杜鹃花总黄酮对缺血/再灌注损伤模型大鼠脑基底动脉TRPV4的作用研究

摘要

目的 探讨杜鹃花总黄酮(TFR)对缺血/再灌注损伤模型大鼠脑基底动脉(CBA)瞬时感受器电位通道香草酸受体亚型Ⅳ(TRPV4)的作用.方法 以改良四血管阻断法(4-VO)建立大鼠缺血/再灌注模型(IR);运用加压灌注法和细胞膜电位记录法,离体观察TFR对KCl预收缩IR大鼠CBA的舒张作用和超极化反应及TRPV4阻断剂钌红(RR)对其的影响;采用实时荧光定量PCR法和蛋白质印迹法,在体观察TFR对IR大鼠脑血管内皮细胞TRPV4 mRNA和蛋白表达以及RR对其的影响.结果 递增浓度的TFR可诱导IR大鼠离体CBA产生明显剂量依赖性的舒张效应和超极化反应.去除血管内皮细胞后,TFR仍能介导CBA产生较弱的舒张作用和超极化反应,与血管内皮完整组比较,差异有显著性(P<0.01);去除一氧化氮(NO)和前列环素(PGI2)舒张作用后,TFR仍能诱导IR大鼠CBA产生明显的舒张效应和超极化作用,此作用可被TRPV4通道阻断剂RR抑制;TFR可明显上调IR大鼠脑血管TRPV4 mRNA和蛋白表达,而阻断TRPV4可明显抑制TFR上调的TRPV4基因表达.结论 TFR能介导IR大鼠CBA产生较强的内皮依赖性和较弱的内皮非依赖性血管舒张反应和超极化作用,其内皮依赖性效应推测可能与TFR促使脑血管内皮激活,促进内皮细胞生成和释放内皮衍生性超极化因子(EDHF)增多,继而激活TRPV4,引发Ca2+内流,导致血管平滑肌细胞膜超极化,产生血管舒张效应有关.%Aim To research the effects of total flavones of rhododendra(TFR)on transient receptor potential vanilloid receptor 4(TRPV4)in cerebral basilar arteries(CBA)of rats subjected to ischemia/reperfusion(IR)injury.Methods The model of total brain IR was established by four-artery occlusion(4-VO)method in rats.Arterial pressure perfusion and cell membrane potential recording methods were used for surveying the dilatation and hyperpolarization of TFR and ruthenium red(RR,an inhibitor of TRPV4)in the KCl-preconstricted CBA ex vivo in rats subjected to IR.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot were utilized to investigate the TRPV4 mRNA and protein expressions of TFR and RR in cerebrovascular endothelial cells of CBA in vivo in rats subjected to IR.Results 11～2 700 mg·L-1 TFR significantly induced concentration-dependent hyperpolarization and dilatation in the KCl-preconstricted CBA in rats subjected to IR.TFR still produced degenerative hyperpolarization and dilatation by removal of endothelium in CBA,which was remarkably attenuated as compared with endothelium-intact group(P<0.01).After removal of NO and PGI2 vasodilatation,TFR obviously elicited the hyperpolarization and dilatation that were further decreased by RR(an inhibitor of TRPV4)in IR CBAs.TFR pretreatment apparently increased the level of TRPV4 mRNA and protein expressions in IR CBAs.These effects were restrained by RR,an inhibitor of TRPV4.Conclusions TFR could mediate endothelium-dependent and endothelium-independent effects.The endothelium-derived dilatation may be related to the increase of endothelium activity and endothelium-derived hyperpolarizing factor(EDHF)generation and release that have been promoted by TFR,and secondarily activating TRPV4,which results in Ca2+ inflow and subsequent hyperpolarization of vascular smooth muscle cell membrane and vasorelaxation.