内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

doi:10.12047/j.cjap.5611.2018.066

中国应用生理学杂志 ›› 2018, Vol. 34 ›› Issue (3): 283-288.doi: 10.12047/j.cjap.5611.2018.066

• 研究论文 • 上一篇

内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

汪晨净¹, 裴淑燕¹, 南晓东², 马艳庆¹

1. 西北民族大学医学院机能教研室, 甘肃兰州 730030;
2. 武警甘肃总队医院重症医学科, 兰州 730050

收稿日期:2017-06-08 修回日期:2017-11-17 出版日期:2018-05-28 发布日期:2018-09-08
通讯作者: 汪晨净,Tel:18909401229;E-mail:1971918893@qq.com E-mail:1971918893@qq.com
基金资助:
国家自然科学基金资助项目（81360490）；甘肃省自然科学基金资助项目（1010RJZA079）

Effects of endothelin-1 on monocyte chemotactic protein-1 production in rat vascular smooth muscle cells and its mechanism

WANG Chen-jing¹, PEI Shu-yan¹, NAN Xiao-dong², MA Yan-qing¹

1. Function Teaching and Research Section, Medical College of Northwest Nationality University, Lanzhou 730030;
2. Department of Intensive Care Unit, Gansu Provincial Corps Hospital of Chinese People's Armed Police Force, Lanzhou 730050, China

Received:2017-06-08 Revised:2017-11-17 Online:2018-05-28 Published:2018-09-08
Supported by:
国家自然科学基金资助项目（81360490）；甘肃省自然科学基金资助项目（1010RJZA079）

摘要/Abstract

摘要： 目的：观察内皮素-1（ET-1）对大鼠血管平滑肌细胞（VSMCs）产生单核细胞趋化蛋白-1（MCP-1）的影响及其机制。方法：培养大鼠血管平滑肌细胞（VSMCs）。细胞分为2组：ET-1刺激组：以不同浓度ET-1刺激VSMCs不同时间；阻断剂干预组：VSMCs分别与不同阻断剂[ET_AR、ET_BR阻断剂BQ123、BQ788，抗氧化剂N-乙酰半胱氨酸（NAC），ERK、p38MAPK、JNK及NF-κB抑制剂PD98059、SB203580、SP600125及PDTC]预先孵育30 min，再加入ET-1刺激24 h。在预定时间，以酶联免疫吸附（ELISA）法、逆转录聚合酶链反应（RT-PCR）法分别测定不同因素下VSMCs MCP-1蛋白质及mRNA表达量。VSMCs分别与不同阻断剂（BQ123、BQ788、NAC、PD98059、SB203580及SP600125预先孵育20 min，再加入ET-1刺激5 min，免疫印迹（WB）法测定VSMCs胞浆中细胞外调节蛋白激酶（ERK）、p38丝裂原活化蛋白激酶（p38MAPK）、c-Jun氨基末端激酶（JNK）及其各自磷酸化蛋白质的水平。各项检测均重复3次。结果：ET-1能刺激VSMCs MCP-1蛋白质及mRNA表达，其表达量随ET-1浓度及刺激时间的增加呈升高趋势（P<0.05，P<0.01）；BQ123、NAC、PD98059、SB203580及PDTC能显著抑制ET-1诱导的大鼠VSMCs MCP-1蛋白质及mRNA表达（P<0.01），而BQ788及SP600125对此作用无明显影响。BQ123、NAC与PD98059或SB203580能分别抑制ET-1刺激后VSMCs胞浆内ERK及p38MAPK的磷酸化（P<0.05，P<0.01），而ET-1对JNK的磷酸化无明显激活作用。结论：ET-1通过ET_AR、ROS、ERK、p38MAPK及NF-κB诱导大鼠VSMCs产生MCP-1。

关键词: 内皮素-1, 单核细胞趋化蛋白-1, 炎症, 动脉粥样硬化, 血管平滑肌细胞, 大鼠

Abstract: Objective: To observe the effects of endothelin-1 (ET-1) on monocyte chemotactic protein-1 (MCP-1) generation and the primary mechanisms in rat vascular smooth muscle cells (VSMCs).Methods: Rats VSMCs were cultured and divided into two groups:ET-1 group and inhibitor group (the latter including BQ123+ET-1, BQ788+ET-1, NAC+ET-1, PD98059+ET-1, SB203580+ET-1, SP600125+ET-1 group). ①The ET-1 group was stimulated with different concentration of ET-1 for indicated time. The VSMCs in inhibitor groups were incubated with various inhibitor, including BQ123 and BQ788 (ET_A and ET_B receptor antagonist), antioxidant NAC (N-acetyl cysteine), PD98059 (ERK inhibitor), SB203580 (p38MAPK inhibitor), SP600125 (JNK inhibitor), and PDTC (NF-κB inhibitor) for 30 minutes, then incubated with ET-1 for 24 hours. At indicated time, concentration of MCP-1 protein and expression of MCP-1 mRNA were determined by ELISA and RT-PCR respectively. ②The VSMCs were incubated with inhibitors (including BQ123, BQ788, NAC, PD98059, SB203580, and SP600125 for 20 minutes, then incubated with ET-1 for 5 minutes. The levels of ERK, p38MAPK, JNK, and corresponding phosphorylated protein of them (i.e. p-ERK, p-p38MAPK, p-JNK) were determined by Western blot. Each assay was repeated by three independent experiments.Results: The results showed that ET-1 could stimulate MCP-1 generation in VSMCs both in protein and in mRNA levels in concentration-dependence manner(P<0.05, P<0.01). BQ123, NAC, PD98059, SB203580 and PDTC, but not BQ788 and SP600125, could inhibite ET-1-stimulated protein and mRNA expressions of MCP-1 in VSMCs (P<0.01). In addition, BQ123, NAC, and PD98059 or SB203580 could suppress ET-1 induced ERK and p38MAPK activation respectively (P<0.05, P<0.01), but there was no increase of phospho-JNK in ET-1 treated VSMCs.Conclusion: The results demonstrate that ET-1 induces MCP-1 production in VSMCs via ET_A receptor and subsequent ROS, ERK, p38MAPK, NF-κB signal pathway.

Key words: endothelin-1, monocyte chemotactic protein-1, inflammation, atherosclerosis, vascular smooth muscle cells, rat

汪晨净, 裴淑燕, 南晓东, 马艳庆. 内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制[J]. 中国应用生理学杂志, 2018, 34(3): 283-288.

WANG Chen-jing, PEI Shu-yan, NAN Xiao-dong, MA Yan-qing. Effects of endothelin-1 on monocyte chemotactic protein-1 production in rat vascular smooth muscle cells and its mechanism[J]. CJAP, 2018, 34(3): 283-288.

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内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

Effects of endothelin-1 on monocyte chemotactic protein-1 production in rat vascular smooth muscle cells and its mechanism

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