槲皮素通过增强电压依赖性钾电流和抑制L-型电压依赖性钙电流舒张大鼠冠状动脉
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摘要
研究背景
槲皮素是一种广泛存在于水果、蔬菜和植物的花、叶、果实中的典型植源性黄酮类化合物，人们日常饮食中的苹果，沙棘，西兰花，洋葱，茶叶等均富含槲皮素。研究表明，槲皮素具有多种重要的生物学活性如抗氧化，抗血小板聚集，抗炎，免疫调节，抗肿瘤，减轻心肌缺血再灌注损伤等。据文献报道，槲皮素对大鼠主动脉，肠系膜动脉，门静脉和猪的冠状动脉有明显的舒张作用，然而对大鼠冠状动脉的舒张作用及其机制却未见报道。
L-型电压依赖性钙通道（L-type voltage gated Ca2+channels, LVGC）介导细胞外钙跨细胞膜内流，对调控血管平滑肌细胞的舒缩生理功能起着至关重要的作用。钾通道具有维持细胞的正常膜电位和调节血管张力的功能。血管平滑肌上主要存在4种类型的钾通道：电压依赖性钾通道（voltage-gated K+channels, Kv），钙激活的钾通道（Ca2+-activated K+channels, KCa），内向整流钾通道（inwardrectifier K+channels, KIR）和ATP敏感的钾通道（ATP-activated K+channels, KATP）。其中，Kv通道是表达最多的钾离子通道，Kv通道在调节细胞静息电位和小动脉直径方面起重要作用。本课题主要研究槲皮素对离体大鼠冠状动脉的舒张作用及其对离体大鼠心脏冠脉流量和血流动力学参数的影响，并进一步探讨槲皮素舒张大鼠冠脉的作用与LVGC和Kv的关系。
本课题分三部分进行研究：
第一部分槲皮素对离体大鼠冠状动脉的肌源性反应
实验目的
1.观察槲皮素对大鼠离体冠状动脉的肌源性反应，研究槲皮素对冠状动脉收缩的抑制作用和对冠状动脉的舒张作用。
2.在肌源性反应实验中，通过利用钾通道阻断剂，一氧化氮合酶抑制剂和去内皮，探讨钾通道、一氧化氮（nitric oxide, NO）、内皮与槲皮素对大鼠冠状动脉舒张作用的关系。
3.通过在无钙液中加入2.5mM CaCl2，观察槲皮素对细胞外钙引起大鼠冠脉血管环收缩的影响。
实验方法
离体大鼠冠状动脉环肌源性实验方法：
1.大鼠麻醉后，断头，立即取出其心脏，放于4℃O2饱和的HEPES液中。钝性分离其冠状动脉，然后剪成长约2mm的血管环。将两根直径为25μm的钢丝平行穿入血管环管腔，将血管环固定于Multi Myograph System-610M微血管张力记录仪（DMT）上。浴槽内含有5.0mLHEPES液体，持续O2饱和，并维持37℃恒温。将血管环前负荷调整到80mmHg，平衡1h，平衡期间每隔15min用新鲜的37℃HEPES液更换一次浴槽内的液体。血管环的张力变化通过Chart7.03记录在计算机上。平衡1h后，用60mM KCl收缩血管达坪台，再加入乙酰胆碱对血管环进行舒张，连续刺激血管环三次，当三次的收缩幅度或舒张幅度相差不超过10%时，认为血管反应良好，选取收缩达到2mN或收缩坪台稳定的血管环进行正式实验。
2.向浴槽中累积加入KCl（20，28，39，55，77，108mM）或U46619（0.03，0.1，0.3，1，3μM），制作KCl或U46619的收缩量效曲线，当其收缩曲线可重复时，向浴液中加入槲皮素（3，10，30μM），孵育15min，然后再次在槲皮素存在的条件下制作KCl或U46619的收缩量效曲线。以未孵育槲皮素前108mMKCl或3μΜ U46619引起的最大收缩幅度为100%，计算未孵育和孵育槲皮素时，不同浓度KCl或U46619引起冠脉收缩张力的百分比。
3.用60mM KCl或1μΜ U46619收缩冠脉达稳定的坪台，向内皮完整或去内皮组冠脉血管环中累积加入浓度梯度的槲皮素（1，3，10，30，100μM）或单纯槲皮素溶剂。以60mM KCl或1μΜ U46619收缩达坪台时的最大收缩幅度为100%，计算槲皮素或溶剂对大鼠冠脉舒张时张力所占的百分比。
4.当60mM KCl或1μΜ U46619收缩达稳定的坪台时，向浴槽中分别加入阻断剂：电压依赖性钾通道阻断剂（4-aminopyridine,4-AP），大电导钙激活钾通道阻断剂（iberiotoxin, Iber），内向整流钾通道阻断剂（BaCl2），ATP敏感的钾通道阻断剂（glibenclamide, Glib）及一氧化氮合酶抑制剂（NG-nitro-L-argininemethylester ester, L-NAME），孵育10min，血管收缩再次达坪台时，向浴槽中累积加入不同浓度的槲皮素（1，3，10，30，100μM）舒张冠脉。以60mM KCl或1μΜ U46619收缩达坪台时的最大收缩幅度为100%，计算不同浓度槲皮素对大鼠冠脉血管舒张所占的百分比。
5.当60mM KCl的收缩幅度可重复时，先用含1mM EGTA的无钙液孵育血管环20min。当血管环张力恢复达基线时，再用不含EGTA的无钙液洗脱血管环，稳定10min后，将浴液换为无钙的60mM KCl或1μΜ U46619孵育10min，向浴槽内加入2.5mM CaCl2。向浴槽内加入槲皮素（此后槲皮素一直存在于浴液中），孵育15min，然后在无钙液中加入60mM KCl或1μΜ U46619，10min后，向浴槽内加入2.5mM CaCl2。记录孵育槲皮素前后，无钙液中60mM KCl或1μΜU46619的收缩幅度以及加入2.5mM CaCl2后冠脉的收缩幅度。
实验结果
1. KCl（20，28，39，55，77，108mM）或U46619（0.03，0.1，0.3，1，3μM）浓度依赖性收缩冠脉，最大收缩幅度分别为4.910.38mN和4.830.41mN，KCl和U46619收缩冠脉的EC50值分别为31.87mM和0.19M。孵育槲皮素（3，10，30M），使KCl和U46619的浓度收缩曲线右下移，槲皮素抑制KCl和U46619收缩量效的IC50值分别为48.8M和88.30M。
当孵育槲皮素浓度为3μM时，KCl或U46619的收缩幅度与对照组相比无显著性差异（P>0.05）；当孵育槲皮素浓度为10μM时，KCl或U46619的最大收缩幅度分别降低了35.4%和21.3%，与对照组相比有显著性差异（P <0.05）；当孵育槲皮素浓度为30μM时，KCl或U46619的最大收缩幅度分别降低了79.9%和54.7%，与对照组相比有显著性差异（P <0.05）。
2.槲皮素（1，3，10，30，100μM）浓度依赖性引起内皮完整组和去内皮组冠脉舒张，且内皮对槲皮素舒张大鼠冠脉无显著影响。
在内皮完整组实验中，溶剂对冠脉张力无显著影响；槲皮素使60mM KCl或1M U46619预收缩大鼠冠脉的最大张力分别下降了（96.34±7.35）%和（82.47±7.24）%，RC50值分别为22.87M和41.48M。
去内皮组与内皮完整组相比，槲皮素对60mM KCl或1M U46619预收缩大鼠冠脉的最大舒张幅度没有发生显著差异（P>0.05）。
