• The Korean Journal of Physiology and Pharmacology
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• 제20권4호
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• pp.347-355
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• 2016

Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha ($TNF-{\alpha}$) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of $TNF-{\alpha}$ on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe $CM-DCFH_2-DA$. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. $TNF-{\alpha}$ induced LOX-1 expression in a dose- and time- dependent manner in endothelial cells. $TNF-{\alpha}$ induced ROS formation, phosphorylation of $NF-{\kappa}B$ p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. $NF-{\kappa}B$ inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited $TNF-{\alpha}-induced$ LOX-1 expression. CPT and NAC suppressed $TNF-{\alpha}-induced$ LOX-1 expression and phosphorylation of $NF-{\kappa}B$ p65 and ERK in rat aorta. These data suggested that $TNF-{\alpha}$ induced LOX-1 expression via ROS activated $NF-{\kappa}B/ERK$ pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.

• Journal of Radiation Protection and Research
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• 제34권2호
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• pp.49-54
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• 2009

Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with $^{137}Cs$ $\gamma$-rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and $H_2O_2$-treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and $H_2O_2$-treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells.

• Molecules and Cells
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• 제39권6호
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• pp.508-513
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• 2016

To investigate the potential therapeutic effects of polyphenols in treating Pb induced renal dysfunction and intoxication and to explore the detailed underlying mechanisms. Wistar rats were divided into four groups: control groups (CT), Pb exposure groups (Pb), Pb plus Polyphenols groups (Pb+PP) and Polyphenols groups (PP). Animals were kept for 60 days and sacrificed for tests of urea, serum blood urea nitrogen (BUN) and creatinine. Histological evaluations were then performed. In vitro studies were performed using primary kidney mesangial cells to reveal detailed mechanisms. Cell counting kit-8 (CCK-8) was used to evaluate cell viability. Pb induced cell apoptosis was measured by flow cytometry. Reactive oxygen species (ROS) generation and scavenging were tested by DCFH-DA. Expression level of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), interleukin-1-${\beta}$ (IL-1-${\beta}$) and IL-6 were assayed by ELISA. Western blot and qPCR were used to measure the expression of ERK1/2, JNK1/2 and p38. Polyphenols have obvious protective effects on Pb induced renal dysfunction and intoxication both in vivo and in vitro. Polyphenols reduced Pb concentration and accumulation in kidney. Polyphenols also protected kidney mesangial cells from Pb induced apoptosis. Polyphenols scavenged Pb induced ROS generation and suppressed ROS-mediated ERK/JNK/p38 pathway. Downstream pro-inflammatory cytokines were inhibited in consistency. Polyphenol is protective in Pb induced renal intoxication and inflammatory responses. The underlying mechanisms lie on the antioxidant activity and ROS scavenging activity of polyphenols.

• 대한예방한의학회지
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• 제16권3호
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• pp.155-165
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• 2012

Objective : The object of this study is to confirm the immune enhancement effect of Rubus coreanus Miquel (RC) (30% EtOH extract) on RAW264.7 mouse macrophage cell line. Methods : RAW264.7 cells were treated with $10-500{\mu}g/mL$ RC for 24 hours. Cell viability was then measured using WST assays. Levels of intracellular NO and ROS were measured by Griess reagent and DCFH-DA staining respectively. Levels of iNOS and COX-2 mRNA was determined by RT-PCR. Secretion levels of IL-$1{\beta}$ and IL-6 cytokines were evaluated by sandwich ELISA assay. Western blot analysis was performed to measure the levels of intracellular molecules related to MAPKs pathways. Results : RC suppressed the growth of RAW264.7 cells. RC increased the production of NO and ROS. RC increased the mRNA and protein levels of COX-2 and iNOS. RC augmented the levels of secreted IL-$1{\beta}$ and IL-6 cytokines. RC increased MAPKs phosphorylation. Conclusion : In summary, our result shows that RC has inflammatory effect increasing the levels of NO, ROS and secreted cytokines and activating MAPKs. Hence, RC seems to have an immune enhancement effect.

• 치위생과학회지
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• 제19권3호
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• pp.198-204
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• 2019

