杜鹃素通过增强Kv1.5和Kv2.1表达抑制尼古丁所致的肺动脉平滑肌细胞增殖  

Farrerol inhibits nicotine-induced proliferation of pulmonary vascular smooth muscle cells by enhancing Kv1.5 and Kv2.1 expression

作　　者：侯晓敏[1] 张明升 秦小江[2] HOU Xiao-min;ZHANG Ming-sheng;QIN Xiao-jiang(Shanxi Medical University School of Basic Medicine,Taiyuan 030001 , China;School of Public Health,Taiyuan 030001 , China)

机构地区：[1]山西医科大学基础医学院,山西太原030001 [2]山西医科大学公共卫生学院,山西太原030001

出　　处：《中国病理生理杂志》2019年第7期1276-1282,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81803282);山西省高等学校科技创新项目(No.2017146;No.2017147);山西省青年科技研究基金资助项目(No.201701D221247;No.201701D221259);山西医科大学博士启动基金资助项目(No.03201510;No.03201521);山西医科大学青年基金资助项目(No.02201604;No.02201613);山西医科大学教育教学改革创新项目(No.XJ2018003);山西医科大学校级教育教学改革研究课题(No.2017-8)

摘　　要：目的:研究杜鹃素(farrerol,Far)对尼古丁所致的大鼠肺动脉平滑肌细胞(pulmonary artery vascular smooth muscle cells,PASMCs)增殖的作用,并进一步探讨其与电压依赖性钾通道(voltage-dependent potassium channels,Kv)1.5和Kv2.1的关系。方法:首先应用细胞计数法检测不同浓度尼古丁对PASMCs细胞数量的影响,筛选出最适的尼古丁浓度。将PASMCs随机分为5组,分别为:正常对照组、尼古丁(1μmol/L)组、尼古丁(1μmol/L)+低浓度(10^-6 mol/L)、中浓度(10^-5 mol/L)和高浓度(10^-4 mol/L)Far组。利用细胞凋亡试剂盒检测细胞caspase-3活性,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡率。并进一步应用RT-qPCR和Western blot法分别检测各组细胞Kv1.5和Kv2.1及凋亡相关因子Bcl-2和Bax的mRNA和蛋白水平。结果:细胞计数结果显示,1μmol/L尼古丁能最大限度地增加PASMCs的数量。1μmol/L尼古丁能显著降低PASMCs的caspase-3活性,并增强细胞活力(P<0.01)。杜鹃素(10^-6-10^-4 mol/L)可浓度依赖性地阻断尼古丁的作用。流式细胞术检测显示,与对照组相比,1μmol/L尼古丁可明显诱导PASMCs增殖,降低其凋亡率;而杜鹃素干预后,PASMCs的凋亡率显著高于尼古丁组(P<0.01)。RT-qPCR和Western blot检测结果显示,1μmol/L尼古丁显著抑制PASMCs Kv1.5、Kv2.1和Bax的表达,增加Bcl-2的表达;10^-5μmol/L杜鹃素可以显著逆转尼古丁对PASMCs的作用(P<0.01)。结论:杜鹃素能够对抗尼古丁所引起的大鼠PASMCs caspase-3和Bax抑制及Bcl-2增强,该作用与促进Kv1.5和Kv2.1表达有关。AIM: To study the effect of farrerol(Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells(PASMCs), and further to explore its relationship with voltage-dependent potassium channels(Kv) 1.5 and Kv2.1. METHODS: Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups: normal control group, nicotine(1 μmol/L)group, nicotine(1 μmol/L)+ Far(10^-6 mol/L, 10^-5 mol/L and 10^-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent(P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs(P<0.01). Farrerol at 10^-6~10^-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs(P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group(P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs(P<0.01). Farrerol at 10^-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION: Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.

关 键 词：杜鹃素 尼古丁 肺动脉平滑肌细胞 细胞增殖 细胞凋亡 电压依赖性钾通道 

分 类 号：R541[医药卫生—心血管疾病] R363[医药卫生—内科学]