Kv7在尼古丁致小鼠肺动脉肌张力增强中的作用研究

doi:10.12092/j.issn.1009-2501.2026.08.001

中国临床药理学与治疗学 ›› 2026, Vol. 31 ›› Issue (8): 1009-1015.doi: 10.12092/j.issn.1009-2501.2026.08.001

• 基础研究 •

Kv7在尼古丁致小鼠肺动脉肌张力增强中的作用研究

孟欣¹^,²(), 秦小江¹^,²^,³, 郭苏²^,⁴, 满志¹^,², 方梦薇²^,⁴, 任晓霞⁵, 郑志发⁶, 杨灵波⁶, 施熠炜⁷, 侯晓敏²^,⁴^,*()

1. 山西医科大学医学科学院，太原 030001，山西
2. 山西医科大学环境暴露血管疾病研究所，太原 030001，山西
3. 山西医科大学公共卫生学院，太原 030001，山西
4. 山西医科大学基础医学院，太原 030001，山西
5. 中日友好医院呼吸与危重症医学科，北京 100020
6. 山西省白求恩医院心胸外科，太原 030032，山西
7. 山西医科大学第一医院呼吸与危重症医学科，太原 030001，山西

收稿日期:2025-11-06 修回日期:2026-01-08 出版日期:2026-08-26 发布日期:2026-09-09
通讯作者: 侯晓敏 E-mail:13271703717@163.com;xiaominhou@sxmu.edu.cn
作者简介:孟欣，女，硕士研究生，研究方向：微血管病变及防治机制研究。E-mail：13271703717@163.com
基金资助:
国家自然科学基金青年基金项目（82204042）；国家自然科学基金面上项目（82373622）；国家卫生健康委员会尘肺病重点实验室开放课题（NHC202307）；山西省科技创新人才团队专项项目（202304051001038）；山西省科技合作交流专项项目（202204041101022）；山西省高等教育“百亿工程”科技引导专项项目（BYJL067）

Effects of Kv7 channels in nicotine-induced enhancement of pulmonary artery tone in mice

Xin MENG¹^,²(), Xiaojiang QIN¹^,²^,³, Su GUO²^,⁴, Zhi MAN¹^,², Mengwei FANG²^,⁴, Xiaoxia REN⁵, Zhifa ZHENG⁶, Lingbo YANG⁶, Yiwei SHI⁷, Xiaomin HOU²^,⁴^,*()

1. Academy of Medical Sciences, Shanxi Medical University, Taiyuan 030001, Shanxi, China
2. Environmental Exposure Vascular Diseases Institute, Shanxi Medical University, Taiyuan 030001, Shanxi, China
3. School of Public Health, Shanxi Medical University, Taiyuan 030001, Shanxi, China
4. School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, Shanxi, China
5. Department of Respiratory and Critical Care Medicine, China-Japan Friendship Hospital, Beijing 100020, China
6. Department of Cardiothoracic Surgery, Shanxi Bethune Hospital, Taiyuan 030032, Shanxi, China
7. Department of Respiratory and Critical Care Medicine, the First Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi, China

Received:2025-11-06 Revised:2026-01-08 Online:2026-08-26 Published:2026-09-09
Contact: Xiaomin HOU E-mail:13271703717@163.com;xiaominhou@sxmu.edu.cn

