• 대한한방내과학회지
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• 제31권1호
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• pp.54-65
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• 2010

Objective : The water extract of Chungsimyeonja-eum (CSYJE) has traditionally been used in treatments of heart diseases and brain diseases in Oriental medicine. However, little is known about the mechanism by which CSYJE protects neuronal cells from injury damages. Therefore, in this study we attempted to elucidate the mechanism of the cytoprotective effect of the CSYJE extract on glutamate-induced C6 glial cell death. Methods : Cultured cells were pretreated with CSYJE and exposed to glutamate, cell damage was assessed by using MTT assay and propidium iodide (PI), probe 2',7'-dichlorofluorescein diacetate (DCF-DA) staining. Western blotting was performed using anti-procaspase-3 and anti-PARP, respectively. Result : We determined the elevated cell viability by CSYJE extract on glutamate-induced C6 glial cell death. Glutamate induced DNA fragmentation on C6 glial cells but pre-treatment with CSYJE inhibited DNA fragmentation. One of the main mediators of glutamate-induced cytotoxicity was known to generation of reactive oxygen species (ROS). Pre-treatment with CSYJE inhibited this ROS generation from glutamate-stimulated C6 glial cells. Also, we identified that the ROS-induced DCF-DA green fluorescence was reduced by CSYJE pre-treatment. The critical markers of apoptotic cell death are the cleavages of procaspase-3 protease and PARP proteins, so we checked the expression level and cleavages of procaspase-3 protease and PARP proteins. Glutamate-treated C6 glial cells showed the cleavages of procaspase-3 protease and PARP proteins and followed the reduction of expression of these proteins. Conclusion : These findings indicate that CSYJE may prevent cell death from glutamate-induced C6 glial cell death by inhibiting the ROS generation and procaspase-3 and PARP expression.

• 동의생리병리학회지
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• 제20권2호
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• pp.365-371
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• 2006

Bee Venom aqua-acupuncture (BVA) simultaneously exerts pharmacological effects of biologically active compounds, existed in the whole bee venom, and medicinal effect of the stimulation of acupuncture points. BVA has been considered as a promising therapeutic method for treating various chronic diseases, mainly accompanying severe pain and inflammation. As a painless injection device, jet injectors have been commercially marketed for various clinical applications including insulin injection and vaccination. Among them, a pressure-driven jet injector system could be used for intradermal delivery of a variety of drugs. The aim of this study was to investigate the analgesic effects of the BVA using a needle-free injector (Biojector $2000^{\circledR}$, Bioject Inc., OR, USA), compared to the conventional BV aqua-acupuncture using a typical syringe. Adult Sprague-Dawley rats were injected with bee venom $(0.08mg/kg,\;50{\mu}l)$ using Biojector $2000^{\circledR}$ (BVA-B) or a syringe (BVA-5) into the Zusanli (ST36) acupoint, 30 minutes before plantar injection of 2% formalin. It was found that BVA-B-, or BVA-5-treated rats, compared to controls, exhibited significantly less licking behavior during the late phase in the rat formalin test, when compared to controls. During early phase, however, those effects were not significant but substantial. The analgesic effect of BVA-B was also compatible with that of the conventional BVA-5. In the immunohistochemical studies, BVA-B significantly suppressed the expression of formalin-evoked c-fos, a biomarker of neuronal activity, in the lumbar dorsal horn of the spinal cord. These results indicated that BVA-B waseffective in the modulation of pain in the rat formalin test, compared to BVA-5. Taken together, the needle-free jet injector system could be substituted for the conventional aqua- acupuncture with the advantage of little pain.

