• Preventive Nutrition and Food Science
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• 제13권4호
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• pp.259-265
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• 2008

This study was designed to investigate the protective effect of fucoidan against AAPH-induced oxidative stress in LLC-PK1 cells (porcine kidney epithelial cells). Oxidative stress was induced by exposing of LLC-PK1 cells to the 1 mM 2,2'-azobis(2-amidino propane) dihydrochloride (AAPH) for 24 hr. Exposure of LLC-PK1 cells to 1 mM AAPH for 24 hr resulted in a significant (p<0.05) decrease in cell viability, but fucoidan treatment protected LLC-PK1 cells from AAPH-induced cell damage in a dose dependant manner. To investigate the protective action of fucoidan against AAPH-induced damage of LLC-PK1 cells, we measured the effects of fucoidan on lipid peroxidation and antioxidant enzymes activities of AAPH treated cells as well as scavenging activities on superoxide anion radical and hydroxyl radical. Fucoidan had protective effect against the AAPH-induced LLC-PK1 cellular damage and decreased lipid peroxidation and increased activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-px). Furthermore, fucoidan showed strong scavenging activity against superoxide anion radical. The $IC_{50}$ value of fucoidan was $48.37{\pm}1.54\;{\mu}g/mL$ for superoxide anion radical scavenging activity. The fucoidan also had high hydroxyl radical scavenging activity ($IC_{50}=32.03\;{\mu}g/mL$). These results indicate that fucoidan protects against AAPH-induced LLC-PK1 cell damage by inhibiting lipid peroxidation, increasing antioxidant enzyme activities and scavenging offree radicals.

• Food Science and Biotechnology
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• 제18권5호
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• pp.1253-1257
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• 2009

Exo-polysaccharide isolated from the culture of Grifola frondosa was modified by sodium periodate ($NaIO_4$) and sodium chlorite ($NaClO_2$) to delete polysaccharide part and phenolic compound, respectively, and was investigated what effect has each part of exo-polysaccharide against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress in porcine kidney epithelial cells (LLC-PK1). Oxidative stress on LLC-PK1 cell was measured by cell viability, lipid peroxidation, superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) activity. Exposure of LLC-PK1 cells to 1 mM AAPH for 24 hr resulted in significant decrease in cell viability, SOD, and GSH-px action, and significant increase in lipid peroxidation. The treatment of exo-polysaccharide and $NaIO_4$ modified sample protected LLC-PK1 cells from AAPH-induced cell damage such as cell viability, lipid peroxidation, SOD, and GSH-px activity in a dose dependant manner (10, 100, and $500{\mu}g/mL$). However, the treatment of $NaClO_2$ modified sample did not affect for cell viability, lipid peroxidation, SOD, and GSH-px activity. The antioxidant activity of exo-polysaccharide was significantly decreased on AAPH-induced LLC-PK1 cell system when phenolic compound was deleted. The antioxidant activity was significantly correlated with the content of phenolic compound of exo-polysaccharide.

• Preventive Nutrition and Food Science
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• 제13권2호
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• pp.84-89
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• 2008

This study was designed to investigate the protective effect of a methanol extract of Chungkookjang (CKJ) on high glucose induced oxidative stress in LLC-$PK_1$ cells (renal tubular epithelial cells), which are susceptible to oxidative stress. Freeze dried CKJ powder was extracted with methanol, and the extract solution was concentrated, and then used in this study. To determine the protective effect of CKJ extract, oxidative stress was induced by exposing of LLC-$PK_1$ cells to high glucose (30 mM) or normal glucose (5 mM) for 24 hr. Exposure of LLC-$PK_1$ cells to high glucose for 24 hr resulted in a significant (p<0.05) decrease in cell viability, catalase, SOD and GSH-px activity and a significant (p<0.05) increase in intracellular ROS level and thiobarbituric acid reactive substances (TBARS) formation in comparison to the cells treated with 5 mM glucose. CKJ extract treatment decreased intracellular ROS level and TBARS formation, and increased cell viability and activities of antioxidant enzymes including catalase, SOD and GSH-px in high glucose pretreated LLC-$PK_1$ cells. These results suggest that CKJ extract may be able to protect LLC-$PK_1$ cells from high glucose-induced oxidative stress, partially through the antioxidative defense systems.