摘要/Abstract

摘要：

目的: 探究Kv7通道在尼古丁致小鼠肺动脉肌张力增强中的作用。方法: 麻醉小鼠，迅速取出肺组织置于4 ℃ Krebs-Henseleit（K-H）缓冲液中，显微镜下分离剪成约2 mm肺动脉环备用。激动剂预刺激条件下尼古丁对小鼠离体肺动脉张力的影响：选取基础张力2 mN以上的小鼠肺动脉环，在静息状态下加入去甲肾上腺素（norepinephrine，NE）、苯肾上腺素（phenylephrine，PE）给予预刺激达到平台后，加入不同浓度的尼古丁（10^?11～10^?5 mol/L），记录张力变化，并计算得出其半数有效量（EC₅₀），后续采用EC₅₀浓度作为最佳干预浓度。肺动脉平滑肌细胞（pulmonary artery smooth muscle cells，PASMCs）内Ca²⁺检测：利用酶解法从小鼠肺动脉中分离出新鲜的PASMCs，置于无钙HBSS溶液中，用Ca²⁺荧光探针Fluo-4 AM染色，加入EC₅₀尼古丁对细胞进行刺激并记录，检测尼古丁刺激前后细胞内相对荧光强度的变化。尼古丁体外孵育时间对小鼠肺动脉肌张力的影响：以EC₅₀浓度的尼古丁分别孵育小鼠肺动脉0、6、12、24 h，用NE、PE刺激，比较不同孵育时间对小鼠肺动脉收缩反应的影响。尼古丁孵育后对肺动脉Kv7通道的影响：选用EC₅₀浓度尼古丁体外孵育肺动脉环24 h，加入Kv7通道抑制剂XE991（10^?8～10^?5 mol/L）诱导小鼠离体肺动脉收缩，并记录肺动脉张力的变化。结果: 在NE、PE给予一个轻度预刺激后，尼古丁可呈浓度依赖性地收缩小鼠肺动脉（P<0.01），并计算得到其EC₅₀为10^?8 mol/L。给予10^?8 mol/L浓度尼古丁进行刺激，可显著增加PASMCs细胞内Ca²⁺浓度（P<0.01）。尼古丁（10^?8 mol/L）分别孵育离体肺动脉0、6、12、24 h后，NE、PE诱导的肺动脉收缩程度逐渐上升。其中，以尼古丁（10^?8 mol/L）孵育24 h孵育组的增强效应最为显著（P<0.01），确定24 h作为最佳干预时间。与对照组相比，尼古丁（10^?8 mol/L）孵育24 h后，Kv7抑制剂XE991诱导的肺动脉收缩张力显著减弱（P<0.01）。结论: 尼古丁可致小鼠肺动脉张力升高，其机制可能与抑制Kv7通道有关。

关键词: 尼古丁, 肺动脉, 肌张力, Kv7通道

Abstract:

AIM: To investigate the role of Kv7 channels in nicotine-induced enhancement of pulmonary arterial tension in mice. METHODS: Mice were anesthetized, and lung tissues were quickly removed and placed in 4 ℃ Krebs–Henseleit (K-H) buffer. Pulmonary arteries were dissected under a microscope and cut into rings approximately 2 mm in length. Effect of nicotine on isolated pulmonary artery tension under agonist prestimulation: pulmonary artery rings with a basal tension >2 mN were precontracted with norepinephrine (NE) or phenylephrine (PE) to reach a plateau. Different concentrations of nicotine (10^?11-10^?5 mol/L) were then added, and changes in tension were recorded. The half-maximal effective concentration (EC₅₀) was calculated and used as the optimal intervention concentration for subsequent experiments. Detection of intracellular Ca²⁺ in pulmonary artery smooth muscle cells (PASMCs): fresh PASMCs were isolated from mouse pulmonary arteries using enzymatic digestion and incubated in Ca²-free HBSS solution. Cells were stained with the Ca²⁺-sensitive fluorescent probe Fluo-4 AM, stimulated with EC₅₀ nicotine, and fluorescence intensity before and after stimulation was recorded to evaluate changes in intracellular Ca²⁺ concentration. Effect of incubation time on nicotine-induced pulmonary arterial tension: pulmonary artery rings were incubated with EC₅₀ nicotine for 0, 6, 12, and 24 h, respectively, followed by NE or PE stimulation. The contractile responses at different incubation times were compared. Effect of nicotine incubation on Kv7 channel function: Pulmonary artery rings were incubated with EC₅₀ nicotine for 24 h, then treated with the Kv7 channel inhibitor XE991 (10^?8-10^?5 mol/L). The induced contractions were recorded to assess Kv7 channel activity. RESULTS: After mild prestimulation with NE or PE, nicotine induced concentration-dependent contractions of mouse pulmonary arteries (P<0.01), with an EC₅₀ of 10^?8 mol/L. Nicotine stimulation at 10^?8 mol/L significantly increased intracellular Ca²⁺ levels in PASMCs (P<0.01). Nicotine (10^?8 mol/L) incubation for 0, 6, 12, and 24 h progressively enhanced NE- and PE-induced contractions, with the 24 h group showing the most pronounced effect (P<0.01), which was selected as the optimal intervention duration. Compared with the control group, nicotine (10^?8 mol/L, 24 h) incubation significantly attenuated XE991-induced pulmonary artery contraction (P<0.01). CONCLUSION: Nicotine increases pulmonary arterial tension in mice, and the underlying mechanism may be related to the inhibition of Kv7 channels.

