• Archives of Plastic Surgery
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• 제40권5호
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• pp.517-521
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• 2013

Background Diabetes is characterized by chronic hyperglycemia, which can increase reactive oxygen species (ROS) production by the mitochondrial electron transport chain. The formation of ROS induces oxidative stress and activates oxidative damage-inducing genes in cells. No research has been published on oxidative damage-related extracellular superoxide dismutase (EC-SOD) protein levels in human diabetic skin. We investigated the expression of EC-SOD in diabetic skin compared with normal skin tissue in vivo. Methods The expression of EC-SOD protein was evaluated by western blotting in 6 diabetic skin tissue samples and 6 normal skin samples. Immunohistochemical staining was also carried out to confirm the EC-SOD expression level in the 6 diabetic skin tissue samples. Results The western blotting showed significantly lower EC-SOD protein expression in the diabetic skin tissue than in the normal tissue. Immunohistochemical examination of EC-SOD protein expression supported the western blotting analysis. Conclusions Diabetic skin tissues express a relatively small amount of EC-SOD protein and may not be protected against oxidative stress. We believe that EC-SOD is related to the altered metabolic state in diabetic skin, which elevates ROS production.

• 생약학회지
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• 제52권1호
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• pp.19-25
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• 2021

We previously reported that lonicerin isolated from Lonicera japonica methanolic extract had potent neuro-protective activities in neuronal cell death injured by excessive glutamate. In this study, we tried to confirm the neuroprotective activities of L. japonica extract and lonicerin in glutamate injured HT22 cells and establish mechanisms of neuroprotective action of lonicerin. We used HT22 cell death injured by glutamate as a bioassay system. The compound decreased reactive oxygen species increased by excessive glutamate treatment in HT22 cells. Also, Ca2+ concentration was decreased by lonicerin treatment. This compound made mitochondrial membrane potential maintain to normal condition. Lonicerin also increased not only glutathione reductase but also peroxidase to the control level. And this compound increased amount of glutathione, an endogenous antioxidant. These results indicated that lonicerin isolated from L. japonica showed potent neuroprotective activity through the anti-oxidative pathway.

• 생약학회지
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• 제53권1호
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• pp.1-7
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• 2022

In the previous study, we reported that luteolin isolated from Lonicera japonica methanolic extract had potent neuroprotective activities in neuronal cell death injured by excessive glutamate. In this study, we tried to confirm the neuroprotective activities of luteolin in glutamate injured HT22 cells and establish mechanisms of neuroprotective action of luteolin. We used HT22 cell death injured by glutamate as a bioassay system. Luteolin decreased reactive oxygen species increased by excessive glutamate treatment in HT22 cells. Also, Ca²⁺ concentration was decreased by luteolin treatment. Luteolin made mitochondrial membrane potential maintain to normal condition. It also increased not only glutathione reductase but also peroxidase to the control level. And it increased amount of glutathione, an endogenous antioxidant. These results suggested that luteolin isolated from L. japonica showed potent neuroprotective activity through the anti-oxidative pathway.

• 생약학회지
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• 제54권1호
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• pp.9-15
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• 2023

In the previous study, we reported that cynaroside isolated from Lysimachia christinae methanolic extract had potent neuroprotective activities in neuronal cell death injured by excessive glutamate. In this study, we tried to confirm the neuroprotective activities of cynaroside in glutamate injured HT22 cells and establish mechanisms of neuroprotective action of cynaroside. We employed HT22 cells damaged by glutamate-induced cell death as a bioassay system. Cynaroside decreased reactive oxygen species increased by excessive glutamate treatment in HT22 cells. Also, Ca²⁺ concentration was decreased by cynaroside treatment. Cynaroside restored mitochondrial membrane potential to normal condition. It also increased not only glutathione reductase but also peroxidase to the control level. And it increased amount of glutathione, an endogenous antioxidant. These results suggested that cynaroside isolated from L. christinae showed potent neuroprotective activity through the anti-oxidative pathway.

• 동의생리병리학회지
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• 제27권6호
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• pp.764-770
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• 2013

Stevia rebaudiana is a traditional herb used as a sweetener in Brazil and Paraguay as well as Korea and China. This study investigated the efficacy of Stevia rebaudiana methanol extract (SRE) to protect cells against the mitochondrial dysfunction and apoptosis in hepatocyte. To determine the effects of SRE on oxidative stress, we used the human derived hepatocyte cell line, HepG2 cell. Treatment of arachidonic acid (AA)+iron in HepG2 cells synergistically amplified cytotoxicity, as indicated by the excess reactive oxygen species (ROS) and mitochondrial permeability transition by fluorescence activated cell sorter (FACS) and immunoblot analysis. Treatment with SRE protected hepatocytes from AA+iron-induced cellular toxicity, as shown by alterations in the protein levels related with cell viability such as procaspase-3. SRE also prevented the mitochondrial dysfunction induced by AA+iron, and showed anti-oxidant effects as inhibition of $H_2O_2$ production and GSH depletion. Moreover, we measured the effects of SRE on AMP-activated protein kinase (AMPK), a key regulator in determining cell survival or death. Acetyl-CoA Carboxylase (ACC), a direct downstream target of AMPK. SRE increased phosphorylation of ACC, and prevented the inhibition of ACC phosphorylation by AA+iron. These results indicated that SRE has the ability to protect cells against AA+iron-induced $H_2O_2$ production and mitochondrial impairment, which may be mediated with AMPK-ACC pathway.