• Preventive Nutrition and Food Science
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• 제16권1호
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• pp.12-17
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• 2011

This study was designed to investigate the protective effect of Sasa borealis leaf extract on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress in LLC-PK1 cells (porcine kidney epithelial cells). The butanol fraction from Sasa borealis leaf extract (SBBF) was used in this study because it possessed strong antioxidant activity and high yield among fractions. Exposure of LLC-PK1 cells to 1 mM AAPH for 24 hr resulted in a significant decrease in cell viability, but SBBF treatment protected LLC-PK1 cells from AAPH-induced cell damage in a dose dependant manner. To determine the protective action of SBBF against AAPH-induced damage of LLC-PK1 cells, we measured the effects of SBBF on lipid peroxidation and antioxidant enzymes activities of AAPH treated cells as well as scavenging activities on superoxide anion radical and hydroxyl radical. SBBF had a protective effect against the AAPH-induced LLC-PK1 cellular damage and decreased lipid peroxidation and increased activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase. Furthermore, SBBF showed strong scavenging activity against superoxide anion radical. The $IC_{50}$ value of SBBF was $28.45{\pm}1.28\;{\mu}g/mL$ for superoxide anion radical scavenging activity. The SBBF also had high hydroxyl radical scavenging activity ($IC_{50}=31.09{\pm}3.08\;{\mu}g/mL$). These results indicate that SBBF protects AAPH-induced LLC-PK1 cells damage by inhibiting lipid peroxidation, increasing antioxidant enzyme activities and scavenging free radicals.

• Toxicological Research
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• 제11권1호
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• pp.1-8
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• 1995

Fumonisins are a family of mycotoxins produced by the fungus Fusarium moniliforme which is a common contaminant in corn. Fumonisins are potent inhibitors of sphingosine and sphinganine N-acyltransferase (ceramide synthase), key enzymes in sphingolipid metabolism. The purpose of this study was to provide the evidence that the elevated levels of free sphingoid bases (primarily sphinganine) and depletion of complex sphingolipids were closely related to the inhibition of cell growth in LLC-$PK_1$ cells exposed to fumonisin $B_1$$(\leq 35 {\mu}M)$. Concentrations of fumonisin $B_1$ between 10 and $35 {\mu}M$ were known to inhibit cell growth without cytotoxicity in $LLC-PK_1$ cells (Yoo et al. Toxicol. Appl. Pharmacol. 114, 9-15, 1992). Cells exposed to 35$\mu M$ fumonisin B$_1$ for 48 and 72 hr developed a fibroblast-like (elongated and spindle-shaped) appearance and were less confluent than normal cells. At between 24 and 48 hr after exposure to fumonisin $B_1$ cells were beginning to show the inhibition of cell growth and at 72 hr the number of viable cells in fumonisin-treated cultures was about 50% of concurrent control cultures. During the 24 hr lag period preceding inhibition of cell growth, the free sphinganine levels in cells exposed to $35 {\mu}M$ fumonisin $B_1$ were highly elevated (approximately 230 fold higher than normal cells). The elevated levels of free sphinganine were $435\pm14$$pmoles/{10^6}$ cells at 48 hr and approximately TEX>$333\pm11$$pmoles/{10^6}$ cells in cells exposed to $35{\mu}M$ fumonisin$B_1$ at 72 hr, while the levels of free sphinganine in normal cells were less than 2$pmoles/{10^6}$ cells. Under the same condition, depletion of intracellular complex sphingolipids as a consequence of fumonisin inhibition of de novo sphingolipid biosynthesis and turnover pathway was appeared. Content of free sphingold bases in dividing cells was more elevated than in confluent cells at 24-48 hr after cells were exposed to $20{\mu}M$ fumonisin $B_1$. The dividing cells were showing the inhibition of cell growth at 48-72 hr and $20{\mu}M$ fumonisin $B_1$. The results of this study support the hypothesis that the inhibition of cell growth is very well related to the disruption of sphingolipid metabolism in $LLC-PK_1$ cells.

