• Title/Summary/Keyword: Oxygen radicals

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Anti-wrinkle Effect of Cambodian Phellinus linteus Extracts (캄보디안 상황버섯 추출물의 주름개선 효과 연구)

  • Cheon, Soon-Ju;Jang, Min-Jung;Jang, Young-Ah;Choi, Eun-Young;Jun, Dong-Ha;Kim, Young-Hun;Cho, Woo-A;Jeong, Yeon-Sook;Kwon, Hyeork-Bum;Kim, Tae-Hoon;Choi, Kyung-Im;Do, Jeong-Ryong;Lee, Chang-Eon;Lee, Jin-Tae
    • Journal of Life Science
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    • v.18 no.12
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    • pp.1718-1721
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    • 2008
  • The skin of human is constantly being exposed to environmental irritants such as ultraviolet, smoke and chemicals. These irritants cause free radicals and reactive oxygen species which leave serious damages on the cells of skin. The water and ethanol extracts of Cambodian Phellinus linteus were investigated for the activities of anti-lipid peroxidation and anti-wrinkle effects to apply as a functional ingredient for cosmetic products. As the result of evaluation of liquid oxidation rate by add $Fe^{2+}$ and $Cu^{2+}$ to Cambodian Phellinus linteus extracts, Cambodian Phellinus linteus ethanol extracts were higher than Cambodian Phellinus linteus water extracts in the chealting ability of $Fe^{2+}$ and $Cu^{2+}$. The Cambodian Phellinus linteus ethanol extracts exhibited that anti-lipid peroxidation higher than butylated hydroxytoluene (BHT) at the concentration of 0.1 mg/ml, 0.5 mg/ml and 1 mg/ml. Cambodian Phellinus linteus water and ethanol extracts showed a higher inhibitory effect on $Fe^{2+}$-induced lipid peroxidation compared to $Cu^{2+}$-induced lipid peroxidation. In the case of anti-wrinkle effect, the elastase inhibition activity of Cambodian Phellinus linteus ethanol extracts was 50.7%, and it is higher than urosolic acid at the concentration of 0.01 mg/ml. Also, in collagenase inhibition activity, Cambodian Phellinus linteus water extract showed low effect, but Cambodian Phellinus linteus ethanol extract was about 50% at a 0.1 mg/ml. concentration. These results proved that Cambodian Phellinus linteus had anti-lipid peroxidation and anti-wrinkle effect. Therefore, Cambodian Phellinus linteus could be useful as an anti-wrinkle cosmetic ingredient.

Comparison of Antioxidant, Cytotoxicity and Flavonoid Content of Stachys sieboldii Miq. vs. Lycopus lucidus Turcz. Leaf Extracts (초석잠 및 택란 잎 추출물의 항산화 및 세포독성 활성과 총 플라보노이드 함량 비교)

  • Na, Eun;Lee, Jung Woo;Lim, Sun Young
    • Journal of Life Science
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    • v.30 no.2
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    • pp.147-155
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    • 2020
  • In this study, the antioxidant and cytotoxic effects and the flavonoid contents of leaf extracts from Stachys sieboldii Miq. and Lycopus lucidus Turcz. were compared. The flavonoid contents of the acetone + methylene chloride (A+M) and methanol (MeOH) extracts of L. lucidus Turcz. leaves were 55.7 and 233.2 mg/g, respectively. In a DPPH assay, A+M and MeOH extracts from L. lucidus Turcz leaves had a greater scavenging effect than those of S. sieboldii Miq. leaves (p<0.05). In an ABTS assay, MeOH extracts from S. sieboldii Miq. and L. lucidus Turcz (0.5 mg/ml concentration) leaves had scavenging effects of 85% and 91%, respectively (p<0.05), suggesting that both of the MeOH extracts had greater scavenging effects than both A+M extracts. In a 120 min ROS production assay, all tested extracts decreased the cellular ROS production induced by H2O2 compared to that produced by exposure to the extract-free control. The MeOH extract from L. lucidus Turcz leaves had a greater inhibitory effect on cellular ROS production (p<0.05). Treatment with A+M and MeOH extracts from both S. sieboldii Miq. and L. lucidus Turcz. leaves showed a dose-dependent increased cytotoxicity against the growth of AGS, HT-29 cancer cells, and HT-1080 (p<0.05). Both A+M extracts had a greater inhibitory effect on the growth of all cancer cells than both MeOH extracts. These results suggest that the MeOH extract of L. lucidus Turcz. leaves is effective in scavenging free radicals and inhibiting cellular oxidation, while the A+M extract inhibits proliferation of three types of cancer cell.