3. Iber、Glib、BaCl2、L-NAME孵育对槲皮素所致的大鼠冠脉舒张均无显著性影响（P>0.05）；而4-AP（1mM）孵育使槲皮素对KCl预收缩冠脉的最大舒张幅度减小42.88%（P <0.05）；4-AP（1mM）孵育使槲皮素对U46619预收缩冠脉的最大舒张幅度减小了28.82%（P <0.05）。
4.在无钙液中，60mM KCl对大鼠冠脉的张力基本没有影响，当加入2.5mMCaCl2后，引起冠脉收缩。分别孵育不同浓度的槲皮素（3，10，30M）可浓度依赖性抑制2.5mM CaCl2引起的冠脉收缩，其IC50值为9.55M。在无钙液中孵育槲皮素（3，10，30M）后，对1M U46619引起的收缩没有显著的抑制作用，可浓度依赖性抑制2.5mM CaCl2引起的冠脉收缩，其IC50值为22.5M。
第二部分槲皮素对大鼠离体冠状动脉血管平滑肌细胞内钙离子浓度和LVGC电流及Kv电流的影响
实验目的
1.观察槲皮素对大鼠冠脉血管平滑肌细胞内钙离子浓度的影响。
2.观察槲皮素对大鼠冠脉血管平滑肌细胞LVGC电流和Kv电流的影响。
实验方法
1.细胞分离
利用两步酶解法分离得到大鼠冠状动脉血管平滑肌细胞：首先将冠脉血管置于I号酶解液中，即低钙的HEPES液（含有0.1mM CaCl2，1mg/mL白蛋白，0.5mg/mL木瓜蛋白酶和1mg/mL二硫丁四醇），在O2饱和且37℃条件下，孵育约30min，然后将冠脉血管转移至II号酶解液中，即无钙的HEPES液（含有1mg/mL白蛋白，0.5mg/mL胶原蛋白F和0.5mg/mL胶原蛋白H），在O2饱和且37℃条件下，孵育约15min，即得大鼠冠脉血管平滑肌细胞。
2.细胞内钙离子浓度的测定
用荧光染料Fluo-4-AM将大鼠冠脉血管平滑肌细胞染色，依次用正常HEPES液，40mM KCl，40mM KCl+10-100μM槲皮素，正常HEPES液，40mM KCl灌流细胞，分别记录不同条件下细胞的荧光值。实验结果用相对荧光密度值（F/F0）来反映细胞内钙离子浓度的变化。
3.电生理学测量
采用全细胞电压钳实验技术，记录并探讨槲皮素对大鼠冠状动脉血管平滑肌细胞LVGC电流和Kv电流的影响。
实验结果
1.槲皮素浓度依赖性降低KCl引起的冠脉血管平滑肌细胞内的钙离子荧光密度增加。40mM KCl使冠脉平滑肌细胞内的钙离子荧光密度增加了97.4%。槲皮素（10，30，100M）降低冠脉平滑肌细胞内钙离子的荧光密度百分比分别为30.1%，60.8%和86.5%。
2. Bay K8644（1μM）增大LVGC电流幅度，使峰值电流增加了31.9%；硝苯地平（1μM）抑制LVGC电流幅度，使峰值电流减少了75.6%，上述实验结果证明所记录电流为LVGC电流。槲皮素（3，10，30μM）浓度依赖性的使冠脉血管平滑肌细胞LVGC电流的I-V曲线右上移。单独给予+10mV单电压刺激时，槲皮素（3，10，30μM）抑制LVGC峰值电流的百分比分别为28.7%，57.1%和76.8%。
3.在测试电压为+60mV时，冠脉血管平滑肌细胞KV电流峰值为345.26±23.57pA，电流密度为32.52±5.24pA/pF（n=12）。槲皮素（10，30，100μM）浓度依赖性的使冠脉血管平滑肌细胞KV电流的I-V曲线上移。单独给予+60mV单电压刺激时，槲皮素（10，30，100μM）增加KV电流的百分比分别为20.1%，38.1%和73.3%。
第三部分槲皮素对大鼠离体心脏冠脉流量及血流动力学参数的影响
实验目的
利用Langendorff实验系统，观察槲皮素对大鼠离体心脏冠脉流量（coronaryflow, CF）及血流动力学参数：左心室发展压（left ventricular developed pressure,LVDP），左心室舒张末压（left ventricular end-diastolic pressure, LVEDP），左心室内压最大上升速率（positive dp/dtmax,+dp/dtmax）和左心室内压最大下降速率（negative dp/dtmax,-dp/dtmax）的影响。
实验方法
利用Langendorff实验系统，先用台式液灌流大鼠心脏，记录冠脉流量及血流动力学参数。然后分别用含不同浓度槲皮素（0.3，1，3，10，30μM）的台式液灌流大鼠心脏，观察冠脉流量及血流动力学参数的变化。
实验结果
槲皮素使大鼠离体心脏CF，LVDP，+dp/dtmax和-dp/dtmax值呈浓度依赖性的增大，但对LVEDP值无显著影响。
1.用正常台式液灌流时，CF值为7.8±1.2mL/min，改用含不同浓度槲皮素的台式液灌流时，槲皮素（0.3，1，3，10，30μM）浓度依赖性的增大CF值，最大值为13.0±1.3mL/min，其EC50值为3.04μM。
2.离体大鼠心脏LVDP和+dp/dtmax的基础值分别为：64±3mmHg，1368±124mmHg/s。随灌流液中槲皮素浓度增加（0.3，1，3，10，30μM），上述两项指标均逐渐增大，最大值分别为：78±2mmHg，1692±196mmHg/s。
3.离体大鼠心脏-dp/dtmax的基础值为：1143±126mmHg/s。随灌流液中槲皮素浓度增加（0.3，1，3，10，30μM），-dp/dtmax值逐渐增大，最大值为：1375±151mmHg/s。离体大鼠心脏LVEDP的基础值为：10±1mmHg，槲皮素对LVEDP值无显著影响。
全文实验结论
1.槲皮素浓度依赖性舒张大鼠冠状动脉，其舒张作用是非内皮依赖性的，且与增强Kv和抑制LVGC有关；
2.槲皮素增加离体大鼠心脏冠脉流量，这一结论与肌源性实验所得槲皮素舒张大鼠冠脉相一致，且槲皮素对离体大鼠心脏有一定的正性肌力作用。
Background
Quercetin is as a typical fruit-derived flavonoid, which is widely distributed infruits, vegetables, botanic flowers and leaves, such as apple, sea buckthorn, broccoli,onions, tea, etc. Quercetin has been reported to possess many biological activities,including antioxidation, antiplatelet aggregation, antiinflammatory,immunomodulation, anticancer as well as reduction of cardiac ischemia-reperfusioninjury. Quercetin could induce vasodilation in rat aorta, rat mesenteric arteries, ratportal vein and porcine coronary arteries, whether quercetin could exert relaxationeffect on coronary artery (CA) of rat has not been studied.