• 약학회지
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• 제40권6호
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• pp.705-712
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• 1996

We have previously shown that determination of glucose uptake using ${\alpha}$-methylglucose(${\alpha}$-MG) is very sensitive and rapid parameter for the assessment of loss of cellular fu nction in renal cell line($LLC-PK_1$). The present study was designed to elucidate the mechanism of inhibition of ${\alpha}$-MG uptake and the intracellular site of toxic action of cisplatin(CIS). $LLC-PK_1$ cells were exposed to various concentrations(5 ${\mu}$M-l00 ${\mu}$M) of CIS for 5 hrs or 24 hrs and ${\alpha}$-MG uptake was determined. Mitochondrial function was evaluated by measuring intracellular ATP content and MTT reduction. The activities of marker enzymes for the basolateral membrane(Na$^+$-K$^+$ ATPase) and brush border membrane (alkaline phosphatase: ALP) were also measured. CIS treatment significantly inhibited the ${\alpha}$-MG uptake in a time- and dose-dependent manner above 25 ${\mu}$M for 5 hrs. Intracellular ATP content and MTT reduction were affected by 24 hr-treatment of 50 ${\mu}$M CIS. The activities of Na$^+$-K$^+$ ATPase and ALP were significantly decreased at 10 ${\mu}$M and 5 ${\mu}$M of CIS for 24 hrs, respectively. The incubation with CIS for 5 hrs had no effects on the intracellular ATP content, MTT reduction and the activities of marker enzymes up to 100 ${\mu}$M. These results partly indicate that inhibition of ${\alpha}$-MG uptake by CIS may not be attributed to the disturbance of mitochondrial function or inhibition of the activity of Na$^+$-K$^+$ ATPase and can be resulted from direct effect of CIS on the Na$^+$/glucose cotransporter in brush border membrane. This study shows that additional mechanistic information, indicating the intracellular site of nephrotoxic action, can be gained by coupling the ${\alpha}$-MG uptake and ATP content or the activity of Na$^+$-K$^+$ ATPase.

• Environmental Analysis Health and Toxicology
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• 제9권1_2호
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• pp.25-35
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• 1994

Many nephrotoxic agents exert their effect primarily on the cells of the proximal tubules. We used the LLC-$PK_1$, kidney epithelial cell line as a model system for studies on nephrotoxicity and investigated whether the uptake of $\alpha$-methylglucose($\alpha$-MG) could serve as a parameter to assess effects of nephrotoxicants on the functional integrity of the cells at an early time of toxicity. The enzyme leakage test which has been used to be as a conventional cytotoxic parameter in vitro, was conducted to compare with $\alpha$-MG uptake. Treatment with cisplatin for 24 and 48 hours significantly increased activities of lactate dehydrogenase and $\gamma$-glutamyltransferase in culture medium at a concentration of 50$\mu$M. However, above 100$\mu$M of concentration, activities of these enzymes in media were dramatically decreased by cisplatin. These observations indicate that cisplatin has direct inhibitory effect on the activities of these enzymes and make it doutful to use enzyme leakage test to demonstrate damage of kidney cells by chemicals such as cisplatin over the appropriate range of concentration. Cisplatin inhibited $\alpha$-MG uptake at a low concentration which enzymes were not leaked. Also cadmium chloride and mercuric chloride which are acutely nephrotoxic in vivo, significantly inhibited $\alpha$-MG uptake at a low concentration. These results indicate that the uptake of $\alpha$-methylglucose in LLC-$PK_1$cell line is a useful biomarker for the study of nephrotoxicity.

• 한국식품영양과학회지
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• 제43권11호
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• pp.1674-1680
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• 2014

잡곡밥 메탄올 추출물이 가지는 산화적 스트레스 개선 효과를 확인하기 위하여 $H_2O_2$로 유도된 산화적 스트레스에 대한 LLC-PK1 세포의 보호 효과를 조사하였다. $250{\mu}M$의 $H_2O_2$에 의한 산화적 스트레스가 유발된 LLC-PK1 세포에 시료추출물을 처리한 결과, WR, TMR, GR 순으로 $H_2O_2$ 처리군에 비해 세포 생존율이 증가하였다(P<0.05). 특히 GRS와 GRK에서 정상군에 가까운 세포 생존율을 나타내어 $H_2O_2$에 의한 산화적 스트레스로 유발된 세포 손상에 대한 잡곡밥 추출물의 보호 효과를 확인할 수 있었다. 세포 내 활성산소(ROS)의 수준과 세포 내 지질과산화물질(MDA)의 생성 억제 효과 역시 WR, TMR, GR, GRK, GRS 순으로 증가하는 것을 관찰하였다. $H_2O_2$로 인하여 세포 내 항산화 효소인 SOD, CAT와 GSH-px 등의 활성이 감소된 LLC-PK1 세포에 잡곡밥 추출물을 처리했을 때 이들 효소의 활성이 증가했으며 특히 GRK, GRS군에서 현저하게 증가되었다. LLC-PK1 세포에서 $H_2O_2$에 의해 발생하는 산화적 스트레스에 대한 보호 효과를 측정한 결과 잡곡밥 추출물은 세포 손상을 억제하고 세포 내 활성산소(ROS)의 수준과 지질과산화물질(MDA)의 생성을 억제하며, 세포 내 항산화 효소의 활성을 증가시키는 효과를 가지는 것으로 보인다. 또한 항산화 효소유전자 발현에서도 위와 비슷한 효과를 나타내었다. 이상의 결과로 잡곡밥 메탄올 추출물, 특히 기능성 물질인 카레가루와 상황버섯 추출물이 첨가된 잡곡밥은 LLC-PK1 세포에 대한 보호 작용과 in vitro에서의 항산화 효과가 높은 것으로 확인되었다.

