【作者】 张建华；

【导师】 陈志武； 张野；

【作者基本信息】 安徽医科大学 ， 药理学， 2009， 博士

【摘要】 内皮依赖性超极化因子(endothelium-derived hyperpolarizing factor, EDHF)是除NO和PGI2外血管内皮细胞合成分泌的第三种舒张血管的物质。目前,在多种属、多部位的血管中都发现存在EDHF反应,但血管中,尤其是在脑血管中,EDHF到底是哪种物质还不清楚。杜鹃花总黄酮(total flavones of Rhododendra,TFR)是从植物杜鹃花中提取的有效部位,本实验室曾发现TFR的抗心脑血管损伤的作用,本实验拟对其抗心脑血管损伤作用的部分机制进行探讨。目的:1.探讨大鼠基底动脉的EDHF反应;2.研究在大鼠基底动脉中,H2S是否具有EDHF样作用;3. TFR对大鼠基底动脉的扩张作用及其EDHF机制研究;4. TFR抗心肌缺血损伤时对iNOS表达的影响。方法:1.采用离体微血管舒缩功能测定实验,检测ACh对基底动脉的舒张作用、基底动脉的EDHF反应、PPG对EDHF舒张作用的影响、NaHS及L-cys对血管的舒张作用、TFR对血管的舒张作用及其EDHF机制。实验结束后,收集各组血管管腔内灌流液,用以检测H2S的含量。2.细胞膜电位记录实验,研究ACh对大鼠基底动脉平滑肌细胞膜电位的超极化作用及内源性和外源性EDHF的超极化作用。3.原代培养大鼠基底动脉内皮细胞,采用RT-PCR的方法检测内源性合成硫化氢的酶的表达,并观察ACh及TFR对酶表达的影响。4.在体结扎冠脉前降支致心肌缺血损伤模型,TFR预处理给药,检测心肌梗死面积、血清中cTnI、NO含量,western blot方法检测心肌组织中iNOS的蛋白表达水平。结果:1.在离体血管舒缩功能测定实验中,用30 mmol/L的KCl或10-7 mol/L的U46619预收缩的大鼠基底动脉,ACh均可产生浓度依赖性的舒张作用,其最大舒张率分别是80.97±1.27 %和80.84±5.10 %,与溶媒对照组比较差异有显著性(P<0.01);而去除血管内皮后,ACh的舒张作用消失。2.应用NO合酶抑制剂(L-NAME,3×10-5mol/L)和PGI2合酶抑制剂(Indo,10-5mol/L)后,ACh对基底动脉还存在部分舒张作用,其最大舒张率分别是31.51±3.55(KCl)%和38.87±3.48(U46619)%,与溶媒对照组比较差异有显著性(P<0.01);在细胞膜电位记录实验中,应用L-NAME和Indo后,ACh对大鼠基底动脉平滑肌细胞的超极化作用并未被完全抑制,其最大超极化作用为-63.11±4.47 mV,与溶媒对照组比较差异有显著性(P<0.01)。此结果表明,在大鼠基底动脉中,这种非NO、非PGI2、内皮依赖的超极化及舒张血管作用可能为EDHF的作用。3.在离体血管舒缩功能测定实验中发现,10-5-10-2.5mol/L NaHS(H2S外源性供体)可产生浓度依赖性的舒张作用;同时,在细胞膜电位记录实验中也发现,10-5-10-2.5mol/L NaHS具有浓度依赖性超极化血管平滑肌细胞的作用。4.在离体血管舒缩功能测定实验中发现,内源性合成硫化氢的酶胱硫醚-γ-裂解酶(cystathionine-γ-lyase, CSE)的阻断剂DL-炔丙基甘氨酸(DL-propargylglycine, PPG, 10-4mol/L)可以抑制EDHF引起的血管舒张作用,与未用阻断剂组比较差异有显著性(P<0.01);同样,在细胞膜电位记录实验中也发现,PPG可以明显抑制EDHF对基底动脉平滑肌细胞的超极化作用,与不加阻断剂组相比差异有显著性(P<0.05 or P<0.01)。5.在离体血管舒缩功能测定实验中发现,合成硫化氢的底物L-半胱氨酸(L-cysteine,L-cys, 10-5-10-2.5mol/L)可产生浓度依赖性的舒张作用;去除血管内皮后L-cys的舒张作用被部分抑制。6.在离体血管舒缩功能测定实验中,收集血管内腔灌流液,用来检测H2S的含量,结果显示大鼠基底动脉基础状态下就有H2S的产生,其含量为3.92±0.25×10-5 mol/L,用ACh联合L-NAME,Indo灌流结束后H2S的含量有明显的增加,与基础含量比较差异有显著性(P<0.01),而加用PPG(10-4mol/L)预灌后H2S的含量明显降低(P<0.01)。7.在原代培养的大鼠基底动脉内皮细胞中,采用RT-PCR的方法检测合成硫化氢的酶的表达,结果显示,血管内皮细胞中表达CSE而不表达CBS;而且ACh(10-4.5mol/L)可以上调基底动脉内皮细胞中CSE的表达。8.在离体血管舒缩功能测定实验中发现,TFR(0.011～2.7g/L)对大鼠基底动脉具有明显的浓度依赖性舒张作用;去除血管内皮细胞后,TFR的舒张作用被部分抑制;在血管内腔灌流液中用L-NAME(3×10-5mol/L)和Indo(10-5mol/L)预灌30min后,TFR舒张作用被部分抑制。结果表明在TFR舒张基底动脉的作用中除NO和PGI2外,尚有EDHF机制的参与。9.在离体血管舒缩功能测定实验中,用L-NAME(3×10-5mol/L),Indo(10-5mol/L)和PPG(10-4mol/L)同时预灌30min后,TFR舒张作用被部分抑制,与仅用L-NAME和Indo预灌组比较差异有显著性;TFR灌流结束后,血管内腔灌流液中H2S的含量有明显的增加,与基础生成量比较差异有显著性,联合L-NAME, Indo预灌组,H2S的含量较基础生成量也明显增加,而加用PPG预灌后H2S的含量则明显降低。RT-PCR半定量结果显示,TFR(2.7g/L)可以上调大鼠基底动脉内皮细胞CSE的mRNA表达水平,与溶媒对照组比较,差异有显著性(P<0.01)。此结果进一步提示,在TFR扩张基底动脉的机制中有EDHF作用的参与。10.在结扎冠脉前降支致心肌缺血损伤模型中发现,TFR预处理具有明显的抗心肌缺血再灌注损伤的作用,可以降低心肌梗死面积,明显抑制急性心肌梗死造成的血清中cTnI的升高,增加血清中NO含量,上调心肌组织中iNOS的蛋白表达水平。结论:1.大鼠基底动脉中,乙酰胆碱可产生非NO、非PGI2、内皮依赖的超极化及舒张血管作用,即EDHF反应;2.在原代培养的大鼠基底动脉内皮细胞中,有内源性合成的酶胱硫醚-γ-裂解酶(cystathionine-γ-lyase, CSE)的表达,而且CSE的抑制剂可以抑制这种非NO、非PGI2、内皮依赖的超极化及舒张血管作用,同时发现NaHS(H2S外源性供体)具有超极化及舒张血管的作用,提示H2S可能是大鼠基底动脉中的EDHF;3. TFR对大鼠基底动脉具有明显的舒张作用,而且TFR对大鼠基底动脉的扩张作用中有EDHF作用的参与,此作用可能是TFR其抗脑缺血损伤的机制之一;TFR可通过增强iNOS基因的表达,增加NO的水平,从而起到抗心肌缺血损伤的作用。更多还原

