晚期糖基化白蛋白通过上调核苷酸结合寡聚结构域样受体蛋白3诱导巨噬细胞焦亡
张昭强¹, 杨一帆², 闫京锐¹, 于飞¹, 王晓旭¹, 王志超³, 田华⁴, 姚树桐^1,*
1山东第一医科大学(山东省医学科学院)基础医学院，泰安 271000 ；²山东第一医科大学(山东省医学科学院)药学院，泰安 271000 ；³山东第一医科大学(山东省医学科学院)护理学院，泰安 271000 ；⁴山东第一医科大学(山东省医学科学院)动脉粥样硬化研究所，泰安 271000
摘要
本研究旨在探讨晚期糖基化白蛋白(advanced glycated albumin, AGE-alb)对巨噬细胞核苷酸结合寡聚化结构域样受体蛋白3 (nucleotide-binding oligomerization domain-like receptor protein 3, NLRP3)-caspase-1途径的影响，以阐明AGE-alb对巨噬细胞焦亡的影响及机制。体外培养RAW264.7巨噬细胞，分别给予AGE-alb (1、2、4和6 g/L)、对照白蛋白(C-alb，4 g/L)处理24 h或以NLRP3抑制剂MCC950 (1 μmol/L)预处理细胞，1 h后以AGE-alb (4 g/L)处理24 h。MTT法检测细胞活力，试剂盒测定caspase-1和培养基乳酸脱氢酶(lactate dehydrogenase, LDH)活性以及白细胞介素-1β (interleukin-1β, IL-1β)、IL-18浓度，TUNEL法和Hoechst 33342/PI双染法检测细胞死亡情况，Western blot分析NLRP3、procaspase-1和cleaved caspase-1表达变化。结果显示：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、TUNEL阳性和PI阳性细胞率显著增加，且呈浓度依赖性，并可促进IL-1β和IL-18分泌。AGE-alb显著上调NLRP3表达和caspase-1活性，尤其在4和6 g/L浓度时更为明显。然而，MCC950预处理可抑制AGE-alb所诱导的RAW264.7巨噬细胞活力降低、LDH漏出、TUNEL阳性和PI阳性细胞率增加以及IL-1β和IL-18分泌，并可抑制AGE-alb所致的caspase-1活化。以上结果表明，AGE-alb可诱导RAW264.7巨噬细胞焦亡，其机制至少部分是通过激活NLRP3-caspase-1信号途径实现的。
关键词： 晚期糖基化白蛋白; 核苷酸结合寡聚化结构域样受体蛋白3; aspase-1; 巨噬细胞; 焦亡
分类号：R332;R363.2;R329.2

Advanced glycated albumin induces macrophage pyroptosis via upregulating nucleotide-binding oligomerization domain-like receptor protein 3
ZHANG Zhao-Qiang¹, YANG Yi-Fan², YAN Jing-Rui¹, YU Fei¹, WANG Xiao-Xu¹, WANG Zhi-Chao³, TIAN Hua⁴, YAO Shu-Tong^1,*
1College of Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271000, China；²College of Pharmacy, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271000, China；³College of Nursing, Shandong First Medical University & Shandong Academy of Medical ciences, Taian 271000, China；⁴Institute of Atherosclerosis, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271000, China
Abstract
The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 μmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1β (IL-1β) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner.Additionally, AGE-alb promoted IL-1β and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1β and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.
Key words: advanced glycated albumin; nucleotide-binding oligomerization domain-like receptor protein 3; caspase-1; Macrophage; pyroptosis

收稿日期：2019-05-14　　录用日期：2019-09-18
通讯作者：姚树桐　　E-mail: yst228@126.com
DOI: 10.13294/j.aps.2019.0072
引用本文：
张昭强, 杨一帆, 闫京锐, 于飞, 王晓旭, 王志超, 田华, 姚树桐. 晚期糖基化白蛋白通过上调核苷酸结合寡聚结构域样受体蛋白3诱导巨噬细胞焦亡[J]. 生理学报 2019; 71 (6): 846-854.
ZHANG Zhao-Qiang, YANG Yi-Fan, YAN Jing-Rui, YU Fei, WANG Xiao-Xu, WANG Zhi-Chao, TIAN Hua, YAO Shu-Tong. Advanced glycated albumin induces macrophage pyroptosis via upregulating nucleotide-binding oligomerization domain-like receptor protein 3. Acta Physiol Sin 2019; 71 (6): 846-854 (in Chinese with English abstract).



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