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《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:

2017年第3期

页码:

253-258

栏目:

基础研究

出版日期:

2017-01-25

文章信息/Info

Title:

Effects and mechanism of ROS/NF-κB signaling on MuRF1-mediated BK-β1 degradation on aortic smooth muscle in diabetic mice

作者:

吴 宾¹; 樊苗苗¹; 郭 涛¹; 胡立中²; 陶 凌¹; 袁 铭¹; 易 甫¹

第四军医大学：1.西京医院心血管内科，2.药学院药剂学教研室，陕西 西安 710032

Author(s):

WU Bin¹;  FAN Miao-miao¹;  GUO Tao¹;  HU Li-zhong²;  TAO Ling¹;  YUAN Ming¹;  YI Fu¹

1.Department of Cardiology, Xijing Hospital; 2.Dpeartment of Pharmacy, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, Shaanxi, China

关键词:

大电导钙激活钾通道; 糖尿病; 肌肉环指蛋白1; 活性氧簇; 核因子-κB

Keywords:

large conductance Ca2+-activated K+ channel;  diabetes;  muscle RING finger protein 1;  reactive oxygen species;  NF-κB

分类号:

R587.1

DOI:

-

文献标识码:

A

摘要:

目的 探讨糖尿病小鼠主动脉平滑肌活性氧簇（ROS）/核因子-κB（NF-κB）信号通路对肌肉环指蛋白（Muscle RING finger，MuRF）1介导大电导钙激活钾通道β1亚基（BK-β1）降解的调控机制。方法 腹腔注射链脲霉素制备1型糖尿病小鼠模型，将雄性C57/BL6J小鼠分为6组（每组15只）：对照组，对照+N-乙酰半胱氨酸（NAC，ROS清除剂）组，对照+吡咯烷二硫代氨基甲酸盐（PDTC，NF-κB抑制剂）组，糖尿病组，糖尿病+NAC组，糖尿病+PDTC组。对照+NAC组和糖尿病+NAC组给予NAC 100 mg/（kg·d）腹腔注射治疗，对照+PDTC组和糖尿病+PDTC组给予PDTC 50 mg/（kg·d）腹腔注射治疗，对照组和糖尿病组给予同等体积的生理盐水腹腔注射。药物治疗12周后，Western blot检测胸主动脉BK-β1、MuRF1、RelA/p65的表达水平；动脉血管舒张反应性实验检测胸主动脉血管环对BK通道的激动剂NS-1619的反应性；酶消化法急性分离小鼠胸主动脉平滑肌细胞，全细胞膜片钳实验技术记录小鼠胸主动脉平滑肌细胞BK通道的电流。结果 与对照组、对照+NAC组和对照+PDTC组相比，糖尿病组的BK-β1的表达显著降低（P<0.05），MuRF1和RelA/p65的表达显著增加（P<0.05），胸主动脉血管环对NS-1619的舒张反应性降低（P<0.05），当刺激电压>50 mV时，糖尿病组胸主动脉平滑肌细胞BK通道电流显著降低（P<0.05）；与糖尿病组相比，糖尿病+NAC组和糖尿病+PDTC组的BK-β1的表达显著增加（P<0.05），MuRF1和RelA/p65的表达显著降低（P<0.05），胸主动脉血管环对NS-1619的舒张反应性显著改善（P<0.05），当刺激电压>50 mV时，糖尿病+NAC组和糖尿病+PDTC组胸主动脉平滑肌细胞BK通道电流显著增加（P<0.05）。结论 NAC和PDTC可抑制糖尿病小鼠胸主动脉平滑肌MuRF1介导的BK-β1降解，ROS/NF-κB信号通路可能是参与调控MuRF1介导的BK-β1降解。

Abstract:

AIM To explore the effects and mechanisms of reactive oxygen species (ROS)/NF-κB signaling on muscle RING finger protein 1 (MuRF1)-mediated β1 subunit of the large conductance Ca2+-activated K+ channel (BK-β1) degradation on aortic smooth muscle in diabetic mice. METHODS Diabetes was induced by a single intraperitoneal (i.p.) injection of streptozotocin. Male C57/BL6J mice were randomly divided into six groups (n=15): control group, control+NAC (ROS scavenger ) group, control+PDTC (NF-κB inhibitor) group, diabetes group, diabetes+NAC group, and diabetes+PDTC group. Control+NAC group and diabetes+NAC group were administered a dose of 100 mg/(kg·day) NAC and control+PDTC group and diabetes+PDTC group were given a dose of 50 mg/(kg·day) PDTC. Control group and diabetes group received normal saline injection i.p. After 12 weeks, Western blot was used to determine the expression of BK-β1, MuRF1 and RelA/p65. Concentration-response of NS-1619 (BK channel activator) to vasodilatation was measured by functional vasoreactivity test. Aortic smooth muscle cells were isolated acutely by enzyme digestion and BK currents were recorded by patch clamp technique in whole cell configuration. RESULTS Compared with those in the control group, control+NAC group and control+PDTC group, expressions of BK-β1 decreased significantly in diabetes group (P<0.05), with significantly increased expressions of MuRF1 and RelA/p65 (P<0.05) as well as reduced NS-1619-mediated aortic dilation (P<0.05). There was a reduction of BK current densities when test potential was >50 mV in diabetes group (P<0.05). Compared with those in the diabetes group, protein levels of BK-β1 increased significantly (P<0.05), whereas protein levels of both MuRF1 and RelA/p65 reduced significantly (P<0.05) with restored NS-1619-mediated aortic dilation (P<0.05) and increased BK currents densities (P<0.05) with treatment with NAC and PDTC in diabetes+NAC group and diabetes+PDTC group. CONCLUSIONN AC and PDTC inhibit MuRF1-mediated BK-β1 degradation on aortic smooth muscle, restore the response to NS-1619 and increase BK current densities in diabetic mice. ROS/NFκB signaling may be involved in the regulation of MuRF1-mediated BK-β1 degradation.

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备注/Memo

备注/Memo:

收稿日期：2016-07-19.基金项目：国家自然科学基金项目资助（81470479）；陕西省科技攻关项目资助（S2016YFSF0666） 通讯作者：袁铭，副主任医师，主要从事冠心病的诊治及机制研究 Email：yuanming@fmmu.edu.cn 共同通讯作者：易甫，副主任医师，主要从事心血管电生理研究 Email:yi12fu56@hotmail.com 作者简介：吴宾，硕士生 Email：wubin0917@163.com

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更新日期/Last Update: 2017-02-20