Effect of Iron Excess-induced Oxidative Stress on Platelet Aggregation (과잉 철로 유도된 산화적 스트레스가 혈소판 활성화에 미치는 작용)

  • Seo, Geun-Young;Park, Hyo-Jin;Jang, Sung-Geun;Park, Young-Hyun
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.35 no.8
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    • pp.979-984
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    • 2006
  • Although iron is essential for many physiological processes, excess iron can lead to tissue damage by promoting the generation of reactive oxygen species (ROS). There is increasing evidence that ROS might play an important role in the pathogenesis of cardiovascular disease. However, the effects of iron excess on platelet function and the thrombotic response to vascular injury are not well understood. We examined the effects of iron excess-induced oxidative stress and the antioxidants on platelet aggregation. Oxidative stress was accessed by either free iron $(Fe^{+2})$ or hydrogen peroxide $(H_2O_2)$, as well as their combination on washed rabbit platelets (WPs) in vitro. When WPs were stimulated with either $Fe^{+2}$ alone or a subthreshold concentration of collagen, which gave an aggregatory curve with a little effect, and a dose dependent increase in platelet aggregation was observed by increasing concentrations of $Fe^{+2}$ with $H_2O_2$. This aggregation was associated with the iron-catalyzed formation of hydroxyl radicals from $H_2O_2$, and were inhibited by NAD/NADP (proton acceptor), catalase $(H_2O_2\;scavenger)$, tiron (iron chelator), mannitol (hydroxyl radical scavenger), and indomethacin (cyclooxygenase inhibitor), but not by NADH/NADPH (proton donor), superoxide mutase, and aspirin. However, NADH/NADPH, an essential cofactor for the antioxidant capacity by the supply of reducing potentials, showed the effect of an enhanced radical formation, suggesting a role for NADH/NADPH-dependent oxidase. These results suggest that iron $(Fe^{+2})$ can directly interact with washed rabbit platelets and this aggregation be mediated by OH formation as in the Fenton reaction, inhibited by radical scavengers.

Photoprotection and Anti-inflammatory Effects of Chinese Medical Plants (약용식물추출물의 광보호 효과와 항염증 효과 연구)