Background: Oxidative stress is a known to be associated with in the pathogenesis of many inflammatory diseases, including periodontitis. Ursolic acid is a pentacyclic triterpenoid with has antimicrobial, antioxidative, and anticancer properties. However, the role of ursolic acid in the regulating of osteogenesis remains undetermined. This study was aimed to elucidate the crucial osteogenic effects of ursolic acid and its ability to inhibit oxidative stress by targeting the immediate early response 3 (IER3)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Methods: Cell proliferation was determined using water-soluble tetrazolium salt assay, cell differentiation was evaluated by alkaline phosphatase (ALP) activity, and formation of calcium nodules was detected using alizarin red S stain. Generation of reactive oxygen species (ROS) was determined using by DCFH-DA fluorescence dye in hydrogen peroxide ($H_2O_2$)-treated MG-63 cells. Expression levels of IER3, Nrf2, and heme oxygenase-1 (HO-1) were analyzed using western blot analysis. Results: Our results showed that ursolic acid up-regulated the proliferation of osteoblasts without any cytotoxic effects, and promoted ALP activity and mineralization. $H_2O_2$-induced ROS generation was found to be significantly inhibited on treatment with ursolic acid. Furthermore, in $H_2O_2$-treated cells, the expression of the early response genes: IER3, Nrf2, and Nrf2-related phase II enzyme (HO-1) was enhanced in the presence of ursolic acid. Conclusion: The key findings of the present study elucidate the protective effects of ursolic acid against oxidative stress conditions in osteoblasts via the IER3/Nrf2 pathway. Thus, ursolic acid may be developed as a preventative and therapeutic agent for mineral homeostasis and inflammatory diseases caused due to oxidative injury.

• Journal of Microbiology and Biotechnology
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• 제31권6호
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• pp.775-783
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• 2021

Sea cucumber, Holothuria scabra, is a well-known traditional Asian medicine that has been used for suppressing inflammation, promoting wound healing, and improving immunity. Moreover, previous studies demonstrated that the extract from H. scabra contains many bioactive compounds with potent inhibitory effect on tumor cell survival and progression. However, the effect of the methanolic extract from the body wall of H. scabra (BWMT) on human prostate cancer cells has not yet been investigated. In this study, we aimed to investigate the effects and underlying mechanism of BWMT on prostate cancer cell viability and metastasis. BWMT was obtained by maceration with methanol. The effect of BWMT on cell viability was assessed by MTT and colony formation assays. The intracellular ROS accumulation was evaluated using a DCFH-DA fluorescence probe. Hoechst 33342 staining and Annexin V-FITC/PI staining were used to examine the apoptotic-inducing effect of the extract. A transwell migration assay was performed to determine the anti-metastasis effect. BWMT significantly reduced cell viability and triggered cellular apoptosis by accumulating intracellular ROS resulting in the upregulation of JNK and p38 signaling pathways. In addition, BWMT also inhibited the invasion of PC3 cells by downregulating MMP-2/-9 expression via the ERK pathway. Consequently, our study provides BWMT from H. scabra as a putative therapeutic agent that could be applicable against prostate cancer progression.

• 대한한의학방제학회지
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• 제31권1호
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• pp.29-39
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• 2023

Objective : Gardeniae Fructus (GF) is the ripe fruit of Gardenia jasminoides Ellisa with a bitter taste and cold properties. Ingredient compounds including geniposide are known to have anti-inflammatory, antioxidant, and neuroprotective effects. The purpose of this study was to investigate the neuroprotective effect of GF on tBHP-induced PC12 cells. Methods : Cell viability was measured by the MTT assay, and apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression level of each protein was monitored by Western blot analysis, and reactive oxygen species (ROS) were analyzed using DCFH-DA. Results : In PC12 cells, tBHP induced cell death through apoptosis with caspase activation and PARP inactivation. Cells treated with tBHP showed an increase in intracellular ROS and depletion of GSH. Pretreatment with GF prevented tBHP-induced apoptosis, reduced ROS, and increased GSH. GF also maintained increased Nrf2 expression in the presence of tBHP. Phosphorylation of JNK and p38 MAPK was increased by tBHP, whereas phosphorylation of ERK was decreased. GF restored changes in ERK and p38 phosphorylation, but not JNK phosphorylation. Conclusion : These results indicate that GF has neuroprotective effects through anti-apoptotic and antioxidant effects mediated by regulation of Nrf2 expression and phosphorylation of ERK and p38. It also demonstrates the potential use of GF as a source of antioxidant and neuroprotective substances.

• 한국식생활문화학회지
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• 제24권6호
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• pp.749-755
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• 2009

We investigated the chemical components of red onion powder dried using the low temperature vacuum method and the inhibitory effects of solvent extracts of the dried red onion powder on the growth of HT-1080 human fibrosarcoma and HT-29 human colon cancer cells and $H_2O_2$-induced oxidative stress. The moisture content of the dried red onion powder was 17.95%, while the vitamin C content was 96 mg/100 g and the total phenols content was 39.1 mg/mL. The inhibitory effects of acetone with methylene chloride (A+M) and methanol (MeOH) extracts of the red onion powder on the growth of HT-1080 and HT-29 cancer cells increased in a dose dependent manner (p<0.05). The inhibitory effect was greater on the growth of HT-29 cells, while the A+M extracts had a higher inhibitory effect than the MeOH extracts. Treatment with the hexane, 85% aq. methanol, butanol and water fractions of the extract led to significant inhibition of the growth of both cancer cell lines (p<0.05). Among the fractions, the hexane and 85% aq. methanol fractions showed a greater inhibitory effect. To determine the protective effect on $H_2O_2$-induced oxidative stress, a DCFH-DA (dichlorodihydrofluorescin diacetate) assay was conducted. All fractions, including the crude extracts of dried red onion, appeared to lead to a significant reduction in the levels of intracellular reactive oxygen species (ROS), and these reductions occurred in a dose dependent fashion (p<0.05). Among the fractions, the 85% methanol fraction showed the greatest protective effect on the production of lipid peroxides.