• 한국발생생물학회지:발생과생식
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• 제23권1호
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• pp.11-19
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• 2019

The study was conducted to investigate the effects of alpha-linolenic acid (ALA) combined with bovine serum albumin (BSA) or methyl-beta-cyclodextrin (MBCD) on plasma and acrosomal membrane damages, mitochondrial activity, morphological abnormality, motility, and oxidative stress in frozen-thawed boar sperm. In previous our study, 3 ng/mL ALA had been shown protective effect during freezing process of boar sperm. Therefore, we used 3 ng/mL ALA in present study and ALA was combined with same molar ratio of BSA or MBCD (ALA+BSA and ALA+MBCD, respectively). To confirm the effect of two carrier proteins, same volume of BSA and MBCD without ALA were added during cryopreservation. Membrane damage, mitochondrial activity, reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were measured using flow cytometry, and movement of sperm tail as motility parameter and morphological abnormality were observed under light microscope. In results, all of sperm parameters were enhanced by ALA combined with BSA or MBCD compared to control groups (p<0.05). Mitochondrial activity, morphological abnormality, ROS and LPO levels in ALA+BSA or MBCD groups were no significant difference compared with ALA, BSA and MBCD treatment groups. On the other hand, plasma and acrosomal membrane intact, and sperm motility in ALA+MBCD group were higher than single treatment groups (p<0.05), whereas ALA+BSA did not differ. Our findings indicate that carrier proteins such as BSA and MBCD could improve the effect of ALA during cryopreservation of boar sperm, and treatment of ALA with carrier proteins enhance membrane integrity, mitochondrial activity through reduction of ROS-induced LPO.

• The Korean Journal of Physiology and Pharmacology
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• 제22권3호
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• pp.311-319
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• 2018

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (${\Delta}{\psi}_m$). Therefore, pharmacological manipulation of ${\Delta}{\psi}_m$ can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ${\Delta}{\psi}_m$ against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity ($100{\mu}M$, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate ($100{\mu}M$)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of $Ca^{2+}$ ($5{\mu}M$). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ${\Delta}{\psi}_m$ were completely abolished in $K^+-free$ medium on pure isolated mitochondria. Taken together, results demonstrate that $K^+$ influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial $K^+$ influx is probably mediated, at least in part, by activation of mitochondrial $K^+$ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

• 생명과학회지
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• 제22권12호
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• pp.1621-1627
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• 2012

세포의 이동은 성장, 면역 작용, 그리고 혈관 신생 등 많은 생리현상에 중요한 역할을 한다. 또한 염증 및 종양 세포 침윤 등의 다양한 병리적 현상과도 밀접한 연관이 있다. 본 연구에서는 lysophosphatidic acid (LPA)는 활성산소의 생성을 통해 SKOV-3 난소암세포의 이동을 조절한다는 것을 관찰하였다. 먼저, 난소 암세포인 SKOV-3에서 LPA에 의한 세포의 이동이 강하게 일어남을 확인하였다. LPA에 의한 SKOV-3 세포의 이동은 phosphatidylinositol 3-kinase (PI3K)/Akt 신호전달체계를 저해시키는 약물에 의해서 완벽히 억제됨을 확인하였으나 ERK 신호전달체계를 저해시키는 약물에 의해서는 전혀 영향을 받지 않았다. 그리고 SKOV-3 세포에서 LPA에 의한 활성산소 형성이 시간에 따라 강하게 일어남을 확인하였다. 더욱이 LPA에 의한 활성산소 형성도 PI3K 또는 Akt의 저해제에 의해서 완벽히 억제됨을 확인하였으나 ERK 신호전달을 억제하였을 때는 거의 영향을 받지 않았다. SKOV-3 세포에서 LPA에 의해 생성된 활성산소는 diphenylene idonium (DPI, $10{\mu}M$), apocyanin (Apo, $10{\mu}M$)과 같은 NADPH oxidase 억제제를 전 처리하였을 때 활성산소가 형성되지 못함을 관찰하였다. 그러나 xanthine oxidase (allopurinol, Allo, $10{\mu}M$), cyclooxygenase (indomethacin, Indo, $10{\mu}M$), 또는 mitochondrial respiratory chain complex I (rotenone, Rot, $10{\mu}M$)를 억제하였을 때는 LPA에 의한 활성산소 형성에 영향을 주지 못함을 확인하였다. 마지막으로 활성산소 억제제인 N-acetylcysteine (NAC, $10{\mu}M$)에 의해서 LPA에 의한 암세포의 이동이 억제됨을 관찰하였다. 이와 더불어 LPA에 의한 SKOV-3 세포의 이동도 NADPH oxidase 억제에 의해 저해가 됨을 확인하였다. 이러한 연구결과로 보아 LPA에 의한 활성산소의 형성에는 PI3K/Akt/NADPH oxidase 신호전달체계가 중추적인 역할을 하며 이를 통해 암세포의 이동을 조절한다는 것을 알 수 있었다.

