杜鹃素下调Cx43改善尼古丁致C57BL/6J小鼠大脑中动脉损伤的机制

The mechanism of farrerol improving middle cerebral arterial injury induced by nicotine in C57BL/6J mice through down-regulating Cx43

目的研究杜鹃素下调Cx43保护尼古丁致C57BL/6J小鼠大脑中动脉损伤的机制。方法40只C57BL/6J小鼠随机分为5组(每组8只),空白对照组、尼古丁干预组(0.3 mg/kg·bw·d尼古丁,腹腔注射)、杜鹃素低剂量组(0.3 mg/kg·bw·d尼古丁,腹腔注射+12.5 mg/kg·bw·d杜鹃素,灌胃)、杜鹃素中剂量组(0.3 mg/kg·bw·d尼古丁,腹腔注射+25 mg/kg·bw·d杜鹃素,灌胃)和杜鹃素高剂量组(0.3 mg/kg·bw·d尼古丁,腹腔注射+50 mg/kg·bw·d杜鹃素,灌胃),造模24周;利用DMT急性离体肌张力测定法,检测并比较血管收缩剂TXA2类似物(9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α,U46619)和苯肾上腺素(Phenylephrine,PE)对各组小鼠离体大脑中动脉的收缩反应;苏木素-伊红(HE)染色观察比较各组小鼠大脑中动脉形态学变化;急性分离各组小鼠大脑中动脉平滑肌细胞,全细胞膜片钳记录大脑中动脉平滑肌细胞的L-型电压依赖性钙电流(L-type voltage-gated calcium currens,LVGC);并分别利用RT-PCR和Western blot检测各组小鼠大脑中动脉Cx43的mRNA和蛋白表达情况。结果肌张力结果显示,与空白对照组相比较,U46619和PE对尼古丁组离体大脑中动脉的收缩均明显增高,差异有统计学意义(P<0.05);使用杜鹃素干预可减弱U46619和PE对尼古丁组大脑中动脉的收缩幅度,其减弱效果随补充杜鹃素浓度的增大而增强。HE染色形态学观察发现,与空白对照组相比较,尼古丁组大脑中动脉管壁明显增厚,且管腔狭窄明显;补充杜鹃素可浓度依赖性改善尼古丁组大脑中动脉病变程度。膜片钳结果显示,与空白对照组相比较,尼古丁组小鼠大脑中动脉平滑肌细胞LVGC电流的最大电流密度明显增大,差异有统计学意义(P<0.05);补充杜鹃素可浓度依赖性抑制其最大电流密度。RT-PCR和Western blot结果显示,与空白对照组相比,尼古丁组小鼠大脑中动脉Cx43的mRNA和蛋白表达明显增高,差异有统计学意义(P<0.05),补充杜鹃素可浓度依赖性抑制其增高幅度。结论杜鹃素可改善尼古丁致C57BL/6J小鼠大脑中动脉的损伤程度,其机制可能与杜鹃素下调Cx43的表达和/或功能有关。

Objective To study the mechanism of farrerol down-regulating Cx43 to protect nicotine-induced middle cerebral artery dysfunction in C57 BL/6 J mice.Methods Forty C57 BL/6 J mice were randomly divided into five groups(eight mice in each group),control group,nicotine intervention group(0.3 mg/kg·bw·d nicotine,ip),farrerol low-dose group(0.3 mg/kg·bw·d nicotine,ip+12.5 mg/kg·bw·d farrerol,ig),farrerol medium-dose group(0.3 mg/kg·bw·d nicotine,ip+25 mg/kg·bw·d farrerol,ig)and farrerol high-dose group(0.3 mg/kg·bw·d nicotine,ip+50 mg/kg·bw·d farrerol,ig).Model preparation time is 24 weeks.DMT was used in vitro to detect and compare the contraction responses of isolated middle cerebral arteries of mice in each group to vasoconstrictors(U46619 or PE).HE staining was used to observe and compare the morphological changes of middle cerebral arteries of mice in each group.The middle cerebral arterial smooth muscle cells of mice were acutely isolated,then the L-type voltage-gated calcium currents were recorded by patch clamp.RT-PCR and Western blot were used to detect the mRNA and protein expression of Cx43 in middle cerebral arteries of mice in each group.Results The result of middle cerebral arterial muscle tension showed that compared with the control group,the isolated middle cerebral arteries were more sensitive to the vasoconstrictor TXA2 analogs(9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α)U46619 and Phenylephrine(PE),the maximum contraction was significantly increased,and the difference was statistically significant(P<0.05).Farrerol can inhibit the contraction amplitude of middle cerebral arteries to U46619 and PE,and its inhibitory effect was enhanced with the increase of farrerol concentration.Morphological observation of HE staining showed that compared with the control group,the isolated middle cerebral artery wall was significantly thickened,and the lumen narrowed significantly after nicotine intervention for 24 weeks.Farrerol could concentration-dependently improve the degree of lesions.Acute isolation of middle cerebral arterial smooth muscle cells,and the L-type voltage-gated calcium currents was recorded by patch clamp.The result showed that,compared with the control group,the maximum current density of L-type voltage-gated calcium currents in middle cerebral arterial smooth muscle cells of mice was significantly increased after nicotine intervention for 24 weeks,and the difference was statistically significant(P<0.05).And its maximum current density was inhibited by farrerol in a concentration-gated manner.RT-PCR and Western blot result showed that compared with the control group,the mRNA and protein expressions of Cx43 in the middle cerebral arteries of the nicotine group were significantly increased,and the difference was statistically significant(P<0.05).Farrerol can inhibit the increased amplitude in a concentration-dependent manner.Conclusion Farrerol could protect nicotine-induced injury in middle cerebral artery function of C57 BL/6 J mice,and the mechanism may be related to the down-regulation of Cx43.