孤啡肽对大鼠左心室功能的影响及其机制研究
发布时间：2019-03-27 21:14
【摘要】： 神经肽是一类广泛存在于机体组织且参与多种器官生理功能调节的肽类物质,其对心血管功能的影响一直是研究的热点,孤啡肽(nociceptin, Orphanin FQ,OFQ)即是其中之一。孤啡肽是一种结构类似于κ阿片受体的内源性配体-强啡肽A的十七肽,但它与阿片类受体的亲和力很低,其特异性受体为ORL1受体,与阿片受体有很高的同源性,同属于G蛋白耦联受体超家族。在中枢及外周应用孤啡肽均可引起一定程度的血压下降和心率减慢,研究认为除与孤啡肽参与中枢和外周神经对心血管的调节有关外,还可能与其直接作用于心血管有关。目前在急性心肌缺血及心衰的研究中发现心肌与血浆中孤啡肽含量增高,提示孤啡肽可能参与上述病理过程,但是目前对其作用机制尚不清楚。孤啡肽是否对心功能有直接的调节作用?以及作用的机制如何?我们对此做了进一步的研究。 目的:研究孤啡肽对大鼠心脏功能的影响及其可能机制,以期进一步阐述对孤啡肽参与调节心脏生理功能的认识。 方法:本实验从四个方面进行研究 1.大鼠应用孤啡肽(静脉注射)的在体研究:健康大鼠分为对照组(静脉给予等体积生理盐水)、不同浓度孤啡肽1nM组、10nM组和50nM组,采用颈动脉插管逆行置入左心室,观察给药前后LVSP、LVDP、+dP/dtmax、-dP/dtmax、HR的变化。进一步选用孤啡肽拮抗剂UFP-101与孤啡肽配伍给药,观察上述指标的变化。 2.孤啡肽及其受体的免疫荧光化学标定实验:采用组织免疫荧光观察观察孤啡肽及其受体在大鼠心肌中的分布;并且采用急性分离大鼠心肌细胞使用Confocal方法观察孤啡肽受体的分布。 3.孤啡肽对心肌细胞的L型钙通道、内向整流钾通道及瞬时外向钾通道研究:采用膜片钳技术,观察不同浓度的孤啡肽对上述离子通道功能的影响,以探讨孤啡肽对心脏作用可能的机制。 4.孤啡肽对心肌细胞PKA活性的影响:提取分别经不同浓度OFQ处理的心肌细胞蛋白,采用PKA活性分析试剂盒测定其活化程度,以探讨孤啡肽作用可能的分子机制。 结果: 1.静脉给予孤啡肽引起大鼠LVSP、LVDP、+dP/dtmax、-dP/dtmax、HR有明显的剂量依赖性抑制(P0.05),预先应用孤啡肽的拮抗剂UFP-101可以阻断此作用。 2.免疫荧光组织化学显示在大鼠心肌组织中有孤啡肽及其受体存在而confocal结果进一步证实心肌细胞分布有孤啡肽受体。 3.膜片钳实验结果显示,孤啡肽对心肌细胞L型钙电流有明显的抑制作用,但没有明显的剂量依赖性,以1nM孤啡肽的作用最为明显(抑制率为22.7%,P0.05),UFP-101可以阻断此抑制作用。而孤啡肽对内向整流钾电流和瞬时外向钾电流均没有明显影响。 4.心肌细胞PKA活性分析显示,孤啡肽对心肌细胞的PKA没有明显影响。给予Forskolin处理心肌细胞可以明显增高PKA活性,而孤啡肽并不能抑制这种PKA活性的增高,提示可能孤啡肽对心肌细胞的影响并不是通过PKA机制。 结论:孤啡肽对大鼠心脏左心室收缩与舒张功能有明显的抑制作用,此作用可能是通过孤啡肽-孤啡肽受体作用,引起心肌细胞L型钙电流的抑制所致。进一步的研究显示孤啡肽对于心肌细胞的PKA活性没有影响,提示可能孤啡肽的这种作用不是通过影响cAMP-PKA通路介导的。对于心肌细胞的内向整流钾电流和瞬时外向钾电流并未见孤啡肽对其有明显影响。
[Abstract]:Neuropeptide is a kind of peptide substance which is widely present in the body tissue and is involved in the physiological function regulation of various organs. Its effect on the cardiovascular function has been the hot spot of the study, and the enkephalin (nociceptin, Orphanoin FQ, OFQ) is one of them. The endorphin is a 17-peptide structure similar to the endogenous ligand-dynorphin A of the opioid receptor, but its affinity to the opioid receptor is very low, and its specific receptor is the ORL1 receptor and has a high homology to the opioid receptor and is the superfamily of the G-protein-coupled receptor. In both the central and peripheral applications, the endorphins can cause a certain degree of blood pressure drop and heart rate to slow down, and the study is that, in addition to the regulation of the central and peripheral nerves involved in the central and peripheral nerves of the enkephalin, it is also possible to directly act on the cardiovascular system. At present, in the study of acute myocardial ischemia and heart failure, the content of enkephalin in the myocardium and plasma is increased, and the enkephalin may be involved in the above-mentioned pathological process, but the mechanism of its action is not clear at present. Does the enkephalin have a direct effect on the heart function? What is the mechanism of action? We made a further study of this. Objective: To study the effect of enkephalin on the heart function of rats and its possible mechanism, with a view to further elucidating the role of enkephalin in regulating the physiological function of the heart Awareness. Method: This lab is from four The rats were divided into control group (intravenous administration of equal volume of physiological saline), different concentrations of enkephalin 1 nM, and 10n. In the M and 50 nM groups, the left ventricle was placed retrograde with a carotid artery, and the LVSP, LVDP, + dP/ dtmax,-dP/ dtm were observed before and after administration. The changes of ax and HR were further selected by the combination of UFP-101 and enkephalin. To observe the change of the above-mentioned index.2. Immunofluorescence chemical calibration of the endorphin and its receptor: the distribution of the enkephalin and its receptor in the myocardium of the rat was observed