Departments of Medical Biochemistry, Akdeniz University Faculty of Medicine, Antalya, Turkey.
Departments of Histology, Akdeniz University Faculty of Medicine, Antalya, Turkey.
Toxicol In Vitro. 2020 Aug;66:104836. doi: 10.1016/j.tiv.2020.104836. Epub 2020 Mar 24.
This study evaluated polyunsaturated fatty acids (PUFAs) in human kidney epithelial cells exposed to diclofenac (DCL) toxicity. Kidney cells were treated with DCL to induce cytotoxicity and thymoquinone (TQ) was administered to decrease cytotoxic effects. Levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were determined by liquid chromatography coupled with tandem mass spectrometry. Cytosolic phospholipase A (cPLA), cyclooxygenase 1 (COX-1) and prostaglandin E (PGE) were measured to evaluate changes in enzyme activity. Immunofluorescence staining and western blot analysis was performed to determine protein levels of COX- 1. Renal cell toxicity was accomplished by DCL and was alleviated by TQ treatment. Diclofenac significantly increased all measured PUFAs while pretreatment with TQ decreased PUFA levels in DCL treated cells. Cytosolic PLA and total COX activity was significantly decreased in DCL treated cells. Immunofluorescence staining and western blot analysis confirmed significantly decreased COX-1 levels in DCL and DCL + TQ treated groups. The results of this study reveal that DCL treatment is associated with accumulation of PUFAs in kidney cells. We suggest that PUFA accumulation in DCL toxicity might be a consequence of both cPLA and COX-1 inhibition. Thymoquinone administration, along with DCL treatment alleviated the buildup of PUFAs and DCL-induced cell death in kidney cells.
本研究评估了暴露于双氯芬酸（DCL）毒性的人肾上皮细胞中的多不饱和脂肪酸（PUFAs）。用 DCL 处理肾细胞以诱导细胞毒性，并给予百里醌（TQ）以减少细胞毒性作用。通过液相色谱-串联质谱法测定花生四烯酸（AA，C20：4n-6），二高γ-亚麻酸（DGLA，C20：3n-6），二十碳五烯酸（EPA，C20：5n-3）和二十二碳六烯酸（DHA，C22：6n-3）的水平。测定胞质型磷脂酶 A（cPLA），环氧化酶 1（COX-1）和前列腺素 E（PGE）的活性，以评估酶活性的变化。进行免疫荧光染色和 Western blot 分析以确定 COX-1 的蛋白水平。 DCL 完成肾细胞毒性，TQ 处理可减轻 TQ 处理。双氯芬酸显著增加了所有测定的 PUFAs，而 TQ 预处理可降低 DCL 处理细胞中的 PUFAs 水平。 DCL 处理的细胞中胞质 PLA 和总 COX 活性明显降低。免疫荧光染色和 Western blot 分析证实 DCL 和 DCL + TQ 处理组中 COX-1 水平明显降低。这项研究的结果表明，DCL 处理与肾细胞中 PUFAs 的积累有关。我们建议，PUFA 在 DCL 毒性中的积累可能是 cPLA 和 COX-1 抑制的结果。 DCL 治疗时给予百里醌可减轻 PUFAs 的堆积并减轻 DCL 诱导的肾细胞死亡。