Calcium influx mainly through L-type voltage gated calcium channels (LVGC),which is prerequisite for the physiological function of vasomotor regulation ofvascular smooth muscle cells (VSMCs). At the same time, potassium channels couldmaintain normal cell membrane potential and regulate of vascular tone. There aremainly four kinds of potassium channels on vascular smooth muscle: voltage-gatedK+channels (Kv), Ca2+-activated K+channels (KCa), inward rectifier K+channels (KIR)and ATP-activated K+channels (KATP). However, Kv channels are the highestexpression, Kv channels play an important role in regulating cell resting potential andsmall artery diameter. The present study was designed to observe the effects ofquercetin on coronary dilation of rat and coronary flow and hemodynamic parametersof isolated rat heart, and whether LVGC and KVare involved in quercetin-inducedvasorelaxation in rat coronary arteries (RCAs).
Part1The myogenic response of quercetin in rat coronary arteryObjectives
1. To observe the myogenic response of quercetin in isolated RCAs, and to elucidatethe effect of quercetin on the inhibitory vasoconstriction and relaxantion in RCAs.
2. In the myogenic experiment, we used the potassium channels blockers, nitric oxidesynthase inhibitor and endothelium denudation to study the relaxant effect ofquercetin on RCAs, and whether potassium channels, nitric oxide (NO) andendothelium are involved in myogenic response of quercetin.
3. In the calcium-free solution, the effect of quercetin on extracellular calcium inducecontraction was observed.
Methods
1. After sacrifice, the heart was removed and transferred immediately into chilled(4°C) HEPES solution bubbled with100%O2. RCAs were isolated, cut into2mm-long rings and mounted on a wire myograph (Multi Myograph System-610M,DMT, Denmark) using two tungsten wires (25μm in diameter) in a tissue chambercontaining of5.0mL HEPES solution bubbled with100%O2,37°C. The rings werenormalized, stretched to a state equal to80mmHg, equilibrated for at least1h andwashed with HEPES solution (37°C) every15min. The tension changes of rings wererecorded by Chart7.03on the computer. After equilibration1h, the rings werecontracted with60mM KCl, then were relaxed with Ach, three times repeatedly.When the contraction or relaxation difference did not exceed10%, we thought that therings were good at the vascular reactivity. We discarded the arterial rings in case thatthe contraction was not repeatable or not sustained, or the contraction was less than2mN.
2. High K+HEPES solution or U46619was cumulatively added to the chamber toconstruct a concentration-contraction curve for KCl (20,28,39,55,77,108mM) orU46619(0.03,0.1,0.3,1,3μM). When successive curves were repeatable, the arterialrings were preincubation with quercetin (3,10,30μM) for15min beforereconstruction of the curves for KCl or U46619in the presence of quercetin(quercetin was always in the chamber in this period). The maximal contraction induced by108mM KCl or3μM U46619in the absence of quercetin was taken as100%and the percentage of contraction by each concentration of stimulator in theabsence or presence of quercetin was calculated.
3. When the contraction induced by60mM KCl or1μM U46619was sustained,quercetin (1,3,10,30,100μM) or vehicle were added cumulatively to theendothelium-intact or-denudation RCAs. The maximal contraction induced by60mM KCl or1μM U46619in the absence of quercetin was taken as100%and thepercentage of contraction by each concentration of stimulator in the absence orpresence of quercetin or vehicle was calculated.
4. When the contraction induced by60mM KCl or1μM U46619reached a sustainedplateau,4-aminopyridine (4-AP, a KVblocker), iberiotoxin (Iber, a KCablocker),BaCl2(a KIRblocker) or glibenclamide (Glib, a KATPblocker) was added respectivelyto the chamber. After10min, based on the arterial tone was stable in the presence ofan inhibitor, quercetin (1,3,10,30,100μM) was cumulatively added to the abovechamber. Relaxation was expressed as a percentage of the precontraction induced by60mM KCl or1μM U46619, respectively.
5. When the contraction induced by60mM KCl was repeatable, the ring was rinsedand incubated with Ca2+-free HEPES solution (containing1mM EGTA) for20min.When the tone of the ring restored to the baseline, the solution in above chamber waschanged to Ca2+-free HEPES solution. After10min, the ring was stimulated with60mM KCl or1M U46619, after10min,2.5mM CaCl2was added to the chamber.Quercetin was added to the chamber (the bath concentration of quercetin was keptconstant through the following experiment), after15min, the ring was stimulated with60mM KCl or1μΜ U46619in the Ca2+-free HEPES solution, after10min,2.5mMCaCl2was added to the chamber. The contraction induced by60mM KCl,1μΜU46619and2.5mM CaCl2were recorded in the absence or presence of quercetin.
Results
1. KCl (20,28,39,55,77,108mM) and U46619(0.03,0.1,0.3,1,3μM) inducedcontraction concentration-dependently. The maximal contractions were4.910.38 mN and4.830.41mN, respectively. The values of EC50were31.87mM for KCland0.19M for U46619, respectively. Pretreatment with quercetin (3,10,30M)shifted the concentration curves downwards to the right. The values of IC50were48.80M for KCl and88.30M for U46619, respectively.
At3μM quercetin, there were no significant difference between quercetin groupand control group on KCl or U46619-contraction (P>0.05). At10μM, quercetindepressed the maximal contraction of KCl or U46619by35.4%and21.3%,respectively. There were significant difference between quercetin group and controlgroup on KCl or U46619-contraction (P <0.05). At30μM, quercetin depressed themaximal contraction of KCl or U46619by79.9%and54.7%, respectively. Therewere significant difference between quercetin group and control group on KCl orU46619-contraction (P <0.05).
