尼古丁上调Cx43致C57BL/6J小鼠肺动脉肌源性异常实验研究

Nicotine Induced Pulmonary Artery Myogenic Abnormalities in C57BL/6J Mice by Up-Regulating Cx43 Expression:An Experimental Study

目的探讨缝隙连接蛋白connexin 43(Cx43)在尼古丁致C57BL/6J小鼠肺动脉肌源性异常中的作用机制。方法将C57BL/6J小鼠随机分成4组,每组12只,分别为空白对照组(正常饮水、摄食)、0.003 mg/kg尼古丁组[腹腔注射尼古丁0.003 mg/(kg·d)]、0.03 mg/kg尼古丁组[腹腔注射尼古丁0.03 mg/(kg·d)]和0.3 mg/kg尼古丁组[腹腔注射尼古丁0.3 mg/(kg·d)]。干预时间为24周。利用微血管张力测定检测各组小鼠离体肺动脉对不同浓度梯度氯化钾KCl(20、28、39、55、77和108 mmol/L)和U46619(3×10^(-8)、10^(-7)、3×10^(-7)、10^(-6)、3×10^(-6) mol/L)的收缩反应,以及对不同浓度硝普钠SNP(10^(-8)、3×10^(-8)、10^(-7)、3×10^(-7)、10^(-6) mol/L)和吡那地尔Pina(10^(-8)、3×10^(-8)、10^(-7)、3×10^(-7)、10^(-6) mol/L)的舒张反应。HE特染观察各组小鼠肺动脉重构程度。免疫荧光检测各组小鼠肺动脉Cx43表达水平。RT-PCR和Western blot测定小鼠肺动脉Cx43的mRNA和蛋白表达水平。结果离体血管张力测定结果显示,与空白对照组相比较,分别使用0.003、0.03和0.3 mg/(kg·d)尼古丁干预C57BL/6J小鼠24周后,小鼠离体肺动脉对浓度梯度KCl和U46619的最大收缩反应均呈现不同程度的增强(P<0.05);同时小鼠离体肺动脉对浓度梯度SNP和Pina的最大舒张反应却呈现不同程度的减弱(P<0.05)。0.003、0.03和0.3 mg/(kg·d)尼古丁组肺动脉重构程度呈现浓度依赖性增加,且Cx43的免疫荧光蛋白表达均显著高于空白对照组。RT-PCR和Western blot实验结果显示,0.003、0.03和0.3 mg/kg尼古丁组肺动脉Cx43 mRNA和蛋白表达均显著高于空白对照组(P<0.05)。结论尼古丁可造成C57BL/6J小鼠肺动脉肌源性反应异常,其机制可能与上调Cx43表达有关。

Objective To investigate the mechanism of action of connexin 43(Cx43)in nicotine-induced pulmonary artery myogenic abnormalities in C57BL/6J mice.Methods The tested C57BL/6J mice were randomly divided into blank control group(n=12,normal drinking and food intake),0.003 mg/kg nicotine group[n=12,intraperitoneal injection of nicotine at a dose of 0.003 mg/(kg·d)],0.03 mg/kg nicotine group[n=12,intraperitoneal injection of nicotine at a dose of 0.03 mg/(kg·d)],and 0.3 mg/kg nicotine group(n=12,intraperitoneal injection of nicotine at a dose of 0.3 mg/kg·d),with an intervention time of 24 weeks.Microvascular tension test was used to determine the contractile response of isolated mouse pulmonary arteries to different concentrations of KCl(20,28,39,55,77 and 108 mmol/L)and U46619(3×10^(-8),10-7,3×10^(-7),10^(-6) and 3×10^(-6) mol/L),and the vasodilation to different concentrations of SNP(10^(-8),3×10^(-8),10-7,3×10^(-7) and 10^(-6) mol/L)and pinacidil(10^(-8),3×10^(-8),10-7,3×10^(-7) and 10-6 mol/L)in each group.Hematoxylin-eosin staining was used to observe the pulmonary artery remodeling of mice in each group.Immunofluorescence assay was used to measure the expression level of Cx43 in the pulmonary arteries of mice in each group.Reverse transcription polymerase chain reaction(RT-PCR)and Western blot were used to measure the mRNA and protein expression levels of Cx43 in the pulmonary arteries of mice.Results The results of in vitro vascular tension measurement showed that compared with the blank control group,after 24 weeks of intervention,the isolated pulmonary arteries of C57BL/6J mice in the 0.003,0.03,and 0.3 mg/kg nicotine groups showed different degrees of enhancement in the maximal contractile response to different concentrations of KCl and U46619(P<0.05),as wella different degrees of weakening in the maximal vasodilation to different concentrations of SNP and pinacidil(P<0.05).In addition,compared with the blank control group,the 0.003,0.03 and 0.3 mg/kg nicotine groups had concentration-dependent increases in the degree of pulmonary artery remodeling,significantly higher Cx43 protein expression according to the immunofluorescence assay results,and significantly higher mRNA and protein expression of Cx43 according to the RT-PCR and Western blot results(P<0.05).Conclusion Nicotine can cause pulmonary artery myogenic abnormalities in C57BL/6J mice,and its mechanism may be related to the up-regulation of Cx43 expression.