慢性尼古丁干预抑制Kv1.5致小鼠肺动脉舒缩异常的机制研究

The mechanism of chronic nicotine intervention in inhibition of Kv1.5-induced abnormal systolic and diastolic function of pulmonary artery

目的研究慢性尼古丁干预抑制Kv1.5致C57BL/6J小鼠肺动脉舒缩异常的机制。方法将40只4周龄的C57BL/6J小鼠随机分成4组,每组10只,分别为空白对照组(腹腔注射等量灭菌注射用水)、低浓度尼古丁组(腹腔注射尼古丁0.003 mg/kg·bw/d)、中浓度尼古丁组(腹腔注射尼古丁0.03 mg/kg·bw/d)和高浓度尼古丁组(腹腔注射尼古丁0.3 mg/kg·bw/d),干预24周。利用四通道微血管张力测定仪测定各组小鼠离体肺动脉对氯化钾(KCl)和TXA2类似物(9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α,U46619)的收缩反应,及对硝普钠(Sodium Nitroprusside,SNP)和乙酰胆碱(Acetylcholine,ACh)的舒张反应;使用原代培养的C57BL/6J小鼠肺动脉平滑肌细胞,利用α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)进行免疫荧光鉴定;采用鼠尾胶原收缩法检测各组肺动脉平滑肌细胞对血管紧张素II(Angiotensin II,AngII)的收缩反应;全细胞膜片钳记录各组肺动脉平滑肌细胞Kv电流;RT-PCR和Western blot检测各组肺动脉Kv1.5的mRNA和蛋白表达量。结果血管张力检测结果显示,与空白对照组相比较,尼古丁干预24周可浓度依赖性的增强C57BL/6J小鼠离体肺动脉对KCl和U46619的最大收缩反应,同时尼古丁可浓度依赖性的减弱C57BL/6J小鼠离体肺动脉对SNP和ACh的最大舒张反应,差异有统计学意义(P<0.05)。胶原收缩法结果显示,与空白对照组相比较,不同浓度尼古丁组肺动脉平滑肌细胞对AngII的收缩呈浓度依赖性的增强,差异有统计学意义(P<0.05)。膜片钳结果显示,与空白对照组相比较,不同浓度尼古丁组肺动脉平滑肌细胞Kv电流逐渐减小,其I-V曲线向右并向下移动。RT-PCR和Western blot结果显示,与空白对照组相比较,不同浓度尼古丁组肺动脉Kv1.5的mRNA和蛋白表达呈浓度依赖性的减小,差异有统计学意义(P<0.05)。结论慢性尼古丁干预致C57BL/6J小鼠肺动脉舒缩功能异常,其机制可能与抑制Kv1.5有关。

Objective To study the mechanism of chronic nicotine intervention in inhibiting Kv1.5-induced pulmonary artery diastolic and contraction abnormalities in C57 BL/6 J mice.Methods Forty C57 BL/6 J mice were randomly divided into four groups,which were a blank control group(intraperitoneal injection of the same amount of sterile water),a low-concentration nicotine group(intraperitoneal injection of nicotine 0.003 mg/kg·bw/d),and a medium-concentration nicotine group(intraperitoneal injection of nicotine 0.03 mg/kg·bw/d)and high-concentration nicotine group(intraperitoneal injection of nicotine 0.3 mg/kg·bw/d),lasting for 24-week intervention.A four-channel microvascular tension meter was used to detect the contractile response of the isolated pulmonary arteries of each group of mice to potassium chloride(KCl)and TXA2 analogs 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α(U46619),and the relaxation response to sodium nitroprusside(SNP)and acetylcholine(ACh).Primary cultured C57 BL/6 J mouse pulmonary artery smooth muscle cells were identified by immunofluorescent assay ofα-smooth muscle actin(α-SMA).Collagen contraction method was used to detect the contractile response of each group of pulmonary artery smooth muscle cells to Angiotensin II(AngII).The whole-cell patch clamp was used to record the Kv currents of each group of pulmonary artery smooth muscle cells.RT-PCR and Western blot were used to detect the Kv1.5 mRNA and protein expression of pulmonary artery in each group.Results The result of vascular tension test showed that compared with the control group,nicotine intervention for 24 weeks could increase the maximum contractile response to KCl and U46619 in a concentration-dependent manner,while nicotine concentration-dependently weaken the maximum diastolic response to SNP and ACh.The difference was statistically significant(P<0.05).The collagen contraction method showed that compared with the blank control group,the pulmonary artery smooth muscle cells of different concentrations of nicotine group showed a concentration-dependent enhancement of the contraction to AngII,and the difference was statistically significant(P<0.05).The patch clamp result showed that compared with the control group,the Kv currents of pulmonary artery smooth muscle cells in the nicotine group with different concentrations gradually decreased,and the I-V curve shifted to the right and down.The result of RT-PCR and Western blot showed that compared with the control group,the mRNA and protein expression of pulmonary artery Kv1.5 in different concentrations of nicotine group decreased in a concentration-dependent manner,and the difference was statistically significant(P<0.05).Conclusion Chronic nicotine intervention could cause abnormal diastolic and contractile function of pulmonary artery in C57 BL/6 J mice.The mechanism may be related to the inhibition of Kv1.5.