Eunjoo H. Lee

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 Alzheimers and Parkinsons 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-1β, 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-β. 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.

Apigenin inhibits the production of NO and PGE2 in microglia and inhibits neuronal cell death in a middle cerebral artery occlusion-induced focal ischemia mice model

Flavonoids have been intensively studied on their pharmacological activities such as anti-cancer, anti-oxidant and anti-inflammation. However, little is known about their neuroprotective effects. Recent studies suggest that inflammation mediated by microglia may play a role in neurodegenerative diseases. In this study, we evaluated the anti-inflammatory effect of various flavonoid compounds by using BV-2, a murine microglia cell line. Of the compounds that were evaluated, apigenin inhibited the production of nitric oxide and prostaglandin E(2) by suppressing the expression of inducible nitric oxide synthase and cyclooxygenase-2 protein, respectively. Moreover, apigenin suppressed p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) phosphorylation without affecting the activity of extracellular signal-regulated kinase (ERK). Apigenin was also found to protect neuronal cells from injury in middle cerebral artery occlusion.

Inhibitory Effects of Arbutin on Melanin Biosynthesis of α-Melanocyte Stimulating Hormone-induced Hyperpigmentation in Cultured Brownish Guinea Pig Skin Tissues

Arbutin has been used as a whitening agent in cosmetic products. Melanin, the major pigment that gives color to skin, may be over-produced with sun exposure or in conditions such as melasma or hyperpigmentary diseases. Tyrosinase is a key enzyme that catalyzes melanin synthesis in melanocytes; therefore, inhibitors of the tyrosinase enzyme could be used for cosmetic skin whitening. A recent study has reported that arbutin decreases melanin biosynthesis through the inhibition of tyrosinase activity. However, this inhibitory mechanism of arbutin was not sufficiently demonstrated in skin tissue models. We found that arbutin both inhibits melanin production in B16 cells induced with α-MSH and decreases tyrosinase activity in a cell-free system. Furthermore, the hyperpigmentation effects of α-MSH were abrogated by the addition of arbutin to brownish guinea pig and human skin tissues. These results suggest that arbutin may be a useful agent for skin whitening.

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 Alzheimers and Parkinsons 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.

Effect of electroacupuncture on the stress-induced changes in brain-derived neurotrophic factor expression in rat hippocampus

Stress induces neuronal atrophy and death especially in the hippocampus. Alterations in the expression of neurotrophic factors are implicated in stress-induced hippocampal degeneration. In the hippocampus, stress decreases brain-derived neurotrophic factor (BDNF) mRNA levels. In oriental medicine, acupuncture has long been employed as a treatment of numerous disorders. The objective of this study was to examine whether electroacupuncture (EA) stimulation can influence BDNF expression in the hippocampus of rats exposed to immobilization stress. Rats were immobilized in plastic bags, and then subjected to EA at ST36 Zusanli. After treatment, the animals were decapitated and the hippocampi were rapidly removed and processed for RNA isolation and reverse transcription. Real-time polymerase chain reaction analysis showed that EA stimulation significantly restored BDNF mRNA expression declined by immobilization stress. The results suggest that EA may relieve neuropathological effects of stress by modulating neurotrophic factor expression.

Transforming growth factor-β-inducible gene-h3 (βig-h3) promotes cell adhesion of human astrocytoma cells in vitro: implication of α6β4 integrin

Beta(ig)-h3 is a secretory protein that is induced by transforming growth factor (TGF)-beta. We have recently found that beta(ig)-h3 expression is induced in cultured astrocytes by TGF-beta1 and in rat cerebral cortex by stab wound. The purpose of this study was to examine the effect of the secreted beta(ig)-h3 on cell adhesion of astrocytes and the underlying mechanisms. When U87 human astrocytoma cells were seeded on dishes coated with recombinant beta(ig)-h3, cell adhesion was significantly enhanced. Blocking experiments using various antibodies to the integrin subunit suggested alpha6beta4 integrin could be involved in the beta(ig)-h3-mediated astrocyte cell adhesion. Cell adhesion to beta(ig)-h3 substrate was substantially blocked by preincubation with the inhibitor to the src kinase. When cells were plated on beta(ig)-h3-coated dishes, tyrosine phosphorylation of focal adhesion kinase was prominently increased within 20 min in a beta4 integrin-dependent manner. The results suggest that alpha6beta4 integrin-mediated interactions of astrocytes with beta(ig)-h3 transduce intracellular signals through the focal adhesion proteins, which may regulate certain aspects of astrocyte response to brain injury.

