Alloxan에 의한 HIT-T15 세포의 산화적 손상에 대한 매실(Prunus mume Sieb. et Zucc.) 주정추출물의 세포보호효과

Abstract

본 연구는 췌장베타세포인 HIT-T15 세포를 이용하여 매실주정추출물(PME)의 alloxan에 의한 산화스트레스로부터의 세포보호, 인슐린 분비능 및 항산화 효소 활성을 평가하였다. PME는 alloxan에 의해 유발된 산화스트레스로부터 세포를 보호하여 세포생존율을 증가시켰다. PME는 세포막 손상지표인 LDH 방출을 억제하였고 <TEX>$NAD^+$</TEX>/NADH ratio를 유의적으로 증가시켜 세포사멸이 억제되어짐을 확인하였다. 또한 alloxan 단독처리군에 비해 250 <TEX>${\mu}g$</TEX>/ml PME 처리군에서 인슐린 분비량이 유의성 있게 증가하였다. Alloxan과 PME를 동시에 처리하여 HIT-T15세포의 항산화효소 활성을 측정했을 때 산화스트레스에 의해 감소되었던 항산화효소 활성이 PME에 의해 보호되는 효과를 확인하였다. 이상의 연구결과로부터 PME는 세포괴사 및 DNA fragmentation을 억제하고 세포내 항산화효소 활성을 증가시켜 alloxan에 의해 유발된 산화스트레스로부터 췌장베타세포를 보호하고 이에 따른 인슐린 분비능 조절 효과가 있는 것으로 생각된다. The present study was designed to examine the potential antidiabetic and antioxidant effect of ethanol extract from <TEX>$Prunus$</TEX> <TEX>$mume$</TEX> fruit (PME) against alloxan-induced oxidative stress in pancreatic <TEX>${\beta}$</TEX>-cells, HIT-T15. To evaluate the antidiabetic effect of PME, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliu bromide (MTT) cell proliferation assay, lactate dehydrogenase (LDH) release assay, <TEX>$NAD^+$</TEX>/NADH ratio and insulin secretion were assessed. We also measured its antioxidant effect against alloxan-induced oxidative stress in the cells by assessing the levels of the antioxidant enzymes including superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx). The results of this analysis showed that alloxan significantly decreased cell viability, increased LDH leakage, and lowered <TEX>$NAD^+$</TEX> /NADH ratio and insulin secretion in HIT-T15 cells. However, PME significantly increased the viability of alloxan-treated cells and lowered LDH leakage. The intracellular <TEX>$NAD^+$</TEX> /NADH ratio and insulin secretion were also increased by 1.5~1.9-fold and 1.4-fold, respectively, after treatment with the PME. The HIT-T15 cells treated with alloxan showed significant decreases in the activities of antioxidant enzymes, while PME significantly elevated the levels of antioxidant enzymes. Based on these results, we suggest that PME could have a protective effect against the cytotoxicity and dysfunction of pancreatic <TEX>${\beta}$</TEX>-cells in the presence of alloxan-induced oxidative stress.