고수온 환경에 의해 유도된 산화 스트레스에 대한 넙치의 항산화 작용과 생리적 변화 Antioxidant Defenses and Physiological Changes in Olive Flounder (Paralichthys olivaceus) in Response to Oxidative Stress Induced by Elevated Water Temperature원문보기
고수온 환경 ($25^{\circ}C$와 $30^{\circ}C$)에 노출시킨 넙치의 산화 스트레스 정도를 알아보기 위하여 넙치의 간 조직에서 항산화 효소 [superoxide dismutase (SOD)와 catalase(CAT)] mRNA의 발현량 및 그 활성을 측정한 결과, $20^{\circ}C$ 대조구보다 $25^{\circ}C$와 $30^{\circ}C$ 실험구에서 증가하는 경향을 보였다. 또한 지질 과산화 지표로 사용되는 lipid peroxidation(LPO)을 측정한 결과, $25^{\circ}C$와 $30^{\circ}C$ 실험구에서 증가하는 경향을 나타내었다. LPO의 증가는 SOD 및 CAT의 증가와 밀접한 관련이 있으며, 체내의 $H_O_2$ 농도 또한 $25^{\circ}C$와 $30^{\circ}C$ 실험구에서 증가하는 것으로 보아 고수온 환경이 넙치의 산화 스트레스를 유발하고 있는 것으로 사료된다. 고수온 환경에 노출시킨 넙치의 혈중 alanine aminotransferase (AlaAT)와 aspartate aminotransferase (AspAT) 값을 측정 한 결과, AlaAT와 AspAT 모두 유의적으로 증가하는 경향을 보였다. 또한 면역 지표로 사용되는 lysozyme 활성도가 $20^{\circ}C$ 대조구보다 $30^{\circ}C$ 실험구에서 유의적으로 낮은 값을 나타낸 점으로 보아, 고수온 환경에 노출된 넙치에서는 간 세포의 손상뿐만 아니라 면역력 또한 저해되고 있는 것으로 사료된다. 고수온 환경에 노출시킨 넙치에서 항산화 효소인 SOD와 CAT mRNA 발현량 및 활성이 증가하였을 뿐만 아니라 활성산소와 LPO 값 또한 증가된 점으로 보아, 고수온 환경은 넙치의 체내에서 산화 스트레스를 유발시키는 동시에 면역 기능을 저해시키고 있는 것으로 사료된다.
고수온 환경 ($25^{\circ}C$와 $30^{\circ}C$)에 노출시킨 넙치의 산화 스트레스 정도를 알아보기 위하여 넙치의 간 조직에서 항산화 효소 [superoxide dismutase (SOD)와 catalase(CAT)] mRNA의 발현량 및 그 활성을 측정한 결과, $20^{\circ}C$ 대조구보다 $25^{\circ}C$와 $30^{\circ}C$ 실험구에서 증가하는 경향을 보였다. 또한 지질 과산화 지표로 사용되는 lipid peroxidation(LPO)을 측정한 결과, $25^{\circ}C$와 $30^{\circ}C$ 실험구에서 증가하는 경향을 나타내었다. LPO의 증가는 SOD 및 CAT의 증가와 밀접한 관련이 있으며, 체내의 $H_O_2$ 농도 또한 $25^{\circ}C$와 $30^{\circ}C$ 실험구에서 증가하는 것으로 보아 고수온 환경이 넙치의 산화 스트레스를 유발하고 있는 것으로 사료된다. 고수온 환경에 노출시킨 넙치의 혈중 alanine aminotransferase (AlaAT)와 aspartate aminotransferase (AspAT) 값을 측정 한 결과, AlaAT와 AspAT 모두 유의적으로 증가하는 경향을 보였다. 또한 면역 지표로 사용되는 lysozyme 활성도가 $20^{\circ}C$ 대조구보다 $30^{\circ}C$ 실험구에서 유의적으로 낮은 값을 나타낸 점으로 보아, 고수온 환경에 노출된 넙치에서는 간 세포의 손상뿐만 아니라 면역력 또한 저해되고 있는 것으로 사료된다. 고수온 환경에 노출시킨 넙치에서 항산화 효소인 SOD와 CAT mRNA 발현량 및 활성이 증가하였을 뿐만 아니라 활성산소와 LPO 값 또한 증가된 점으로 보아, 고수온 환경은 넙치의 체내에서 산화 스트레스를 유발시키는 동시에 면역 기능을 저해시키고 있는 것으로 사료된다.
