• 대한통합의학회지
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• 제11권2호
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• pp.169-179
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• 2023

Purpose : Although human periodontal ligament stem cells (hPDLSCs) are a supportive factor for tissue engineering, oxidative stress during cell culture and transplantation has been shown to affect stem cell viability and mortality, leading to failed regeneration. The aim of this study was to evaluate the antioxidant and protective effects against cell damage of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, and the antioxidant signal of hPDLSCs in H₂O₂-induced oxidative stress. Methods : To induce oxidative stress in cultured hPDLSCs, H₂O₂ was used as an exogenous reactive oxygen species (ROS). Dose-dependent celecoxib (.1, 1, 10, or 100 µM) was administered after H₂O₂ treatment. WST-1 assay was used to assess cell damage and western blot was used to observe antioxidant activity of hPDLSCs in oxidative stress. Immunohistochemistry was performed for inverting the localization of the SOD and Nrf2 antibody. Results : We found that progressive cell death was induced in hPDLSCs by H₂O₂ treatment. However, low-dose celecoxib reduced H₂O₂-induced cellular damage and eventually enhanced the SOD activity and Nrf2 signal of hPDLSCs. Oxidative stress-induced morphological change in hPDLSCs included lowered the survival and number of spindle-shaped cells, and shrinkage and shortening of cell fibers. Notably, celecoxib promoted cell survival function and activated antioxidants such as SOD and Nrf2 by positively regulating the cell survival signal pathway, and also reduced the number of morphological changes in hPDLS. Immunohistochemistry results showed a greater number of SOD- and Nrf2-stained cells in the celecoxib-treated group following oxidative stress. Conclusion : By increasing SOD and Nrf2 expression at the antioxidant system, the findings suggest that celecoxib enhanced the antioxidative ability of hPDLSCs and protected cell viability against H₂O₂-induced oxidative stress by increasing SOD and Nrf2 expression in the antioxidant system.

• The Korean Journal of Physiology and Pharmacology
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• 제25권3호
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• pp.239-249
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• 2021

The present study explored the therapeutic potential of hydrogen sulfide (H2S) in restoring aging-induced loss of cardioprotective effect of remote ischemic preconditioning (RIPC) along with the involvement of signaling pathways. The left hind limb was subjected to four short cycles of ischemia and reperfusion (IR) in young and aged male rats to induce RIPC. The hearts were subjected to IR injury on the Langendorff apparatus after 24 h of RIPC. The measurement of lactate dehydrogenase, creatine kinase and cardiac troponin served to assess the myocardial injury. The levels of H2S, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), nuclear factor erythroid 2-related factor 2 (Nrf2), and hypoxia-inducible factor (HIF-1α) were also measured. There was a decrease in cardioprotection in RIPC-subjected old rats in comparison to young rats along with a reduction in the myocardial levels of H2S, CBS, CSE, HIF-1α, and nuclear: cytoplasmic Nrf2 ratio. Supplementation with sodium hydrogen sulfide (NaHS, an H2S donor) and l-cysteine (H2S precursor) restored the cardioprotective actions of RIPC in old hearts. It increased the levels of H2S, HIF-1α, and Nrf2 ratio without affecting CBS and CSE. YC-1 (HIF-1α antagonist) abolished the effects of NaHS and l-cysteine in RIPC-subjected old rats by decreasing the Nrf2 ratio and HIF-1α levels, without altering H2S. The late phase of cardioprotection of RIPC involves an increase in the activity of H2S biosynthetic enzymes, which increases the levels of H2S to upregulate HIF-1α and Nrf2. H2S has the potential to restore aging-induced loss of cardioprotective effects of RIPC by upregulating HIF-1α/Nrf2 signaling.

