In Vitro Activity of Diphenyleneiodonium toward Multidrug-Resistant Helicobacter pylori Strains

( Jun-won Chung ),( Su Young Kim ),( Hee Jung Park ),( Chang Su Chung ),( Hee Woo Lee ),( Sun Mi Lee ),( Inki Kim ),( Jhang Ho Pak ),( Gin Hyug Lee ),( Jin-yong Jeong ) 대한간학회 2017 Gut and Liver Vol.11 No.5

Background/Aims: The increased resistance of Helico-bacter pylori to antibiotics has increased the need to develop new treatments for this bacterium. The aim of our study was to identify new drugs with anti-H. pylori activity. Methods: We screened a small molecule library―the library of pharmaco-logically active compounds (LOPAC), which includes 1,280 pharmacologically active compounds―to identify inhibitors of H. pylori growth. The minimal inhibitory concentrations (MICs) of antibiotics against multidrug-resistant H. pylori strains were determined using the agar dilution method. Results: We identified diphenyleneiodonium (DPI) as a novel anti- H. pylori agent. The MIC values for DPI were <0.03 μg/mL against all tested H. pylori strains. DPI also exhibited strong antibacterial activity against common gram-negative and gram-positive pathogenic bacteria. Conclusions: DPI may be a candidate anti-H. pylori drug for future development. (Gut Liver 2017;11:648-654)

Diphenyleneiodonium Inhibits Apoptotic Cell Death of Gastric Epithelial Cells Infected with Helicobacter pylori in a Korean Isolate

조순옥,김혜영,임주원 연세대학교의과대학 2015 Yonsei medical journal Vol.56 No.4

NADPH oxidase produces a large amount of reactive oxygen species (ROS) in Helicobacterpylori (H. pylori)-induced gastric epithelial cells. Even though ROS mediateapoptotic cell death, direct involvement of NADPH oxidase on H. pylori-inducedapoptosis remains unclear. Besides, H. pylori isolates show a high degree of genetic variability. The predominant genotype of H. pylori in Korea has been reportedas cagA+, vacA s1b, m2, iceA genotype. Present study aims to investigate whether NADPH oxidase-generated ROS mediate apoptosis in human gastric epithelialAGS cells infected with H. pylori in a Korean isolate. AGS cells were pretreatedwith or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) and cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Cell viability,hydrogen peroxide level, DNA fragmentation, and protein levels of p53, Bcl-2, and Bax were determined. Results showed that H. pylori inhibited cell viability with the density of H. pylori added to the cells. Inhibition of NADPH oxidase by DPI suppressed H. pylori-induced cell death, increased hydrogen peroxide, DNA fragmentation, and the ratio of Bax/Bcl-2, and p53 induction in AGS cells dose-dependently. The results suggest that targeting NADPH oxidase may prevent the developmentof gastric inflammation associated with H. pylori infection by suppressingabnormal apoptotic cell death of gastric epithelial cells.

Control of JNK for an Activation of NADPH Oxidase in LPS-stimulated BV2 Microglia

Jung Eun Han,최지웅 대한약학회 2012 Archives of Pharmacal Research Vol.35 No.4

NADPH oxidase is a main regulator for H2O2 productivity in neuroinflammatory cells, including microglia, under various CNS diseases and its activity is controlled by mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK). However, little is known about the link between NADPH oxidasedriven H2O2 productivity and JNK in microglia. The purpose of this study is to uncover the link using lipopolysaccharide (LPS)-stimulated BV2 microglia. LPS-stimulated BV2 microglia produced H2O2 that was decreased by NADPH oxidase inhibitors, including 4-(2-aminoethyl benzenesulfonylfluoride and diphenyleneiodonium chloride. In addition, NADPH oxidase was activated in LPS-stimulated BV2 cells. These results suggest that NAPDH oxidase is a main factor for H2O2 productivity in LPS-stimulated BV2 microglia. Based on a semi-quantitative PCR analysis,two of NADPH oxidase components, p47phox and gp91phox, were involved in the activation of NADPH oxidase because transcriptional levels of both components were upregulated by LPS. Role of JNK in NADPH oxidase-regulated H2O2 productivity was pursued using specific inhibitors,including SP600125 and JNK inhibitory peptide (JIP). Inhibition of the JNK pathways significantly reduced H2O2 productivity, which was closely related to the attenuation of NADPH oxidase activation and the upregulation of components. We conclude that JNK pathways are involved in NADPH oxidase-mediated H2O2 productivity in BV2 microglia.

