二甲基乙二酰基甘氨酸对缺氧复氧诱导的肾小管上皮细胞损伤的保护作用及其机制  

Protective mechanism of dimethyloxallyl glycine on human renal tubular epithelial cells from hypoxia reoxygenation injury

作　　者：张晓丽[1] 盛蔚文[1] 陈伟军[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：《中华肾脏病杂志》2010年第3期210-214,共5页Chinese Journal of Nephrology

基　　金：基金项目：上海市国际科技合作项目（09410705800）;上海市卫生局青年科研项目（2008Y029）

摘　　要：目的通过脯氨酸羟化酶抑制剂二甲基乙二酰基甘氨酸（DMOG）稳定缺氧诱导因子1α（HIF—1α）表达，探讨其对缺氧复氧诱导的肾小管上皮细胞（HKC）损伤的保护作用及其机制。方法制作无糖缺氧复氧细胞损伤模型，用不同浓度的DMOG预处理，锥虫蓝染色和乳酸脱氢酶（LDH）活性方法检测细胞活力及损伤；AnnexinV和PI染色流式细胞仪技术检测细胞凋亡；实时荧光定量PCR方法检测红细胞生成素（EPO）、热休克蛋白70（HSP70）和血红素氧合酶1（HO—1）mRNA的表达；Western印迹法检测HIF—1α、活性caspase-3和Bcl-2蛋白表达。结果正常情况下HKC细胞内几乎无HIF-1α蛋白表达，DMOG刺激6h后HIF-10【蛋白及其靶基因EPO、HSP70和HO-1mRNA表达均显著上调（均P〈0．01），且呈浓度依赖性。500μmol／L或1mmol／LDMOG预处理可明显改善缺氧复氧诱导的细胞损伤，表现为细胞存活率升高（95．6％±1．8％、96．1％±1．0％比83．3％±3．1％）；培养上清液中LDH活性下降；细胞凋亡减少（8．6％±2．7％、6．1％±2．3％比19．2％±4．0％）（均P〈0．05）。另外，细胞内活性caspase-3蛋白表达显著下调，而Bcl-2蛋白表达则显著上调（均P〈0．05）。结论DMOG预处理可稳定肾小管上皮细胞内HIF—1α表达，对缺氧复氧诱导的肾小管上皮细胞损伤具有-定保护作用。其机制可能与促进EPO、HSP70和HO—1表达，抑制caspase-3活化，上调Bcl-2表达有关。Objective To explore the possible mechanism of prolyl hydroxylases inhibitor-dimethyloxallyl glyeine （DMOG） in protecting human renal tubular cells （HKC） from hypoxia reoxygenation injury throngh stabilizing hypoxia inducible factor 1 （HIF-1）. Methods Hypoxia reoxygenation injury and pretreatment with different concentrations of DMOG on HKC were studied. Cell activitiy and cell death were evaluated by trypan blue staining and lactate dehydrogenase （LDH） activity respectively. Cell apoptosis was detected by Annexin V/PI staining and flow cytometry. The expression of EPO, HSP70 and HO-1 were examined by real-time PCR. The expressions of HIF-1α, cleaved caspase-3 and Bcl-2 were examined by Western blotting.Results The expression of HIF-1α protein was very weak in normal HKC cells. Pretreatment with DMOG for 6 h, the expressions of HIF-1α and its target gene such as EPO, HSP70 and HO-1 were all significantly up-regulated in protein or mRNA levels （all P〈0.01）, and these up-regulations were concentrationdependent. Ceils pretreated with 500 μmol/L or 1 mmol/L DMOG exhibited less injury compared to hypoxia reoxygenation group, with elevated cell survival （95.6%±1.8% or 96.1%±1.0% vs 83.3%±3.1%）, decreased LDH activity and reduced cell apoptosis ratio （8.6%±2.7% or 6.1%±2.3% vs 19.2%±4.0%） （all P〈0.01）. In DMOG-treated cells, the protein expression of cleaved caspase-3 was significantly inhibited and the protein expression of Bcl-2 was obviously up-regulated （all P〈0.05）. Conclusions Preconditioning with DMOG can stabilize HIF-1α and protect HKC from hypoxia reoxygenation injury. The mechanism may be via up-regulating the expression of HIF-1α and its target genes EPO, HSP70 and HO-1, suppressing the expression of cleaved caspase-3, increasing the expression of Bcl-2, and finally reduce cell apoptosis.

关 键 词：缺氧诱导因子1α亚基 细胞凋亡 肾小管 上皮细胞 缺氧 复氧损伤 

分 类 号：R96[医药卫生—药理学]