• 생명과학회지
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• 제27권3호
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• pp.310-317
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• 2017

본 연구진은 HaCaT-ARE-luciferase 세포를 이용하여 400 여개의 약용식물 에탄올 추출물 중 NRF2/ARE 유도효과가 있는 신규 추출물을 검색하였고 이를 통하여 종대황(Rheum undulatum)과 선복화(Inula japonica)의 주정 추출물이 HaCaT-ARE-luciferase 세포에서 ARE 활성을 강하게 유도하는 것을 관찰하였다. 종대황과 선복화 에탄올 추출물은 HaCaT 세포에서 생존(viability)을 증가시켰고 NRF2/ARE에 의존적인 phase II cytoprotective 효소인 heme oxygenase-1 (HO-1)와 NADPH:quinone oxidoreductase-1 (NQO1)의 전사 및 단백질 발현을 강하게 유도하였다. 또한 종대황과 선복화 추출물은 HaCaT 세포에서 TPA로 유도한 세포 내 활성 산소 및 이를 통하여 생성되는 스트레스 마커인 8-hydroxydeoxyguanosine (8-OH-dG)과 4-hydroxynonenal (4HNE)의 발생을 강하게 억제하였다. 본 연구는 종대황과 선복화의 에탄올 추출물이 인간 피부 세포주인 HaCaT 세포에서 NRF2/ARE에 의존적인 유전자 발현의 유도를 통하여 강력한 항산화 효과를 발휘한다는 것을 증명한다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권1호
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• pp.299-304
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• 2014

Cholangiocarcinoma (CCA), a malignancy of biliary duct with a very poor prognosis, is the leading cause of cancer death in countries of the Mekong subregion. Liver fluke infection is the main etiological factor, but genetic variation has been recognized as also important in conferring susceptibility to CCA risk. Nuclear factor (erythroid derived 2)-like 2 (NRF2) is a key transcription factor in detoxification and antioxidant defense. Emerging evidence has demonstrated that genetic polymorphisms in the NRF2 gene may be associated with cancer development. The objectives of this study were to investigate the association of NRF2 genetic polymorphism with CCA risk and to evaluate the influence of the NRF2 genotype on survival time of affected patients. Single nucleotide polymorphisms (SNPs) of the NRF2 gene, including rs6726395: A/G, rs2886161: C/T, rs1806649: C/T, and rs10183914: C/T, were analyzed using TaqMan$^{(R)}$ SNP genotyping assays. Among 158 healthy northeastern Thai subjects, the allele frequencies were 41, 62, 94, and 92%, respectively. The correlation of NRF2 SNPs and CCA risk was analyzed in the 158 healthy subjects and 198 CCA patients, using unconditional logistic regression. The results showed that whereas the NRF2 SNPs were not associated with CCA risk (p>0.05), Kaplan-Meier analysis of 88 intrahepatic CCA patients showed median survival time with rs6726395 genotypes of GG and AA/AG to be $344{\pm}138$ (95%CI: 73-615) days and $172{\pm}37$ (95%CI: 100-244) days, respectively, (p<0.006). On multivariate Cox proportional hazard analysis, the GG genotype of rs6726395 was found to be associated with longer survival with a hazard ratio of 0.54 (95%CI: 0.31-0.94). In addition, non-papillary adenocarcinoma was associated with poor survival with a hazard ratio of 2.09 (95%CI: 1.16-3.75). The results suggest that the NRF2 rs6726395 polymorphism can be a potential prognostic biomarker for CCA patients.

• Biomolecules & Therapeutics
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• 제27권2호
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• pp.178-184
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• 2019

Parkinson's disease is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons within the substantia nigra pars compacta. In the present study, we investigated whether ${\beta}-Lapachone$ (${\beta}-LAP$), a natural naphthoquinone compound isolated from the lapacho tree (Tabebuia avellanedae), elicits neuroprotective effects in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease mouse model. ${\beta}-LAP$ reduced the tyrosine hydroxylase (TH)-immunoreactive fiber loss induced by MPTP in the dorsolateral striatum, and alleviated motor dysfunction as determined by the rotarod test. In addition, ${\beta}-LAP$ protected against MPTP-induced loss of TH positive neurons, and upregulated B-cell lymphoma 2 protein (Bcl-2) expression in the substantia nigra. Based on previous reports on the neuroprotective role of nuclear factor-E2-related factor-2 (Nrf2) in neurodegenerative diseases, we investigated whether ${\beta}-LAP$ induces upregulation of the Nrf2-hemeoxygenae-1 (HO-1) signaling pathway molecules in MPTP-injected mouse brains. Western blot and immunohistochemical analyses indicated that ${\beta}-LAP$ increased HO-1 expression in glial fibrillary acidic protein-positive astrocytes. Moreover, ${\beta}-LAP$ increased the nuclear translocation and DNA binding activity of Nrf2, and the phosphorylation of upstream adenosine monophosphate-activated protein kinase (AMPK). ${\beta}-LAP$ also increased the localization of p-AMPK and Nrf2 in astrocytes. Collectively, our data suggest that ${\beta}-LAP$ exerts neuroprotective effect in MPTP-injected mice by upregulating the p-AMPK/Nrf2/HO-1 signaling pathways in astrocytes.

