NGAL对肾小管上皮HK-2细胞的缺氧与复氧损伤保护作用

Protective role of NGAL against hypoxia-reoxygenation injury in HK-2 cell

目的 探究NGAL对肾小管上皮HK-2细胞缺氧与复氧损伤的作用及其机制.方法 以HK-2细胞为模型,体外模拟缺氧与复氧环境（缺氧时氧浓度＜0.1％）.先用WB和实时定量RT-PCR检测正常对照组、缺氧与复氧组细胞内NGAL蛋白和基因水平.用噻唑蓝（MTT）法和磷脂结合蛋白V-异硫氰酸荧光素（ Annexin V-FITC）/碘化丙啶双染法检测并比较缺氧与复氧同时加入不同浓度NGAL（ 20、100、200、400、1 000 ng/ml）处理组、正常对照组、缺氧与复氧组细胞增殖和凋亡情况,确定加入重组NGAL的适宜浓度.用流式细胞术和实时定量RT-PCR检测并比较正常对照组、缺氧与复氧组、缺氧与复氧同时加适宜浓度NGAL组细胞周期及凋亡相关基因bax、bcl-2、caspase-3.平行实验重复3次,比较各组检测结果.结果 WB法检测细胞内NGAL蛋白/肌动蛋白缺氧与复氧组为5.88±0.08,与正常对照组（1.51±0.03）比较差异有统计学意义（t=96.89,P＜0.05）.RT-PCR法检测NGAL/肌动蛋白基因水平缺氧与复氧组为12.32±1.27,正常对照组为1.00±0.00,两组差异有统计学意义（t=15.40,P＜0.05）.MTT结果正常对照组吸光度（A）值为0.97±0.03,缺氧与复氧组为0.56±0.04,两组差异有统计学意义（=18.68,P＜0.05）.缺氧与复氧同时加入不同浓度的NGAL（50、100、200、400、1 000 ng/ml）组A值分别为0.56 ±0.04、0.53±0.03、0.56±0.04、0.53±0.03、0.54±0.02,缺氧与复氧组和不同浓度NGAL处理组间A值水平相近,差异无统计学意义（F =0.978,P＞0.05）,但其与正常对照组比较A值差异均有统计学意义（F=105.20,P＜0.05）.Annexin VFITC/碘化丙啶双染检测细胞早期凋亡率（Annexin V阳性,碘化丙啶阴性的细胞群）,正常对照组为（1.0±0.2）％、缺氧与复氧组为（27.6±1.4）％,两组细胞早期凋亡率差异有统计学意义（t=33.590,P＜0.05）；加入重组NGAL50、100 ng/ml后分别为（27.8±1.1）％、（26.4±1.3）％,和缺氧与复氧组比较,其差异无统计学意义（t分别为0.250、1.520,p均＞0.05）；加入NGAL 200 ng/ml组为（19.4±0.6）％,明显低于缺氧与复氧组,且早期凋亡率差异有统计学意义（t=10.350,P＜0.05）;而NGAL400、1 000 ng/ml组为（19.3±1.1）％、（18.9±0.5）％,与NGAL 200 ng/ml组间早期凋亡率差异均无统计学意义（t分别为0.130、0.630,P均＞0.05）；因此选取NGAL浓度为200 ng/ml.流式细胞术检测PI正常对照组为（30.2±0.4）％,而缺氧与复氧组下降至（22.1±2.7）％,两组PI差异有统计学意义（t=3.750,P＜0.05）,而缺氧与复氧同时加入NGAL 200 ng/ml组PI为（23.2±3.7）％,和缺氧与复氧组比较,差异无统计学意义（t=0.510,P＞0.05）.实时定量RT-PCR法检测各组细胞内bax/bcl-2、caspase-3基因水平,正常对照组分别为1.00±0.00、1.00±0.00,缺氧与复氧组为5.83±0.33、8.13±0.20,加入NGAL 200 ng/ml后为2.52 ±0.07、1.89±0.02,三组bax/bcl-2（f=485.30,P＜0.05）和caspase-3基因水平（F=3 456.78,P＜0.05）差异有统计学意义.结论 NGAL在缺氧与复氧损伤的肾小管上皮HK-2细胞中通过调节凋亡相关基因表达发挥抗凋亡作用,且为缺血再灌注致急性肾损伤治疗提供了新思路和实验室依据.

