• Nutrition Research and Practice
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• v.8 no.2
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• pp.138-145
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• 2014

BACKGROUND/OBJECTIVES: This study was performed to investigate the in vitro antioxidant and cytoprotective effects of fermented sesame sauce (FSeS) against hydrogen peroxide ($H_2O_2$)-induced oxidative damage in renal proximal tubule LLC-PK1 cells. MATERIALS/METHODS: 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical ($^{\bullet}OH$), and $H_2O_2$ scavenging assay was used to evaluate the in vitro antioxidant activity of FSeS. To investigate the cytoprotective effect of FSeS against $H_2O_2$-induced oxidative damage in LLC-PK1 cells, the cellular levels of reactive oxygen species (ROS), lipid peroxidation, and endogenous antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) were measured. RESULTS: The ability of FSeS to scavenge DPPH, $^{\bullet}OH$ and $H_2O_2$ was greater than that of FSS and AHSS. FSeS also significantly inhibited $H_2O_2$-induced ($500{\mu}M$) oxidative damage in the LLC-PK1 cells compared to FSS and AHSS (P < 0.05). Following treatment with $100{\mu}g/mL$ of FSeS and FSS to prevent $H_2O_2$-induced oxidation, cell viability increased from 56.7% (control) to 83.7% and 75.6%, respectively. However, AHSS was not able to reduce $H_2O_2$-induced cell damage (viability of the AHSS-treated cells was 54.6%). FSeS more effectively suppressed $H_2O_2$-induced ROS generation and lipid peroxidation compared to FSS and AHSS (P < 0.05). Compared to the other sauces, FSeS also significantly increased cellular CAT, SOD, and GSH-px activities and mRNA expression (P < 0.05). CONCULUSIONS: These results from the present study suggest that FSeS is an effective radical scavenger and protects against $H_2O_2$-induced oxidative damage in LLC-PK1 cells by reducing ROS levels, inhibiting lipid peroxidation, and stimulating antioxidant enzyme activity.

• The Korean Journal of Pain
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• v.23 no.1
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• pp.1-10
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• 2010

Background: Recent studies indicate that reactive oxygen species (ROS) are involved in persistent pain, including neuropathic and inflammatory pain. Since the data suggest that ROS are involved in central sensitization, the present study examines the levels of activated N-methyl-D-aspartate (NMDA) receptors in the dorsal horn after an exogenous supply of three antioxidants in rats with chronic post-ischemia pain (CPIP). This serves as an animal model of complex regional pain syndrome type-I induced by hindpaw ischemia/reperfusion injury. Methods: The application of tight-fitting O-rings for a period of three hours produced CPIP in male Sprague-Dawley rats. Allopurinol 4 mg/kg, allopurinol 40 mg/kg, superoxide dismutase (SOD) 4,000 U/kg, N-nitro-L-arginine methyl ester (L-NAME) 10 mg/kg and SOD 4,000 U/kg plus L-NAME 10 mg/kg were administered intraperitoneally just after O-ring application and on the first and second days after reperfusion. Mechanical allodynia was measured, and activation of the NMDA receptor subunit 1 (pNR1) of the lumbar spinal cord (L4-L6) was analyzed by the Western blot three days after reperfusion. Results: Allopurinol reduced mechanical allodynia and attenuated the enhancement of spinal pNR1 expression in CPIP rats. SOD and L-NAME also blocked spinal pNR1 in accordance with the reduced mechanical allodynia in rats with CPIP. Conclusions: The present data suggest the contribution of superoxide, produced via xanthine oxidase, and the participation of superoxide and nitric oxide as a precursor of peroxynitrite in NMDA mediated central sensitization. Finally, the findings support a therapeutic potential for the manipulation of superoxide and nitric oxide in ischemia/reperfusion related pain conditions.