  • Jin-Hwa, Kim;Sung-Min, Park;Gwan-Sub, Sim;Bum-Chun , Lee;Hyeong-Bae, Pyo
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.30 no.2
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    • pp.227-233
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    • 2004
  • Chronic exposure to solar radiation, particularly ultraviolet (UV) light, causes a variety of adverse reactions on human skin, such as sunburn, photoaging and photocarcinogenesis. Free radicals and reactive oxygen species (ROS) caused by UV exposure or other environmental facts play critical roles in cellular damage. And, repeated-UV irradiation activated the expression of the matrix metalloproteinase (MMP) and induced skin irritation. Therefore, the development of effective and safe photoprotectants that can reduce and improve the skin damage has been required. The purpose of this study was to investigate the photo-protective effect of several chinese medical plants (Juniperus chinensis) on the UV -induced skin cell damages. We tested free radical and superoxide scavenging effect in vitro. Fluorometric assays of the proteolytic activities of MMP-1 (collagenase) were performed using fluorescent collagen substrates. UVA induced MMP-1 synthesis and activity were analyzed by enzyme-linked immunosorbent assay (ELISA) and gelatin-based zymography in skin fibroblasts. We also examined anti-inflammatory effects by the determination test of proinflammatory cytokine, interleukin 6 in HaCaT keratinocytes. Expression of prostaglandin E$_2$ (PGE$_2$) after UVB irradiation was measured by competitive enzyme immunoassay(EIA) using PGE$_2$ monoclonal antibody. In the human skin we tested anti-irritation effect on the SLS-induced damage skin after appling the extract containing emulsion. We found that Juniperus chinensis extract had potent radical scavenging effect by 98% at 100$\mu\textrm{g}$/mL. The extract of Juniperus chinensis showed strong inhibitory effect on MMP-1 activities by 97% at 100 $\mu\textrm{g}$/mL and suppressed the UVA induced expression of MMP-1 by 79% at 25$\mu\textrm{g}$/mL. This extract also showed strong inhibition on MMP-2 activity in UVA irradiated fibroblast by zymography. In the test of proinflammatory cytokines of human keratinocytes Juniperus chinensis extract decreased expression of interleukin 6 about 30%. The amount of PGE$_2$ by HaCaT keratinocytes was significantly increased at the doses of above 10 mJ/$\textrm{cm}^2$ of UVB (p < 0.05). At the concentrations of 3.2-25$\mu\textrm{g}$/mL of this extract, the production of PGE$_2$ by HaCaT keratinocytes (24 h after 10mJ/$\textrm{cm}^2$ UVB irradiation) was significantly inhibited in culture supernatants (p < 0.05). In SLS-induced skin irritation model in vivo, we found to reduce skin erythema and improve barrier recovery after appling Juniperus chinensis extract containing emulsion when compared to irritated non-treated and placebo-treated skin. Our results suggest that Juniperus chinensis extract can be effectively used for the prevention of UV and SLS-induced adverse skin reactions and applied as anti-aging and anti-irritation cosmetics.

Study on the Antioxidant and Human Neutrophil Elastase Inhibitory Activities of Mushroom Ramaria formosa Extracts (붉은싸리버섯 추출물의 항산화 및 Human Neutrophil Elastase 저해활성)

  • Kim, Kwan-Chul;Kwon, Yong-Beom;Jang, Hae-Dong;Kim, Jae Wha;Jeong, Jae Cheol;Lee, Ik-Soo;Ha, Byung-Jo;Yoo, Ick-Dong
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.42 no.3
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    • pp.269-278
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    • 2016
  • In searching for novel agents for skin anti-aging from natural resources, we found that the extract of the fruiting bodies of Ramaria formosa (R. formosa) had significant antioxidant and human neutrophil elastase (HNE) inhibitory activities. R. formosa extract exhibited a considerable DPPH radical scavenging activity with an antioxidant content of 117.0mg/mL (ascorbic acid equivalents) at the concentration of $500{\mu}g/mL$. The capacity of R. formosa extract to scavenge peroxy radicals measured by ORAC assay also showed dose-dependent antioxidant effect with $ORAC_{Roo}$ (trolox equivalents, $1{\mu}M$) values of 0.8, 5.2, and 7.8 at the concentrations of 1, 10, and $20{\mu}g/mL$. The cellular antioxidant capacity of R. formosa extract was investigated by assaying the cellular fluorescence intensity using dichlorodihydrofluorescein (DCF). The cellular oxidative stress induced by AAPH, $Cu^{2+}$ or $H_2O_2$ in HepG2 cells was significantly attenuated by more than 30% at $20{\mu}g/mL$ of R. formosa extract. HNE activity was reduced by treatment with R. formosa extract in a dose-dependent manner, and the $ED_{50}$ value for the ethanol extract of R. formosa was $42.9{\mu}g/mL$. R. formosa extract did not exhibited antimicrobial activity against four microorganisms including Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), Candida albicans (C. albicans), Aspergillus oryzae (A. oryzae). Furthermore, the extract did not affect the inflammatory cytokine production of interleukin-10 and interferon-${\gamma}$ in NK92 cells. From the above results, we found that R. formosa extract has considerable antioxidant and elastase inhibitory effects, and does not stimulate immune cells. These findings suggest that R. formosa extract may be used as a bioactive component in cosmetic composition.