• 대한한의학방제학회지
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• 제31권2호
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• pp.111-124
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• 2023

Objectives : Oxidative stress is an important cause of many diseases including liver injury. Therefore, adequate regulation of oxidative stress plays a pivotal role in maintaining liver function. Until recently, there has been no studies on the hepatoprotective effect of Samchulgeonbi-tang (SCGBT). Therefore, the hepatoprotective effect of SCGBT was investigated in HepG2 cells. In this study, oxidative stress was induced by arachidonic acid (AA) and iron. Methods : To analyze the hepatoprotective effects of SCGBT against oxidative stress induced by AA + iron, the cell viability, apoptosis-related proteins and intracellular ROS, glutathione (GSH), and mitochondrial membrane permeability (MMP) were measured. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) transcription activation and expressions of Nrf2 target gene were analyzed through immunoblot analysis. Results : SCGBT increased the cell viability from AA + iron - induced cell death and inhibited apoptosis by regulating apoptosis related proteins. SCGBT protected cells by inhibiting ROS production, GSH depletion, and MMP degradation against AA + iron induced oxidative stress. Furthermore, Nrf2 activation was increased by SCGBT, and the Nrf2 target genes were also activated by SCGBT. Conclusions : These results suggest that the SCGBT has a hepatocyte protection effect and antioxidant effect from AA + iron induced oxidative stress.

• Biomolecules & Therapeutics
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• 제21권1호
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• pp.60-65
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• 2013

3,4,5-Trihydroxycinnamic acid (THC) is a derivative of hydroxycinnamic acids, which have been reported to possess a variety of biological properties such as anti-inflammatory, anti-tumor, and neuroprotective activities. However, biological activity of THC has not been extensively examined. Recently, we reported that THC possesses anti-inflammatory activity in LPS-stimulated BV2 microglial cells. However, its precise mechanism by which THC exerts anti-inflammatory action has not been clearly identified. Therefore, the present study was carried out to understand the anti-inflammatory mechanism of THC in BV2 microglial cells. THC effectively suppressed the LPS-induced induction of pro-inflammatory mediators such as NO, TNF-${\alpha}$, and IL-$1{\beta}$. THC also suppressed expression of MCP-1, which plays a key role in the migration of activated microglia. To understand the underlying mechanism by which THC exerts these anti-inflammatory properties, involvement of Nrf2, which is a cytoprotective transcription factor, was examined. THC resulted in increased phosphorylation of Nrf2 with consequent expression of HO-1 in a concentration-dependent manner. THC-induced phosphorylation of Nrf2 was blocked with SB203580, a p38 MAPK inhibitor, indicating that p38 MAPK is the responsible kinase for the phosphorylation of Nrf2. Taken together, the present study for the first time demonstrates that THC exerts anti-inflammatory properties through the activation of Nrf2 in BV2 microglial cells, suggesting that THC might be a valuable therapeutic adjuvant for the treatment of inflammation-related disorders in the CNS.

• Biomolecules & Therapeutics
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• 제21권1호
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• pp.49-53
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• 2013

A number of naturally-occurring or synthetic chemicals have been reported to exhibit prostate chemopreventive effects. Synthetic $5{\alpha}$-reductase (5-AR) inhibitors, e.g. finasteride and durasteride, gained special interests as possible prostate chemopreventive agents. Indeed, two large-scale epidemiological studies have demonstrated that finasteride or durasteride significantly reduced the incidence of prostate cancer formation in men. However, these studies have raised an unexpected concern; finasteride and durasteride increased the occurrence of aggressive prostate tumor formation. In the present study, we have observed that treatment of finasteride did not affect the growth of androgen-refractory PC-3 prostate cancer cells. Finasteride also failed to induce apoptosis or affect the expression of proto-oncogenes in PC-3 cells. Interestingly, we found that treatment of finasteride induced the expression of Nrf2 and HO-1 proteins in PC-3 cells. In particular, basal level of Nrf2 protein was higher in androgen-refractory prostate cancer cells, e.g. DU-145 and PC-3 cells, compared with androgen-responsive prostate cancer cells, e.g. LNCaP cells. Also, treatment of finasteride resulted in a selective induction of Nrf2 protein in DU-145 and PC-3 cells, but not in LNCaP cells. In view of the fact that upregulation of Nrf2-mediated phase II cytoprotective enzymes contribute to attenuating tumor promotion in normal cells, but, on the other hand, confers a selective advantage for cancer cells to proliferate and survive against chemical carcinogenesis and other forms of toxicity, we propose that finasteride-mediated induction of Nrf2 protein might be responsible, at least in part, for an increased risk of high-grade prostate tumor formation in men.