• Toxicological Research
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• 제18권4호
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• pp.355-362
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• 2002

Pathophysiological elevation of intracellular calcium concentration ($[Ca^{2+}]_1$) in the neuron has been considered as an important responsible factor in the neuronal cell damages. However the mechanism of increase of $[Ca^{2+}]_1$ and the relationship between $[Ca^{2+}]_1$ level and cytotocixity have not been fully demonstrated. In the present study, real-time alteration of $[Ca^{2+}]_1$and cellular response (cell damages) in the pheochromocytoma cells (PC12) stimulated by glutamate were investigated. Glutamate dose dependently decreased cell viability determined propidium iodide fluorescence method and morphology change. Conversely related with cell damages, glutamate dose dependently increased the level of［Ca$^{2+}$］$_{i}$ . To investigate the mechanism of glutamate-induced increase of $[Ca^{2+}]_1$,$[Ca^{2+}]_1$, was first measured in the cell cultured in calcium free media and in the presence of dantrolene, an inhibitor of calcium release from ryanodine receptor located in endoplasmic reticulum (ER). Similar to the increase$[Ca^{2+}]_1$ in the calcium-containing media, glutamate dose dependently increased $[Ca^{2+}]_1$ in the cell cultured in free calcium media. However pretreatment (2 hr) with 20~50 $\mu\textrm{M}$ dantrolene substantial lowered glutamate-induced increase of $[Ca^{2+}]_1$, suggesting that release of calcium from ER may be major sourse of increase of $[Ca^{2+}]_1$ in PC12 cells. Dantrolene-induced inhibition of $[Ca^{2+}]_1$ resulted in recovery of cytotoxicity by glutamate. Relevance of N-methy-D-aspartate (NMDA) receptor, a type of glutamte receptor on glutamate-induced incense of $[Ca^{2+}]_1$,$[Ca^{2+}]_1$ was also determined in the cells pretreated (2 hr) with NMDA receptor antagonist MK-80l. Glutamate-induced increase of $[Ca^{2+}]_1$ was reduced by MK-801 dose dependently. Furthermore, glutamate-induced cytotoxicity was also prevented by MK-80l. These results demonstrate that glutamte increase $[Ca^{2+}]_1$ dose dependently and thereby cause cytotoxicity. The increase of $[Ca^{2+}]_1$ may release from ER, especially through ryanodine receptor and/or through NMDA receptor Alteration of calcium homeostasis through disturbance of ER system and/or calcium influx through NMDA receptor could contribute glutamate-induced cell damages.s.

• Biomolecules & Therapeutics
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• 제18권1호
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• pp.39-47
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• 2010

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor involved in neuronal differentiation, plasticity, survival and regeneration. BDNF draws massive attention mainly due to the potential as a therapeutic target in neurological diseases such as depression and Alzheimer's disease. In a primary screening for the natural compounds enhancing BDNF release from cultured rat primary cortical neuron, we found that compounds such as baicalein, tanshinone IIa, cinnamic acid, epiberberine, genistein and wogonin among many others increased BDNF release. All the compounds at $0.1{\mu}M$ of concentration barely showed stimulatory effect on BDNF induction, however, their combination (mixture 1; baicalein, tanshinone IIa and cinnamic acid, mixture 2; epiberberine, genistein and wogonin) showed synergistic increase in BDNF release as well as mRNA and protein expression. The level of BDNF expression was comparable to the maximum BDNF stimulation attainable by a positive control oroxylin A ($20{\mu}M$) without cell toxicity as determined by MTT analysis. Both mixtures synergistically increased the phosphorylation of extracellular signal-regulated kinase (ERK) as well as cAMP response element binding protein (CREB), an immediate and essential regulator of BDNF expression. Similar to these results, mixture of these compounds synergistically inhibited the up-regulation of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide treatments in rat primary astrocytes. These results suggest that the combinatorial treatment of natural compounds in lower concentration might be a useful strategy to obtain sufficient BDNF stimulation in neurological disease condition such as depression, while minimizing potential side effects and toxicity of higher concentration of a single compound.

