• The Korean Journal of Physiology and Pharmacology
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• v.18 no.5
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• pp.397-402
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• 2014

Microglia are activated by inflammatory and pathophysiological stimuli in neurodegenerative diseases, and activated microglia induce neuronal damage by releasing cytotoxic factors like nitric oxide (NO). Activated microglia synthesize a significant amount of vitamin $D_3$ in the rat brain, and vitamin $D_3$ has an inhibitory effect on activated microglia. To investigate the possible role of vitamin $D_3$ as a negative regulator of activated microglia, we examined the effect of 25-hydroxyvitamin $D_3$ on NO production of lipopolysaccharide (LPS)-stimulated microglia. Treatment with LPS increased the production of NO in primary cultured and BV2 microglial cells. Treatment with 25-hydroxyvitamin $D_3$ inhibited the generation of NO in LPS-activated primary microglia and BV2 cells. In addition to NO production, expression of 1-${\alpha}$-hydroxylase and the vitamin D receptor (VDR) was also upregulated in LPS-stimulated primary and BV2 microglia. When BV2 cells were transfected with 1-${\alpha}$-hydroxylase siRNA or VDR siRNA, the inhibitory effect of 25-hydroxyvitamin $D_3$ on activated BV2 cells was suppressed. 25-Hydroxyvitamin $D_3$ also inhibited the increased phosphorylation of p38 seen in LPS-activated BV2 cells, and this inhibition was blocked by VDR siRNA. The present study shows that 25-hydroxyvitamin $D_3$ inhibits NO production in LPS-activated microglia through the mediation of LPS-induced 1-${\alpha}$-hydroxylase. This study also shows that the inhibitory effect of 25-hydroxyvitamin $D_3$ on NO production might be exerted by inhibiting LPS-induced phosphorylation of p38 through the mediation of VDR signaling. These results suggest that vitamin $D_3$ might have an important role in the negative regulation of microglial activation.

• Development and Reproduction
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• v.17 no.4
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• pp.469-475
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• 2013

In mammals, puberty is a process of acquiring reproductive competence, triggering by activation of hypothalamic kisspeptin (KiSS)-gonadotropin releasing hormone (GnRH) neuronal circuit. During peripubertal period, not only the external genitalia but the internal reproductive organs have to be matured in response to the hormonal signals from hypothalamic-pituitary-gonadal (H-P-G) axis. In the present study, we evaluated the maturation of male rat accessory sex organs during the peripubertal period using tissue weight measurement, histological analysis and RT-PCR assay. Male rats were sacrificed at 25, 30, 35, 40, 45, 50, and 70 postnatal days (PND). The rat accessory sex organs exhibited differential growth patterns compared to those of non-reproductive organs. The growth rate of the accessory sex organs were much higher than the those of non-reproductive organs. Also, the growth spurts occurred differentially even among the accessory sex organs; the order of prepubertal organ growth spurts is testis = epididymis > seminal vesicle = prostate. Histological study revealed that the presence of sperms in seminiferous tubules and epididymal ducts at day 50, indicating the puberty onset. The number of duct and the volume of duct in epididymis and prostate were inversely correlated during the experimental period. Our RT-PCR revealed that the levels of hypothalamic GnRH transcript were increased significantly on PND 40, suggesting the activation of hypothalamic GnRH pulse-generator before puberty onset. Studies on the peripubertal male accessory sex organs will provide useful references on the growth regulation mechanism which is differentially regulated during the period in androgen-sensitive organs. The detailed references will render easier development of endocrine disruption assay.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.4
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• pp.371-376
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• 2017

The caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) receives direct inputs from small diameter primary afferent fibers that predominantly transmit nociceptive information in the orofacial region. Recent studies indicate that reactive oxygen species (ROS) is involved in persistent pain, primarily through spinal mechanisms. In this study, we aimed to investigate the role of xanthine/xanthine oxidase (X/XO) system, a known generator of superoxide anion ($O_2{^-}$), on membrane excitability in the rat MDH neurons. For this, we used patch clamp recording and confocal imaging. An application of X/XO ($300{\mu}M/30mU$) induced membrane depolarization and inward currents. When slices were pretreated with ROS scavengers, such as phenyl N-tert-butylnitrone (PBN), superoxide dismutase (SOD), and catalase, X/XO-induced responses decreased. Fluorescence intensity in the DCF-DA and DHE-loaded MDH cells increased on the application of X/XO. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), significantly decreased X/XO-induced depolarization. X/XO elicited an inward current associated with a linear current-voltage relationship that reversed near -40 mV. X/XO-induced depolarization reduced in the presence of $La^{3+}$, a nonselective cation channel (NSCC) blocker, and by lowering the external sodium concentration, indicating that membrane depolarization and inward current are induced by influx of $Na^+$ ions. In conclusion, X/XO-induced ROS modulate the membrane excitability of MDH neurons, which was related to the activation of NSCC.