by the aid of tissue immunofluorescence; and the use of Confcal in the myocardial cells of the rats with acute separation was used. The distribution of the endorphin receptor was observed.3. The L-type calcium channel, the inward rectifier potassium channel and the transient outward potassium channel of the cardiac myocyte were studied by using the technique of patch clamp, and the effect of the endorphin on the function of the ion channels was observed. The effect of enkephalin on the activity of PKA in the cardiac muscle cells was described.4. The effect of the enkephalin on the activity of the PKA in the cardiac muscle cells: the cardiac myocyte protein treated by different concentration of OFQ was extracted, and the activation range was determined by the PKA activity analysis kit. degree, in order to The possible molecular mechanism of enkephalin was discussed. Results:1. The VSP, LVDP, + dP/ dtmax,-dP/ dtmax and HR were significantly inhibited by the intravenous administration of enkephalin (P0.05). UFP-101, an antagonist of endorphin, can block this effect. The results of the experiment of the patch clamp showed that the enkephalin had an obvious inhibitory effect on the L-type calcium current in the cardiac muscle cells, but there was no significant dose-dependence, and the effect of 1 nM of the enkephalin was the most obvious (the inhibition rate was the inhibition rate). 22.7%, P 0.05), UFP-101 can block this inhibition. The enkephalin does not have a significant effect on the inward rectifier potassium current and the transient outward potassium current. The activity of PKA in the cardiac muscle cells showed that the PKA had no significant effect on the PKA of the cardiomyocytes. Conclusion: The effect of the enkephalin on the left ventricular contraction and the diastolic function of the rat heart is obviously inhibited by the increase of the activity of A. Conclusion: The effect of the enkephalin on the left ventricular contraction and the diastolic function of the rat heart is obviously inhibited. The effect of enkephalin on the activity of the L-type calcium current in the cardiac muscle cell may be caused by the effect of the enkephalin-enkephalin receptor. The further study shows that the PKA activity of the isolated enkephalin in the cardiac muscle cells is not There is an effect suggesting that this effect of enkephalin is not mediated by the effect of the cAMP-PKA pathway.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R33
本文编号：2448569
[Abstract]:Neuropeptide is a kind of peptide substance which is widely present in the body tissue and is involved in the physiological function regulation of various organs. Its effect on the cardiovascular function has been the hot spot of the study, and the enkephalin (nociceptin, Orphanoin FQ, OFQ) is one of them. The endorphin is a 17-peptide structure similar to the endogenous ligand-dynorphin A of the opioid receptor, but its affinity to the opioid receptor is very low, and its specific receptor is the ORL1 receptor and has a high homology to the opioid receptor and is the superfamily of the G-protein-coupled receptor. In both the central and peripheral applications, the endorphins can cause a certain degree of blood pressure drop and heart rate to slow down, and the study is that, in addition to the regulation of the central and peripheral nerves involved in the central and peripheral nerves of the enkephalin, it is also possible to directly act on the cardiovascular system. At present, in the study of acute myocardial ischemia and heart failure, the content of enkephalin in the myocardium and plasma is increased, and the enkephalin may be involved in the above-mentioned pathological process, but the mechanism of its action is