脯氨酸羟化酶抑制剂对人肾小管上皮细胞缺氧/复氧损伤的影响  

Effect of prolyl hydroxylase inhibitor on hypoxia/reoxygenation injury of human proximal tubular epithelial cells

作　　者：张晓丽[1] 司徒碧颖[1] 肖婧[1] 张振兴[1] 叶志斌[1] 

机构地区：[1]复旦大学附属华东医院肾内科,上海200040

出　　处：《复旦学报（医学版）》2010年第5期560-564,共5页Fudan University Journal of Medical Sciences

基　　金：上海市非政府间国际科技合作项目(09410705800);上海市卫生局青年科研项目(2008Y029)

摘　　要：目的探讨脯氨酸羟化酶抑制剂——二甲基乙二酰基甘氨酸(DMOG)通过稳定人肾小管上皮细胞(HKC)中低氧诱导因子-1α(HIF-1α)表达抗缺氧/复氧损伤(HRI)的作用及其机制。方法无糖缺氧12 h,复氧6 h制作HKC细胞HRI模型,分别于缺氧前2、4、6、12 h给予DMOG预处理,四甲基偶氮唑蓝比色法(MTT法)检测细胞增殖活性,试剂盒检测细胞培养液中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,Annexin/PI染色流式细胞仪技术检测细胞凋亡,实时荧光定量PCR方法检测bcl-2和bax mRNA的表达,Western印迹检测HIF-1α、bcl-2和bax蛋白的表达。结果 DMOG预处理使HKC中HIF-1α表达增加,细胞损伤明显改善,表现为细胞增殖活性提高,培养上清液中MDA含量下降、SOD活性增强,细胞凋亡率下降(P<0.05);同时baxmRNA和蛋白表达下调(P<0.05),bcl-2 mRNA和蛋白表达上调(P<0.05)。结论 DMOG可通过稳定HIF-1α,影响凋亡相关基因的表达,改善HRI诱导的HKC细胞损伤。Objective To explore the effect of prolyl hydroxylases inhibitor-dimethyloxallyl glycine（DMOG） on hypoxia/reoxygenation injury（HRI） of human proximal tubular epithelial cells（HKC） by preconditioning stabilize HIF-1α. Methods Hypoxia reoxygenation injury and time course of HIF-1α expression induced by DMOG in HKC cells were studied.Cell proliferative activitiy was evaluated by methyl thiazolyl tetrazolium（MTT） assay.The content of malondialdehyde（MDA） and the activity of superoxide dismutase（SOD） of the cells were respectively detected.Cell apoptosis was detected by Annexin V/PI staining and flow cytometry.The expression of bcl-2 mRNA and bax mRNA levels were examined by Real-time PCR.The expression of HIF-1α protein,bcl-2 protein and bax protein levels were examined by Western blot. Results The expression of HIF-1α protein was very weak in normal HKC cells.Pretreated with DMOG for either 2,4,6 or 12 h,the expressions of HIF-1α were all significantly up-regulated（all P0.05） and these up-regulations were time-dependent.Cells pretreated with 500 μmol/L DMOG for either 4,6 or 12 h exhibited less injury compared with hypoxia/reoxygenation injury group,expressed as decreased content of MDA,increased activity of SOD and proliferative activity of cells and reduced cell apoptosis ratio（all P0.05）,which was concomitant with up-regulation of HIF-1α.In DMOG pretreated cells,bax was significantly inhibited and bcl-2 was obviously up-regulated（all P0.05）. Conclusions Prolyl hydroxylases domain-containing protein inhibitor-DMOG could protect HKC from HRI via up-regulating the expression of HIF-1α and influencing the expression of genes related to apoptosis.

关 键 词：脯氨酸羟化酶 低氧诱导因子 缺氧/复氧 肾小管上皮细胞 

分 类 号：R692.5[医药卫生—泌尿科学]