2. Quercetin (1,3,10,30,100μM) elicited concentration-dependent relaxation inendothelium-intact or-denudation RCAs, and endothelium has no effect on relaxationof quercetin in RCAs.
Vehicle did not significantly affect the tone of endothelium-intact RCAs; themaximal relaxation of quercetin in RCAs precontracted by either60mM KCl or1M U46619were (96.34±7.35)%and (82.47±7.24)%, respectively. The values ofRC50were22.87M for KCl and41.42M for U46619, respectively.
Compared endothelium-denudation RCAs with endothelium-intact RCAs, themaximal relaxation of quercetin did not change significantly (P>0.05).
3. Preincubation with Iber, Glib, BaCl2or L-NAME, there have no effect on therelaxation of quercetin in RCAs, but preincubation with4-AP (1mM) attenuated therelaxation on KCl-precontraction by42.88%(P <0.05). Preincubation with4-AP (1mM) attenuated the relaxation on U46619-precontraction by28.82%(P <0.05).
4. In Ca2+-free HEPES solution,60mM KCl failed to produce significant contraction(phasic) and restoration of2.5mM CaCl2evoked a full contraction (tonic). Quercetin(3,10,30μM) inhibited2.5mM CaCl2-induced contraction in aconcentration-dependent manner and the calculated IC50was9.55M. Quercetin didnot significantly affect the phasic for U46619, but reduced tonic component of the contraction and the calculated IC50was22.5M.
Part2Effects of quercetin on [Ca2+]inand LVGC currents and Kvcurrents of rat coronary artery smooth muscle cells
Objectives
1. To study the effect of quercetin on [Ca2+]influorescence intensity of RCA VSMCs.
2. In order to clarify the mechanism of quercetin relaxation in RCAs, we recordedLVGC currents and Kv currents of RCA VSMCs.
Methods
1. Rat coronary artery smooth muscle cells isolation:
Coronary artery smooth muscle cells were obtained using two steps enzymatic:Firstly, RCAs were placed in enzymatic solution I (HEPES solution containing0.1mM CaCl2,1mg/mL albumin,0.5mg/mL papain and1mg/mL dithioerythritol)bubbled with100%O2and incubated for30min at37oC. The RCAs were transferredto enzymatic solution II (HEPES solution containing1mg/mL albumin,0.5mg/mLcollagenase F and0.5mg/mL collagenase H) bubbled with100%O2and incubatedfor15min at37oC.
2.[Ca2+]inmeasurement:
Isolated RCA VSMCs were loaded with Fluo-4-AM and perfused with thedifferent solutions (HEPES solution,40mM KCl,40mM KCl+quercetin (10,30,100μM), HEPES solution and40mM KCl) in order of time precedence. Thefluorescence intensity of RCA VSMCs were recorded under various conditions.Fluorescence ratios (F/F0) were calculated to reflect the [Ca2+]in.
3. Electrophysiological measurements:
Using whole-cell voltage clamp, we recorded and investigated the effects ofquercetin on LVGC currents and Kv currents of RCA VSMCs.
Results
1. Quercetin reduced KCl-induced elevation of the intracellular Ca2+concentration ina concentration-dependent manner.40mM KCl increased fluorescence intensity ratio (F/F0, fluorescence after vs before40mM KCl) by97.4%. Application of quercetin(10,30,100M) decreased the intracellular Ca2+fluorescence intensity increment by30.1%,60.8%and86.5%, respectively.
2. Bay K8644(1M) increased LVGC currents and increased the peak currents by31.9%; nifedipine (1M) decreased LVGC currents and decreased the peak currentsby75.6%. These results proved that the currents recorded were LVGC currents.
Quercetin (3,10,30μM) shifted the I-V curve of LVGC currents of RCA VSMCsupwards to the right in a concentration-dependent manner. At test potential of+10mV,quercetin (3,10,30M) decreased LVGC currents by28.7%,57.1%and76.8%,respectively.
3. At test potential of+60mV, the stable peak of Kv currents was345.26±23.57pAand current density was32.52±5.24pA/pF (n=12). Quercetin (10,30,100μM)shifted the I-V curve of KVcurrents upward in a concentration-dependent manner. Attest potential of+60mV, quercetin (10,30,100M) increased the KVcurrents ofRCA VSMCs by20.1%,38.1%and73.3%, respectively.
Part3Effects of quercetin on coronary flow and hemodynamicparameters of isolated rat heart
Objective
To study the effects of quercetin on coronary flow (CF) and hemodynamicparameters (LVDP，LVEDP，+dp/dtmax，-dp/dtmax) of isolated rat heart by Langendorffapparatus.
Methods
The isolated rat heart was perfused firstly with Tyrode’s solution usingLangendorff apparatus, subsequently, it was perfused with Tyrode’s solutioncontaining different concentrations of quercetin (0.3,1,3,10,30μM). At the sametime, coronary flow and hemodynamics parameters were recorded.
Results
This experiment showed that quercetin increased the vaule of CF, LVDP, +dp/dtmaxand-dp/dtmax, but it had no effect on LVEDP.
1. When the isolated rat hearts were perfused with Tyrode’s solution, the vaule of CFwas7.8±1.2mL/min, then they were perfused with Tyrode’s solution containingdifferent concentration of quercetin (0.3,1,3,10,30μM), quercetin increased the CFin a concentration-dependent manner, the maximal value of CF was13.0±1.3mL/min and the EC50was3.04μM.
2. Quercetin increased LVDP and+dp/dtmaxof isolated rat hearts in aconcentration-dependent manner, the basal value were64±3mmHg (LVDP) and1368±124mmHg/s (+dp/dtmax), respectively. With increasing concentrations ofquercetin (0.3,1,3,10,30μM), the value of LVDP and+dp/dtmaxwere graduallyincreased, the maximal value were78±2mmHg and1692±196mmHg/s,respectively.
3. Quercetin increased-dp/dtmaxof isolated rat hearts in a concentration-dependentmanner, the basal value was1143±126mmHg/s. With increasing concentrations ofquercetin (0.3,1,3,10,30μM), the value of-dp/dtmaxwas gradually increased, themaximal value was1375±151mmHg/s. The basal value of LVEDP was10±1mmHg, quercetin had no effect on LVEDP.
Conclusions
1. Quercetin elicited relaxation in RCA in a concentration-dependent manner. Therelaxation effect of quercetin on RCA was non-endothelial dependent, and wasassociated with increased Kv currents and depressed LVGC currents of RCA VSMCs.
2. Quercetin increased coronary flow of isolated rat heart, this result was consistentwith the relaxation effect of quercetin on RCA in myogenic experiment. Quercetinhad a positive inotropic effect on isolated rat heart.