not clear at present. Does the enkephalin have a direct effect on the heart function? What is the mechanism of action? We made a further study of this. Objective: To study the effect of enkephalin on the heart function of rats and its possible mechanism, with a view to further elucidating the role of enkephalin in regulating the physiological function of the heart Awareness. Method: This lab is from four The rats were divided into control group (intravenous administration of equal volume of physiological saline), different concentrations of enkephalin 1 nM, and 10n. In the M and 50 nM groups, the left ventricle was placed retrograde with a carotid artery, and the LVSP, LVDP, + dP/ dtmax,-dP/ dtm were observed before and after administration. The changes of ax and HR were further selected by the combination of UFP-101 and enkephalin. To observe the change of the above-mentioned index.2. Immunofluorescence chemical calibration of the endorphin and its receptor: the distribution of the enkephalin and its receptor in the myocardium of the rat was observed by the aid of tissue immunofluorescence; and the use of Confcal in the myocardial cells of the rats with acute separation was used. The distribution of the endorphin receptor was observed.3. The L-type calcium channel, the inward rectifier potassium channel and the transient outward potassium channel of the cardiac myocyte were studied by using the technique of patch clamp, and the effect of the endorphin on the function of the ion channels was observed. The effect of enkephalin on the activity of PKA in the cardiac muscle cells was described.4. The effect of the enkephalin on the activity of the PKA in the cardiac muscle cells: the cardiac myocyte protein treated by different concentration of OFQ was extracted, and the activation range was determined by the PKA activity analysis kit. degree, in order to The possible molecular mechanism of enkephalin was discussed. Results:1. The VSP, LVDP, + dP/ dtmax,-dP/ dtmax and HR were significantly inhibited by the intravenous administration of enkephalin (P0.05). UFP-101, an antagonist of endorphin, can block this effect. The results of the experiment of the patch clamp showed that the enkephalin had an obvious inhibitory effect on the L-type calcium current in the cardiac muscle cells, but there was no significant dose-dependence, and the effect of 1 nM of the enkephalin was the most obvious (the inhibition rate was the inhibition rate). 22.7%, P 0.05), UFP-101 can block this inhibition. The enkephalin does not have a significant effect on the inward rectifier potassium current and the transient outward potassium current. The activity of PKA in the cardiac muscle cells showed that the PKA had no significant effect on the PKA of the cardiomyocytes. Conclusion: The effect of the enkephalin on the left ventricular contraction and the diastolic function of the rat heart is obviously inhibited by the increase of the activity of A. Conclusion: The effect of the enkephalin on the left ventricular contraction and the diastolic function of the rat heart is obviously inhibited. The effect of enkephalin on the activity of the L-type calcium current in the cardiac muscle cell may be caused by the effect of the enkephalin-enkephalin receptor. The further study shows that the PKA activity of the isolated enkephalin in the cardiac muscle cells is not There is an effect suggesting that this effect of enkephalin is not mediated by the effect of the cAMP-PKA pathway.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R33
【参考文献】
相关期刊论文 前1条
1 孟美金,Ting Wee Lee,Michael George Ricos,吴新民,Lee Tat Leang,Tachibana Shinro;鞘内注射孤啡肽对不同小鼠所致痛觉超敏的比较[J];中华麻醉学杂志;2003年09期
，本文编号：2448569
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