• Journal of Chest Surgery
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• 제49권4호
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• pp.232-241
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• 2016

Background: Paraplegia is a devastating complication following operations on the thoracoabdominal aorta. We investigated whether histidine-tryptophan-ketoglutarate (HTK) solution could reduce the extent of ischemia/reperfusion (IR) spinal cord injuries in a rat model using a direct delivery method. Methods: Twenty-four Sprague-Dawley male rats were randomly divided into four groups. The sham group (n=6) underwent a sham operation, the IR group (n=6) underwent only an aortic occlusion, the saline infusion group (saline group, n=6) underwent an aortic occlusion and direct infusion of cold saline into the occluded aortic segment, and the HTK infusion group (HTK group, n=6) underwent an aortic occlusion and direct infusion of cold HTK solution into the occluded aortic segment. An IR spinal cord injury was induced by transabdominal clamping of the aorta distally to the left renal artery and proximally to the aortic bifurcation for 60 minutes. A neurological evaluation of locomotor function was performed using the modified Tarlov score after 48 hours of reperfusion. The spinal cord was harvested for histopathological and immunohistochemical examinations. Results: The spinal cord IR model using direct drug delivery in rats was highly reproducible. The Tarlov score was 4.0 in the sham group, $1.17{\pm}0.75$ in the IR group, $1.33{\pm}1.03$ in the saline group, and $2.67{\pm}0.81$ in the HTK group (p=0.04). The histopathological analysis of the HTK group showed reduced neuronal cell death. Conclusion: Direct infusion of cold HTK solution into the occluded aortic segment may reduce the extent of spinal cord injuries in an IR model in rats.

• Journal of Periodontal and Implant Science
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• 제27권2호
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• pp.425-441
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• 1997

The neuropeptide substance P(SP) has been implicated in the mediation of inflammation and immune-mediated disease such as arthritis. Recently, it was reported that SP was markedly increased around the blood vessels in inflamed gingiva as well as in close association with the inflammatory cell infiltrate. These results support that SP may contribute to the pathophysiology of neuronal inflammation in human periodontal tissues. SP may regulate inflammatory/immune responses by stimulating the proliferation of human T cells, differentiation and antibody-secreting potential of B cells, macrophage respiratory burst, connective tissue proliferation, and the secretion of cytokines from monocytes and T cells. Here, I studied potential role of SP as a costimulatory chemical signal in inflammatory/immune responses, by determining the released proinflammatory cytokines such as $MIP-1{\alpha}$, $IL-1{\beta}$, and IL-6 from culture supernatants of homogeneous immune cell lines. Serum free cell supernatants were concentrated with TCA precipitation, fractionated with SDS-PAGE, and subjected into western blot analysis. Among 15 cell lines tested, macrophage/monocyte cell line RAW264.7 and WRl9m.1 showed the highest level of induction of $MIP-1{\alpha}$ when stimulated with LPS. Discrete IL-6 bands with multiple forms of molecular mass were detected from supernatants of B cell lines A20(32kDa), Daudi(32, 35kDa), and SKW6.4(29kDa), which were expressed constitutively. $IL-1{\beta}$ could not be detected by the method of western blot analysis from supernatants of all cell lines tested except RAW264.7, WRl9m.1, and erythroid cell line K562 which showed the least amount of $IL-{\beta}$ secretion. SP $10^{-9}M$ with suboptimal dose of LPS treatment showed synergistic induction of $MIP-1{\alpha}$ release from RAW264.7 or WR19m.1, and also IL-6 release from A20, but this synergism is not the case in costimulation of RAW264.7 or WRl9m.1 with SP $10^{-9}M$ and TPA. Although treatment of T cell line CTLL-R8 with SP $10^{-7}M$ or PHA+TPA induced modest level of $MIP-1{\alpha}$ secretion, synergism was not observed when they are applied together. These findings all together suggest the possibility of a regulatory role of SP in inflammatory/immune reaction through differential modulation of bioactivities of other chemical cosignals.

