Division of Pediatric Nephrology, Department for Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
Ren Fail. 2012;34(4):487-94. doi: 10.3109/0886022X.2012.655684.
Apoptosis plays a critical role in the pathogenesis of gentamicin (Gen)-induced nephrotoxicity. However, the underlying molecular mechanisms still remain unclear. In this study, we addressed the role of p38 mitogen-activated protein kinase (MAPK)/inducible nitric oxide synthase (iNOS) signaling pathway in Gen-induced nephrotoxicity and evaluated the protective effect of the free-radical scavenger N-acetylcysteine amide (NACA).
Pig kidney epithelial cells (LLC-PK1) cells were exposed to Gen for variable times and doses. Cytotoxicity was assessed by morphology and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Protein expression was assessed by Western blotting.
Exposure to Gen-induced apoptosis in a dose-dependent and time-dependent manner was assessed by DNA content analysis and poly ADP ribose polymerase (PARP) cleavage. Gen caused increased phosphorylation of p38 MAPK and induction of iNOS. This was accompanied by a significant upregulation of Bax and nuclear factor κB (NF-κB) and a downregulation of Bcl-2 expression. Pretreatment with SB203580, aminoguanidine (AG), and NACA inhibited apoptosis. Furthermore, pretreatment with SB203580 and NACA not only attenuated the pro-apoptotic effect of Gen, but also significantly reversed its effects on p38 MAPK phosphorylation and iNOS induction. The Gen-induced effects on Bcl-2, Bax, and NF-κB expression were also reversed by SB203580, AG, and NACA.
In conclusion, NACA can attenuate Gen-induced apoptotic injury in LLC-PK1 cells through inhibiting p38 MAPK/iNOS signaling pathway.
细胞凋亡在庆大霉素（Gen）诱导的肾毒性发病机制中起关键作用。然而，其潜在的分子机制仍不清楚。在这项研究中，我们研究了 p38 丝裂原活化蛋白激酶（MAPK）/诱导型一氧化氮合酶（iNOS）信号通路在 Gen 诱导的肾毒性中的作用，并评估了自由基清除剂 N-乙酰半胱氨酸酰胺（NACA）的保护作用。
将猪肾上皮细胞（LLC-PK1）细胞暴露于 Gen 中不同时间和剂量。通过形态学和 3-(4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐（MTT）测定法评估细胞毒性。通过 Western 印迹评估蛋白表达。
通过 DNA 含量分析和多聚 ADP 核糖聚合酶（PARP）切割评估 Gen 诱导的细胞凋亡呈剂量和时间依赖性。Gen 引起 p38 MAPK 磷酸化和 iNOS 诱导增加。这伴随着 Bax 和核因子 κB（NF-κB）的显著上调和 Bcl-2 表达的下调。SB203580、氨基胍（AG）和 NACA 的预处理抑制了细胞凋亡。此外，SB203580 和 NACA 的预处理不仅减弱了 Gen 的促凋亡作用，而且显著逆转了其对 p38 MAPK 磷酸化和 iNOS 诱导的作用。Gen 对 Bcl-2、Bax 和 NF-κB 表达的影响也被 SB203580、AG 和 NACA 逆转。
总之，NACA 通过抑制 p38 MAPK/iNOS 信号通路，可减轻 Gen 诱导的 LLC-PK1 细胞凋亡损伤。