Objective To explore the role and mechanism of NGAL in the process of hypoxia/ reoxygenation （H/R） injury in human renal tubular epithelial cell （HK-2）.Methods The H/R model of HK-2 cells was established in vitro （oxygen concentration 〈 0.1% ）.The expressions of NGAL in the cells of H/R group and control group were determined by WB and real-time RT-PCR.The cell models were treated with 50,100,200,400 and 1 000 ng/ml of recombinant NGAL respectively.Cell proliferation and apoptosis of the treated groups and control group were detected by the MTT assay and Annexin V-FITC/PI staining to determine the appropriate NGAL concentration.The cell cycle distributions in the H/R group,NGAL treated H/R group and control group were detected by flow cytometry,and the expressions of bax/bcl-2 and caspase-3 were measured by real-time RT-PCR.The experiments were triplicated to obtain the mean values.Results The protein expression of NGAL/actin in H/R group （5.875 ±0.081 ） was higher than that of the control group （ 1.513 ±0.032）,the differences between the two groups had statistic significance （t =96.89,P 〈0.05）. While the gene expressions of NGAL/actin in HL/R groups （12.32 ± 1.27 ） and control group （ 1.00 ±0.00） had statistical significance of difference （ t =15.40,P 〈 0.05 ）.In MTT assay,the absorption values of the H/R group and control group were 0.97 ± 0.03 and 0.56 ± 0.04,the differences had statistic significance（ t =18.680,P 〈 0.05 ）.And the absorption value of cells treated with different concentrations of NGAL （50,100,200,400,1 000 ng/ml） were 0.56±0.04,0.53 ±0.03,0.56 ±0.04,0.53 ± 0.03,0.54 ± 0.02,respectively.There were no significant differences between the H/R group and NGAL treated groups （ F =0.978,P 〉 0.05 ）,but the differences of absorption values in the control group and other groups had statistical significance （ F =105.20,P 〈 0.05 ）.The ratios of early apoptotic cells （ Annexin V positive,PI negative） in the control group and the H/R group were （ 1.0 ±0.2） % and （ 27.6 ± 1.4 ） % respectively with a statistical significance of difference （ t =33.590,P 〈0.05 ）.After the treatment of NGAL （ 50,100 ng/ml）,the ratios were （ 27.8 ± 1.1 ） % and （ 26.4 ±1.3 ） % and there were no significant differences compared to the H/R group （ t =0.250,P 〉 0.05 ）.When the H/R model was treated with 200 ng/ml of NGAL,the ratio of early apoptotic cells dropped to （ 19.4 ± 0.6） %,leading to a statistical significance of difference （ t =10.350,P 〈 0.05 ）.However,in the H/R model treated with high concentration of NGAL （400,1 000 ng/ml）,the ratios were （ 19.3 ± 1.1 ）% and （ 18.9 ±0.5 ） %,which had no significant differences compared to the cells treated with 200 ng/ml of NGAL （t =0.130,0.630; P 〉0.05）.Thus the study chose 200 ng/ml as the appropriate treating concentration of NGAL.In the control group,H/R group and 200 ng/ml,NGAL treated HR group,PI values were （30.2 ±0.4）%,（22.1 ± 2.7 ）% and （23.2 ± 3.7 ）% respectively.There were no significant differences between the H/R group and NGAL treated group （t =0.510,P 〉0.05）,but there was still statistical significance in difference among the control group and the treated groups （ F =8.28,P 〈 0.05 ）.The ratio of bax/bcl-2 and the expression of caspase-3 detected by real-time RT-PCR were 1.00 ± 0.00,1.00 ± 0.00 in the control group,5.83 ±0.33,8.13 ±0.20 in the H/R group and 2.52 ±0.07,1.89 ±0.02 in the NGAL treated H/R group.The bax/bcl-2 （ F =485.30,P 〈 0.05 ） and caspase-3 （ F =3456.78,P 〈 0.05 ） did have statistical significances of difference among the three groups.Conclusions NGAL acts as a protective factor against hypoxia-reoxygenation injury by regulating pro-apoptotic genes.It provides a new idea and evidences for the treatment of AKI caused by ischemia-reperfusion injury.