ADMA可以通过自噬引起内皮细胞死亡  

Study on the death of endothelial cells due to autophagy caused by ADMA

作　　者：臧强[1] 郭维康[1] 王丽妍[1] 刘文虎[1] 

机构地区：[1]首都医科大学附属北京友谊医院肾内科首都医科大学肾病学系,北京100050

出　　处：《临床和实验医学杂志》2014年第13期1045-1049,共5页Journal of Clinical and Experimental Medicine

基　　金：北京市自然科学基金(BJNSF)资助项目(7132091);北京市科学技术委员会基金资助项目(D09050704310903);博士学科点专项科研基金的高等教育资助项目(SRFDP)(20101107120003);北京友谊医院院启动基金资助项目(YYQD)(2011-17)

摘　　要：目的探讨非对称性二甲基精氨酸(ADMA)在体外培养的条件下,是否可以通过自噬引起脐静脉内皮细胞死亡。方法首先,将细胞分为阴性对照组,ADMA(25μmol/L),ADMA(50μmol/L),ADMA(100μmol/L)及阳性对照组雷帕霉素(RAPA,自噬的激动剂,20 nmol/L)刺激人脐静脉内皮细胞24 h。然后应用ADMA(100μmol/L)分别刺激人脐静脉内皮细胞0 h,12 h,24 h,36 h,48 h。最后引入自噬抑制剂3-甲基腺嘌呤(3-MA,5 mmol/L),分为阴性对照组,ADMA(100μmol/L),3-MA(5 mmol/L)和ADMA+3-MA(100μmol/L ADMA+5 mmol/L 3-MA)。免疫印迹方法检测微管相关蛋白Ⅰ轻链3(LC3Ⅱ/Ⅰ)和酵母自噬相关基因Atg6的哺乳动物同源基因beclin-1的表达水平;实时荧光定量方法检测beclin-1的mRNA表达水平;应用流式细胞仪检测细胞死亡。结果随着ADMA浓度的增加,脐静脉内皮细胞内的LC3Ⅱ/LC3Ⅰ和beclin-1的表达增加(P<0.05),细胞死亡的百分比也逐渐增加(P<0.05)。在ADMA浓度为100μmol/L时,随着时间的延长,脐静脉内皮细胞内的LC3Ⅱ/LC3Ⅰ和beclin-1的表达增加(P<0.05),细胞死亡的百分比也逐渐增加(P<0.05)。在ADMA(100μmol/L)的基础上应用自噬抑制剂3-MA后,脐静脉内皮细胞内的LC3Ⅱ/LC3Ⅰ和beclin-1的表达较ADMA组减低(P<0.05),同时加入3-MA后细胞死亡的百分比较ADMA组减低(P<0.05)。结论 ADMA可以引起人脐静脉内皮细胞发生自噬,自噬的强度具有浓度及时间的依赖性,并可以通过自噬引起人脐静脉内皮细胞的死亡,当应用自噬抑制剂后可以减少这种由于ADMA所致细胞自噬引起的细胞死亡,这说明ADMA可以通过自噬引起内皮细胞死亡,从而引起内皮损伤,这可能是引起内皮功能不全的机制之一。Objective The aim of this study is to explore the mechanism of death of endothelial cells in culture of asymmetric dimethylarginine（ ADMA） in vitro. ADMA is accumulated in plasma during chronic kidney disease（ CKD） and it is known as a risk factor for cardiovascular disease（ CVD）. The present study is designed to determine whether ADMA can cause the death of endothelial cells through autophagy in vitro.Methods Firstly,human umbilical vein endothelium cells（ HUVECs） were exposed to various concentrations of ADMA（ 0 μmol /L,25 μmol /L,50 μmol /L and 100 μmol /L） and rapamycin（ 20 nmol /L） for 24 h. Then HUVECs were exposed in the same concentration of ADMA（ 100μmol/L） at different periods（ 0 h,12 h,24 h,36 h,48 h）,and the expression of autophagy and cell death were detected. At last,HUVECs were incubated with 3- methyladenine,and then the expression of autophagy and cell death were detected and compared with those of ADMA group and control group. Western blot was used to determine protein expression of TLC3Ⅰ /Ⅱ and beclin- 1. RNA level of beclin- 1 was detected by quantitative real- time PCR. Cell death was detected by flow cytometry. Results Autophagy was enhanced along with the increase of concentration of ADMA,and the percentage of cell death（ analyzed by PI cell staining） was increased along with the increase of concentration of ADMA. Meanwhile autophagy was enhanced as time went on when using ADMA of 100 μmol /L,and the percentage of cell death increased with time lasted when using ADMA of 100 μmol /L. By using autophagy inhibitor 3- MA,which could block autophagy triggered by ADMA,and then the percentage of cell death had been decreased. Conclusion ADMA can cause the autophagy of endothelium cells,then leading to cell death. Autophagy was enhanced and cells death was increased along with the increase of concentration of AMDA,autophagy is enhanced and cells death is increased,but it can be reversed by using autophagy inhibitor 3- MA. This study suggests that ADMA

关 键 词：非对称二甲基精氨酸 自噬 内皮细胞死亡 

分 类 号：R363[医药卫生—病理学]