Antioxidative Activity of Rumex crispus L. Extract (소리쟁이 추출물의 항산화 효능)

  • Rhim, Tae-Jin;Choi, Moo-Young;Park, Hee-Juhn
    • Korean Journal of Plant Resources
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    • v.25 no.5
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    • pp.568-577
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    • 2012
  • The objective of this study was to investigate the antioxidative capacity of ethanol extracts from Rumex crispus L. The concentration of R. crispus L. extract at which DPPH radical scavenging activity was inhibited by 50% was 2.15 mg/mL, which was lower than that of ${\alpha}$-tocopherol (0.43 mg/mL), as compared to 100% by pyrogallol as a reference. Total antioxidant status was examined by total antioxidant capacity against ABTS radical reactions. Total antioxidant capacities of R. crispus L. extract at concentrations of 0.1 and 1 mg/mL were 0.47 and 2.33 mM Trolox equivalents, respectively, which were higher than those of ${\alpha}$-tocopherol. Superoxide scavenging activities of R. crispus L. extract at concentrations of 0.1 and 1 mg/mL were 21.5 and 78.9%, respectively, which were not significantly (p>0.05) different from those of catechin. Oxygen radical absorbance capacities of R. crispus L. extract at concentrations of 20 and 100 ${\mu}g/mL$ were 62.5 and 156.4 ${\mu}M$ Trolox equivalents, respectively, which were lower than those of ascorbic acid. Cupric reducing antioxidant capacities of R. crispus L. extract at concentrations of 0.1 and 1 mg/mL were 0.28 and 1.88 mM Trolox equivalents, which were similar or significantly (p<0.05) higher than those of ${\alpha}$-tocopherol, respectively. R. crispus L. extract prevented supercoiled DNA strand breakage induced by hydroxyl radical and peroxyl radical. Total phenolic contents of R. crispus L. extract at concentrations of 0.5 and 5 mg/mL were 0.58 and 3.85 mM gallic acid equivalents, respectively. R. crispus L. extract at concentration of 0.1 and 0.5 mg/mL inhibited 0.2 mM tert-butyl hydroperoxide-induced cytotoxicity by 38.5 and 63.5%, respectively, in HepG2 cell culture system. Thus, strong antioxidant and cytotoxicity-inhibiting effects of R. crispus L. extract seem to be due to, at least in part, the prevention from free radicals-induced oxidation as well as high levels in total phenolic contents.

Selectivity of Bleaching Herbicides Caused by Physiological Differences between Rice and Barnyardgrass (벼와 피의 생리적(生理的) 차이(差異)에 의한 백화형(白化型) 제초제(除草劑)의 선택성(選擇性))

  • Na, J.Y.;Kim, J.S.;Kim, T.J.;Cho, K.Y.;Pyon, J.Y.
    • Korean Journal of Weed Science
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    • v.12 no.2
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    • pp.89-101
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    • 1992
  • In this study, various physiological and biochemical experiments were conducted to know whether the selectivity between rice and barnyardgrass treated with bleaching herbicides containing diphenyl ether compounds was also partly based on their basic physiological proterties such as peroxidation ability, membrane stability or antioxidant system. It seemed to be partly based on the differences of their physiological characteristics that barnyardgrass was commonly more susceptible to most of the bleaching herbicides than rice. The scenescence of intact leaf segment was more rapid in barnyardgrass than in rice, indicating that barnyardgrass is weak at early stage. Also pigment metabolic ability, antioxidant enzyme activities(peroxidase, catalase, superoxide dismutase, glutathione reductase) and antioxidant content (tocopherol, ascorbic acid, glutathione, carotenoids) were lower in barnyardgrass on the basic of fresh weight. However, lipoxygenase activity and the content of unsaturated fatty acid which is vulnerable to oxygen radicals were higher in barnyardgrass, suggesting that barnyardgrass seedling bave a properties easy to be peroxidized. The differences of PPIX (protoporphyrin IX) or carotenoid content, which are the primary substances inducing herbicide activity, were not related to the selectivity between rice and barnyardgrass.