• 생명과학회지
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• 제29권9호
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• pp.1034-1046
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• 2019

미토콘드리아는 세포 에너지 공급을 위한 에너지 대사의 주요 세포 소기관으로 칼슘 조절, 활성 산소(ROS) 생성, 세포 사멸(apoptosis)을 조절하는데도 중요한 역할을 한다. 이러한 미토콘드리아에 발생한 기능 이상은 신경퇴행질환, 루게릭병, 심혈관계 질환, 정신 질환, 당뇨, 암과 같은 다양한 질병과 연관이 있다. 미토콘드리아 기능 이상 관련 질병들은 노화와 관련된 질병이 주를 차지하며, 이 논문에서는 그 중에서도 암에 초점을 맞춰 서술하고자 한다. 미토콘드리아 기능 이상은 발암을 유도하며, 많은 암 종에서 발견된다. 암종에 따라 미토콘드리아 기능 이상을 일으키는 요인들이 다르며, 이러한 변화는 치료 내성, 전이와 같은 암 악성화도 유발한다. 미토콘드리아 기능 이상의 요인으로는 미토콘드리아 수 부족, 주요 물질 제공 불능, ATP 합성 기능 이상 등이 존재하나, 암 발병과 악성화에 영향을 미치는 주요 원인으로 미토콘드리아 DNA (mtDNA)의 감소(depletion)를 들 수 있다. 미토콘드리아 기능 이상은 분자 활성 변화 혹은 발현 변화를 통해 암 악성화를 일으키나, 어떠한 변화가 암 악성화를 야기하는지 구체적으로 알려진 바가 없다. 미토콘드리아 기능 이상과 암의 상관관계는 대부분 미토콘드리아 기능 이상 세포를 이용하여 연구하는데, 그 제작 방법으로는 EtBr에 의한 화학적 방법과 shRNA, Crispr/Cas9과 같은 유전자 수선 (gene editing) 방법 등이 있다. 이러한 기법으로 제작된 미토콘드리아 기능 이상 세포주는 암을 비롯한 미토콘드리아 기능 이상에 의한 다양한 질병 연구에 이용되고 있다.

• 생명과학회지
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• 제19권5호
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• pp.598-606
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• 2009

본 연구는 저산소증에서 반하가 대뇌신경세포에 미치는 영향을 알아보기 위하여 $E_18$의 배양 흰쥐 대뇌신경세포를 반하로 전처리한 후, LDH assay와 tryphan blue 염색으로 세포 생존율을 측정하였고, $H_2DCF-DA$, JC-1 염색으로 MMP, ROS 및 RNS 변화를 조사하였다. 이에 반하는 저산소증으로 유발된 대뇌신경세포를 2.5 ${\mu}g/ml$까지 농도의존적으로 세포 생존률을 증가시켰으며, 시간에 따른 생존율을 살펴보면 저산소증 유발 후 1 시간에는 별 차이를 보이지 않았지만 3 일, 5 일 후에는 각각 10.2%, 17.8%로 매우 유의한 증가를 보였다. 저산소증에서 반하가 MMP에 미치는 영향을 보기 위해 저산소증 유발직전과 유발 후 1 일, 3 일, 5 일에 JC-1으로 염색하고 미토콘드리아의 염색강도를 측정한 결과 적색형광은 실험군에서 전반적으로 대조군에 비하여 강하게 염색되는 미토콘드리아의 비율을 증가시킨 반면 녹색형광은 대조군과 뚜렷한 차이를 보이지 않았다. 즉 반하가 저산소증으로 유발된 MMP의 소실을 감소시킴을 알 수 있다. 또한 반하는 전반적으로 $H_2DCF-DA$에 염색되는 세포 비율을 현저하게 낮추는 것으로 나타나 저산소증으로 유발된 ROS 및 RNS의 생성을 유의성 있게 감소시켰다. 따라서 반하는 저산소증에서 ROS의 생성을 억제하고 MMP의 소실을 막아 세포의 에너지고갈을 방지함으로서 신경세포를 보호하는 것으로 이해된다.