• Biomolecules & Therapeutics
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• 제27권5호
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• pp.442-449
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• 2019

This study sought to evaluate the effects of Asiatic acid in LPS-induced BV2 microglia cells and 1-methyl-4-phenyl-pyridine ($MPP^+$)-induced SH-SY5Y cells, to investigate the potential anti-inflammatory mechanisms of Asiatic acid in Parkinson's disease (PD). SH-SY5Y cells were induced using $MPP^+$ to establish as an in vitro model of PD, so that the effects of Asiatic acid on dopaminergic neurons could be examined. The NLRP3 inflammasome was activated in BV2 microglia cells to explore potential mechanisms for the neuroprotective effects of Asiatic acid. We showed that Asiatic acid reduced intracellular production of mitochondrial reactive oxygen species and altered the mitochondrial membrane potential to regulate mitochondrial dysfunction, and suppressed the NLRP3 inflammasome in microglia cells. We additionally found that treatment with Asiatic acid directly improved SH-SY5Y cell viability and mitochondrial dysfunction induced by $MPP^+$. These data demonstrate that Asiatic acid both inhibits the activation of the NLRP3 inflammasome by downregulating mitochondrial reactive oxygen species directly to protect dopaminergic neurons from, and improves mitochondrial dysfunction in SH-SY5Y cells, which were established as a model of Parkinson's disease. Our finding reveals that Asiatic acid protects dopaminergic neurons from neuroinflammation by suppressing NLRP3 inflammasome activation in microglia cells as well as protecting dopaminergic neurons directly. This suggests a promising clinical use of Asiatic acid for PD therapy.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1885-1895
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• 2020

Rumex japonicus Houtt (RJH) is a valuable plant used in traditional medicine to treat several diseases, such as scabies and jaundice. In this study, Jurkat cell growth inhibitory extracts of R. japonicus roots were subjected to bioassay-guided fractionation, resulting in the isolation of three naphthalene derivatives (3-5) along with one anthraquinone (6) and two phenolic compounds (1 and 2). Among these compounds, 2-methoxystypandrone (5) exhibited potent anti-proliferative effects on Jurkat cells. Analysis by flow cytometry confirmed that 2-methoxystypandrone (5) could significantly reduce mitochondrial membrane potential and promote increased levels of mitochondrial reactive oxygen species (ROS), suggesting a strong mitochondrial depolarization effect. Real-time quantitative polymerase chain reaction (qPCR) analysis was also performed, and the results revealed that the accumulation of ROS was caused by reduced mRNA expression levels of heme oxygenase (HO-1), catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). In addition, 2-methoxystypandrone (5) triggered strong apoptosis that was mediated by the arrest of the G0/G1 phase of the cell cycle. Furthermore, 2-methoxystypandrone (5) downregulated p-IκB-α, p-NF-κB p65, Bcl2, and Bcl-xl and upregulated BAX proteins. Taken together, these findings revealed that 2-methoxystypandrone (5) isolated from RJH could potentially serve as an early lead compound for leukemia treatment involving intracellular signaling by increasing mitochondrial ROS and exerting anti-proliferative effects.

• Molecules and Cells
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• 제43권12호
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• pp.989-1001
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• 2020

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes salmonellosis and mortality worldwide. S. Typhimurium infects macrophages and survives within phagosomes by avoiding the phagosome-lysosome fusion system. Phagosomes sequentially acquire different Rab GTPases during maturation and eventually fuse with acidic lysosomes. Lysophosphatidylcholine (LPC) is a bioactive lipid that is associated with the generation of chemoattractants and reactive oxygen species (ROS). In our previous study, LPC controlled the intracellular growth of Mycobacterium tuberculosis by promoting phagosome maturation. In this study, to verify whether LPC enhances phagosome maturation and regulates the intracellular growth of S. Typhimurium, macrophages were infected with S. Typhimurium. LPC decreased the intracellular bacterial burden, but it did not induce cytotoxicity in S. Typhimurium-infected cells. In addition, combined administration of LPC and antibiotic significantly reduced the bacterial burden in the spleen and the liver. The ratios of the colocalization of intracellular S. Typhimurium with phagosome maturation markers, such as early endosome antigen 1 (EEA1) and lysosome-associated membrane protein 1 (LAMP-1), were significantly increased in LPC-treated cells. The expression level of cleaved cathepsin D was rapidly increased in LPC-treated cells during S. Typhimurium infection. Treatment with LPC enhanced ROS production, but it did not affect nitric oxide production in S. Typhimurium-infected cells. LPC also rapidly triggered the phosphorylation of IκBα during S. Typhimurium infection. These results suggest that LPC can improve phagosome maturation via ROS-induced activation of NF-κB pathway and thus may be developed as a therapeutic agent to control S. Typhimurium growth.