We determined oxidative stress caused by thermal stress in olive flounder Paralichthys olivaceus based on the altered-mRNA expression and enzymatic activity of two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), along with monitoring of several other biomarkers. When the fish...
We determined oxidative stress caused by thermal stress in olive flounder Paralichthys olivaceus based on the altered-mRNA expression and enzymatic activity of two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), along with monitoring of several other biomarkers. When the fish were exposed to acute thermal change (from $20^{\circ}C$ to $25^{\circ}C$ and $30^{\circ}C$), the expression and activity of both enzymes were significantly higher at elevated temperatures ($25^{\circ}C$ and $30^{\circ}C$) than at $20^{\circ}C$. Lipid peroxidation (LPO) was also higher at $25^{\circ}C$ and $30^{\circ}C$ than at $20^{\circ}C$. In addition, the plasma $H_2O_2$ concentration was significantly increased by thermal stress. Furthermore, we investigated changes due to thermal stress by measuring levels of plasma alanine aminotransferase (AlaAT) and aspartate aminotrasferase (AspAT). Both were significantly increased by thermal stress. As an immune indicator, the lysozyme concentration was lower at $30^{\circ}C$ than at $20^{\circ}C$, indicating that thermal stress decreases immune function. Therefore, thermal stress could induce oxidative stress and suppress immune function and can cause physiological stress.
We determined oxidative stress caused by thermal stress in olive flounder Paralichthys olivaceus based on the altered-mRNA expression and enzymatic activity of two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), along with monitoring of several other biomarkers. When the fish were exposed to acute thermal change (from $20^{\circ}C$ to $25^{\circ}C$ and $30^{\circ}C$), the expression and activity of both enzymes were significantly higher at elevated temperatures ($25^{\circ}C$ and $30^{\circ}C$) than at $20^{\circ}C$. Lipid peroxidation (LPO) was also higher at $25^{\circ}C$ and $30^{\circ}C$ than at $20^{\circ}C$. In addition, the plasma $H_2O_2$ concentration was significantly increased by thermal stress. Furthermore, we investigated changes due to thermal stress by measuring levels of plasma alanine aminotransferase (AlaAT) and aspartate aminotrasferase (AspAT). Both were significantly increased by thermal stress. As an immune indicator, the lysozyme concentration was lower at $30^{\circ}C$ than at $20^{\circ}C$, indicating that thermal stress decreases immune function. Therefore, thermal stress could induce oxidative stress and suppress immune function and can cause physiological stress.
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제안 방법
, 1995). In this study, we investigated the mRNA expression and/or enzymatic activities of the antioxidant enzymes SOD and CAT as well as the plasma H2O2 concentration and lipid peroxidation (LPO) in order to examine the oxidative stress in olive flounder (Paralichthys olivaceus) exposed to high temperatures. We also examined potential change of immune function during thermal stress based on the analysis of plasma lysozyme concentration, and determine plasma alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) concentrations to measure general stress levels arisen from the thermal changes.
In this study, we measured plasma H2O2 and LPO levels, as well as the expression and activity of the antioxidant enzymes SOD and CAT to understand the oxidative stress and mechanism in olive flounder exposed to the high temperature environment. '快 also examined lysozyme-related immune function, AlaAT and AspAT to investigate physiological changes induced by oxidative stress.
