晚期糖基化终末产物诱导肾小球系膜细胞线粒体途径凋亡  

Advanced glycation end products induce mitochondrial pathway apoptosis in glomerularmesangial cells

作　　者：鲁伶旭 徐莉[1] 范秋灵[1] 汪旭[1] 曹旭[1] 王力守[1] Lu Lingxu;Xu Li;Fan Qiuling;Wang Xu;Cao Xu;Wang Lining(Department of Nephrology, the First Hospital, China Medical University, Shenyang 110001, China)

机构地区：[1]中国医科大学附属第一医院肾内科,沈阳110001

出　　处：《中华肾脏病杂志》2018年第2期122-129,共8页Chinese Journal of Nephrology

基　　金：国家自然科学基金(81270808);辽宁省自然科学基金(201602821);辽宁省高等学校重大科技平台免疫皮肤病学重点实验室自主创新课题基金(201303);沈阳市科技计划项目(F16-205-1)

摘　　要：目的探索高糖环境下晚期糖基化终末产物（AGEs）是否通过改变肾小球系膜细胞Ros、JC-1及其凋亡相关蛋白的表达，诱导肾小球系膜细胞线粒体途径凋亡及肾小球系膜细胞的损伤。方法体外培养大鼠肾小球系膜细胞株HBZY-1，使用不同浓度的AGEs分别培养0、12、24、48h后，MTT法观察细胞增殖能力。选择AGEs最佳作用时间和浓度后，加入caspase酶抑制剂Z．VAD—fmk及活性氧（ROS）清除剂乙酰半胱氨酸（NAC）进行培养，使用细胞凋亡检测试剂盒和AnnexinV—FITC／PI试剂盒检测细胞的凋亡率，JC-1染色检测线粒体膜电位（MMP）的变化，使用细胞ROX深红流式细胞仪检测细胞内总ROS水平，Westem印迹检测抗凋亡蛋白Bcl-2和促凋亡蛋白BAX表达，并检测caspase-9、caspase-3和聚腺苷二磷酸核糖聚合酶（PARP）活化片段（cleaved）的表达。结果AGEs可呈时间和浓度依赖性降低肾小球系膜细胞活力，诱导细胞死亡。250mg／LAGEs作用24h可显著增加肾小球系膜细胞凋亡率（P〈0．01），Z—VAD—fmk可显著缓解AGEs诱导的肾小球系膜细胞凋亡（P〈0．01）。与对照组相比，AGEs增加细胞内活性氧水平，降低线粒体MMP，并呈时间依赖性，AGEs引起两者显著改变的作用时间分别为1h和2h（均P〈0．01）。AGEs还可减少细胞抗凋亡蛋白Bcl-2表达（P〈0．01），增加促凋亡蛋白Bax、cleavedcaspase-9、cleavedcaspase-3和cleavedPARP的表达（均P〈0．01）。与AGEs组相比，NAC可显著稳固线粒体膜电位（P〈0．01），增加Bcl-2表达（P〈0．01），减少BAX、cleavedcaspase-9、cleavedcaspase-3和cleavedPARP的表达（均P〈0．01）。结论AGEs通过增加细胞内ROS水平，破坏线粒体膜势能，从而诱导肾小球系膜细胞线粒体途径凋亡。Objective To investigate whether advanced glycation end products （AGEs） can induce the expression of Ros, JC - 1 and its apoptosis - related proteins in glomerular mesangial cells under high glucose environment, induce apoptosis and injury of glomerular mesangial cells. Methods Rat glomerular mesangial cell line HBZY- 1 was cultured in vitro. The cells were cultured with different concentrations of AGEs for 0, 12, 24 and 48 hours respectively. MTT assay was used to observe the cell proliferation ability. After the optimal time and concentration of AGEs were selected, the caspase enzyme inhibitor Z-VAD-fmk and reactive oxygen species （ROS） scavenger N-acetyl-L-cysteine （NAC）were cultured and the apoptosis rate was detected by cell death detection apoptosis ELISA plus and Annexin V - FITC/PI kit. JC - 1 staining was used to detect the changes of mitochondrial membrane potential （MMP）. Cell ROX deep red flow cytometry was used to detect the total ROS level. The expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein BAX, caspase-9, caspase-3 and poly ADP- ribose polymerase （PARP）- activated fragments was detected by Western blotting. Results AGEs could decrease the activity of glomerular mesangial cells in a time and concentration-dependent manner, and induce cell death. The percentage of apoptotic cells in glomerular mesangial cells was significantly increased after treatment with 250 mg/L AGEs for 24 h （P 〈 0.01）, and Z-VAD-fmk could significantly alleviate AGEs- induced glomerular mesangial cell apoptosis （P 〈 0.01）. Compared with the control group, AGEs increased the level of intracellular reactive oxygen species and decreased MMP in a time-dependent manner, and the two time points that AGEs significantly caused the change were 1 h and 2 h （all P 〈 0.01）. AGEs also reduced the expression of antiapoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP （all P 〈 0.01）. Compared with

关 键 词：肾小球系膜细胞 细胞凋亡 活性氧 晚期糖基化终末产物 线粒体 途径 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]