韩国产芝麻酱油对AAPH诱发LLC-PK1细胞氧化应激的保护作用

以芝麻酿造酱油乙醇提取物(ethanol extract sesame sauce,SSE)为研究对象,探讨其对1 mmol/L 2,2’-偶氮二异丁基脒二盐酸盐(2,2’-azobis(2-methylpropionamidine)dihydrochloride,AAPH)引发的LLC-PK1猪肾近曲小管上皮细胞氧化应激损伤的保护作用。方法:LLC-PK1细胞与不同质量浓度(10~100μg/m L)的SSE预先共同培养24 h后,用含AAPH的DMEM培养液继续培养4 h建立细胞损伤模型。细胞存活率用四甲基偶氮唑蓝法检测,细胞内丙二醛(malondialdehyde,MDA)含量和总活性氧簇(reactive oxygen species,ROS)水平分别以硫代巴比妥酸比色法和2’,7’-二氢二氯荧光黄双乙酸钠探针法测定。细胞内过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化酶(glutathione peroxidase,GSH-Px)、谷胱甘肽巯基转移酶(glutathione S-transferase,GST)、γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase,γ-GCS)活力和谷胱甘肽(glutathione,GSH)含量按试剂盒说明书以比色法测定。结果表明,SSE预处理可以提高受损细胞存活率,降低细胞内总ROS水平和MDA含量。同时,SSE还能提高受损细胞内抗氧化酶(CAT、SOD、GSH-Px)及γ-GCS活力并提高细胞内GSH含量。实时荧光定量聚合酶链式反应分析也提示SSE能上调细胞内抗氧化物酶(CAT、SOD和GSH-Px)的m RNA表达量。此外,SSE可以通过提高细胞内源性抗氧化系统能力来降低细胞内MDA和ROS水平,并由此缓解AAPH对LLC-PK1细胞所造成的氧化应激损伤。

Protective Effect of Korean Sesame Sauce on AAPH-Induced Oxidative Stress in LLC-PK1 Cells

The present study aimed to investigate the protective effect of ethanol extract from sesame sauce (SSE) on 2,2’-azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative stress in pig renal epithelial LLC-PK1 cells. LLC-PK1 cells were incubated with different concentrations of SSE (10–100 μg/mL) for 24 h, and then exposed to AAPH (1 mmol/L) for 4 h. Cell viability was determined by methyl thiazolyl tetrazolium assay. The levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were determined by thiobarbituric acid reactive substances assay (TBARS) and DCFHDA assay, respectively. The activities of cellular antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), γ-glutamylcysteine synthetase (γ-GCS) and glutathione (GSH) were measured by colorimetric assay. In addition, the mRNA levels of these antioxidant enzymes (SOD, GSH-Px and CAT) were determined by quantitative real-time polymerase chain reaction assay. SSE was able to increase cell viability, and decrease ROS and MDA level, as well as increase the activities and mRNA expression of antioxidant enzymes (CAT、SOD and GSH-Px) compared with the control group. SSE treatment also increased the activity of γ-GCS and GSH levels in AAPH-treated LLC-PK1 cells. These results suggested that SSE showed a protective effect against AAPH-induced oxidative stress in LLC-PK1 cells through inhibiting lipid peroxidation, deceasing ROS levels, and increasing the activity of the endogenous antioxidant system.