• Journal of Applied Biological Chemistry
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• v.54 no.1
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• pp.15-20
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• 2011

Red ginseng is one of the most popular traditional medicines in Korea. In this study, we developed a new technique in which ethanol extract of $\underline{r}$ed $\underline{g}$inseng (HRG) was exposed to the $Co^{60}$ gamma radiation ranging from 1~5 kGy. The irradiated HRG (IHRG) were analyzed by high performance liquid chromatography (HPLC) to determine any compositional changes of ginsenosides due to irradiation. No appreciable difference was observed in the HPLC pattern of ginsenosides of HRG. Using MTT assay, the cytotoxicity effect was significantly increased by IHRG compared to HRG. The $LD_{50}$ concentration was $30{\mu}g/mL$ for IHRG-1 (1 kGy), and $15{\mu}g/mL$ for IHRG-5 (5 kGy). The evidences of apoptosis, such as nuclei cleavage and Annexin V staining, were observed in the human prostate cancer PC-3 cells treated with the IHRG. Additionally, the level of reactive oxygen species (ROS) was apparently elevated by IHRG. We also studied the inhibitory effect of IHRG on the growth rate of tumor xenografts in BALB/c male mice. The tumor growth rates were inhibited by 56.9 and 76.1% in mice treated with 10 mg/kg of IHRG-1 and IHRG-5, respectively, compared with control group (21.1%). These results suggest that some biologically active and soluble components in HRG can be more effectively enhancement of anti-tumor effects using irradiation.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.6
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• pp.325-331
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• 2003

3-Nitropropionic acid (3-NP) inhibits electron transport in mitochondria, leading to a metabolic failure. In order to elucidate the mechanism underlying this toxicity, we examined a few biochemical changes possibly involved in the process, such as metabolic inhibition, generation of reactive oxygen species (ROS), DNA strand breakage, and activation of Poly(ADP-ribose) polymerase (PARP). Exposure of SK-N-BE(2)C neuroblastoma cells to 3-NP for 48 h caused actual cell death, while inhibition of mitochondrial function was readily observed when exposed for 24 h to low concentrations (0.2${\sim}$2 mM) of 3-NP. The earliest biochemical change detected with low concentration of 3-NP was an accumulation of ROS (4 h after 3-NP exposure) followed by degradation of DNA. PARP activation by damaged DNA was also detectable, but at a later time. The accumulation of ROS and DNA strand breakage were suppressed by the addition of glutathione or N-acetyl-L-cysteine (NAC), which also partially restored mitochondrial function and cell viability. In addition, inhibition of PARP also reduced the 3-NP-induced DNA strand breakage and cytotoxicity. These results suggest that oxidative stress and activation of PARP are the major factors in 3-NP-induced cytotoxicity, and that the inhibition of these factors may be useful in protecting neuroblastoma cells from 3-NP-induced toxicity.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.1
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• pp.25-32
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• 2014

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.2
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• pp.65-70
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• 2007

Chinese herb medicines have traditionally been used to treat or alleviate the symptom of various diseases. The rationale for use of certain herbs to certain disorder is now getting unveiled by modern technology. In the present study, we investigated whether herb mix extract(HMX), which is alleged to be useful for gastric ulcer, protects stomach from oxidative stress. Rats were allowed to normal diet with and without HMX (1, 5, 10 mg/kg) for 30 days. To induce gastric ulcer, ethanol (75%, 1.5 ml) or acidified aspirin (100 mg/kg in 0.2 N HCl) was administered by oral route in 24 h-fasted rats and examined the gastric ulceration(bleeding) by measuring the size 1 h after the treatment. Results indicated the area of gastric bleeding was significantly less in HMX fed rats than in normal diet fed ones, and it was dependent on the duration and amount of HMX. To investigate the underlying mechanism by which HMX protects stomach from oxidative stress, expression of enzymes like heme oxygenase (HO), cyclooxygenase (COX), and inducible nitric oxide (iNOS) were investigated in MKN-74 cells, where aspirin or H. pylori was introduced. The results were compared with RAW 264.7 cells to check if there's cell specificities exist. The expression of HO-1 but not COX-2, iNOS was significantly increased by HMX. Furthermore, HO-1 inhibitor, SnPP IX reduced the HO-1 activity and reversed the survival rate in HMX-treated MKN-74 cells. There's no difference between RAW 264.7 cells and MKN-74 cells. We, thus, concluded that HMX is beneficial for protection from oxidative injury, and induction of HO-1 by HMX in gastric cells is, at least, responsible for protection from oxidative stress such as ethanol, aspirin and possibly H. pylori infection.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.2
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• pp.57-64
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• 2007