Key words: nicotine, pulmonary artery, vascular tension, Kv7 channel

中图分类号:

R971.1
R969

孟欣, 秦小江, 郭苏, 满志, 方梦薇, 任晓霞, 郑志发, 杨灵波, 施熠炜, 侯晓敏. Kv7在尼古丁致小鼠肺动脉肌张力增强中的作用研究[J]. 中国临床药理学与治疗学, 2026, 31(8): 1009-1015.

Xin MENG, Xiaojiang QIN, Su GUO, Zhi MAN, Mengwei FANG, Xiaoxia REN, Zhifa ZHENG, Lingbo YANG, Yiwei SHI, Xiaomin HOU. Effects of Kv7 channels in nicotine-induced enhancement of pulmonary artery tone in mice[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2026, 31(8): 1009-1015.

图/表 5

图 1

Fig.1 Isolation and mounting of mouse pulmonary arteries A: mouse in supine position; B: exposure of the thoracic cavity; C: mouse lung tissue; D: observation of the lung under microscope after removal of veins; E: pulmonary artery during the separation process; F: complete isolation of the pulmonary artery; G: pulmonary artery cut into vascular rings; H: vascular rings suspended in the bath of the microvascular tension recording system.

图 2

Fig.2 Concentration-dependent contraction of mouse pulmonary arteries induced by nicotine following NE or PE pre-stimulation ($\bar x $±s, n=6) A-D: representative tension tracings of mouse pulmonary arteries exposed to concentration-gradient stimulation with vehicle (A,C) or nicotine (B, D) following pre-stimulation with 10−9 mol/L NE(A,B) or 3×10−10 mol/L PE (C,D). E,F: concentration–response curves of nicotine-induced contraction in mouse pulmonary arteries pre-stimulated with 10−9 mol/L NE (E) or 3×10−10 mol/L PE(F). cP<0.01, compared with control group.

图 3

Fig.3 Nicotine-induced changes in intracellular calcium concentration in PASMCs ($\bar x $±s, n=20) A: (a,d) acutely isolated mouse PASMCs by enzymatic digestion. (b,e) cells stained with Fluo-4 AM calcium fluorescent probe for 30 minutes. (c,f) after staining, cells were stimulated with 10−8 mol/L nicotine or an equal volume of vehicle and observed under a 20×objective. B: quantitative analysis of relative fluorescence intensity changes in PASMCs induced by 10−8 mol/L nicotine. cP<0.01, compared with control group.

图 4

Fig.4 Effect of incubation time with nicotine on the tension of mouse pulmonary artery ($\bar x $±s, n=20) A: representative tension tracings of mouse pulmonary arteries after incubation with nicotine at EC50(10−8 mol/L) for 0, 6, 12, or 24 h, followed by stimulation with NE or PE. (B,C): quantitative analysis of pulmonary artery tension induced by NE or PE after different incubation times with nicotine (10−8 mol/L); bP<0.05, cP<0.01, compared with control group.

图 5

Fig.5 Nicotine-induced contraction of mouse pulmonary arteries and its association with Kv7 channels ($\bar x $±s, n=6) A,B: Representative tension tracings of mouse pulmonary arteries incubated for 24 h with vehicle or 10−8 mol/L nicotine, followed by concentration-gradient stimulation with XE991. C: Quantitative analysis of pulmonary artery contraction induced by XE991 after 24 h incubation with vehicle or 10−8 mol/L nicotine. cP<0.01, compared with control group.

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Kv7在尼古丁致小鼠肺动脉肌张力增强中的作用研究

Effects of Kv7 channels in nicotine-induced enhancement of pulmonary artery tone in mice

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