过表达P21减轻顺铂诱导的HK-2细胞损伤

目的:探讨P21在顺铂诱导的肾小管上皮细胞损伤中的作用。方法:实时荧光定量聚合酶链式反应(RT-q PCR)及Western blot法检测顺铂诱导的肾小管上皮细胞(HK-2细胞)中P21的表达水平。在HK-2细胞中过表达P21后,采用CCK-8法和流式细胞术检测细胞活力及细胞凋亡;Western blot检测急性肾损伤标志物肾损伤因子1(KIM-1)、中性粒细胞明胶酶相关载脂蛋白(NGAL)的表达及细胞凋亡效应蛋白caspase-3的表达;此外,还同时检测葡萄糖调节蛋白78(GRP78)、CCAAT增强子结合蛋白同源蛋白(CHOP)的蛋白水平及蛋白激酶R样内质网激酶(PERK)、真核翻译起始因子2α(e IF2α)的磷酸化水平以反映细胞内质网应激相关信号通路的活性。结果:在肾小管上皮细胞中,顺铂可剂量及时间依赖性上调P21的mRNA及蛋白表达。过表达P21可逆转顺铂诱导的HK-2细胞凋亡,并使KIM-1、NGAL、GRP78、p-PERK、p-e IF2α、CHOP和cleaved caspase-3的蛋白水平明显减少。结论:过表达P21可减轻顺铂诱导的肾小管上皮细胞急性损伤,其机制可能与调控HK-2细胞内质网应激信号通路,抑制细胞凋亡有关。

Over-expression of P21 attenuates cisplatin-induced HK-2 cells injury

AIM:To investigate the effect of P21 on cisplatin-induced renal tubular epithelial cells injury.METHODS:The expression of P21 at mRNA and protein levels in cisplatin treated human renal tubular epithelial cells (HK-2) cells was determined by RT-qPCR and Western blot. Over-expression of P21in the HK-2 cells was induced by the transfection of pcDNA3-P21. The cell viability and cell apoptosis were detected by CCK-8 assay and flow cytometry, respectively. Furthermore, the protein expression of kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), caspase-3, glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP), and phosphorylation level of eucaryotic translation initiation factor 2α (eIF2α) and protein kinase R-like endoplasmic reticulum kinase (PERK) were detected by Western blot.RESULTS:Cisplatin increased the mRNA and protein levels of P21 in a time-and concentration-dependent manner in the HK-2 cells. Over-expression of P21 inhibited cisplatin-induced cell apoptosis, and down-regulated the expression of KIM-1 and NGAL. Furthermore, Over-expression of P21 decreased the protein levels of GRP78, p-PERK, p-eIF2α, CHOP and cleaved caspase-3.CONCLUSION:Over-expression of P21 attenuates cisplatin-induced HK-2 cells injury, and the mechanism may be related to the modulation of endoplasmic reticulum stress pathway and inhibition of cell apoptosis.