Role of nitric oxide synthase inhibitors and NMDA receptor antagonist in nicotine-induced behavioral sensitization in the rat

Repeated injections of nicotine are well known to produce progressively larger increases in locomotor activity, an effect defined as behavioral sensitization. This study was carried out to investigate the role of nitric oxide (NO) and N-methyl-D-aspartate (NMDA) receptors in nicotine-induced behavioral sensitization. Rats were given repeated injections of nicotine (0.4 mg/kg, s.c., twice daily for 7 days) followed by one challenge injection on the fourth day after the last daily injection. Systemic challenge with nicotine produced a much larger increase in locomotor activity in nicotine-pretreated rats. Rats were pretreated with the nonselective nitric oxide synthase (NOS) inhibitor, N(G)-nitro-arginine-methyl-ester (L-NAME; 75 mg/kg, i.p.), the selective constitutive NOS inhibitor, N-nitro-L-arginine (L-NNA; 15 mg/kg, i.p.), the prototypical selective inducible NOS inhibitor, aminoguanidine (100 mg/kg, i.p.) or NMDA receptor antagonist, MK-801 ((5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine; 0.3 mg/kg, i.p.), 30 min before injections of nicotine during a 7-day development or a 3-day withdrawal phase after which challenged with nicotine on day 11. Pretreatment with L-NAME, L-NNA and MK-801, but not aminoguanidine, blocked the development of nicotine-induced sensitization to subsequent nicotine challenge. Injections of MK-801 twice daily during 3-day withdrawal periods after a 7-day induction period of nicotine attenuated nicotine-induced behavioral sensitization, whereas injections of L-NAME, L-NNA or aminoguanidine had no effects on the expression of sensitization produced by repeated nicotine. This study demonstrates that NMDA receptors can play a major role in the expression as well as development of nicotine-induced behavioral sensitization, and that NO is also involved in the development, but not critically involved in the expression of behavioral sensitization to nicotine.

Lipopolysaccharide induces hypoxia-inducible factor-1 alpha mRNA expression and activation via NADPH oxidase and Sp1-dependent pathway in BV2 murine microglial cells.

Hypoxia-inducible factor-1 (HIF-1), the key transcription factor of hypoxia-inducible genes, is known to be involved in inflammation and immune response, but little is known about the regulation of HIF-1 during microglial activation. Thus, we examined effect of lipopolysaccharide (LPS) on HIF-1 activation and its signaling mechanism in BV2 microglial cells. LPS induced HIF-1alpha mRNA and protein expression as well as HIF-1 transcriptional activation. Moreover, HIF-1alpha knockdown by small interfering RNA (siRNA) decreased LPS-induced expression of hypoxia responsive genes, VEGF, iNOS, and COX-2. We then showed that LPS-induced HIF-1alpha mRNA expression was blocked by an antioxidant, NADPH oxidase inhibitors, and siRNA of gp91phox, a subunit of NADPH oxidase. In addition, we showed that specific pharmacological inhibitors of PI 3-kinase and protein kinase C decreased LPS-induced HIF-1alpha mRNA expression. Finally, we showed that inhibition of transcription factor Sp1 by mithramycin A or Sp1 siRNA decreased LPS-induced HIF-1alpha mRNA and protein expression. Consistently, LPS increased Sp1 DNA binding and its transcriptional activity. Taken together, these results suggest that LPS induces HIF-1alpha mRNA expression and activation via NADPH oxidase and Sp1 in BV2 microglia.

Immobilization stress induces cell death through production of reactive oxygen species in the mouse cerebral cortex

Prolonged stress has been shown to impair brain function and increase vulnerability to neuronal injury. To elucidate the in vivo response of neuronal cells to induced stress, we immobilized mice by binding their legs. Levels of reactive oxygen species (ROS) in the cerebral cortex were increased after stress induction. NADPH oxidase, interleukin-1beta (IL-1beta) and cyclooxygenase 2 mRNA (COX-2) expression levels were upregulated, and Fas levels were also increased. The increased expression of these factors was associated with neuronal death, which was confirmed by TUNEL and NeuN staining. OX42 staining was also evident around the TUNEL-stained lesions. From these findings, it appears that immobilization stress induces neuronal death in the mouse cerebral cortex, a process mediated by NADPH oxidase, IL-1beta, COX-2, ROS and Fas. However, this could be inhibited by pretreating the animals with antioxidants such as ebselen or pyrrolidine dithiocarbamate.

Induction of TGF-β-inducible gene-h3 (βig-h3) by TGF-β1 in astrocytes: implications for astrocyte response to brain injury

Abstract Transforming growth factor (TGF)-β-inducible gene-h3 (βig-h3) product is a secreted protein that is induced by TGF-β in several cell types and implicated in various tissue pathologies. The aims of this study were to determine the effect of TGF-β1 on βig-h3 expression in cultured astrocytes and to examine whether βig-h3 is expressed in the brain after traumatic injury. The results showed that βig-h3 mRNA and protein increased in response to TGF-β1 in U87 human astrocytoma cells and mouse cortical astrocytes. Treatment with other cytokines, including tumor necrosis factor-α and fibroblast growth factor-2, did not enhance the expression of βig-h3 in astrocytes. βig-h3 was significantly expressed in reactive astrocytes at the site of a stab wound in the cerebral cortex of adult rats. These results provide an insight into understanding a novel role for βig-h3 protein in the response of astrocytes to brain injury.