemistry. These results demonstrate the neuroprotective effects of CHD in PD models that are mediated through inhibition of ROS generation and associated mitochondrial dysfunction.

Inhibitory effects of 5-chloroacetyl-2-piperidino-1,3-selenazole, a novel selenium-containing compound, on skin melanin biosynthesis.

OBJECTIVES: Increased production and accumulation of melanin leads to many hyperpigmentation disorders such as melasma, freckles and geriatric pigment spots. Thus, there is a need for the development of depigmenting agents. Based on our previous reports, selenium derivatives as anti-melanogenic lead compounds could be very important. The aim of this study was to investigate the depigmenting effect of novel selenium-containing compounds. METHODS: The inhibitory effects of 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1), a novel selenium-containing compound, on melanogenesis were investigated in B16F10 melanoma cells and cultured brownish guinea pig skin tissue with alpha-melanocyte-stimulating hormone stimulation. KEY FINDINGS: We found that CS1 inhibited melanin production in B16F10 cells by suppressing tyrosinase activity and its protein expression. In addition, Western blotting analysis revealed that CS1 suppressed the expression of tyrosinase-related protein (TRP)-1 and TRP-2. Therefore, the depigmenting effect of CS1 might have been due to inhibition of tyrosinase activity and expression of melanogenic enzymes. Furthermore, CS1 had inhibitory effects on melanin biosynthesis of primary cultured skin of brownish guinea pig. CONCLUSIONS: The results suggested that CS1 could be a useful candidate for the treatment of skin hyperpigmentation.

Ginsenoside rb1 and rg3 attenuate glucocorticoid-induced neurotoxicity.

Glucocorticoid (GC) hormones, increased in response to stress, can cause neuronal loss. We tested the effect of GC hormone on cell viability of neural SHSY-5Y cells and protective effects of ginsenoside Rb1 and Rg3 on the action of GC. We treated SHSY-5Y cells with increasing concentrations of synthetic GC dexamethasone (DEX; 10, 25, 50, and 100 nM) for 24 and 48 h, and then determined cell viability by using MTT assay. We then treated SHSY-5Y cells with DEX (100 nM) with or without the ginsenosides to examine their preventive effects on the cytotoxicity. To explore the underlying molecular mechanisms, we measured mRNA expression of bax and bcl-2 by using reverse transcriptase real-time PCR. SHSY-5Y cells treated with DEX significantly reduced cell viability as compared with control cells. In the presence of Rb1 or Rg3, DEX-induced cytotoxicity was effectively blocked. DEX considerably increased pro-apoptotic bax mRNA expression as compared with control cells. However, Rb1 and Rg3 completely blocked DEX-mediated up-regulation of bax expression. DEX significantly increased neuronal death in organotypic hippocampal slice cultures of rat brain with enhanced generation of ROS, which was effectively inhibited by ginsenoside Rb1 and Rg3. This suggests a potential role of the ginsenosides to target GC action in the brain.

Genipin inhibits the inflammatory response of rat brain microglial cells.

Microglia are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). Under pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. Genipin, the aglycon of geniposide found in gardenia fruit has long been considered for treatment of various disorders in traditional oriental medicine. Genipin has recently been reported to have diverse pharmacological functions, such as antimicrobial, antitumor, and anti-inflammatory effects. The specific aim of this study was to examine whether genipin represses brain microglial activation. Genipin was effective at inhibiting LPS-induced nitric oxide (NO) release from cultured rat brain microglial cells. Genipin reduced the LPS-stimulated production of tumor necrosis factor-alpha, interleukin-1beta, prostaglandin E(2), intracellular reactive oxygen species, and NF-kappaB activation. In addition, genipin reduced NO release from microglia stimulated with interferon-gamma and amyloid-beta. Both pretreatment and post-treatment of genipin to LPS-stimulated microglia were effective at decreasing NO release. Furthermore, genipin effectively inhibited microglial activation in a mouse model of brain inflammation. These results suggest that genipin provide neuroprotection by reducing the production of various neurotoxic molecules from activated microglia.

The multi-herbal medicine Gongjin-dan enhances memory and learning tasks via NGF regulation.

Nerve growth factor (NGF) decreases degeneration of cholinergic neurons, improves memory loss, and increases long-term potentiation and learning tasks. Therefore, NGF mimetics or NGF inducers may be important targets for the treatment of various neurodegenerative disorders. Traditionally, Gongjin-dan (GJD) has been used clinically for the treatment of central nervous system disorders. In this study, we examined the effects of GJD on NGF mimetic activity in PC12 cells and the induction of NGF secretion in primary astrocytes. Moreover, we also measured neuron survival by MAP-2 staining in an immobilization stress rat model and induction of long-term potentiation by the MEA system in rat hippocampus slices treated with dexamethasone. The behavioral syndrome by novel object test was also performed in mice. GJD increased neurite outgrowth in PC12 cells and NGF secretion in primary astrocytes. Also, it reduced neuronal cell death and increased long-term potentiation in the rat hippocampus. Moreover, the number of entries, the time spent and the distance moved in the center area of the test region by the mice was increased by oral administration of GJD in comparison with the distance moved over the total area. These data suggest that administration of GJD may improve memory and learning tasks via NGF regulation, and that it may have a potential for multiple function neuroprotection via NGF regulation.

Mechanism of glucocorticoid-induced oxidative stress in rat hippocampal slice cultures.

Prolonged stress results in elevation of glucocorticoid (GC) hormones, which can have deleterious effects in the brain. The hippocampus, which has a high concentration of glucocorticoid receptors, is especially vulnerable to increasing levels of GCs. GCs have been suggested to endanger hippocampal neurons by exacerbating the excitotoxic glutamate-calcium-reactive oxygen species (ROS) cascade. In an effort to reveal the mechanisms underlying GC-mediated hippocampal neurotoxicity, we aimed to clarify the molecular pathway of GC-induced ROS increase by using organotypic hippocampal slice cultures. Assays for ROS, using 2',7'-dichlorodihydrofluorescein diacetate fluorescence, showed that treatment of synthetic GC, dexamethasone (DEX) significantly enhanced ROS levels. Time course and dose response analyses indicated that peak amount of ROS was generated at 4 h after treatment with 50 micromol/L DEX. By contrast, other steroid hormones, progesterone and estradiol did not influence ROS production. N-acetyl-L-cysteine completely suppressed ROS produced by DEX. Propidium iodide staining exhibited prominent cell death in the hippocampal layer after 96 h of DEX treatment. RU486, a GC receptor antagonist, almost completely blocked the effect of DEX on ROS production and cell death, indicating that DEX-induced ROS overproduction and hippocampal death are mediated via GC receptors. Real-time reverse transcriptase PCR analysis demonstrated that after DEX treatment the level of glutathione peroxidase mRNA was decreased whereas that of NADPH oxidase mRNA was significantly enhanced. These findings suggest that excess GCs cause hippocampal damage by regulating genes involved in ROS generation.