• Journal of Ginseng Research
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• 제45권4호
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• pp.473-481
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• 2021

Background: Mitochondrial dysfunction is one of the significant reasons for Alzheimer's disease (AD). Ginsenosides, natural molecules extracted from Panax ginseng, have been demonstrated to exert essential neuroprotective functions, which can ascribe to its anti-oxidative effect, enhancing central metabolism and improving mitochondrial function. However, a comprehensive analysis of cellular mitochondrial bioenergetics after ginsenoside treatment under Aβ-oxidative stress is missing. Methods: The antioxidant activities of ginsenoside Rb₁, Rd, Re, Rg₁ were compared by measuring the cell survival and reactive oxygen species (ROS) formation. Next, the protective effects of ginsenosides of mitochondrial bioenergetics were examined by measuring oxygen consumption rate (OCR) in PC12 cells under Aβ-oxidative stress with an extracellular flux analyzer. Meanwhile, mitochondrial membrane potential (MMP) and mitochondrial dynamics were evaluated by confocal laser scanning microscopy. Results: Ginsenoside Rg₁ possessed the strongest anti-oxidative property, and which therefore provided the best protective function to PC12 cells under the Aβ oxidative stress by increasing ATP production to 3 folds, spare capacity to 2 folds, maximal respiration to 2 folds and non-mitochondrial respiration to 1.5 folds, as compared to Aβ cell model. Furthermore, ginsenoside Rg1 enhanced MMP and mitochondrial interconnectivity, and simultaneously reduced mitochondrial circularity. Conclusion: In the present study, these results demonstrated that ginsenoside Rg₁ could be the best natural compound, as compared with other ginsenosides, by modulating the OCR of cultured PC12 cells during oxidative phosphorylation, in regulating MMP and in improving mitochondria dynamics under Aβ-induced oxidative stress.

• The Korean Journal of Physiology and Pharmacology
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• 제26권5호
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• pp.325-333
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• 2022

Heart failure (HF) has become one of the severe public health problems. The detailed role of mitochondrial function in HF was still unclear. Benzoylaconine (BAC) is a traditional Chinese medicine, but its role in HF still needs to be explored. In this study, oxygen-glucose deprivation and reperfusion (OGD/R) was executed to mimic the injury of H9C2 cells in HF. The viability of H9C2 cells was assessed via MTT assay. OGD/R treatment markedly decreased the viability of H9C2 cells, but BAC treatment evidently increased the viability of OGD/R-treated H9C2 cells. The apoptosis of H9C2 was enhanced by OGD/R treatment but suppressed by BAC treatment. The mitochondrial membrane potential was evaluated via JC-1 assay. BAC improved the mitochondrial function and suppressed oxidative stress in OGD/R-treated H9C2 cells. Moreover, Western blot analysis revealed that the protein expression of p-AMPK and PGC-1α were reduced in OGD/R-treated H9C2 cells, which was reversed by BAC. Rescue assays indicated that AMPK attenuation reversed the BAC-mediated protective effect on OGD/R-treated cardiomyocytes. Moreover, BAC alleviated myocardial injury in vivo. In a word, BAC modulated the mitochondrial function in OGD/R-induced cardiomyocyte injury by activation of the AMPK/PGC-1 axis. The findings might provide support for the application of BAC in the treatment of HF.