• 대한의생명과학회지
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• 제13권3호
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• pp.197-206
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• 2007

P2X receptors are membrane-bound ion channels that conduct $Na^+,\;K^+$, and $Ca^{2+}$ in response to ATP and its analogs. There are seven subunits identified so far ($P2X_1-P2X_7$). $P2X_2$ receptors are known to be expressed in a wide range of organs including brains and adrenal grands. PC12 cells are originated from adrenal grand and differentiated by nerve growth factor or pituitary adenylate cyclase activating poly peptide (PACAP). Previous studies indicate that $P2X_2$ receptor activation in PC12 cells couples to $Ca^{2+}-dependent$ release of catecholamine and ATP. It is known that acidic pH potentiates ATP currents at $P2X_2$ receptors. This leads to a hypothesis that $P2X_2$ receptors may play an important role in PC12 cell differentiation, one of the characteristics of which is neurite outgrowth, induced by the hormones under lower pH. In the present study, we isolated several clones which potentiate neurite outgrowth by PACAP in acidic pH (6.8), but not in alkaline pH (7.6). RT-PCR and electrophysiology data indicate that these clones express only functional $P2X_2$ receptors in the absence or presence of PACAP for 3 days. Potentiation of neurite outgrowth resulted from PACAP (100 nM) in acidic pH is inhibited by the two P2X receptor antagonists, suramin and PPADS ($100\;{\mu}M)$ each), and exogenous exprerssion of ATP-binding mutant $P2X_2$ receptor subunit ($P2X_2[K69A]$). However, acid sensing ion channels (ASICs) are not involved in PACAP-induced neurite outgrowth potentiation in lower pH since treatments of an inhibitor of ASICs, amyloride ($10\;{\mu}M$), did not give any effects to neurite extension. The vesicular proton pump ($H^+-ATPase$) inhibitor, bafilomycin (100 nM), reduced neurite extension indicating that ATP release resulted from $P2X_2$ receptor activation in PC12 cells is needed for neurite outgrowth. These were confirmed by activation of mitogen activated protein kinases, such as ERKs and p38. These results suggest roles of ATP and $P2X_2$ receptors in hormone-induced cell differentiation or neuronal synaptogenesis in local acidic environments.

• Investigative Magnetic Resonance Imaging
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• 제16권2호
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• pp.124-135
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• 2012

목적: 본 연구에서는 숙달된 택시운전기사들을 대상으로 내비게이션 시스템을 이용하여 운전하는 한국인 대조군에 비교하여 해마의 부피와 모양 그리고 기능적 변화를 알아보고자 하였다. 대상과 방법: 총 8명의 대상군(4명의 숙달된 택시운전기사들과 4명의 같은 나이대 일반인 대조군)에 대해 삼차원 T1강조영상과 혈류산소 수준에 따른 기능성 자기공명영상을 촬영하였다. 해마의 용적과 모양, 기능성 자기공명영상 자료를 분석하여 두 군간에 신경 활성도 차이를 비교하였다. 결과: 해마의 용적은 두 군간에 통계적으로 유의한 차이를 보이지 않았다(p > 0.05). 해마의 모양을 보면, 택시운전기사군의 왼쪽 해마가 대조군에 비해 전체 길이가 약간 길고 머리와 꼬리부분이 약간 더 컸다(p < 0.05, uncorrected). 기능성 자기공명영상에서는 택시운전기사군에서 대조군에 비해 방추상회가 특히 활성화되어 있었다. 결론: 택시운전기사군의 해마가 보이는 이러한 구조적, 기능적 변화는 그들의 직업과 관련하여 공간적 탐색능력을 지속적으로 사용하면서 해마와 관련 뇌영역이 기능적으로 분화했음을 나타내는 소견으로 추측할 수 있겠다.

• 대한한의학회지
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• 제26권3호
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• pp.1-12
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• 2005

