硫化氢对大鼠心功能的影响及其离子通道机制

发布时间：2018-05-11 03:27

本文选题：硫化氢 + 心功能　；参考：《宁夏医科大学》2010年硕士论文

【摘要】：H_2S是一种内源性气体信号分子,在心血管系统中发挥着重要的生理和病理作用。已有报道H_2S可抑制心肌细胞L型钙通道,还可浓度依赖性地降低离体灌流大鼠心脏的心功能。但灌流实验并未钳制离体大鼠心脏的心率,而心率的变化对心功能也会产生影响,此外我们在预实验中发现低于生理浓度的H_2S有改善心脏的舒张功能。因此研究低浓度H_2S对心脏的保护作用和机制具有重要意义。目的本工作在离体灌流大鼠心脏观察在固定心率情况下,硫化氢对大鼠心脏冠脉流量及心功能的影响;以急性分离的大鼠心肌细胞为模型,应用膜片钳技术,观察硫化氢对心肌细胞L型钙通道电流(ICa,L)、钠钙交换电流(INaCa)、ATP敏感钾通道(I_(KATP))等电流的影响,以进一步明确硫化氢对心肌舒缩功能的影响及其离子电流机制。方法离体SD大鼠心脏采用Langendorff系统用KH液进行逆行恒压(80cmH2O)灌流,并固定离体心脏心率。观察不同剂量NaHS对左室收缩压(LVSP)、左室舒张末压(LVEDP)、左心室内压最大上升/下降速率(±dp/dtmax),冠脉流量(CF)的影响。以LVSP和LVEDP差值作为左室发展压(LVDP)。应用酶解法分离心肌细胞,用膜片钳技术记录单个心肌细胞ICa,L、INaCa、I_(KATP),给药组以不同浓度NaHS的细胞外液灌流心肌细胞5 min;对照组直接以细胞外液灌流5 min,记录并观察电流曲线的变化。结果1、硫化氢对离体大鼠心脏功能的影响:给药前平衡灌流期间,各实验组冠脉流量和各项心功能观察指标基本保持稳定,各组间所测指标无显著差异。对照组灌流KH液15min,LVEDP为7.06±0.42mmHg,12.5、25μmol/L(低于生理浓度)NaHS组,灌流15min,LVEDP分别为5.88±0.97mmHg和5.42±0.88 mmHg,显著性低于对照组(P0.01,n=8); 50μmol/L(生理浓度)NaHS组,LVEDP无明显变化,与对照组相比无显著差异(P0.05,n=6),100和200μmol/L NaHS组, LVEDP显著升高,分别达15.22±3.34 mmHg、24.81±3.61 mmHg。NaHS 12.5、25μmol/L组冠脉流量增加,NaHS 50、100和200μmol/L组冠脉流量呈时间依赖性下降。以上提示,在心率固定的条件下,低浓度的NaHS可能对冠脉有扩张作用,因而冠脉流量增加。NaHS 25μmol/L(n=8),50、100和200μmol/L(n=6)灌流15 min,LVDP均降低,分别下降10±6%、19±2%、51±5%和76±6%;+dp/dtmax的抑制百分数分别为16±4%、23±10%、52±10%和65±9%;-dp/dtmax的抑制百分数分别为17±4%、23±11%、61±6%、74±5%,分别与空白对照组相比较,有显著性差异(P0.05)。以上结果表明,NaHS能够浓度依赖性地抑制离体大鼠心脏的收缩功能。2、硫化氢对大鼠心肌细胞L型钙电流的影响:硫化氢呈浓度依赖性抑制大鼠心肌细胞ICa,L,6.25μmol/LNaHS灌流1 min和5min后,ICa,L分别下降4±2%、8±3%,12.5μmol/LNaHS灌流1 min和5min后,ICa,L分别下降8±3%、14±5%,与空白对照组相比虽无统计学意义,但此浓度对ICa,L已经有一定的抑制作用。25、50、100和200μmol/L NaHS灌流1 min后,ICa,L分别下降13±2%、19±2%、23±4%和27±5%,与对照组的3±1%相比均有统计学差异(P 0.05, n=8);灌流5 min后,ICa,L分别下降22±4%、25±3%、29±7%和52±9%,均显著高于对照组的6±2%(P 0.01, n=8)。200μmol/L NaHS灌流1 min后IV曲线明显上移,但不改变ICa,L的电压依赖关系和反转电位。对照组ICa,L激活曲线的半激活电位为-19.52±0.54 mV,斜率为4.85±0.52 mV;200μmol/L NaHS灌流灌流1 min后半激活电位为-19.03±0.59 mV,斜率为5.43±0.55 mV,二者比较尚无统计学意义(P 0.05, n=8)。对照组ICa,L失活曲线半失活电位为-23.61±0.15 mV,斜率为5.32±0.13 mV;200μmol/L NaHS灌流灌流1 min后半失活电位为-24.57±0.15 mV,斜率为5.44±0.13,二者无明显区别(P0.05, n=8)。上述结果表明上述浓度硫化氢不改变L型钙通道激活和失活的门控特点。3、硫化氢对大鼠心肌细胞钠钙交换电流的影响:硫化氢对正、反向钠钙交换电流均有增大作用。给予浓度为6.25、12.5、25、50、100、200μmol/L的NaHS 5min后,正向INaCa比给药前分别增大15±6%、20±5%、33±8%、16±4%、25±3%、29±7%,均显著高于对照组((P0.05,n=10),6.25、12.5、25、50、100、200μmol/L组反向INaCa分别增大2±1%、6±3%、10±2%、11±2%、21±4%、25±3%,后五组与对照组比较显著增大(P0.05,n=10),对照组单纯给予细胞外液5 min后正、反向INaCa分别减小5±2%和3±2%。4、硫化氢对大鼠心肌细胞ATP敏感性钾通道电流的影响:在由50 mV到-100 mV持续125 ms的斜坡刺激下记录心肌细胞KATP通道电流。给予pinacidil后该电流明显增加,并且能被glibenclamide阻断(说明该电流是I_(KATP))。50、100和200μmol/L NaHS不能使KATP通道开放。