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Development of Natural Antioxidants and Whitening Agents for Cosmeceuticals

  • Kim, Jong-Pyung
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 2007.11a
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    • pp.79-92
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    • 2007
  • Oxidative stress have known to be a risk factor for the degenerative processes and closely related to a lot of diseases. It is well established that antioxidants are good in protection and therapeutic means against oxidative damage. There is increasing interest in natural antioxidants and many natural antioxidants have been found and utilized as the possible protection for various diseases and skin aging. We have screened natural antioxidant agents for cosmeceuticals, nutraceuticals, and drugs as therapeutic and preventive means against oxidative stress, and have developed a number of novel antioxidants from various natural sources. A novel melanin synthesis inhibitor, Melanocin A, isolated from the metabolite of a fungal strain Eupenicillium shearii F80695 inhibited mushroom tyrosinase and melanin biosynthesis of B16 melanoma cells with $IC_{50}$ value of 9.0 nM and MIC value of $0.9\;{\mu}M$, respectively. Melanocin A also exhibited potent antioxidant activity by scavenging of DPPH and superoxide anion radicals. UV was found to increase the level of hydrogen peroxides and other reactive oxygen species (ROS) in skin tissues. This increase in ROS may not only alter the structure and function of many genes and proteins directly but may also modulate their expressions through signal transduction pathways and, ultimately, lead to skin damage. We investigated the effect of Melanocin A on UV-induced premature skin aging. Firstly, the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT in vitro was investigated. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated UV-induced skin changes in hairless mice in vivo by Melanocin A. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. These results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging. Terrein is a bioactive fungal metabolite isolated from Penicillium species. Terrein has a relatively simple structure and can be easily synthesized. However, the biologic effects of terrein are comparatively unknown. We found for the first time that terrein potently inhibit melanin production in melanocytes and has a strong hypopigmentary effect in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment of Mel-Ab cells with terrein (10-100 mM) for 4 days significantly reduced melanin levels in a dose-dependent manner. In addition, terrein at the same concentration also reduced tyrosinase activity. We then investigated whether terrein influences the extracellular signal-regulated protein kinase (ERK) pathway and the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. Terrein was found to induce sustained ERK activation and MITF down-regulation, and luciferase assays showed that terrein inhibits MITF promoter activity in a dose-dependent manner. To elucidate the correlation between ERK pathway activation and a decreased MITF transcriptional level, PD98059, a specific inhibitor of the ERK pathway, was applied before terrain treatment and found to abrogate the terrein-induced MITF attenuation. Terrein also reduced the tyrosinase protein level for at least 72 h. These results suggest that terrain reduces melanin synthesis by reducing tyrosinase production via ERK activation, and that this is followed by MITF down-regulation.

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Effect of Exercise on Antioxidant Enzyme Activities of Skeletal Muscle and Liver in STZ-diabetic Rats (STZ-당뇨쥐에서 운동부하가 골격근 및 간의 항산화효소 활성도에 미치는 영향)

  • Seok, Kwang-Ho;Lee, Suck-Kang
    • Journal of Yeungnam Medical Science
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    • v.17 no.1
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    • pp.21-30
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    • 2000
  • Background: The purpose of the present study was to investigate the effect of exercise on the activities of antioxidant enzymes, super oxide dismutase(SOD), glutathione peroxidase(GPX) and catalase(CAT) of skeletal muscle(gastrocnemius) and liver in streptozotocin(STZ) induced diabetic rats. The malondialdehyde(MDA) concentration was also measured as an index of lipid poroxidation of tho tissues by exercise-induced oxidative stresses in diabetic rats. Material and Methods: Male Sprague-Dawley rats were randomly divided into control and STZ-induced diabetic rats. The STZ in citrate buffer solution was injected twice at S days intervals intraperitoneally(50, 70 mg/kg respectively). On the 28th day after the first STZ injection, the diabetic animals were randomly divided into pre- and post-exercise groups, The exercise was introduced to the rats of post-exercise group by treadmill running until exhaution with moderate intensity ($V_{O2max}$: 50-70%) of exercise. The duration of average running time was 2 hours and 19 minutes. Results: The blood glucose concentration was increased(p<0.001) and plasma insulin concentration was decreased(p<0.001) in the diabetic rats. The glycogen concentration in the muscle and liver was decreased by exhaustive exercise in the diabetic rats(p<0.001), In the skeletal muscle, the activities of GPX was increased(p<0.05) and the activities of SOD and CAT were not changed in the diabetic rats compare to those of the control rats. The activities of GPX was not changed by exercise but the activities of SOD(p<0.01) and CAT(p<0.01) were decreased by exercise in the diabetic rats, The concentration of MDA was not changed by exercise in diabetic rats, and the values of pre-exercise and post-exercise diabetic rats were not different from the value those of control rats, In the liver, the activities of SOD was decreased(p<0.01), and the activities of GPX and CAT were not changed in diabetic rats compared to the values of control rats, The activities of SOD, GPX and CAT were not changed by exercise in diabetic rats but the activity of SOD seemed to decrease slightly, The MDA concentration was increased in the diabetic rats compared to the values of control rats(p<0.001), but there was no change of MDA concentration by exercise in diabetic rats, Conclusions: In summary, exhaustive physical exercise did not seem to impose oxidative stress on the skeletal muscle because of due to oxygen free radicals, regardless of the decrease in SOD and CAT in the diabetic rats, In liver tissue, the tissue damage by oxidative stress was observed in diabetic rats but the additional tissue damage by exhaustive physical exercise was not observed.