Ischemic preconditioning (IPC) is known to protect the heart against ischemia/reperfusion (IR)-induced injuries, and regional differences in the mitochondrial antioxidant state during IR or IPC may promote the death or survival of viable and infarcted cardiac tissues under oxidative stress. To date, however, the interplay between the mitochondrial antioxidant enzyme system and the level of reactive oxygen species (ROS) in the body has not yet been resolved. In the present study, we examined the effects of IR- and IPC-induced oxidative stresses on mitochondrial function in viable and infarcted cardiac tissues. Our results showed that the mitochondria from viable areas in the IR-induced group were swollen and fused, whereas those in the infarcted area were heavily damaged. IPC protected the mitochondria, thus reducing cardiac injury. We also found that the activity of the mitochondrial antioxidant enzyme system, which includes manganese superoxide dismutase (Mn-SOD), was enhanced in the viable areas compared to the infarcted areas in proportion with decreasing levels of ROS and mitochondrial DNA (mtDNA) damage. These changes were also present between the IPC and IR groups. Regional differences in Mn-SOD expression were shown to be related to a reduction in mtDNA damage as well as to the release of mitochondrial cytochrome c (Cyt c). To the best of our knowledge, this might be the first study to explore the regional mitochondrial changes during IPC. The present findings are expected to help elucidate the molecular mechanism involved in IPC and helpful in the development of new clinical strategies against ischemic heart disease.

• Journal of Pharmacopuncture
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• v.19 no.1
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• pp.51-58
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• 2016

Objectives: Oldenlandia diffusa is traditionally used to relieve the symptoms of and to treat various diseases, but its anti-cancer activity has not been well studied. In the present study, the authors investigated the anti-cancer effects of an ethanol extract of Oldenlandia diffusa (EOD) on HT-29 human adenocarcinoma cells. Methods: Cells were treated with different concentrations of an EOD, and cell death was assessed by using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Analyses of the sub G1 peak, the caspase-3 and -9 activities, and the mitochondrial membrane depolarizations were conducted to confirm cell death by apoptosis. Also, intracellular reactive oxygen species (ROS) generation was determined using carboxy-H2DCFDA (5-(and-6)-carboxy-20,70-dichlorodihydrofluorescein diacetate). Results: EOD inhibited the proliferation of HT-29 cells for 24 hours by $78.6%{\pm}8.1%$ at $50{\mu}g/mL$, $74.4%{\pm}4.6%$ at $100{\mu}g/mL$, $65.9%{\pm}5.2%$ at $200{\mu}g/mL$, $51.4%{\pm}6.2%$ at $300{\mu}g/mL$, and by $41.7%{\pm}8.9%$ at $400{\mu}g/mL$, and treatment for 72 hours reduced the proliferation at the corresponding concentrations by $43.3%{\pm}8.8%$, $24.3{\pm}5.1mV$, $13.5{\pm}3.2mV$, $6.5{\pm}2.3mV$, and by $2.6{\pm}2.3mV$. EOD increased the number of cells in the sub-G1 peak in a dose-dependent manner. The mitochondrial membrane depolarization was elevated by EOD. Also, caspase activities were dose-dependently elevated in the presence of EOD, and these activities were repressed by a pan-caspase inhibitor (zVAD-fmk). The ROS generation was significantly increased by EOD and N-acetyl-L-cysteine (NAC; a ROS scavenger) remarkably abolished EOD-induced cell death. In addition, a combination of sub-optimal doses of EOD and chemotherapeutic agents noticeably suppressed the growth of HT-29 cancer cells. Conclusion: These results indicate that EOD might be an effective chemotherapeutic for the treatment of human colorectal cancer.