Effect of NADPH Oxidase Inhibition on Heme Oxygenase-1 Expression in Human Hepatoma Cell Line HepG2

이상권(Sang Kwon Lee),김강미(Kang Mi Kim),박광훈(Kwang Hoon Park),박영철(Young Chul Park) 한국생명과학회 2011 생명과학회지 Vol.21 No.11

CoPP는 다양한 세포에서 HO-1의 유전자 발현과 활성을 증가시키는 강력한 유도제로 알려져 있다. HO-1는 세포 및 조직의 손상을 보호한다는 연구가 활발히 진행되고 있으나, 그 작용 기전에 대해서는 아직 잘 모르고 있다. 본 논문에서는 porphyrin 계열의 CoPP의 자극에 의해 유도되는 HO-1 유전자 발현에서 NADPH oxidase의 활성이 미치는 영향을 인간 간암세포주 HepG2에서 조사하였다. 배양 중인 HepG2 세포에서 CoPP는 HO-1의 발현을 농도의존적으로 증가시키는 것을 확인하였다. NADPH oxidase 저해제로 잘 알려져 있는 DPI를 전처리한 후 CoPP로 자극한 세포에서는 HO-1의 발현이 강력하게 억제되는 것으로 나타났다. DPI의 이런 억제 효과가 HO-1의 전사 조절인자 Nrf2의 활성에도 영향을 줄 수 있기 때문에 DPI를 전처리 한 후 CoPP 자극에 의한 Nrf2의 핵으로의 이동을 분석하였다. 그 결과, DPI는 CoPP에 의해 유도되는 Nrf2의 핵으로의 이동과 세포 내 존재하는 양을 감소시키는 것을 확인하였다. 다른 HO-1 발현 유도제로 알려져 있는 hemin에 의한 자극의 경우에도 DPI는 HepG2 세포의 HO-1의 발현을 억제하는 효과를 나타내었다. 그리고, p47<SUP>phox</SUP>에 대한 siRNA를 사용하여 효과적으로 p47<SUP>phox</SUP> 유전자 발현을 knockdown 시켜서 NADPH oxidase의 활성을 억제시키는 방법을 사용하였다. 그 결과, p47<SUP>phox</SUP> silencing한 세포에 CoPP를 처리한 경우는 control siRNA를 처리한 세포와 비교할 때 HO-1 발현이 현저히 감소됨을 관찰할 수 있었다. 마지막으로, 세포 내 ROS 생성을 억제하는 GSHmee가 처리된 세포에서는 CoPP나 hemin이 Nrf2의 활성을 증가시키지 못하였고, 그 결과 HO-1의 발현을 유도하지 못하는 것을 알 수 있었는데, 이는 ROS가 CoPP나 hemin에 의한 HO-1 유전자 발현 과정에 중요한 역할을 한다는 것을 의미한다. 이를 종합해 볼 때, 인간 간암세포주 HepG2에서 CoPP나 hemin의 자극에 의한 HO-1 유전자의 발현에는 NADPH oxidase의 활성이 요구된다는 것을 알 수 있고, 그 활성은 세포 내 ROS를 생성시키는 것으로 역할을 한다고 여겨진다. Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. In this study, we investigated the role of NADPH oxidase on the expression of HO-1 in human liver hepatoma cell line HepG2. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, markedly inhibited HO-1 expression and the nuclear translocation of transcription factor Nrf2 in cobalt protoporphyrin (CoPP) or hemin-treated HepG2 cells. Similarly, the knockdown of p47<SUP>phox</SUP>, a cytosolic factor for NADPH oxidase activity, by siRNA inhibited the CoPP-induced expression of HO-1. In addition, GSHmee, an intracellular anti-oxidant, blocked the expression of HO-1 in CoPP-treated cells. Based on these results, we conclude that the blockage of NADPH oxidase with DPI or p47<SUP>phox</SUP> siRNA inhibits CoPP-induced HO-1 expression in HepG2 cells, and also suggest that the expression of HO-1 in CoPP-induced HepG2 cells is associated with increase of intracellular ROS by NADPH oxidase activity.