• Fisheries and Aquatic Sciences
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• 제26권2호
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• pp.133-144
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• 2023

Auranofin is a US Food and Drug Administration (FDA)-approved anti-arthritis medication that functions as a thioredoxin reductase inhibitor. Spermidine, a polyamine present in marine algae, can exert various physiological functions. Herein, we examined the synergistic anticancer activity of auranofin and spermidine in hepatocellular carcinoma (HCC). Combined treatment with auranofin and spermidine suppressed cell viability more efficiently than either treatment alone in HCC Hep3B cells. The isobologram plotted by calculating the half maximal inhibitory concentration (IC₅₀) values of each drug indicated that the two drugs exhibited a synergistic effect. Based on the analysis of annexin V and cell cycle distribution, auranofin and spermidine markedly induced apoptosis in Hep3B cells. Moreover, auranofin and spermidine increased mitochondria-mediated apoptosis by promoting mitochondrial membrane potential (Δψm) loss. Auranofin and spermidine significantly increased reactive oxygen species (ROS) production in Hep3B cells, and the blocking ROS suppressed apoptosis induced by spermidine and auranofin. In addition, auranofin and spermidine reduced the expression of phosphorylated phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), and PI3K inhibitor accelerated auranofin- and spermidine-induced apoptosis. Using ROS scavenger and PI3K inhibitor, we revealed that ROS acts upstream of auranofin- and spermidine-induced apoptosis. Collectively, our study suggests that combination treatment with auranofin and spermidine could afford synergistic anticancer activity via ROS overproduction and reduced PI3K/Akt signaling pathway.

• Tuberculosis and Respiratory Diseases
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• 제40권3호
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• pp.236-249
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• 1993

연구배경 : 정상 폐상피세포에서는 항상 생성되고 있는 활성산소(oxygen radical)에 의한 유해작용에 노출되어 있고, 이들 유해 산소들은 폐기종과 같은 폐질환의 원인 기전으로 생각되고 있다. 본 연구에서는 이 방법을 이용하여 만든 폐상피세포 단일막에서 전기생리학적인 관점에서 물질의 이동지표인 short-circuit current(Isc)와 조직저항(R)에 대한 활성산소의 하나인 $H_2O_2$(hydrogen peroxide)가 어떤 영향을 미치는지를 연구함으로서 세포생리학적 기전을 구명하고자 한다. 방법 : Tissue culture-treated polycarbonant membrane filter 에서 배양시킨 쥐 제 2 형 폐상피세포 배양 단일막에서 $H_2O_2$의 능동적 이온 이동 (Isc) 과 수동적 용질이동에 대한 조직저항(R)에 미치는 효과를 관찰하였다. 배양 제 3 일과 제 4 일째 단일막을 수정된 Ussing chamber에 설치하고 막 양측에 HEPES-buffered Ringer 용액으로 incubation 하였다. 외부에서 0~100 mM 농도의 $H_2O_2$를 apical 또는 basolateral side에 작용시켜 Isc와 R의 변화를 관찰하였다. 폐상피세포 장벽이 외부의 $H_2O_2$에 대하여 방어작용을 가지는 세포내 catalase 활성도를 측정하고, catalase 억제제인 aminotriazol(ATAZ) 20 mM의 효과도 함께 관찰하였다. 결과 : 이 단일막은 형태학적으로 보아서 in vivo 에서의 포유류 제 1 형 폐상피세포 장벽의 특성을 나타내고 세포들 사이는 tight junction을 이루며(조직저항 R: 2,000 ohm-$cm^2$ 이상) sodium ion의 능동적 이동 (Isc: 5 ${\mu}A/cm^2$)을 보였다. $H_2O_2$는 dose-dependent 양식으로 Isc와 R 모두 감소시켰다. Apical side에 작용하는 $H_2O_2$에 있어서는 60분에 50% 억제하는 농도인 $ED_{50}$는 Isc와 R은 약 4mM 이었으나 basolateral side의 경우는 약 0.04mM 로서 그 작용 강도는 apical에 비하여 약 100배 정도 더 컸다. ATAZ 존재시 apical side의 $ED_{50}$는 0.4mM로 감소하였으나 basolateral side의 경우 변화가 없었다. $H_2O_2$의 제거율은 apical 또는 basolateral side 어느 쪽에 존재하든 같았으며, 세포내 catalase 활성도는세포배양기간이 길어짐에 따라 증가함을 보였다. 결론 : 이상의 실험결과는 basolateral side에 작용하는 $H_2O_2$는 세포내 막구성성분 중 basolateral 측에 존재하는 곳에(예, $Na^+,\;K^+$-APTase) 직접 장애를 미칠 것으로 생각된다. 한편 apical side에 작용하는 $H_2O_2$는 막성분에 도달하기 전에 세포내에 존재하는 catalase에 의하여 대부분 그 작용을 잃게 된다. 결론적으로 Isc와 R로 측정된 폐상피세포 장벽의 특성은 $H_2O_2$에 의하여 손상을 받고 apical side 보다는 basolateral side 측정이 더 손상을 잘 받게 된다.