Objectives : Substantial evidence suggests that reinforcing effects of nicotine can be mediated by the mesolimbic dopaminergic system. It has been shown that repeated injections of nicotine produce an increase in locomotor activity and expression of the immediate-early gene, c-fos, in the dopaminergic target areas. Herbal medicine as a therapeutic intervention has been widely used for the treatment of mental dysfunction. Many studies have shown that Radix Scutellariae (RS) can affect the biochemical balance in the central nervous system. Tn order to investigate whether RS has an influence on nicotine-induced behavioral sensitization, we examined the effect of RS on nicotine-induced locomotor activity and c-fos expression in the striatum and nucleus accumbens utilizing the fos-tike immunohistochemistry (FLI). Methods : Male SD rats received RS (200mg/kg, i.p.) 30min before repeated daily injections of nicotine (0.4mg/kg, s.c.) for 7 days. This was followed by withdrawal for 3 days and one challenge for 1 day. Results : System challenge with nicotine produced a much larger increase in locomotor activity and accumbal FLI. Pretreatment with RS significantly inhibited nicotine-induced locomotor activity and FLI in the striatum and nucleus accumbens. Conclusions : These results demonstrate that reduction in locomotor activity by RS may be reflected by reduction of dopamine release and postsynaptic neuronal activity in the striatum and nucleus accumbens. Our results suggest that RS may have therapeutic effect on nicotine addiction.

• 대한한의학회지
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• 제24권2호
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• pp.66-80
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• 2003

Objectives : Recently, herbal acupuncture has been developed in the Korea since the earlier 1960' applied to various diseases including the cerebrovascular accident. The main characteristics of herbal acupuncture are a combination the merit of acupuncture and herbal medicine. It was not well known the therapeutic effect and the mechanism of herbal acupuncture on transient forebrain ischemic injury, although it has been used frequently in clinics. Ramulus Uncariae Cum Uncis (釣鉤藤) has been claimed to possess sedative, anti-spasmodic actions, and treat childhood epilepsy in oriental medical preparation. Also, it has been stated that Ramulus Uncariae Cum Uncis was antioxidatvie effect and neuroprotection against glutamateinduced neuronal death. Methods : In this study, effects of Ramulus Uncariae Cum Uncis' herbal acupuncture on the $GV_{20}{\;}or{\;}LR_3$, named Baek-hue or Tae-chung, on neuroprotection after the transient forebrain ischemia were investigated in Sprague-Dawely rats. Expressions of cFos, FosB and BDNF protein in the hippocampus and cortex were observed at 2 hrs and 48 hrs after transient forebrain ischemia by immunohistochemistry and ELISA technique. Results : Expression of cFos protein was increased slightly in the hippocampus and cortex at 2 hrs after transient forebrain ischemia, but FosB protein was increased highly comparing to cFos protein. However, pretreatment with Ramulus Uncariae Cum Uncis' herbal acupuncture on $GV_{20}{\;}or{\;}LR_3$ significantly increased expression of cFos protein and significantly decreased expression of FosB protein compared to control group, respectively. These features were observed in the motor cortex and retrosplenial granular cortex as well as the hippocampus. Especially, cFos expression was more increased at the herbal acupuncture on $GV_{20}{\;}than{\;}LR_3$, but FosB expression was more decreased in $LR_3$ group than $GV_{20}$ group. Also, pretreatment with Ramulus Uncariae Cum Uncis' herbal acupuncture on $GV_{20}{\;}or{\;}LR_3$ significantly increased the expression of BDNF protein in the hippocampus ($254.88{\pm}12{\;}pg/ml{\;}in{\;}GV_{20}$ group, $245.93{\pm}44.4{\;}pg/ml{\;}in{\;}LR_3$ group) and the cortex ($85.81{\pm}3.45{\;}pg/ml{\;}in{\;}GV_{20}$ group, $111.51{\pm}15.79{\;}pg/ml{\;}in{\;}LR_3$, group) compared to the hippocampus ($134.07{\pm}2.96{\;}pg/ml$) and the cortex ($61.16{\pm}4.11{\;}pg/ml$) in control group at 48 hrs after transient forebrain ischemia. Conclusion : These results suggest that pretreatment with Ramulus Uncariae Cum Uncis' herbal acupuncture on $GV_{20}{\;}or{\;}LR_3$ has neuroprotective effect on transient forebrain ischemia and the herbal acupunture on $GV_{20}{\;}or{\;}LR_3$ may be related to antioxidative effect and calcium channel block of Ramulus Uncariae Cum Uncis. Also, it could be mentioned there is specificity of acupoints treating ischemic injury through the difference between the herbal acupuncture of $GV_{20}{\;}and{\;}LR_3$.