사람 폐 섬유아세포의 전환성장인자-β1에 의한 fibronectin 분비와 α-smooth muscle actin 표현에 있어서 활성산소족의 역할

하헌주,유미라,어수택,박춘식,이희발,Ha, Hunjoo,Yu, Mi-Ra,Uh, Soo-taek,Park, Choon Sik,Lee, Hi Bahl 대한결핵및호흡기학회 2005 Tuberculosis and Respiratory Diseases Vol.58 No.3

연구배경 : 전환성장인자-${\beta}1$(transforming growth factor-${\beta}1$: $TGF-{\beta}1$)은 폐 섬유화를 매개하는 주된 인자이지만 $TGF-{\beta}1$에 의한 폐 섬유화의 발생과 진행기전의 이해는 아직 불완전하다. $TGF-{\beta}1$은 다양한 세포에서 활성산소족(reactive oxygen species: ROS)을 통하여 세포내 신호를 전달하고 ${\alpha}$-smooth muscle actin (${\alpha}$-SMA)의 신생합성을 통하여 상피세포와 폐 섬유아세포를 근 섬유아세포 표현형으로의 변화를 유도하는 주된 인자이다. ROS는 또 다양한 세포에서 세포외기질 (extracellular matrix: ECM) 축적을 유발하는 것이 알려져 있음으로 본 연구에서는 폐 섬유아세포인 MRC-5 세포에서 $TGF-{\beta}1$이 ROS를 매개하여 fibronectin 분비와 ${\alpha}-SMA$ 표현의 증가에 관여하는지를 검색하였다. 방 법 : 성장이 동일화된 MRC-5 세포를 $TGF-{\beta}1$ (0.2-10ng/ml)으로 96 시간까지 자극하였고, 필요에 따라 항산화제인 N-acetylcysteine (NAC)이나 NADPH oxidase 억제제인 diphenyleniodonium (DPI)을 $TGF-{\beta}1$ 투여 1 시간 전부터 전처리하였다. Dichlorofluorescein (DCF)에 민감한 세포내 ROS는 FACS로, 분비된 fibronectin과 세포의 ${\alpha}-SMA$ 표현은 Western blot 분석으로 측정하였다. 결 과 : $TGF-{\beta}1$은 용량의존적으로 fibronectin 분비와 ${\alpha}-SMA$ 표현을 상향조절하였다. NAC와 DPI는 $TGF-{\beta}1$에 의한 fibronectin 분비 증가와 ${\alpha}-SMA$ 상향조절을 유의하게 억제하였다. $TGF-{\beta}1$에 의한 세포내 ROS의 증가도 NAC나 DPI에 의하여 유의하게 억제되었다. 결 론 : 본 연구의 결과는 폐 섬유아세포에서 NADPH oxidase 에 의하여 생산된 ROS가 $TGF-{\beta}1$에 의한 fibronectin 분비와 ${\alpha}-SMA$ 표현을 상향조절함으로써 폐 섬유화의 발생과 진행에 관여할 수 있음을 증명하였다. Background : The transforming growth $factor-{\beta}1$ ($TGF-{\beta}1$) plays a key role in lung fibrosis. However, the molecular mechanisms involved in $TGF-{\beta}1$-induced lung fibrosis are unclear. $TGF-{\beta}1$ is the key inducer of myofibroblast transdifferentiation via de novo synthesis of ${\alpha}-smooth$ muscle actin (${\alpha}-SMA$). Since $TGF-{\beta}1$ signals through reactive oxygen species (ROS) and ROS have been shown to induce accumulation of extracellular matrix (ECM) in various tissues, this study examined if ROS play a role in $TGF-{\beta}1$-induced fibronectin secretion and ${\alpha}-SMA$ expression in human lung fibroblasts, MRC-5 cells. Methods : Growth arrested and synchronized MRC-5 cells were stimulated with $TGF-{\beta}1$ (0.2-10 ng/ml) in the presence or absence of N-acetylcysteine (NAC) or diphenyleneiodonium (DPI) for up to 96 hours. Dichlorofluorescein (DCF)-sensitive cellular ROS were measured by FACScan and secreted fibronectin and cellular ${\alpha}-SMA$ by Western blot analysis. Results : $TGF-{\beta}1$ increased the level of fibronectin secretion and ${\alpha}-SMA$ expression in MRC-5 cells in a dosedependent manner. Both NAC (20 and 30 mM) and DPI (1 and $5{\mu}M$) significantly inhibited $TGF-{\beta}1$-induced fibronectin and ${\alpha}-SMA$ upregulation. The $TGF-{\beta}1$-induced cellular ROS level was also significantly reduced by NAC and DPI. Conclusions : The results suggest that NADPH oxidase-dependent ROS play an important role in $TGF-{\beta}1$-induced fibronectin secretion and ${\alpha}-SMA$ expression in MRC-5 cells, which leads to myofibroblast transdifferentiation and progressive lung fibrosis.