400、800和1600μmol/L NaHS能使KATP通道开放,ATP敏感钾通道开放后的电流密度分别为17.25±8.45 pA/pF、20.33±8.56 pA/pF和22.87±6.23 pA/pF。各组间比较无显著差异(P0.05, n=8)。表明较高浓度的NaHS才能开放KATP通道,但此浓度已经达到显著抑制心肌细胞膜L型钙通道。结论高于正常生理浓度的NaHS可以浓度依赖性地抑制离体灌流大鼠心脏心功能,低浓度的NaHS能改善心肌舒张功能。NaHS可以浓度依赖性地抑制急性分离的大鼠心肌细胞膜上的L型钙通道电流,在没有改变钙离子通道动力学特征的情况下减少了通道开放时的外钙内流,从而减弱了心肌细胞的收缩功能。低浓度的NaHS可以增大大鼠心肌细胞的正向钠钙交换电流,提示低浓度的NaHS对心肌舒张功能的改善可能部分是通过增加正向钠钙交换电流实现的。高浓度的NaHS可以使急性分离的大鼠心肌细胞ATP敏感性钾通道开放,而低浓度的NaHS能抑制心肌细胞L型钙通道从而抑制心肌细胞收缩可能和其是ATP敏感性钾通道开放剂的作用没有关系。
[Abstract]:H_2S is an endogenous gas signal molecule, which plays an important physiological and pathological role in the cardiovascular system. It has been reported that H_2S can inhibit the L calcium channel in cardiac myocytes and reduce the cardiac function of isolated rat heart in a concentration dependent manner. However, perfusion test did not clamp the heart rate of the isolated rat heart, and the heart rate changes to the heart. Functions also have an impact. In addition, we have found that H_2S, which is lower than the physiological concentration, can improve the heart diastolic function in the pre experiment. Therefore, it is important to study the protective effect and mechanism of low concentration of H_2S on the heart.
Objective To observe the effect of hydrogen sulfide on cardiac coronary flow and cardiac function in rat heart under the condition of fixed heart rate, with acute isolated rat cardiac muscle cells as model, and using patch clamp technique to observe L type calcium channel (ICa, L), sodium calcium exchange current (INaCa) and ATP sensitive potassium in cardiac myocytes. The effect of current (I_ (KATP)) on the effect of hydrogen sulfide on myocardial contractile function and its ionic current mechanism were further clarified.
Methods the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (LVEDP), left ventricular pressure (+ dp/dtmax) and coronary flow rate (CF) were observed at different doses of NaHS. The difference between LVSP and LVEDP was taken as the effect of the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and left ventricular pressure (LVEDP). Left ventricular development pressure (LVDP). Myocardial cells were separated by enzyme hydrolysis, and single myocardial cells ICa, L, INaCa, I_ (KATP) were recorded by patch clamp technique. The 5 min in the administration group was administered with different concentrations of NaHS, and the control group was injected with 5 min directly from the extracellular fluid and recorded and observed the changes of the current curve.