• 대한약침학회지
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• 제19권1호
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• pp.59-69
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• 2016

Objectives: The mushroom Ganoderma lucidum has been widely used as a traditional herbal medicine for many years. Although several studies have focused on the anti-oxidative activity of this mushroom, the molecular mechanisms underlying its activity have not yet been clearly established. The present study investigated the cytoprotective effect of ethanol extract of Ganoderma lucidum (EGL) against oxidative stress (hydrogen peroxide, $H_2O_2$) and elucidated the underlying mechanisms in a C2C12 myoblast cell line. Methods: Oxidative stress markers were determined by using the comet assay to measure reactive oxygen species (ROS) generation and deoxyribonucleic acid (DNA) damage. Cell viability and Western blotting analyses were employed to evaluate the cellular response to EGL and $H_2O_2$ in C2C12 cells. Transfection with nuclear factor erythroid 2-related factor 2 (Nrf2)-specific small interfering ribonucleic acid (siRNA) was conducted to understand the relationship between Nrf2 expression and $H_2O_2$-induced growth inhibition. Results: The results showed that EGL effectively inhibited $H_2O_2$-induced growth and the generation of ROS. EGL markedly suppressed $H_2O_2$-induced comet-like DNA formation and phosphorylation of histone H2AX at serine 139 ($p-{\gamma}H2AX$), a widely used marker of DNA damage, suggesting that EGL prevented $H_2O_2$-induced DNA damage. Furthermore, the EGL treatment effectively induced the expression of Nrf2, as well as heme oxygenase-1 (HO-1), with parallel phosphorylation and nuclear translocation of Nrf2 in the C2C12 myoblasts. However, zinc protoporphyrin IX, a HO-1 inhibitor, significantly abolished the protective effects of EGL against $H_2O_2$-induced accumulation of ROS and reduced cell growth. Notably, transient transfection with Nrf2-specific siRNA attenuated the cytoprotective effects and HO-1 induction by EGL, indicating that EGL induced the expression of HO-1 in an Nrf2-dependent manner. Conclusion: Collectively, these results demonstrate that EGL augments the cellular anti-oxidant defense capacity through activation of Nrf2/HO-1, thereby protecting C2C12 myoblasts from $H_2O_2$-induced oxidative cytotoxicity.

• The Korean Journal of Physiology and Pharmacology
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• 제24권6호
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• pp.481-492
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• 2020

The present study aimed to examine the effect of allyl isothiocyanate (AITC) on chronic obstructive pulmonary disease and to investigate whether upregulation of multidrug resistance-associated protein 1 (MRP1) associated with the activation of the PARK7 (DJ-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis. Lung function indexes and histopathological changes in mice were assessed by lung function detection and H&E staining. The expression levels of Nrf2, MRP1, heme oxygenase-1 (HO-1), and DJ-1 were determined by immunohistochemistry, Western blotting and reverse transcription-quantitative polymerase chain reaction. Next, the expression of DJ-1 in human bronchial epithelial (16HBE) cells was silenced by siRNA, and the effect of DJ-1 expression level on cigarette smoke extract (CSE)-stimulated protein degradation and AITC-induced protein expression was examined. The expression of DJ-1, Nrf2, HO-1, and MRP1 was significantly decreased in the wild type model group, while the expression of each protein was significantly increased after administration of AITC. Silencing the expression of DJ-1 in 16HBE cells accelerated CSE-induced protein degradation, and significantly attenuated the AITC-induced mRNA and protein expression of Nrf2 and MRP1. The present study describes a novel mechanism by which AITC induces MRP1 expression by protecting against CS/CSE-mediated DJ-1 protein degradation via activation of the DJ-1/Nrf2 axis.