槲皮素是一种广泛存在于水果、蔬菜和植物的花、叶、果实中的典型植源性黄酮类化合物，人们日常饮食中的苹果，沙棘，西兰花，洋葱，茶叶等均富含槲皮素。研究表明，槲皮素具有多种重要的生物学活性如抗氧化，抗血小板聚集，抗炎，免疫调节，抗肿瘤，减轻心肌缺血再灌注损伤等。据文献报道，槲皮素对大鼠主动脉，肠系膜动脉，门静脉和猪的冠状动脉有明显的舒张作用，然而对大鼠冠状动脉的舒张作用及其机制却未见报道。
L-型电压依赖性钙通道（L-type voltage gated Ca2+channels, LVGC）介导细胞外钙跨细胞膜内流，对调控血管平滑肌细胞的舒缩生理功能起着至关重要的作用。钾通道具有维持细胞的正常膜电位和调节血管张力的功能。血管平滑肌上主要存在4种类型的钾通道：电压依赖性钾通道（voltage-gated K+channels, Kv），钙激活的钾通道（Ca2+-activated K+channels, KCa），内向整流钾通道（inwardrectifier K+channels, KIR）和ATP敏感的钾通道（ATP-activated K+channels, KATP）。其中，Kv通道是表达最多的钾离子通道，Kv通道在调节细胞静息电位和小动脉直径方面起重要作用。本课题主要研究槲皮素对离体大鼠冠状动脉的舒张作用及其对离体大鼠心脏冠脉流量和血流动力学参数的影响，并进一步探讨槲皮素舒张大鼠冠脉的作用与LVGC和Kv的关系。
本课题分三部分进行研究：
第一部分槲皮素对离体大鼠冠状动脉的肌源性反应
实验目的
1.观察槲皮素对大鼠离体冠状动脉的肌源性反应，研究槲皮素对冠状动脉收缩的抑制作用和对冠状动脉的舒张作用。
2.在肌源性反应实验中，通过利用钾通道阻断剂，一氧化氮合酶抑制剂和去内皮，探讨钾通道、一氧化氮（nitric oxide, NO）、内皮与槲皮素对大鼠冠状动脉舒张作用的关系。
3.通过在无钙液中加入2.5mM CaCl2，观察槲皮素对细胞外钙引起大鼠冠脉血管环收缩的影响。
实验方法
离体大鼠冠状动脉环肌源性实验方法：
1.大鼠麻醉后，断头，立即取出其心脏，放于4℃O2饱和的HEPES液中。钝性分离其冠状动脉，然后剪成长约2mm的血管环。将两根直径为25μm的钢丝平行穿入血管环管腔，将血管环固定于Multi Myograph System-610M微血管张力记录仪（DMT）上。浴槽内含有5.0mLHEPES液体，持续O2饱和，并维持37℃恒温。将血管环前负荷调整到80mmHg，平衡1h，平衡期间每隔15min用新鲜的37℃HEPES液更换一次浴槽内的液体。血管环的张力变化通过Chart7.03记录在计算机上。平衡1h后，用60mM KCl收缩血管达坪台，再加入乙酰胆碱对血管环进行舒张，连续刺激血管环三次，当三次的收缩幅度或舒张幅度相差不超过10%时，认为血管反应良好，选取收缩达到2mN或收缩坪台稳定的血管环进行正式实验。
2.向浴槽中累积加入KCl（20，28，39，55，77，108mM）或U46619（0.03，0.1，0.3，1，3μM），制作KCl或U46619的收缩量效曲线，当其收缩曲线可重复时，向浴液中加入槲皮素（3，10，30μM），孵育15min，然后再次在槲皮素存在的条件下制作KCl或U46619的收缩量效曲线。以未孵育槲皮素前108mMKCl或3μΜ U46619引起的最大收缩幅度为100%，计算未孵育和孵育槲皮素时，不同浓度KCl或U46619引起冠脉收缩张力的百分比。
3.用60mM KCl或1μΜ U46619收缩冠脉达稳定的坪台，向内皮完整或去内皮组冠脉血管环中累积加入浓度梯度的槲皮素（1，3，10，30，100μM）或单纯槲皮素溶剂。以60mM KCl或1μΜ U46619收缩达坪台时的最大收缩幅度为100%，计算槲皮素或溶剂对大鼠冠脉舒张时张力所占的百分比。
4.当60mM KCl或1μΜ U46619收缩达稳定的坪台时，向浴槽中分别加入阻断剂：电压依赖性钾通道阻断剂（4-aminopyridine,4-AP），大电导钙激活钾通道阻断剂（iberiotoxin, Iber），内向整流钾通道阻断剂（BaCl2），ATP敏感的钾通道阻断剂（glibenclamide, Glib）及一氧化氮合酶抑制剂（NG-nitro-L-argininemethylester ester, L-NAME），孵育10min，血管收缩再次达坪台时，向浴槽中累积加入不同浓度的槲皮素（1，3，10，30，100μM）舒张冠脉。以60mM KCl或1μΜ U46619收缩达坪台时的最大收缩幅度为100%，计算不同浓度槲皮素对大鼠冠脉血管舒张所占的百分比。
5.当60mM KCl的收缩幅度可重复时，先用含1mM EGTA的无钙液孵育血管环20min。当血管环张力恢复达基线时，再用不含EGTA的无钙液洗脱血管环，稳定10min后，将浴液换为无钙的60mM KCl或1μΜ U46619孵育10min，向浴槽内加入2.5mM CaCl2。向浴槽内加入槲皮素（此后槲皮素一直存在于浴液中），孵育15min，然后在无钙液中加入60mM KCl或1μΜ U46619，10min后，向浴槽内加入2.5mM CaCl2。记录孵育槲皮素前后，无钙液中60mM KCl或1μΜU46619的收缩幅度以及加入2.5mM CaCl2后冠脉的收缩幅度。
实验结果
1. KCl（20，28，39，55，77，108mM）或U46619（0.03，0.1，0.3，1，3μM）浓度依赖性收缩冠脉，最大收缩幅度分别为4.910.38mN和4.830.41mN，KCl和U46619收缩冠脉的EC50值分别为31.87mM和0.19M。孵育槲皮素（3，10，30M），使KCl和U46619的浓度收缩曲线右下移，槲皮素抑制KCl和U46619收缩量效的IC50值分别为48.8M和88.30M。
当孵育槲皮素浓度为3μM时，KCl或U46619的收缩幅度与对照组相比无显著性差异（P>0.05）；当孵育槲皮素浓度为10μM时，KCl或U46619的最大收缩幅度分别降低了35.4%和21.3%，与对照组相比有显著性差异（P <0.05）；当孵育槲皮素浓度为30μM时，KCl或U46619的最大收缩幅度分别降低了79.9%和54.7%，与对照组相比有显著性差异（P <0.05）。
2.槲皮素（1，3，10，30，100μM）浓度依赖性引起内皮完整组和去内皮组冠脉舒张，且内皮对槲皮素舒张大鼠冠脉无显著影响。
在内皮完整组实验中，溶剂对冠脉张力无显著影响；槲皮素使60mM KCl或1M U46619预收缩大鼠冠脉的最大张力分别下降了（96.34±7.35）%和（82.47±7.24）%，RC50值分别为22.87M和41.48M。
去内皮组与内皮完整组相比，槲皮素对60mM KCl或1M U46619预收缩大鼠冠脉的最大舒张幅度没有发生显著差异（P>0.05）。
3. Iber、Glib、BaCl2、L-NAME孵育对槲皮素所致的大鼠冠脉舒张均无显著性影响（P>0.05）；而4-AP（1mM）孵育使槲皮素对KCl预收缩冠脉的最大舒张幅度减小42.88%（P <0.05）；4-AP（1mM）孵育使槲皮素对U46619预收缩冠脉的最大舒张幅度减小了28.82%（P <0.05）。