• Molecular & Cellular Toxicology
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• v.4 no.2
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• pp.113-123
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• 2008

Microglia, which is the primary immune effector cells in the central nervous system, constitutes the first line of defense against infection and injury in the brain. The goal of this study was to determine the protective (anti-inflammation) mechanisms of forsythia suspense (FS) on LPS-induced activation of BV-2 microglial cells. The effects of FS on gene expression profiles in activated BV-2 microglial cells were evaluated using microarray analysis. BV-2 microglial cells were cultured in a 100mm dish $(1{\times}10^7/dish)$ for 24hr and then pretreated with $1{\mu}g/mL$ FS or left untreated for 30 min. Next, $1{\mu}g/mL$ LPS was added to the samples and the cells were reincubated at $37^{\circ}C$ for 30 min, 1hr, and 3hr. The gene expression profiles of the BV-2 microglial cells varied depending on the FS. The oligonucleotide microarray analysis revealed that MAPK pathway-related genes such as Mitogen activated protein kinase 1 (Mapk1), RAS protein activator like 2 (Rasal2), and G-protein coupled receptor 12 (Gpr12) and nitric oxide biosynthesis-related genes such as nitric oxide synthase 1 (neuronal) adaptor protein (Nos1ap), and dimethylarginine dimethylaminohydrolase 1 (Ddah1) were down regulated in FS-treated BV-2 microglial cells. FS can affect the MAPK pathway and nitric oxide biosynthesis in BV-2 microglial cells.

• The Journal of Internal Korean Medicine
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• v.31 no.3
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• pp.586-599
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• 2010

Objective : The water extract of Yukgunja-tang(YGJT) has been traditionally used in treatment of qi deficiency and phlegm in Oriental medicine. However, little is known about the mechanism by which YGJT protects neuronal cells from injury damages. Therefore, this study was designed to evaluate the protective effects of YGJT on C6 glial cells by glutamate-induced cell death. Methods : The present study describes glutamate, which is known as an excitatory neurotransmitter, related with oxidative damages, and YGJT, which shows protective effects against glutamate-induced C6 glial cell death. One of the main mediators of glutamate-induced cytotoxicity was known on the generation of reactive oxygen species(ROS) via activation of NADPH oxidase (NOX). The protective effects of antioxidant(NAC) and NOX inhibitor(apocynin) on the glutamate-induced C6 glial cells were determined by a MTT reduction assay. Result : YGJT inhibited glutamate-induced ROS generation via inhibition of NOX expression on glutamate-stimulated C6 glial cells. Furthermore, YGJT attenuated glutamate-induced caspase activation. These results suggest that YGJT could be a new potential candidate against glutamate-induced oxidative stress and cell death. Conclusion : These findings indicate that in C6 glial cells, ROS plays an important role of glutamate-induced cell death and that YGJT may prevent cell death from glutamate-induced cell death by inhibiting the ROS generation.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.2
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• pp.107-112
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• 2012

Although various derivatives of caffeic acid have been reported to possess a wide variety of biological activities such as neuronal protection against excitotoxicity and anti-inflammatory property, the biological activity of 3,4,5-trihydroxycinnamic acid (THC), a derivative of hydroxycinnamic acids, has not been clearly examined. The objective of the present study is to evaluate the anti-inflammatory effects of THC on lipopolysaccharide (LPS)-stimulated BV2 microglial cells. THC significantly suppressed LPS-induced excessive production of nitric oxide (NO) and expression of iNOS, which is responsible for the production of iNOS. THC also suppressed LPS-induced overproduction of pro-inflammatory cytokines such as IL-$1{\beta}$and TNF-${\alpha}$ in BV2 microgilal cells. Furthermore, THC significantly suppressed LPS-induced degradation of $I{\kappa}B$, which retains NF-${\kappa}B$ in the cytoplasm. Therefore, THC attenuated nuclear translocation of NF-${\kappa}B$, a major pro-inflammatory transcription factor. Taken together, the present study for the first time demonstrates that THC exhibits antiinflammatory activity through the suppression of NF-${\kappa}B$ transcriptional activation in LPS-stimulated BV2 microglial cells.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.1
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• pp.51-58
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• 2006

The promoting effect of hydrogen peroxide ($H_2O_2$) against the cytotoxicity of 1-methyl-4-phenylpyridinium ($MPP^+$) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with $MPP^+$ resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. Addition of $H_2O_2$ enhanced the $MPP^+-induced$ nuclear damage and cell death. Catalase, Carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the cytotoxic effect of $MPP^+$ in the presence of $H_2O_2$. Addition of $H_2O_2$ promoted the change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to $MPP^+$ in PC12 cells. The results show that the $H_2O_2$ treatment promotes the cytotoxicity of $MPP^+$ against PC12 cells. $H_2O_2$ may enhance the $MPP^+$-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that $H_2O_2$ as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by neurotoxins.

• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.3
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• pp.463-469
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• 2010

This research is performed to obtain positive evidences of Mori cortex, a kind of oriental medicinal herbs, in the cellular levels. The extracts of M. cortex have shown anti-inflammatory effects against cutaneous inflammation and clinical effects on pulmonary asthma and congestion in oriental medicine. Thus BV2 cells were chosen because microglia are considered as the main immunocompetent cells in the central nervous system. Lipopolysaccharide (LPS)-induced microglial activation of cultured BV2 cells and subsequent release of nitric oxide (NO) and Prostaglandin E2 (PGE2) were effectively suppressed by methylene chloride extract of Morus alba L. (MEMA). From the inflammation-mediated mRNA and protein analyses, we showed that inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-$1{\beta}$ (IL-$1{\beta}$) and tumor necrosis alpha (TNF-${\alpha}$) induced by LPS were markedly decreased by MEMA treatment. From the observation of nuclear factor-kB (NF-${\kappa}B$) which is controlling and mediating inflammation through COX-2 and iNOS, there showed that p65, a subunit of NF-${\kappa}B$, was increased in nuclear and $I{\kappa}B$, a competitor of NF-${\kappa}B$, was recovered in cytosol after MEMA treatment. These are corresponding with results of iNOS, COX-2, IL-$1{\kappa}$ and TNF-${\alpha}$, and confirm some suppressive effect against transcriptional activation of NF-${\kappa}B$. In conclusion, the anti-inflammatory action of M. cortex against BV2 microglia cells is expected to protect nerve tissues through suppression of neuronal inflammation in various neurodegenerative diseases.

• YAKHAK HOEJI
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• v.60 no.2
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• pp.92-99
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• 2016

Parkinson's disease (PD) is one of the representative neurodegenerative movement disorders with the selective loss of dopaminergic neurons in the substantia nigra. 6-Hydroxydopamine (6-OHDA) is widely used as an experimental model system to mimic PD and has been reported to cause neuronal cell death via oxidative and/or nitrosative stress. Therefore, daily intake of dietary or medicinal plants which fortifies cellular antioxidant capacity can exert neuroprotective effects in PD. In the present study, we have investigated the protective effect of Korean red ginseng (KRG) against 6-OHDA-induced nitrosative death in C6 glioma cells. Treatment of C6 cells with 6-OHDA decreased cell viability and increased expression of inducible nitric oxide synthase, production of nitric oxide as well as peroxynitrite, and formation of nitrotyrosine. 6-OHDA led to apoptotic cell death as determined by decreased Bcl-2/Bax, phosphorylation of JNK, activation of caspase-3, and cleavage of PARP. Conversely, pretreatment of C6 cells with KRG attenuated 6-ODHA-induced cytotoxicity, apoptosis, and nitrosative damages. To further elucidate the molecular mechanism of KRG protection against 6-OHDA-induced nitrosative cell death, we have focused on the cellular self-defense molecules against exogenous noxious stimuli. KRG treatment up-regulated heme oxygenase-1 (HO-1), a key antioxidant enzyme essential for cellular defense against oxidative and/or nitrosative stress via activation of Nrf2. Taken together, these findings suggest KRG may have preventive and/or therapeutic potentials for the management of PD.

• The Journal of Korean Medicine
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• v.31 no.2
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• pp.1-18
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• 2010

Objective: The water extract of Chilgi-tang (CGT) has been traditionally used in treatment of heart diseases caused by stress in Oriental Medicine. However, little is known about the mechanism by which CGT rescues neuronal cells from injury damage. Therefore, this study was designed to evaluate the protective effects of CGT on Neuro-2A cells by glucose deprivation-induced cell death. Methods: We investigated how cell death induced by glucose deprivation was associated with increased reactive oxygen species (ROS) generation. Result: The CGT treatment prior to glucose deprivation insult significantly reduced the number of cell deaths and the glucose deprivation-induced increase in ROS. Nitric oxide (NO) was also attenuated by CGT treatment. In addition, we demonstrated that the anti-cell death effect of CGT was blocked by heme oxygenase-1 (HO-1) activation. Finally, pretreatment of cells with a hemin, HO-1 inducer, reduced glucose deprivation-induced cell death. In contrast, pretreatment of cells with a ZnPP, HO-1 activity inhibitor, attenuated CGT-induced inhibition of cell death. Conclusions: These findings indicate that ROS plays an important role in glucose deprivation-induced cell death and that CGT may prevent glucose deprivation-induced cell death by inhibiting the ROS generation through HO-1 activation in Neuro-2A cells.