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晚期糖基化白蛋白通过激活caspase-12途径诱导巨噬细胞凋亡

李金国, 郝奇, 刘映雪, 李鹏, 邵夏炎, 田华, 方永奇, 姚树桐

泰山医学院1基础医学院；人口与计划生育学院；动脉粥样硬化研究所，泰安 271000

摘要

本文旨在研究晚期糖基化白蛋白(advanced glycated albumin, AGE-alb)对巨噬细胞内质网应激(endoplasmic reticulum stress, ERS)凋亡途径关键分子caspase-12的影响，以阐明AGE-alb对巨噬细胞凋亡的诱导作用及机制。体外培养RAW264.7巨噬细胞，分别给予AGE-alb (2、4和6 g/L)、正常对照白蛋白(control albumin, C-alb, 4 g/L)、ERS诱导剂衣霉素(tunicamycin, TM, 4 mg/L)处理，或以ERS抑制剂4-苯丁酸 (4-phenylbutyric acid, PBA, 5 mmol/L)预处理细胞1 h，然后与AGE-alb (4 g/L)共孵育24 h。采用MTT法检测细胞活力，TUNEL法检测细胞凋亡情况，试剂盒测定培养基乳酸脱氢酶(lactate dehydrogenase, LDH)活性，免疫印迹法检测caspase-12表达变化。结果显示：与TM相似，AGE-alb显著诱导RAW264.7巨噬细胞损伤，表现为细胞活力降低，LDH漏出和细胞凋亡率明显增加，且呈浓度依赖性。AGE-alb明显上调caspase-12活性，尤其在4和6 g/L浓度时更为显著 (P < 0.01)。然而，PBA可抑制AGE-alb所致的巨噬细胞活力降低以及LDH漏出和凋亡增加，且减轻AGE-alb诱导的caspase-12活化(P < 0.05)。上述结果提示，AGE-alb可诱导RAW264.7巨噬细胞凋亡，其机制可能与激活caspase-12 介导的ERS凋亡途径有关。

关键词： 晚期糖基化白蛋白; 内质网应激; caspase-12; 巨噬细胞; 凋亡

分类号：R332;R363.2;R329.2

[Advanced glycated albumin induces macrophage apoptosis via activating caspase-12 pathway.] [Article in Chinese]

LI Jin-Guo, HAO Qi, LIU Ying-Xue, LI Peng, SHAO Xia-Yan, TIAN Hua, FANG Yong-Qi , YAO Shu-Tong

College of Basic Medical Sciences； College of Population and Family Planning； Institute of Atherosclerosis, Taishan Medical University, Taian 271000, China

Abstract

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on the activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms of macrophage apoptosis. RAW264.7 macrophages were treated with AGE-alb (2, 4 and 6 g/L), control albumin (C-alb, 4 g/L), tunicamycin (TM, 4 mg/L), or pretreated with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then treated with AGE-alb (4 g/L). After incubation for 24 h, the cell viability and apoptosis were determined by using MTT assay and TUNEL detection kit, respectively. Lactate dehydrogenase (LDH) activity in media was determined by using an assay kit. The protein levels of caspase-12 were examined by Western blot analysis. The results showed that like TM (an ERS inducer), incubation with AGE-alb led to significant decrease in viability and increase in LDH activity in media and apoptotic rate in a dose-dependent manner. In addition, AGE-alb induced activation of caspase-12 especially at the concentration of 4 and 6 g/L (P < 0.01), which was similar to TM. However, PBA (an ERS inhibitor) protected RAW264.7 macrophages from AGE-alb-induced decrease in viability and increases in LDH activity and apoptosis. Moreover, PBA also inhibited the caspase-12 activation induced by AGE-alb (P < 0.05). These results suggest that AGE-alb may induce apoptosis in RAW 264.7 macrophages, and the mechanism may be related to the activation of ERS-associated apoptotic pathway mediated by caspase-12.

Key words: advanced glycated albumin; endoplasmic reticulum stress; caspase-12; Macrophage; apoptosis

收稿日期：2016-04-27　　录用日期：2016-06-29

通讯作者：姚树桐　　E-mail: yst228@126.com

引用本文：

李金国, 郝奇, 刘映雪, 李鹏, 邵夏炎, 田华, 方永奇, 姚树桐. 晚期糖基化白蛋白通过激活caspase-12途径诱导巨噬细胞凋亡[J]. 生理学报 2016; 68 (6): 733-739.

LI Jin-Guo, HAO Qi, LIU Ying-Xue, LI Peng, SHAO Xia-Yan, TIAN Hua, FANG Yong-Qi , YAO Shu-Tong. [Advanced glycated albumin induces macrophage apoptosis via activating caspase-12 pathway.] [Article in Chinese]. Acta Physiol Sin 2016; 68 (6): 733-739 (in Chinese with English abstract).