4.在无钙液中，60mM KCl对大鼠冠脉的张力基本没有影响，当加入2.5mMCaCl2后，引起冠脉收缩。分别孵育不同浓度的槲皮素（3，10，30M）可浓度依赖性抑制2.5mM CaCl2引起的冠脉收缩，其IC50值为9.55M。在无钙液中孵育槲皮素（3，10，30M）后，对1M U46619引起的收缩没有显著的抑制作用，可浓度依赖性抑制2.5mM CaCl2引起的冠脉收缩，其IC50值为22.5M。
第二部分槲皮素对大鼠离体冠状动脉血管平滑肌细胞内钙离子浓度和LVGC电流及Kv电流的影响
实验目的
1.观察槲皮素对大鼠冠脉血管平滑肌细胞内钙离子浓度的影响。
2.观察槲皮素对大鼠冠脉血管平滑肌细胞LVGC电流和Kv电流的影响。
实验方法
1.细胞分离
利用两步酶解法分离得到大鼠冠状动脉血管平滑肌细胞：首先将冠脉血管置于I号酶解液中，即低钙的HEPES液（含有0.1mM CaCl2，1mg/mL白蛋白，0.5mg/mL木瓜蛋白酶和1mg/mL二硫丁四醇），在O2饱和且37℃条件下，孵育约30min，然后将冠脉血管转移至II号酶解液中，即无钙的HEPES液（含有1mg/mL白蛋白，0.5mg/mL胶原蛋白F和0.5mg/mL胶原蛋白H），在O2饱和且37℃条件下，孵育约15min，即得大鼠冠脉血管平滑肌细胞。
2.细胞内钙离子浓度的测定
用荧光染料Fluo-4-AM将大鼠冠脉血管平滑肌细胞染色，依次用正常HEPES液，40mM KCl，40mM KCl+10-100μM槲皮素，正常HEPES液，40mM KCl灌流细胞，分别记录不同条件下细胞的荧光值。实验结果用相对荧光密度值（F/F0）来反映细胞内钙离子浓度的变化。
3.电生理学测量
采用全细胞电压钳实验技术，记录并探讨槲皮素对大鼠冠状动脉血管平滑肌细胞LVGC电流和Kv电流的影响。
实验结果
1.槲皮素浓度依赖性降低KCl引起的冠脉血管平滑肌细胞内的钙离子荧光密度增加。40mM KCl使冠脉平滑肌细胞内的钙离子荧光密度增加了97.4%。槲皮素（10，30，100M）降低冠脉平滑肌细胞内钙离子的荧光密度百分比分别为30.1%，60.8%和86.5%。
2. Bay K8644（1μM）增大LVGC电流幅度，使峰值电流增加了31.9%；硝苯地平（1μM）抑制LVGC电流幅度，使峰值电流减少了75.6%，上述实验结果证明所记录电流为LVGC电流。槲皮素（3，10，30μM）浓度依赖性的使冠脉血管平滑肌细胞LVGC电流的I-V曲线右上移。单独给予+10mV单电压刺激时，槲皮素（3，10，30μM）抑制LVGC峰值电流的百分比分别为28.7%，57.1%和76.8%。
3.在测试电压为+60mV时，冠脉血管平滑肌细胞KV电流峰值为345.26±23.57pA，电流密度为32.52±5.24pA/pF（n=12）。槲皮素（10，30，100μM）浓度依赖性的使冠脉血管平滑肌细胞KV电流的I-V曲线上移。单独给予+60mV单电压刺激时，槲皮素（10，30，100μM）增加KV电流的百分比分别为20.1%，38.1%和73.3%。
第三部分槲皮素对大鼠离体心脏冠脉流量及血流动力学参数的影响
实验目的
利用Langendorff实验系统，观察槲皮素对大鼠离体心脏冠脉流量（coronaryflow, CF）及血流动力学参数：左心室发展压（left ventricular developed pressure,LVDP），左心室舒张末压（left ventricular end-diastolic pressure, LVEDP），左心室内压最大上升速率（positive dp/dtmax,+dp/dtmax）和左心室内压最大下降速率（negative dp/dtmax,-dp/dtmax）的影响。
实验方法
利用Langendorff实验系统，先用台式液灌流大鼠心脏，记录冠脉流量及血流动力学参数。然后分别用含不同浓度槲皮素（0.3，1，3，10，30μM）的台式液灌流大鼠心脏，观察冠脉流量及血流动力学参数的变化。
实验结果
槲皮素使大鼠离体心脏CF，LVDP，+dp/dtmax和-dp/dtmax值呈浓度依赖性的增大，但对LVEDP值无显著影响。
1.用正常台式液灌流时，CF值为7.8±1.2mL/min，改用含不同浓度槲皮素的台式液灌流时，槲皮素（0.3，1，3，10，30μM）浓度依赖性的增大CF值，最大值为13.0±1.3mL/min，其EC50值为3.04μM。
2.离体大鼠心脏LVDP和+dp/dtmax的基础值分别为：64±3mmHg，1368±124mmHg/s。随灌流液中槲皮素浓度增加（0.3，1，3，10，30μM），上述两项指标均逐渐增大，最大值分别为：78±2mmHg，1692±196mmHg/s。
3.离体大鼠心脏-dp/dtmax的基础值为：1143±126mmHg/s。随灌流液中槲皮素浓度增加（0.3，1，3，10，30μM），-dp/dtmax值逐渐增大，最大值为：1375±151mmHg/s。离体大鼠心脏LVEDP的基础值为：10±1mmHg，槲皮素对LVEDP值无显著影响。
全文实验结论
1.槲皮素浓度依赖性舒张大鼠冠状动脉，其舒张作用是非内皮依赖性的，且与增强Kv和抑制LVGC有关；
2.槲皮素增加离体大鼠心脏冠脉流量，这一结论与肌源性实验所得槲皮素舒张大鼠冠脉相一致，且槲皮素对离体大鼠心脏有一定的正性肌力作用。
Background
Quercetin is as a typical fruit-derived flavonoid, which is widely distributed infruits, vegetables, botanic flowers and leaves, such as apple, sea buckthorn, broccoli,onions, tea, etc. Quercetin has been reported to possess many biological activities,including antioxidation, antiplatelet aggregation, antiinflammatory,immunomodulation, anticancer as well as reduction of cardiac ischemia-reperfusioninjury. Quercetin could induce vasodilation in rat aorta, rat mesenteric arteries, ratportal vein and porcine coronary arteries, whether quercetin could exert relaxationeffect on coronary artery (CA) of rat has not been studied.