【Abstract】 9 Endothelium-dependent hyperpolarizing factor (EDHF) is the third relaxing factor derived from vascular endothelium besides NO and PGI2. At present, EDHF responses are reported in a wide variety of arteries from different species including humans. One purpose of the present article was to evaluate whether or not there is the role of EDHF in the basilar artery in rats. From the proposed EDHF to now, the chemical nature of EDHF was also a puzzle. Especially in cerebral arteries, there are still unkown. Total flavones of Rhododendra (TFR) was extracted from the flower of Cuckoo. The essential components of TFR are quercetin, hyperin and other flavonoids. It has been preliminary assessed to be of protection against myocardial and cerebral ischemia. In the present study the partial mechanism of the effects of TFR against myocardial and cerebral were studied in this paper.Purposes:1. To evaluate whether there was EDHF in the rat basilar arteries.2. To evaluate whether H2S was the chemical nature of EDHF.3. To evaluate the relaxation effects of TFR on rat basilar arteries, and to assess whether the efffects of EDHF were involved in the mechanism.4. To evaluate the effects of TFR on the expression of iNOS in myocardial ischemia reperfusion rats.Methods: 1. The basilar arteries in vitro were used to observe the relaxation effects to drugs or blockers. In this model it was observed that the relaxation effects of ACh, EDHF, NaHS, L-cys, and TFR. And the influence of PPG to the relaxation of EDHF and TFR was also observed. The luminal perfusate of each artery was collected and the content of H2S was evaluated.2. Transmembrane potentials were recorded to evaluate the hyperpolarization effects of drugs and the blockers. In this model the hyperpolarization effects of ACh, EDHF and NaHS were observed. And the influence of PPG to the effects of EDHF was also observed.3. Endothelial cells of rat basilar arteries were primary cultured, and used to detect the expression of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). And the effect of ACh and TFR on the expression of CSE was also evaluated.4. On the model of myocardial ischemia and reperfusion in rat, all animals were subjected to 30 minutes occlusion of the left anterior descending coronary artery (LAD). The protective effects of TFR preconditioning were observed in this model. In this model the IS/AAR, and the contents of cTnI and NO in rat serum were evaluated. The expression of iNOS in myocardium were evaluated by the western blot method.Results:1. In the rat basilar arteries, preconstricted by 30mmol/L KCl or 10-7mol/L U46619 in vitro, ACh (10-7-10-4.5mol/L) had the concentration-dependent relaxations, and the effects disappeared by removal of the vascular endothelial cells.2. 3×10-5mol/L L-NAME and 10-5mol/L Indo could partly inhibit the relaxation of ACh to the rat basilar arteries, but there were still partial relaxation effect (P<0.01 vs Vehicle). After preperfusion of L-NAME and Indo there also had the effects of ACh to hyperpolarize the smooth muscle cells. These results suggested that the effect of EDHF existed in rat basilar arteries.3. 10-5-10-2.5mol/L NaHS had concentration-dependent relaxations on KCl- or U46619- preconstricted rat basilar arteries. And NaHS could induce concentration-dependent hyperpolarization to the smooth muscles of rat basilar arteries.4. 