NLRP3炎症小体在糖化终末产物诱导的小鼠胰岛β细胞损伤中的作用研究  

On the effects of NLRP3 inflammasome on mice pancreatic β-cell damage induced by advanced glycation end products

作　　者：孔祥[1] 苏青[1] 张洪梅[1] 林宁[1] 李晓永[1] 杨震[1] 邓儒元 刘冲霄[1] 金杰[1] 孟广勋[2] Kong Xiang;Su Qing;Zhang Hongmei;Lin Ning;Li Xiaoyong;Yang Zhen;Deng Ruyuan;Liu Chongxiao;Jin Jie;Meng Guangxun(Department of Endocrinology,Xinhua Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200092,China)

机构地区：[1]上海交通大学医学院附属新华医院内分泌科,200092 [2]中国科学院上海巴斯德研究所分子病毒与免疫重点实验室,200031

出　　处：《中华内分泌代谢杂志》2018年第8期690-695,共6页Chinese Journal of Endocrinology and Metabolism

基　　金：国家自然科学基金（81670743、81370935、81600645）;安徽省自然科学基金（1708085MH186）;安徽省高校优秀青年人才支持计划重点项目（gxyqZD2016182） （孑L祥、苏青、张洪梅、林宁、李晓永、畅震、邓儒元、刘冲霄、金杰）;（孟广勋）;

摘　　要：目的探讨NLRP3炎症小体（pyrin domain-containing 3 inflammasome）在糖化终末产物（advanced glycation end products，AGEs）诱导小鼠胰岛β细胞损伤中的作用及机制。方法NLRP3基因敲除和C57BL/6J小鼠腹腔注射AGEs 6周。胰腺组织切片行HE染色及F4/80和NLRP3免疫荧光染色。ELISA法测胰腺组织单核细胞趋化蛋白1（monocyte chemotactic protein 1，MCP-1）和白细胞介素1β（interleukin-1β，IL-1β）含量。AGEs孵育MIN6细胞，抗氧化剂N-乙酰半胱氨酸（N-acetyl-L-cystein，NAC）和腺病毒NLRP3 shRNA干预后，实时荧光定量PCR测NLRP3 mRNA表达，ELISA法测胰岛素及IL-1β水平，流式细胞术测活性氧水平及细胞凋亡。AGEs孵育小鼠腹腔巨噬细胞，NAC和NLRP3基因敲除干预后，比色法测caspase 1活性，ELISA法测IL-1β水平。结果AGEs注射升高小鼠胰腺组织IL-1β和MCP-1蛋白水平，增高胰岛炎评分及NLRP3和F4/80蛋白表达，且胰岛中NLRP3蛋白主要表达于巨噬细胞。NLRP3基因敲除小鼠上述损伤得到改善。AGEs孵育上调MIN6细胞活性氧水平，激活NLRP3炎症小体，增加细胞凋亡，减少胰岛素分泌。NAC改善上述损伤，但NLRP3 shRNA干预对MIN6细胞活性氧水平，细胞凋亡和胰岛素分泌无影响。NAC及NLRP3基因敲除抑制AGEs诱导的小鼠腹腔巨噬细胞NLRP3炎症小体激活。结论NLRP3基因敲除改善AGEs诱导的胰岛β细胞损伤，与AGEs上调MCP-1表达促进巨噬细胞浸润胰岛，并诱导氧化应激，激活巨噬细胞中NLRP3炎症小体，分泌IL-1β损伤胰岛β细胞有关。ObjectiveTo explore the role of the pyrin domain-containing 3 （NLRP3） inflammasome in advanced glycation end products （AGEs）-induced mice pancreatic β-cell damage.MethodsAGEs were administered intraperitoneally for 6 weeks in NLRP3 knockout mice or C57BL/6J mice. Intraperitoneal glucose tolerance test and insulin releasing test were performed. Pancreatic sections were stained with haematoxylin and eosin, or with F4/80 and NLRP3 antibodies. Insulin and pancreatic tissue monocyte chemotactic protein 1 （MCP-1） as well as interleukin-1β （IL-1β） levels were measured with ELISA kits. Expression of MCP-1 protein was determined by western blot. MIN6 cells and mouse peritoneal macrophages cells were treated with AGEs and different interventions （antioxidant NAC, adenovirus NLRP3 shRNA or NLRP3 knockout）. Reactive oxygen species production, NLRP3 mRNA expression, IL-1β secretion, caspase 1 activity, apoptosis and glucose stimulated insulin release were determined.ResultsInjection of AGEs induced an abnormal response to glucose, enhanced the insulitis score, and increased the levels of pancreatic tissue MCP-1 and IL-1β, as well as raised the expression of NLRP3 and F4/80 in pancreatic islet. Remarkably, co-localization of NLRP3 and macrophage marker F4/80 was observed in islet. The damages were improved in NLRP3 knockout mice. After incubation with AGEs, reactive oxygen species production and cell apoptosis was enhanced, NLRP3 inflammasome activated, with glucose-stimulated insulin release impaired in MIN6 cells. NAC treatment alliviated the above damages, but NLRP3 gene silencing had no effect on ROS level, apoptosis, and insulin secretion. Finally NAC treatment and NLRP3 gene knockout inhibited activation of NLRP3 inflammasome induced by AGEs in mouse peritoneal macrophages cells.ConclusionNLRP3 knockout ameliorates the islet β-cell damage induced by AGEs. These effects were associated with AGEs-induced islets macrophage infiltrating by up-regulation of MCP-1 expression, and AGEs-induced activati

关 键 词：糖化终末产物 NLRP3炎症小体 Β细胞损伤 

分 类 号：R587.1[医药卫生—内分泌]