Calcium influx mainly through L-type voltage gated calcium channels (LVGC),which is prerequisite for the physiological function of vasomotor regulation ofvascular smooth muscle cells (VSMCs). At the same time, potassium channels couldmaintain normal cell membrane potential and regulate of vascular tone. There aremainly four kinds of potassium channels on vascular smooth muscle: voltage-gatedK+channels (Kv), Ca2+-activated K+channels (KCa), inward rectifier K+channels (KIR)and ATP-activated K+channels (KATP). However, Kv channels are the highestexpression, Kv channels play an important role in regulating cell resting potential andsmall artery diameter. The present study was designed to observe the effects ofquercetin on coronary dilation of rat and coronary flow and hemodynamic parametersof isolated rat heart, and whether LVGC and KVare involved in quercetin-inducedvasorelaxation in rat coronary arteries (RCAs).
Part1The myogenic response of quercetin in rat coronary arteryObjectives
1. To observe the myogenic response of quercetin in isolated RCAs, and to elucidatethe effect of quercetin on the inhibitory vasoconstriction and relaxantion in RCAs.
2. In the myogenic experiment, we used the potassium channels blockers, nitric oxidesynthase inhibitor and endothelium denudation to study the relaxant effect ofquercetin on RCAs, and whether potassium channels, nitric oxide (NO) andendothelium are involved in myogenic response of quercetin.
3. In the calcium-free solution, the effect of quercetin on extracellular calcium inducecontraction was observed.
Methods
1. After sacrifice, the heart was removed and transferred immediately into chilled(4°C) HEPES solution bubbled with100%O2. RCAs were isolated, cut into2mm-long rings and mounted on a wire myograph (Multi Myograph System-610M,DMT, Denmark) using two tungsten wires (25μm in diameter) in a tissue chambercontaining of5.0mL HEPES solution bubbled with100%O2,37°C. The rings werenormalized, stretched to a state equal to80mmHg, equilibrated for at least1h andwashed with HEPES solution (37°C) every15min. The tension changes of rings wererecorded by Chart7.03on the computer. After equilibration1h, the rings werecontracted with60mM KCl, then were relaxed with Ach, three times repeatedly.When the contraction or relaxation difference did not exceed10%, we thought that therings were good at the vascular reactivity. We discarded the arterial rings in case thatthe contraction was not repeatable or not sustained, or the contraction was less than2mN.
2. High K+HEPES solution or U46619was cumulatively added to the chamber toconstruct a concentration-contraction curve for KCl (20,28,39,55,77,108mM) orU46619(0.03,0.1,0.3,1,3μM). When successive curves were repeatable, the arterialrings were preincubation with quercetin (3,10,30μM) for15min beforereconstruction of the curves for KCl or U46619in the presence of quercetin(quercetin was always in the chamber in this period). The maximal contraction induced by108mM KCl or3μM U46619in the absence of quercetin was taken as100%and the percentage of contraction by each concentration of stimulator in theabsence or presence of quercetin was calculated.
3. When the contraction induced by60mM KCl or1μM U46619was sustained,quercetin (1,3,10,30,100μM) or vehicle were added cumulatively to theendothelium-intact or-denudation RCAs. The maximal contraction induced by60mM KCl or1μM U46619in the absence of quercetin was taken as100%and thepercentage of contraction by each concentration of stimulator in the absence orpresence of quercetin or vehicle was calculated.
4. When the contraction induced by60mM KCl or1μM U46619reached a sustainedplateau,4-aminopyridine (4-AP, a KVblocker), iberiotoxin (Iber, a KCablocker),BaCl2(a KIRblocker) or glibenclamide (Glib, a KATPblocker) was added respectivelyto the chamber. After10min, based on the arterial tone was stable in the presence ofan inhibitor, quercetin (1,3,10,30,100μM) was cumulatively added to the abovechamber. Relaxation was expressed as a percentage of the precontraction induced by60mM KCl or1μM U46619, respectively.
5. When the contraction induced by60mM KCl was repeatable, the ring was rinsedand incubated with Ca2+-free HEPES solution (containing1mM EGTA) for20min.When the tone of the ring restored to the baseline, the solution in above chamber waschanged to Ca2+-free HEPES solution. After10min, the ring was stimulated with60mM KCl or1M U46619, after10min,2.5mM CaCl2was added to the chamber.Quercetin was added to the chamber (the bath concentration of quercetin was keptconstant through the following experiment), after15min, the ring was stimulated with60mM KCl or1μΜ U46619in the Ca2+-free HEPES solution, after10min,2.5mMCaCl2was added to the chamber. The contraction induced by60mM KCl,1μΜU46619and2.5mM CaCl2were recorded in the absence or presence of quercetin.
Results
1. KCl (20,28,39,55,77,108mM) and U46619(0.03,0.1,0.3,1,3μM) inducedcontraction concentration-dependently. The maximal contractions were4.910.38 mN and4.830.41mN, respectively. The values of EC50were31.87mM for KCland0.19M for U46619, respectively. Pretreatment with quercetin (3,10,30M)shifted the concentration curves downwards to the right. The values of IC50were48.80M for KCl and88.30M for U46619, respectively.
At3μM quercetin, there were no significant difference between quercetin groupand control group on KCl or U46619-contraction (P>0.05). At10μM, quercetindepressed the maximal contraction of KCl or U46619by35.4%and21.3%,respectively. There were significant difference between quercetin group and controlgroup on KCl or U46619-contraction (P <0.05). At30μM, quercetin depressed themaximal contraction of KCl or U46619by79.9%and54.7%, respectively. Therewere significant difference between quercetin group and control group on KCl orU46619-contraction (P <0.05).