• Archives of Pharmacal Research
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• 제24권2호
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• pp.136-143
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• 2001

Fumonisins are specific inhibitors of ceramide synthase in sphingolipid metabolism. An alteration in sphingolipid metabolism as a result of fumonisin exposure is related to cell death (Yoo et al., 1992). The objective of this study was to investigate whether elevated free sphinganine levels are related to the sensitivity of cultured cells to fumonisin exposure. Fumonisin $B_1$ elevated the intracellular free sphinganine concentraions in both LLC-$PK_1$ and Chinese hamster ovary (CHO) cells. However, CHO cells are resistant to fumonisin cytotoxicity at 50${u}m$, while LLC-$PK_1$ cells are sensitive at concentrations greater than 357M. The intracellular concentration of free sphinganine in LLC-$PK_1$ cells treated at 50${u}m$ fumonisin $B_1$ for 72 h was approximately 1450 pmol/mg protein relative to the 37 pmol observed in the control culture. Under the same conditions, the population of apoptotic cells in the 50${u}m$ fumonisin $B_1$-treated culture was approximately 37% of the total compared to 12% in the control. The caspase III-like activity after 72 h in the 50${\mu}$M fumonisin $B_1$-exposed culture Increased to approximately 50 $pmol/mg$ protein/hr compared to 6 $pmol/mg$ protein/hr in the control. L-cycloserine, a serine palmitoyltransferase inhibitory reduced the fumonisin $B_1$-stimulated caspase III-like activity down to the control level. Under the same culture conditions, the intracellular concentration of free sphinganine after-cycloserine plus fumonisin $B_1$ treatment was 140 pmol/mg protein compared to 1450 $pmol/mg$ protein in fumonisin $B_1$ alone. The intracellular concentration of free sphinganine in CHO cells treated with 50${u}m$ fumonisin $B_1$ for 72 h was al)proximately 460 pmol/mg protein, indicating that the mass amount of elevated free sphinganine in the CHO cells was about 32% of that in LLC-$PK_1$ cells. Adding exogenous sphinganine to the CHO cells along with 50${u}m$ fumonisin $B_1$ treatment for 72 h caused both necrosis and apoptosis. In conclusion, the elevated endogenous sphinganine acts as a contributing factor to the fumonisin-induced cell death.

• 한국식품영양과학회지
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• 제34권8호
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• pp.1151-1156
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• 2005

채소류의 산화방지 지표로서 ORAC측정결과 산화방지 능력이 우수하다고 보고되어진 13가지 채소류를 선정하여 DPPH 라디칼 소거능을 관찰하고 이들의 phenol 함량을 분석한 결과, 비트, 가지, 케일의 DPPH 라디칼 소거능이 우수한 것으로 나타났으며, 이들은 총 phenol 함량 또한 높아 이들 사이의 양의 상관관계가 있음을 알 수 있었다 반면, 알팔파 스프라우트(alfalfa sprout), 브로컬리, 시금치 등에서는 phenol 함량은 높은 것에 비해 DPPH 라디칼 소거능은 그다지 우수하지 못했다. 또한 세포모델에서 LLC-$PK_{1}$ 세포에 radical generator인 AAPH 처리를 함으로써 채소류의 산화적 스트레스에 대한 개선효과 살펴본 결과, 가지와 컬리플라워가 높은 세포 생존율을 보여 가장 우수한 산화적 스트레스 개선 효과를 가진 것으로 나타났으며, 그 다음으로 비트,당근 등의 산화적 스트레스 개선효과가 높은 것으로 나타났다. 특히 가지는 총 phenol 함량도 높을 뿐 아니라, DPPH 라디칼 소거능과 세포모델에서의 산화적 스트레스에 대한 개선효과도 뛰어나 우수한 항산화기능성을 지닌 채소류로 사료된다.