钠-氢交换蛋白1小干扰RNA对抗霉素A诱导人肾小管上皮细胞缺血再灌注损伤的保护机制

Protective mechanism of NHE-I-siRNA on human renal tubular epithelial cell from ischemic reperfusion injury induced by antimycin A

目的通过构建钠-氢交换蛋白1（NHE-1）小干扰RNA（siRNA）抑制肾小管上皮细胞NHE-1的表达，探讨其对细胞缺血再灌注损伤的保护作用机制。方法根据人NHE-1全长基因序列设计并合成NHE-1-siRNA，转染人肾小管上皮细胞系（HKC），以无关siRNA转染组作为对照。利用10μmol／L抗霉素A诱导细胞来模拟缺血缺氧环境。用RT—PCR、Western印迹法检测NHE-1的表达。用BCECF／AM、Fluo-3／AM和SBFI-AM标记HKC，通过激光共聚焦显微镜分别检测其细胞内pHi、Ca^2＋和Na^＋浓度的变化。用Hoechst 33342染色技术及AnnexinV／PI染色结合流式细胞仪技术检测细胞凋亡情况。利用荧光探针JC-1检测细胞线粒体膜电位的变化。结果特异性NHE-1-siRNA能有效抑制HKC的NHE-1表达，与无关siRNA转染组相比，NHE-1-siRNA转染组NHE-1 mRNA和蛋白表达水平明显下调（均P〈0．05）。抗霉素A刺激后，2组细胞NHE-1 mRNA及蛋白表达水平显著上调，而NHE-1-siRNA转染组低于无关siRNA转染组；同时NHE-1-siRNA转染组细胞凋亡率（8．9％±2．9％）明显低于抗霉素A处理组（18．8％±3．2％）和抗霉素A＋无关siRNA转染组（17．4％±3．6％）（均P〈0．05）；NHE-1-siRNA转染组线粒体膜电位明显增高；与无关siRNA转染组相比较，NHE-1-siRNA转染组经抗霉素A处理后，细胞内钠离子、氢离子及钙离子增高的幅度较低（P〈0．05）。结论NHE-1-siRNA可抑制肾小管上皮细胞内NHE-1的表达，对抗霉素A诱导肾小管上皮细胞的缺血再灌性损伤有一定的保护作用。其机制可能通过抑制NHE-1表达，延缓细胞内Na^＋的积聚，减轻细胞内钙离子的超载，抑制损伤细胞的线粒体膜电位下降，减少细胞的凋亡。

Objective To explore the mechanism of protecting cells from ischemic reperfusion injury by constructing specific small interference RNA （siRNA） to inhibit Na^＋-H^＋ exchanger-1 （NHE-1） expression in human renal tubular epithelial cell （HKC）. Methods The siRNA was designed and synthesized based on human NHE-1 complete sequence, and was transfected into HKC. The irrespective siRNA transfected group was used as control. The cells were treated with 10 μmol/L antimycin A to induce ischemia and anoxyaemia environment. NHE-1 expression was examined by RT-PCR and Western blot. The intracellular pH （pHi）, Ca^2＋ or Na^＋ concentrations were detected by BCECF/AM, Fluo-3/AM and SBFI-AM, respectively, combining with laser confocal assay system. Nucleic morphology was determined by Hoechst 33342. Cellular apoptosis was examined by Annexin V/PI staining and flow cytometry. Fluorescent probe JC-1 was used to detect the change of mitochondrial transmembrane potential. Results The specific siRNA could efficiently inhibit NHE-1 expression in HKC. Compared with the irrespective siRNA transfected group, the mRNA and protein expression of NHE-1 was significantly down-regulated in NHE-1 siRNA transfected group （all P〈0.05）. After treatment with antimycin A, the mRNA and protein expression of NHE-1 was significantly up-regulated in both groups, however, it was less than that in irrespective siRNA transfected group. At the same time, the ratio of apoptosis decreased （8.9% ±2.9% vs 18.8% ±3.2%, 17.4% ±3.6%, P〈0.05） and mitochondrial transmembrane potential rose significantly in NHE-1 siRNA transfected group as compared to irrespective siRNA transfected group and antimycin A group. The intracellular Na^＋, H^＋ and Ca^2＋ concentrations increased in NHE-1 siRNA transfected group treated with antimycin A, but their levels were lower than those in irrespective siRNA transfected group with the same treatment （P〈 0.05）. Conclusions The synthesized siRNA can inhibit the expression of NHE-1 and can protect HKC from ischemia reperfusion injury induced by antimycin A. The mechanism might be via suppressing the expression of NHE-1 to delay intracelluar Na^＋ accumulation, attenuate intracellular Ca^2＋ overloading, and inhibit the decrease of mitochondrion transmembrane potential and reduce cellular apoptosis.