2. Quercetin (1,3,10,30,100μM) elicited concentration-dependent relaxation inendothelium-intact or-denudation RCAs, and endothelium has no effect on relaxationof quercetin in RCAs.
Vehicle did not significantly affect the tone of endothelium-intact RCAs; themaximal relaxation of quercetin in RCAs precontracted by either60mM KCl or1M U46619were (96.34±7.35)%and (82.47±7.24)%, respectively. The values ofRC50were22.87M for KCl and41.42M for U46619, respectively.
Compared endothelium-denudation RCAs with endothelium-intact RCAs, themaximal relaxation of quercetin did not change significantly (P>0.05).
3. Preincubation with Iber, Glib, BaCl2or L-NAME, there have no effect on therelaxation of quercetin in RCAs, but preincubation with4-AP (1mM) attenuated therelaxation on KCl-precontraction by42.88%(P <0.05). Preincubation with4-AP (1mM) attenuated the relaxation on U46619-precontraction by28.82%(P <0.05).
4. In Ca2+-free HEPES solution,60mM KCl failed to produce significant contraction(phasic) and restoration of2.5mM CaCl2evoked a full contraction (tonic). Quercetin(3,10,30μM) inhibited2.5mM CaCl2-induced contraction in aconcentration-dependent manner and the calculated IC50was9.55M. Quercetin didnot significantly affect the phasic for U46619, but reduced tonic component of the contraction and the calculated IC50was22.5M.
Part2Effects of quercetin on [Ca2+]inand LVGC currents and Kvcurrents of rat coronary artery smooth muscle cells
Objectives
1. To study the effect of quercetin on [Ca2+]influorescence intensity of RCA VSMCs.
2. In order to clarify the mechanism of quercetin relaxation in RCAs, we recordedLVGC currents and Kv currents of RCA VSMCs.
Methods
1. Rat coronary artery smooth muscle cells isolation:
Coronary artery smooth muscle cells were obtained using two steps enzymatic:Firstly, RCAs were placed in enzymatic solution I (HEPES solution containing0.1mM CaCl2,1mg/mL albumin,0.5mg/mL papain and1mg/mL dithioerythritol)bubbled with100%O2and incubated for30min at37oC. The RCAs were transferredto enzymatic solution II (HEPES solution containing1mg/mL albumin,0.5mg/mLcollagenase F and0.5mg/mL collagenase H) bubbled with100%O2and incubatedfor15min at37oC.
2.[Ca2+]inmeasurement:
Isolated RCA VSMCs were loaded with Fluo-4-AM and perfused with thedifferent solutions (HEPES solution,40mM KCl,40mM KCl+quercetin (10,30,100μM), HEPES solution and40mM KCl) in order of time precedence. Thefluorescence intensity of RCA VSMCs were recorded under various conditions.Fluorescence ratios (F/F0) were calculated to reflect the [Ca2+]in.
3. Electrophysiological measurements:
Using whole-cell voltage clamp, we recorded and investigated the effects ofquercetin on LVGC currents and Kv currents of RCA VSMCs.
Results
1. Quercetin reduced KCl-induced elevation of the intracellular Ca2+concentration ina concentration-dependent manner.40mM KCl increased fluorescence intensity ratio (F/F0, fluorescence after vs before40mM KCl) by97.4%. Application of quercetin(10,30,100M) decreased the intracellular Ca2+fluorescence intensity increment by30.1%,60.8%and86.5%, respectively.
2. Bay K8644(1M) increased LVGC currents and increased the peak currents by31.9%; nifedipine (1M) decreased LVGC currents and decreased the peak currentsby75.6%. These results proved that the currents recorded were LVGC currents.
Quercetin (3,10,30μM) shifted the I-V curve of LVGC currents of RCA VSMCsupwards to the right in a concentration-dependent manner. At test potential of+10mV,quercetin (3,10,30M) decreased LVGC currents by28.7%,57.1%and76.8%,respectively.
3. At test potential of+60mV, the stable peak of Kv currents was345.26±23.57pAand current density was32.52±5.24pA/pF (n=12). Quercetin (10,30,100μM)shifted the I-V curve of KVcurrents upward in a concentration-dependent manner. Attest potential of+60mV, quercetin (10,30,100M) increased the KVcurrents ofRCA VSMCs by20.1%,38.1%and73.3%, respectively.
Part3Effects of quercetin on coronary flow and hemodynamicparameters of isolated rat heart
Objective
To study the effects of quercetin on coronary flow (CF) and hemodynamicparameters (LVDP，LVEDP，+dp/dtmax，-dp/dtmax) of isolated rat heart by Langendorffapparatus.
Methods
The isolated rat heart was perfused firstly with Tyrode’s solution usingLangendorff apparatus, subsequently, it was perfused with Tyrode’s solutioncontaining different concentrations of quercetin (0.3,1,3,10,30μM). At the sametime, coronary flow and hemodynamics parameters were recorded.
Results
This experiment showed that quercetin increased the vaule of CF, LVDP, +dp/dtmaxand-dp/dtmax, but it had no effect on LVEDP.
1. When the isolated rat hearts were perfused with Tyrode’s solution, the vaule of CFwas7.8±1.2mL/min, then they were perfused with Tyrode’s solution containingdifferent concentration of quercetin (0.3,1,3,10,30μM), quercetin increased the CFin a concentration-dependent manner, the maximal value of CF was13.0±1.3mL/min and the EC50was3.04μM.
2. Quercetin increased LVDP and+dp/dtmaxof isolated rat hearts in aconcentration-dependent manner, the basal value were64±3mmHg (LVDP) and1368±124mmHg/s (+dp/dtmax), respectively. With increasing concentrations ofquercetin (0.3,1,3,10,30μM), the value of LVDP and+dp/dtmaxwere graduallyincreased, the maximal value were78±2mmHg and1692±196mmHg/s,respectively.
3. Quercetin increased-dp/dtmaxof isolated rat hearts in a concentration-dependentmanner, the basal value was1143±126mmHg/s. With increasing concentrations ofquercetin (0.3,1,3,10,30μM), the value of-dp/dtmaxwas gradually increased, themaximal value was1375±151mmHg/s. The basal value of LVEDP was10±1mmHg, quercetin had no effect on LVEDP.
Conclusions
1. Quercetin elicited relaxation in RCA in a concentration-dependent manner. Therelaxation effect of quercetin on RCA was non-endothelial dependent, and wasassociated with increased Kv currents and depressed LVGC currents of RCA VSMCs.
2. Quercetin increased coronary flow of isolated rat heart, this result was consistentwith the relaxation effect of quercetin on RCA in myogenic experiment. Quercetinhad a positive inotropic effect on isolated rat heart.
引文
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