Reactive Oxygen Species Mediate ET-1-Induced Activation of ERK1/2 Signaling in Cultured Feline Esophageal Smooth Muscle Cells

Hyun Ju Song,Ji Soo Kim,Myong Jae Lee,Yoon Sung Nam,손의동 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.9

Reactive oxygen species (ROS) have been shown to play a critical role in propagating the signals of several growth factors, peptide hormones, and cytokines, such as epidermal growth factor, insulin, and interleukin-1. We investigated a possible role for ROS generation in mediating the action of ET-1 on activation of ERK1/2 in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated by 10nM ET-1; activation of ERK was examined by western blot analysis with phospho-specific antibodies of ERKs. ET-1 induced ERK1/2 phosphorylation in a dose- and time- dependent manner. ERK1/2 activation by ET-1 reached the maximal levels at 5min showing slight activation up to 20min, and then slowly declined. It was confirmed that the activation of ERK1/2 was reduced by MEK inhibitor PD98059. We observed the dose-dependent inhibitory effect of diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the ET- 1-enhanced ERK1/2 phosphorylation in ESMC. Pretreatment of ESMC with N-acetylcysteine, a ROS scavenger, also attenuated the ET-1-induced ERK1/2 activation. In addition, DPI significantly inhibited the ET-1- induced ROS production when ROS was measured as a function of DCF fluorescence. The results suggest that ROS might be critical mediators of the ET-1- induced ERK1/2 signaling events in ESMC.

Dipenyleneiodonium Induces Growth Inhibition of Toxoplasma gondii through ROS Induction in ARPE-19 Cells

Pu Reum Sun,Fei Fei Gao,Hei Gwon Choi,Wei Zhou,Jae-Min Yuk,Jaeyul Kwon,Young-Ha Lee,Guang-Ho Cha 대한기생충학열대의학회 2019 The Korean Journal of Parasitology Vol.57 No.2

사람 폐 섬유아세포의 전환성장인자-β1에 의한 fibronectin 분비와 α-smooth muscle actin 표현에 있어서 활성산소족의 역할

하헌주 ( Hun Joo Ha ),유미라 ( Mi Ra Yu ),어수택 ( Soo Taek Uh ),박춘식 ( Choon Sik Park ),이희발 ( Hi Bahl Lee ) 대한결핵 및 호흡기학회 2005 Tuberculosis and Respiratory Diseases Vol.58 No.3

연구배경 : 전환성장인자-β1(transforming growth factor-β1: TGF-β1)은 폐 섬유화를 매개하는 주된 인자이지만 TGF-β1에 의한 폐 섬유화의 발생과 진행기전의 이해는 아직 불완전하다. TGF-β1은 다양한 세포에서 활성산소족(reactive oxygen species: ROS)을 통하여 세포내 신호를 전달하고 α-smooth muscle actin (α-SMA)의 신생합성을 통하여 상피세포와 폐 섬유아세포를 근 섬유아세포 Background : The transforming growth factor-β1 (TGF-β1) plays a key role in lung fibrosis. However, the molecular mechanisms involved in TGF-β1-induced lung fibrosis are unclear. TGF-β1 is the key inducer of myofibroblast transdifferentiation via de novo