Results 1, the effect of hydrogen sulfide on the cardiac function of rats in vitro: the observation indexes of coronary flow and heart function in each experimental group remained stable during the period of balance perfusion, and there was no significant difference between the groups. The control group was 15min, LVEDP was 7.06 + 0.42mmHg, 12.5,25 mu mol/L (below the physiological concentration) NaHS group, perfusion of 15min, LVED P was 5.88 + 0.97mmHg and 5.42 + 0.88 mmHg respectively, significantly lower than the control group (P0.01, n=8), and 50 mu mol/L (physiological concentration) NaHS group, there was no significant change in LVEDP (P0.05, n=6), 100 and 200 mu mol/L NaHS group, which was significantly higher than 15.22 + 3.34, 24.81 + 3.61 In addition, the coronary flow of NaHS 50100 and 200 mu group was time dependent. The above suggested that under the condition of heart rate fixation, the low concentration of NaHS might have dilation effect on the coronary artery, so the coronary flow rate increased by.NaHS 25 mu mol/L (n=8), 50100 and 200 micron mol/L (n=6) perfusion 15 min, LVDP decreased, respectively, 10 + 6%, 19 + 2%, 51 + 5% and 76 + 6%; +d The inhibitory percentages of p/dtmax were 16 + 4%, 23 + 10%, 52 + 10% and 65 + 9% respectively. The inhibitory percentages of -dp/dtmax were 17 + 4%, 23 + 11% and 61 + 23, respectively. Compared with the blank control group, there were significant differences (P0.05). The results showed that NaHS could inhibit the contractile function.2 of the isolated rat heart in a concentration dependent manner, and the hydrogen sulfide could be used in rats The effect of L type calcium current in cardiac myocytes: hydrogen sulfide was concentration dependent inhibition of rat myocardial cells ICa, L, 6.25 mu mol/LNaHS perfusion 1 min and 5min, ICa, L decreased 4 + 2%, 8 + 3%, 12.5 micron 1 min and 5min, ICa, respectively, 8 + 3%, 14 + 5%, although no statistical significance compared with the empty white control group, but this concentration was already After.25,50100 and 200 mol/L NaHS perfusion 1 min, ICa and L were decreased by 13 + 2%, 19 + 2%, 23 + 4% and 27 + 5%, respectively, compared with the control group's 3 + 1% (P 0.05, n=8). After 1 min perfusion, the IV curve obviously moved up, but did not change the voltage dependence and reverse potential of ICa, L. The semi activated potential of ICa, L activation curve was -19.52 + 0.54 mV, and the slope was 4.85 + 0.52 mV in the control group. 200 mu mol/L NaHS perfusion flow 1 min half activation potential was 0.59, and the slope was 5.43 + 0.55. Two there was no statistical significance. Meaning (P 0.05, n=8). The semi inactivation potential of the control group ICa, L inactivation curve is -23.61 + 0.15 mV, the slope is 5.32 + 0.13 mV, and the second half inactivation potential of 200 u mol/L NaHS perfusion flow 1 min is -24.57 + 0.15 mV, and the slope is 5.44 + 0.13. The above results show that the above concentration of hydrogen sulfide does not change the activation and inactivation of the calcium channel. .3, the effect of hydrogen sulfide on the sodium and calcium exchange current of rat cardiac myocytes: hydrogen sulfide has an increasing effect on positive and reverse sodium calcium exchange current. After NaHS 5min with a concentration of 6.25,12.5,25,50100200 mol/L, the positive INaCa is increased by 15 +, 20 +, 33 + 8%, 16 + 4%, 25 + 3%, 29 + 7%, respectively. ( P0.05, n=10), the reverse INaCa of group 6.25,12.5,25,50100200 mu mol/L increased by 2 + 1%, 6 + 3%, 10 + 2%, 11 + 2%, 21 + 4%, 25 + 3%, then significantly increased (P0.05, n=10) in the five group compared with the control group (P0.05, n=10). Effect of flow: slope stab at 125 ms from 50 mV to -100 mV
The current of KATP channel of myocardial cells was stimulated. After pinacidil, the current increased obviously and could be blocked by glibenclamide (indicating that the current was I_ (KATP)).50100 and 200 mu mol/L NaHS did not open the KATP channel open.400800 and 1600 mu mol/L NaHS. The current density of the sensitive potassium channel was 17.25 + 8, respectively. There was no significant difference in.45 pA/pF, 20.33 + 8.56 pA/pF and 22.87 + 6.23 pA/pF. (P0.05, n=8). It showed that a higher concentration of NaHS could open the KATP channel, but this concentration had significantly inhibited the L type calcium channel in the myocardial cell membrane.
Conclusion NaHS, which is higher than normal physiological concentration, can inhibit the cardiac cardiac function of rat perfusion in vitro. The low concentration of NaHS can improve the myocardial diastolic function.NaHS, which can inhibit the L type calcium channel current on the membrane of acute isolated rat cardiac myocytes, without changing the dynamic characteristics of calcium ion channel. The low concentration of NaHS can increase the positive sodium calcium exchange current in rat cardiac myocytes, suggesting that the low concentration of NaHS may be partly achieved by increasing the positive sodium calcium exchange current. The high concentration of NaHS can make it urgent. The ATP sensitive potassium channel of isolated rat cardiomyocytes is open, and the low concentration of NaHS can inhibit the L calcium channel in cardiac myocytes and inhibit the contraction of cardiac myocytes, which may not be related to the effect of the ATP sensitive potassium channel opener.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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