• Journal of Pharmacopuncture
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• v.15 no.3
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• pp.13-18
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• 2012

Objectives: The purpose of this study is to examine whether Hominis Placental pharmacopuncture solution (HPPS) combined with zinc-oxide nanoparticles (ZnO NP) activates RAW 264.7 cells. Methods: We soaked ZnO nanoparticles in the Hominis Placenta pharmacopuncture solution, thereby making a combined form (ZnO NP HPPS). The effect of ZnO NP HPPS on the intracellular reactive oxygen species (ROS) production was measured by 2', 7'-dichlorofluorescin diacetate (DCFH-DA) assay. The effect of ZnO NP HPPS on NF-${\kappa}B$ was measured by using a luciferase assay. The effect of ZnO NP HPPS on the cytokine expression was assessed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cellular uptake of ZnO NP HPPS was measured by using a flow cytometric analysis, and cellular structural alterations were analyzed by using transmission electron microscopy (TEM). Results: Neither the HPPS nor the ZnO NPs induced intracellular ROS production in RAW 264.7 cells. Neither of the materials activated NF-${\kappa}B$ or it's dependent genes, such as TNF-${\alpha}$, IL-1, and MCP-1. However, ZnO NP HPPS, the combined form of ZnO NPs and HPPS, did induce the intracellular ROS production, as well as prominently activating NF-${\kappa}B$ and it's dependent genes. Also, compared to ZnO NPs, it effectively increa-sed the uptake by RAW 264.7 cells. In addition, cellular structural alterations were observed in groups treated with ZnO NP HPPS. Conclusions: Neither ZnO NP nor HPPS activated RAW 264.7 cells, which is likely due to a low cellular uptake. The ZnO NP HPPS, however, significantly activated NF-${\kappa}B$ and up-regulated its dependent genes such as TNF-${\alpha}$, IL-1, and MCP-1. ZnO NP HPPS was also more easily taken into the RAW 264.7 cells than either ZnO NP or HPPS.

• Nutrition Research and Practice
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• v.4 no.1
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• pp.3-10
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• 2010

This study was performed to investigate the effect of desalinated underground seawater (named as 'magma seawater', MSW) of Jeju Island in Korea on lipid metabolism and antioxidant activity. MSW was collected from underground of Han-Dong in Jeju Island, and freely given to high fat diet (HFD)-fed C57BL/6 mice for 10 weeks. Although there were no significant differences in the body weight changes and plasma lipid levels, hepatic triglyceride levels were significantly lower in the MSW group than in the normal tap water (TW)-drunken control group. Furthermore, the activity of fatty acid synthase (FAS) was significantly decreased and carnitine palmitoyltransferase (CPT) activity was increased in MSW group compared to TW group. Similarly, real-time PCR analysis revealed that mRNA expressions of lipogenic genes were lowered in MSW groups compared to the control group. In a morphometric observation on the liver tissue, accumulation of fats was remarkably reduced in MSW group. Meanwhile, in vitro assay, tree radical scavenging activity measured by using diphenylpicrylhydrazyl (DPPH) was increased in MSW group. The 2'-7'-dichlorofluorescein diacetate (DCF-DA) staining followed with fluorescent microscopy showed a low intensity of fluorescence in MSW-treated HepG2 cells, compared to TW-treated HepG2 cells, which indicated that the production of reactive oxygen species by tert-butyl hydroperoxide (t-BHP) in HepG2 cells was decreased by MSW treatment. The antioxidant effect of MSW on t-BHP-induced oxidative stress in HepG2 cells was supported by the increased activities of intracellular antioxidant enzymes such as catalase and glutathione reductase. From these results, we speculate that MSW has an inhibitory effect on lipogenesis in liver and might play a protective role against cell damage by t-BHP-induced oxidative stress.