10-4mol/L PPG, the inhibitor of CSE, could markedly inhibit the effects of relaxation and hyperpolarization to EDHF.5. 10-5-10-2.5mol/L L-cys could induce concentration-dependent relaxations on KCl- or U46619- preconstricted endothelium-intact rat basilar arteries. And the relaxation was significantly attenuated by removal of endothelium.6. The luminal perfusate was collected to measure the contents of H2S after the end of perfusion of drugs or vehicle. It was found that there had the produce of H2S in the normal condition, and after perfusion of ACh the contents of H2S were increased, which was also found after perfusion in combination of L-NAME and Indo. PPG could inhibit the increase of H2S contents.7. The method of RT-PCR was used to evaluate the expression of CSE and CBS in the primary cultured endothelial cells of rat basilar arteries. It was found that in the primary cultured endothelial cells there was CSE not CBS expressing. And the expression of CSE in the primary cultured endothelial cells could be increased by ACh.8. TFR could induce concentration-dependent relaxations on U46619- preconstricted endothelium-intact rat basilar arteries. The relaxation of TFR on rat basilar arteries was significantly attenuated by removal of endothelium or pre-perfusion of L-NAME and Indo. Which suggestted that the effect of EDHF was included in the relaxation of TFR. 9. After perfusion of TFR the contents of H2S were increased, which was also found after perfusion in combination of L-NAME and Indo. PPG could inhibit the increase of H2S contents. It was found that TFR could increase the expression of CSE in the primary cultured endothelial cells. These results further suggested that the effect of EDHF was included in the relaxation of TFR.10. In the model of myocardial ischemia and reperfusion in rat, all animals were subjected to 30 minutes occlusion of the left anterior descending coronary artery(LAD) and 60 minutes of reperfusion. TFR preconditioning could markedly inhibit the increase of cTnI in rat serum, and the reduction of serum NO contents. TFR (20, 40mg/kg) preconditioning could markedly decrease IS/AAR, and TFR (40 mg/kg) preconditioning could significantly increase the expression of inducible nitricoxide synthase (iNOS) in rat myocardium.Conclusions:1. In rat basilar arteries, acetylcholine could induce the no-NO, no-PGI2, endothelium-dependent relaxation, which suggested EDHF may be existing in rat basilar arteries.2. It was found that it was CSE not CBS expressing in the primary cultured endothelial cell. And the inhibitor of CSE could markedly inhibit the effects of hyperpolarization and relaxation of EDHF. It was also found that NaHS had concentration-dependent hyperpolarization and relaxation effect in rat basilar arteries. Theses results suggested that H2S may be the chemical nature of EDHF.3. TFR has the effects of relaxation in rat basilar arteries and the effect of EDHF was included in the relaxation of TFR, which may be one of the mechanisms of TFR against cerebral ischemia injury. TFR preconditioning could induce the expression of iNOS in myocardium and increase the content of NO in rat serum, which may be the mechanism of TFR against myocardial ischemia injury.更多还原