EF681883(SOD); GQ229479(CAT); EU090804 (|3-actin)]: SOD forward primer (5Z-CGT TGG AGA CCT GGG GAA TGT G-3Z), SOD reverse primer (5Z-ATC GTC AGC CTT CTC GTG GAT C-3Z), CAT forward primer (5Z-GGC TGA GAA GTT CCA GTT CAA TCC-37), CAT reverse primer (5Z-CTC CAC CTC TGC AAA GTA GTT GAC-3'), P-actin forward primer (5‘-GCA AGA GAG GTA TCC TGA CC-3') and P-actin reverse primer (5Z-CTC AGC TCG TTG TAG AAG G-3‘). PCR amplification was conducted using an iCycler iQ Multicolor Real-Time PCR Detection System (BioRad, Hercules, CA, USA) and iQ™ SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions. QPCR was performed as follows: denaturation at 95℃ for 5 min, 35 cycles of denaturation at 95℃ fo호 20 s, and annealing at 55℃ for 20 s, and extension at 72℃ for 20 s.
5 |ig) was reverse transcribed in a total volume of 20 μL using an oligo-d(T)15 anchor primer and M-MLV reverse transcriptase (Bioneer, Korea) according to the manufacturer's instructions. Primers for QPCR were designed with reference to the known sequences of olive flounder as follows [GenBank accession no. EF681883(SOD); GQ229479(CAT); EU090804 (|3-actin)]: SOD forward primer (5Z-CGT TGG AGA CCT GGG GAA TGT G-3Z), SOD reverse primer (5Z-ATC GTC AGC CTT CTC GTG GAT C-3Z), CAT forward primer (5Z-GGC TGA GAA GTT CCA GTT CAA TCC-37), CAT reverse primer (5Z-CTC CAC CTC TGC AAA GTA GTT GAC-3'), P-actin forward primer (5‘-GCA AGA GAG GTA TCC TGA CC-3') and P-actin reverse primer (5Z-CTC AGC TCG TTG TAG AAG G-3‘). PCR amplification was conducted using an iCycler iQ Multicolor Real-Time PCR Detection System (BioRad, Hercules, CA, USA) and iQ™ SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions.
9 and ammonia was no detected (0 ppm) in water. The fish were fed a commercial feed formed as extruded pellet (jeilfeed company, kyoungnam, Korea) twice daily (09:00 and 17:00).
, Boocheon, Korea), we sampled at 25℃ and 30℃ after 5 days and 10 days started elevating temperature, respectively. The thermal experiment was performed with three replications per group, and five fish from each group [control group (20℃), experimental groups (25℃ and 30℃)] were randomly selected for blood and tissue sampling. The fish were anesthetized with 200 mg/L tricaine methanesulfonate (MS-222; Sigma, St.
대상 데이터
Olive flounder (n=60; length, 10±0.5 cm; weight, 19.9± 1.3g) were obtained from a commercial fish farm (Hwanam fishery, Gijanggun, Busan, Korea) and allowed to acclimate to the experimental conditions for 2 weeks in three 300-L flow-through tank system. The water temperature and photoperiod were maintained at 20± 1℃, and 12-h light: 12-h dark, respectively.
데이터처리
, USA). One way ANOVA followed by Dunnette post hoc test was used to compare the differences to 20℃ group in the data (P<0.05).
이론/모형
QPCR was conducted to determine the relative expression of SOD and CAT mRNA in the total RNA extracted from the liver using the Trizol method according to the man너facturer's instructions (Gibco/BRL, Grand Island, NY, USA). The concentration and purity of the RNA samples was determined by UV spectroscopy at 260 and 280 nm.
성능/효과
In conclusion, in the present study, the expression and activity of the antioxidant e교zymes (SOD and CAT) in olive flounder exposed to high temperature environments were increased, and induced oxidative stress. These results indicate that antioxidant enzymes operated against thermal stress by increasing plasma H2O2 concentrations and LPO levels in olive flounder exposed to high temperature environments.
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