• 대한의용생체공학회:의공학회지
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• 제24권2호
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• pp.121-126
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• 2003

본 연구는 진동형 장치의 활용을 통해 혈관 내 폐 보조장치의 기체전달 효율을 향상시키고자 시도하였으며, 진동에 따른 혈액의 용혈 문제에 있어서 한계 용혈이 발생하는 영역을 선정하고자 하였다. 가진 주파수가 0 Hz일 때를 기준으로 하여 중공사 수의 변화에 따라 산소전달속도를 측정하였다. 최대의 산소전달속도를 나타내는 중공사 모듈에서 가진 주파수의 변화에 따른 산소전달속도를 측정하고 혈액의 용혈도를 측정하였다. 측정결과 액체 유속의 변화에 따라 최대 산소전달속도를 나타내는 중공사 모듈은 type 6으로 이때의 중공사 개수는 675개이다. 또한, 중공사를 가진하지 않았을 때 최대의 산소전달속도를 보여주는 모듈은 type 6이었다. 모듈 type 6의 가진 주파수의 변화에 따른 산소전달속도는 7 Hz에서 최대산소전달속도를 나타내었으며 최대산소전달속도를 나타내는 7 Hz의 가진 주파수에서의 혈액 용혈도를 측정한 결과 혈액의 용혈도는 낮았다. 그러므로 최대 흔들림이 일어나는 7 Hz를 한계 용혈 주파수로 결정할 수 있었다.

• Molecules and Cells
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• 제24권1호
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• pp.95-104
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• 2007

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition ($IC_{50}$) of MCF-7 cells at $26.4{\pm}0.7{\mu}M$ over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with $100{\mu}M$ acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun $NH_4$-terminal kinase 1/2 (SAPK/JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.

• 생명과학회지
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• 제18권11호
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• pp.1513-1520
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• 2008

4-Hydroxynonenal (4-HNE)는 세포내 레독스의 균형을 깨뜨려 혈관 기능 손상을 일으킨다. 본 연구자들은 HNE의 축적이 야기하는 혈관 기능 손상기전을 더 잘 이해하기 위하여 혈관 내피 세포의 미토콘드리아 세포사 메커니즘을 규명하였다. HNE를 처리한 세포에서는 미토콘드리아 막전위 소실과 그에 따른 cytochrome C의 방출이 유도되었으며, Bax의 증가 및 Bcl-2의 감소가 관찰되었다. ROS 제거제인 NAC와 peroxynitrite 제거제인 페니실라민은 HNE가 유도하는 ROS 생성을 차단하여 cytochrome C 방출과 세포사를 억제하였다. 세포에 HNE와 zVAD-fmk (caspase 저해제)를 같이 처리하면 HNE가 유도하는 세포사를 억제하지 못하는데 이는 HNE에 의한 세포사가 caspase에 비의존적 단계일 가능성을 시사하였다. 위의 결과들은 HNE가 유도하는 혈관 내피 세포의 세포사 매커니즘은 미토콘드리아 막전위의 탈분극에 의해 촉발되며 이는 혈관계 항상성의 악화와 노화에 의해 수반되는 혈관기능 손상을 유도할 것으로 사료된다.