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The Evaluation of IL-8 in the Serum of Pneumoconiotic patients (진폐증 환자에서의 혈청내 IL-8 농도)

  • Ahn, Hyeong Sook;Kim, Ji Hong;Chang, Hwang Sin;Kim, Kyung Ah;Lim, Young
    • Tuberculosis and Respiratory Diseases
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    • v.43 no.6
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    • pp.945-953
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    • 1996
  • Background : Many acute and chronic lung diseases including pneumoconiosis are characterized by the presence of increased numbers of activated macrophages. These macrophages generate several inflammatory cell chemoattractants, by which neutrophil migrate from vascular compartment to the alveolar space. Recruited neutrophils secrete toxic oxygen radicals or proteolytic enzymes and induce inflammatory response. Continuing inflammatory response results in alteration of the pulmonary structure and irreversible fibrosis. Recently, a polypeptide with specific neutrophil chemotactic activity, interleukin-8(IL-8), has been cloned and isolated from a number of cells including : monocytes, macrophages and fibroblasts. IL-1 and/or TNF-${\alpha}$ preceded for the synthesis of IL-8, and we already observed high level of IL-1 and TNF-${\alpha}$ in the pneumoconioses. So we hypothesized that IL-8 may be a central role in the pathogenesis of pneumoconiosis. In order to evaluate the clinical utility of IL-8 as a biomarker in the early diagnosis of pneumoconiosis, we investigated the increase of IL-8 in the pneumoconiotic patient and the correlation between IL-8 level and progression of pneumoconiosis. Method : We measured IL-8 in the serum of 48 patients with pneumoconiosis and 16 persons without dust exposure history as a control group. Pneumoconiotic cases were divided into 3 groups according to ILO Classification : suspicious group(n=16), small opacity group(n=16) and large opacity group(n=16). IL-8 was measured by a sandwich enzytne immunoassay technique. All data were expressed as the $mean{\pm}standard$ deviation. Results: 1) The mean value of age was higher in the small opacity and large opacity group than comparison group, but smoking history was even. Duration of dust exposure was not different among 3 pneumoconiosis groups. 2) IL-8 level was $70.50{\pm}53.63pg/m{\ell}$ in the suspicious group, $107.50{\pm}45.88pg/m{\ell}$ in the small opacity group, $132.50{\pm}73.47pg/m{\ell}$ in the large opacity group and $17.85{\pm}33.85pg/m{\ell}$ in the comparison group. IL-8 concentration in all pneumoconiosis group was significant higher than that in the comparison group(p<0.001). 3) IL-8 level tended to increase with the progression of pneumoconiosis. Multiple comparison test using Anova/Scheffe analysis showed a significant difference between suspicious group and large opacity group(p<0.05). 4) The level of IL-8 was correlated with the progression of pneumoconiosis(r=0.4199, p<0.05). Conclusion : IL-8 is thought to be a good biomarker for the early diagnosis of pneumoconiosis.

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