摘要
本文旨在研究内质网应激(endoplasmic reticulum stress,ERS)感受器活化转录因子6(activating transcription factor 6,ATF6)是否介导晚期糖基化白蛋白(advanced glycated albumin,AGE-alb)所诱导的巨噬细胞凋亡,并阐明其可能的分子机制。体外培养RAW264.7巨噬细胞,给予AGE-alb(2、4和6 g/L)处理24 h,以正常白蛋白和ERS诱导剂衣霉素(tunicamycin,TM)处理24 h的巨噬细胞分别作为阴性和阳性对照组,并采用小干扰RNA(small interfering RNA,si RNA)技术沉默ATF6表达。分别采用MTT法和Annexin V-FITC/碘化丙碇双染法检测细胞活力和凋亡情况;试剂盒测定培养基乳酸脱氢酶(lactate dehydrogenase,LDH)和细胞内caspase-3活性;免疫荧光细胞化学法检测ATF6核转位情况;Western blot法检测ATF6和促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein,CHOP)蛋白表达变化,实时荧光定量聚合酶链反应法检测CHOP m RNA表达变化。结果显示:与TM相似,AGE-alb在蛋白和m RNA水平均显著上调ERS凋亡途径关键分子CHOP表达,该作用呈浓度依赖性;Western blot和免疫荧光细胞化学法均显示AGE-alb处理细胞后ATF6明显由胞浆向细胞核内转移;采用si RNA技术沉默ATF6则明显减轻AGE-alb所致的细胞活力降低、LDH漏出、凋亡率增加及caspase-3活化,并可抑制AGE-alb所诱导的CHOP表达上调。上述结果表明,ATF6及其下游分子CHOP介导AGE-alb所诱导的巨噬细胞凋亡。
The purpose of this study was to investigate whether activating transcription factor 6 （ATF6）, a sensor to endoplasmic reticulum stress （ERS）, would mediate advanced glycated albumin （AGE-alb）-induced macrophage apoptosis and to elucidate the possible molecular mechanisms. RAW264.7 macrophages were cultured in vitro and treated with AGE-alb （2, 4 and 6 g/L）, normal control albumin or tunicamycin （TM, 4 mg/L） for 24 h. ATF6 small interfering RNA （siRNA） was transfected to RAW264.7 cells by Lipofectamine 2000. Cell viability and apoptosis were determined by MTT method and Annexin V-FITC/propidium iodide apoptosis detection kit, respectively. The activities of lactate dehydrogenase （LDH） in medium and caspase-3 in cells were measured by corresponding detection kits. ATF6 nuclear translocation was detected by Western blot and immunofluorescence cytochemistry. Protein and mRNA levels of C/EBP homologous protein （CHOP, a key-signaling component of ERS-induced apoptosis） were detected by Western blot and real-time fluorescence quantitative PCR, respectively. The results showed that similar to TM, AGE-alb increased the expression of CHOP at both the protein and mRNA levels in a concentration dependent manner. ATF6, as a factor that positively regulates CHOP expression, was activated by AGE-alb in a concentration dependent manner. siRNA-mediated knockdown of ATF6 significantly inhibited AGE-alb-induced macrophage injury, as indicated by the increased cell viability and the decreased LDH release, apoptosis and caspase-3 activation. Additionally, ATF6 siRNA attenuated AGE-alb-induced CHOP upregulation at both the protein and mRNA levels. These results suggest that ATF6 and its downstream molecule CHOP are involved in AGE-alb-induced macrophage apoptosis.
作者
康攀攀
姚树桐
郭甜甜
王志超
田华
焦鹏
周健
秦树存
KANG Pan-Pan;YAO Shu-Tong;GUO Tian-Tian;WANG Zhi-Chao;TIAN Hua;JIAO Peng(Affiliated Hospital of Chengde Medical University, Chengde 067000, China;Institute of Atherosclerosis, Key Laboratory of Atherosclerosis in Universities of Shandong, Taishan Medical University, Taian 271000, China;College of Basic Medical Sciences, Taishan Medical University, Taian 271000, China;School of Dentistry and Oral Health, Taishan Medical University, Taian 271000, China)
出处
《生理学报》
CAS
CSCD
北大核心
2017年第6期767-774,共8页
Acta Physiologica Sinica
基金
supported by the National Natural Science Foundation of China(No.81570410,81370381)
the Taishan Scholars Foundation of Shandong Province,China(No.ts201511057)