• Korean Circulation Journal
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• 제52권9호
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• pp.680-696
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• 2022

Background and Objectives: Circular RNAs were known to play vital role in myocardial ischemia reperfusion injury (MIRI), while the role of CircZNF609 in MIRI remains unclear. This study was aimed to investigate the function of CircZNF609 in MIRI. Methods: Hypoxia/reoxygenation (H/R) model was established to mimic MIRI in vitro. Quantitative polymerase chain reaction was performed to evaluate gene transcripts. Cellular localization of CircZNF609 and miR-214-3p were visualized by fluorescence in situ hybridization. Cell proliferation was determined by CCK-8. TUNEL assay and flow cytometry were applied to detect apoptosis. Lactate dehydrogenase was determined by commercial kit. ROS was detected by DCFH-DA probe. Direct interaction of indicated molecules was determined by RIP and dual luciferase assays. Western blot was used to quantify protein levels. In vivo model was established to further test the function of CircZNF609 in MIRI. Results: CircZNF609 was upregulated in H/R model. Inhibition of CircZNF609 alleviated H/R induced apoptosis, ROS generation, restored cell proliferation in cardiomyocytes and human umbilical vein endothelial cells. Mechanically, CircZNF609 directly sponged miR-214-3p to release PTGS2 expression. Functional rescue experiments showed that miR-214-3p/PTGS2 axis was involved in the function of circZNG609 in H/R model. Furthermore, data in mouse model revealed that knockdown of CircZNF609 significantly reduced the area of myocardial infarction and decreased myocardial cell apoptosis. Conclusions: CircZNF609 aggravated the progression of MIRI via targeting miR-214-3p/PTGS2 axis, which suggested CircZNF609 might act as a vital modulator in MIRI.

• 생명과학회지
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• 제21권10호
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• pp.1377-1384
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• 2011

Astaxanthin (ATX)은 다양한 생명체에서 생성되는 카로티노이드 색소이다. 본 연구에서는 ATX가 RAW264.7 cell에서 LPS에 의한 inducible nitric oxide synthase (iNOS), nitric oxide (NO), 염증성 사이토카인, nuclear factor-kappa B (NF-${\kappa}B$)와 reactive oxygen species (ROS)의 생성을 억제 시키는 지 또한, superoxide radical 소거능이 있는 지를 조사하였다. iNOS와 NF-${\kappa}B$는 immunoblot analysis로, interleukin (IL)-6와 tumour necrosis factor-${\alpha}$ (TNF-${\alpha}$)는 ELISA 법으로 분석하였다. NO 양은 nitrite의 양을 측정하였고, ROS는 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) 법으로 superoxide radical 소거능은 superoxide radical scavenging activity assay로 검증하였다. 100 ${\mu}M$의 ATX 농도에서 LPS로 유도된 NO, IL-6 및 TNF-${\alpha}$ 같은 염증성 사이토카인의 생성 뿐만 아니라 iNOS 및 NF-${\kappa}B$의 발현도 억제되었다. 특히, IL-6 및 TNF-${\alpha}$ 생성에 있어 ATX의 최대 억제율은 각각 65.2% 및 21.2% 이었으며 LPS로 유도된 NF-${\kappa}B$의 전사활성을 억제하였다. 이러한 현상은 세포질에서 핵으로 NF-${\kappa}B$의 전위를 억제하는 것과 관련이 있다. 또한, 25-100 ${\mu}M$의 ATX 농도에서 세포 내 ROS 생성을 억제하였으며, 5 mg/ml 농도의 ATX는 동일농도의 ${\alpha}$-tocopherol에 비해 superoxide radical 소거능이 1.33배 높았다. 이러한 결과들은 ATX가 대식세포에서 ROS 생성 및 NF-${\kappa}B$ 활성을 저해하므로 iNOS의 발현, NO 및 염증성 사이토카인의 생성을 억제하며, 또한 우수한 superoxide radical 소거능을 보유한다는 것을 나타내었다. 결론적으로, ATX가 항염증제 및 항산화제로서 유용하게 사용될 수 있을 것으로 사료된다.