Nod样受体蛋白3炎性体在2型糖尿病脑微血管内皮细胞中的变化及变化机制

doi:10.6040/j.issn.1671-7554.0.2016.1591

摘要/Abstract

摘要： 目的探讨Nod样受体蛋白3(NLRP3)炎性体在2型糖尿病脑微血管内皮中的变化及机制。方法 3月龄Wistar大鼠和自发性2型糖尿病(GK)大鼠各5只,采用免疫组织化学法观察NLRP3在脑组织中的表达;体外培养小鼠脑微血管内皮细胞(CMEC)。不同糖浓度培养基模拟2型糖尿病内环境,活性氧(ROS)阻断剂N-乙酰半胱氨酸(NAC)作为抑制剂,将细胞分为对照组(糖浓度5.6 mmol/L)、高糖1组(HG1组,培养基糖浓度10 mmol/L)、高糖2组(HG2组,培养基糖浓度20 mmol/L)、高糖3组(HG3,培养基糖浓度30 mmol/L)及高糖+NAC组(HG+NAC组,目的蛋白表达较明显组的糖浓度)。采用Western blotting法检测各组硫氧还蛋白交互蛋白(TXNIP)和NLRP3的表达,并对TXNIP和NLRP3是否存在相关性进行分析;采用ELISA法检测白介素1β(IL-1β)的含量;采用流式细胞术检测对照组、HG3组、HG+NAC组ROS的含量。结果 NLRP3在脑微血管壁聚集,与Wistar大鼠相比,GK大鼠的阳染强度,阳染血管数均有增加(P<0.05);与对照组相比,HG1组、HG2组、HG3组TXNIP、NLRP3的表达增加(P<0.01),HG3组最明显,细胞内IL-1β水平增高(P<0.01),ROS的含量增加(P<0.01);细胞内TXNIP和NLRP3的表达呈正相关性(r=0.993;P<0.05);给予ROS清除剂后,与HG3组相比,HG+NAC组细胞内ROS含量下降(P<0.01),TXNIP、NLRP3表达水平下降(P<0.01),细胞内IL-1β水平下降(P<0.01)。结论 NLRP3在2型糖尿病脑微血管内皮细胞中经ROS-TXNIP途径被激活,并且促进炎性因子IL-β的释放,在2型糖尿病脑微血管损伤中发挥重要作用。

关键词: Nod样受体蛋白3炎性体, 2型糖尿病, 脑微血管内皮细胞, 硫氧还蛋白交互蛋白, 活性氧

Abstract: Objective To investigate the expression changes and mechanism of Nod-like receptor protein 3(NLRP3)inflammasome in cerebral microvascular endothelial cells of type2 diabetes mellitus. Methods A total of 3-month old Wistar rats(n=5)and spontaneous type 2 diabetic rats(GK, n=5)were enrolled. The expression of NLRP3 in the brain tissues was observed with immunohistochemical method. After cerebral microvascular endothelial cells(CMECs)were cultivated in vitro, they were divided into 5 groups: normal control group, high glucose group 1(HG1 group, glucose concentration=10 mmol/L), high glucose group 2(HG2 group, glucose concentration=20 mmol/L), high 山东大学学报 (医学版)55卷3期 -李帅,等.Nod样受体蛋白3炎性体在2型糖尿病脑微血管内皮细胞中的变化及变化机制 \=-glucose group 3(HG3 group, glucose concentration=30 mmol/L), and HG+NAC group. The protein expressions of thioredoxin interacting protein(TXNIP)and NLRP3 were detected with Western blotting. The relationship between NLRP3 and TXNIP was analyzed. The content of interleukin-1β(IL-1β)was evaluated with ELISA kits. The content of intracellular reactive oxygen species(ROS)was measured with flow cytometry. Results NLRP3 gathered in microvascular endothelial cells. Compared with Wistar rats, GK rats had increased strength and number of vascular with positive staining(P<0.05). Compared with normal control group, HG1, HG2 and HG3 groups had higher expressions of TXNIP and NLRP3(P<0.01), especially in the HG3 group, in which the level of IL-1β and ROS also increased (P<0.01). TXNIP and NLRP3 was positively correlated(r=0.993; P<0.05). Lompared with HG3 group, in the HG+NAC group, the level of intracellular ROS, TXNIP, NLRP3 and IL-1β decreased(P<0.01). Conclusion NLRP3 inflammasome is activated in type 2 diabetes mellitus by ROS-TXNIP pathway. It promotes the release of inflammatory factor IL-1β and plays an important role in the microvascular injury.

Key words: Type 2 diabetes mellitus, Reactive oxygen species, Nod-like receptor protein 3 inflammasome, Cerebral microvascular endothelial cells, Thioredoxin interacting protein

中图分类号:

李帅,王雅琳,孙忠文,朱梅佳. Nod样受体蛋白3炎性体在2型糖尿病脑微血管内皮细胞中的变化及变化机制[J]. 山东大学学报（医学版）, 2017, 55(3): 6-11.

LI Shuai, WANG Yalin, SUN Zhongwen, ZHU Meijia. Expression changes and mechanism of Nod-like receptor protein 3 inflammasome in the cerebral microvascular endothelial cells in type 2 diabetes mellitus[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2017, 55(3): 6-11.

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