N-乙酰半胱氨酸通路对AGT-REN双转基因高血压小鼠心肌成纤维细胞中电导钙激活钾离子通道蛋白表达的影响及其意义

doi:10.13481/j.1671-587x.20170505

吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (05): 881-886.doi: 10.13481/j.1671-587x.20170505

N-乙酰半胱氨酸通路对AGT-REN双转基因高血压小鼠心肌成纤维细胞中电导钙激活钾离子通道蛋白表达的影响及其意义

王丽萍, 王建辉, 李树民, 杨秀红

华北理工大学基础医学院生理学系, 河北唐山 063000

收稿日期:2016-11-03 出版日期:2017-09-28 发布日期:2017-09-29
通讯作者: 杨秀红,教授,硕士研究生导师(Tel:0315-8805520,E-mail:1508133811@qq.com) E-mail:1508133811@qq.com
作者简介:王丽萍(1977-),女,河北省唐山市人,讲师,医学博士,主要从事心血管疾病和休克方面的研究。
基金资助:
国家自然科学基金资助课题（81600316，81372029）；河北省科技厅自然科学基金资助课题（H2015209153）

Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca²⁺-activated K⁺ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice

WANG Liping, WANG Jianhui, LI Shumin, YANG Xiuhong

Department of Physiology, School of Basic MedicalSciences, North China University of Science and Technology, Tangshan 063210, China

Received:2016-11-03 Online:2017-09-28 Published:2017-09-29

摘要/Abstract

摘要： 目的：探讨高血压过程中心肌成纤维细胞（CFs）氧化应激水平与中电导钙激活钾离子通道（K_Ca3.1）蛋白表达的关系，阐明K_Ca3.1在心肌纤维化中的作用及机制。方法：原代培养雄性AGT-REN双转基因高血压小鼠（dTH） CFs，另培养同品系野生C57B6小鼠CFs作为对照。dTH小鼠CFs随机分为高血压组（dTH）和N-乙酰半胱氨酸组（NAC），dTH小鼠CFs给予不同浓度NAC干预24 h。MTT法检测细胞增殖情况，双氯荧光素（DCFH-DA）探针检测细胞活性氧（ROS）分子表达，Western blotting法检测细胞培养上清collagen Ⅰ、collagen Ⅲ和细胞中K_Ca3.1通道蛋白表达、PI3K信号通道蛋白磷酸化改变。结果：4、8和12月龄dTH小鼠CFs中ROS和K_Ca3.1通道蛋白表达水平与2月龄dTH小鼠比较均增加（P < 0.05或P < 0.01）；MTT检测，应用1×10^-6、1×10^-5、1×10^-4和1×10^-3 mol·L^-1 NAC干预后，dTH小鼠CFs增殖率分别为165.9%、138.72%、110.92%和109.82%，与dTH组比较，1×10^-4和1×10^-3 mol·L^-1NAC对CFs增殖抑制作用明显（P < 0.01）。与对照组比较，1×10^-4 mol·L^-1 NAC组小鼠CFs中Ⅰ、Ⅲ型胶原合成明显减少（P < 0.01）；Western blotting检测，与对照组比较，1×10^-4 mol·L^-1 NAC组小鼠CFs中K_Ca3.1通道蛋白表达水平下降（P < 0.01）；dTH小鼠CFs中p-Akt/T-Akt表达水平较对照组增加（P < 0.01），而1×10^-4 mol·L^-1 NAC组小鼠CFs中p-Akt/T-Akt表达水平下降（P < 0.01）。结论：ROS清除剂NAC抑制dTH高血压小鼠CFs中K_Ca3.1通道蛋白表达，可能与其上调细胞中PI3K信号通道磷酸化水平有关。

关键词: 心肌纤维化, 活性氧, 心肌成纤维细胞, 中电导钙激活钾离子通道

Abstract: Objective: To investigate the relationship between the level of oxidative stress and the expression of intermediate conductance calcium activated potassium channel (K_Ca3.1) protein in the cardiac fibroblasts (CFs) during hypertension process,and to clarify the role of K_Ca3.1 in cardiac fibrosis and its mechanism. Methods: The CFs of male C57B6 and AGT-REN double transgenic hypertension (dTH) mice were cultured and the wild C57B6 mouse CFs were used as control group. The CFs of dTH mice were randomly divided into high blood pressure group (dTH) and N-acetyl cysteine group (NAC). The CFs were treated with different concentrations of NAC for 24 h. The cell proliferation was detected by MTT and double dichlorofluorescein (DCFH-DA) probe was used for the detection of cellular reactive oxygen species (ROS) expression; Western blotting was employed to detect the expressions of collagen Ⅰ, collagen Ⅲ, K_Ca3.1 channel protein and the changes of PI3K signaling pathway protein phosphorylation. Results: The ROS production and protein expression of K_Ca3.1 channel of the dTH mice on 4, 8, 12 months were increased compared with 2 months(P<0.05 or P<0.01); the results of MTT suggested that the proliferation rates of CFs were 165.9%,138.72%,110.92% and 109.82% after administration of 1×10^-6,1×10^-5,1×10^-4 and 1×10^-3 mol·L^-1 NAC in the dTH mice, and 1×10^-4 and 1×10^-3 mol·L^-1 NAC significantly inhibited the proliferation of CFs.Compared with control group,the secretion of collagen Ⅰ and Ⅲ of CFs in the TH mice was decreased in 1×10^-4 mol·L^-1 NAC group(P<0.01).The results of Western blotting showed that compared with control group, the expression level of K_Ca3.1 channel protein in CFs of the TH mice in 1×10^-4 mol·L^-1 NAC group was decreased(P<0.01). Compared with control group,the p-AKT/T-AKt in CFs of the dTH mice was increased (P<0.01); but in NAC group,the p-AKT/T-AKt was lower than that in dTH group (P<0.01). Conclusion: NAC can inhibit the expression of K_Ca3.1 channel protein in CFs of the dTH hypertensive mice, which may be related to increasing the phosphorylation of AKt/PI3K signaling pathway.

Key words: myocardial fibrosis, cardiac fibroblasts, intermediatec onductance Ca²⁺-activated K⁺ channels, reactive oxygen species

中图分类号:

R542.2

王丽萍, 王建辉, 李树民, 杨秀红. N-乙酰半胱氨酸通路对AGT-REN双转基因高血压小鼠心肌成纤维细胞中电导钙激活钾离子通道蛋白表达的影响及其意义[J]. 吉林大学学报(医学版), 2017, 43(05): 881-886.

WANG Liping, WANG Jianhui, LI Shumin, YANG Xiuhong. Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca²⁺-activated K⁺ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice[J]. Journal of Jilin University Medicine Edition, 2017, 43(05): 881-886.

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N-乙酰半胱氨酸通路对AGT-REN双转基因高血压小鼠心肌成纤维细胞中电导钙激活钾离子通道蛋白表达的影响及其意义

Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca²⁺-activated K⁺ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice

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N-乙酰半胱氨酸通路对AGT-REN双转基因高血压小鼠心肌成纤维细胞中电导钙激活钾离子通道蛋白表达的影响及其意义

Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca2+-activated K+ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice

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Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca²⁺-activated K⁺ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice