中枢内源性硫化氢对血流动力学的作用及机制研究

发布时间：2018-01-20 07:19

本文关键词： 中枢神经系统硫化氢平均动脉血压心率心功能洋地黄类因子 K_(ATP)通道 Na~+-K~+-ATP酶活性　出处：《武汉大学》2012年博士论文　论文类型：学位论文

【摘要】：目的：内源性硫化氢(Hydrogen Sulfide,H2S)作为一个新的神经调质,参与了中枢神经系统的生理功能的调节和疾病过程。本课题的目的是探讨中枢神经系统内源性H2S对正常大鼠血流动力学的调节作用及其机制。方法：侧脑室给予外源性H2S饱和溶液和H2S生成酶胱硫醚-p-合成酶(cystathionine β-synthase, CBS)或3-巯基丙酮酸(3-mercaptopyruvate, MP)或H2S前体半胱氨酸氨或胱硫醚-p-合成酶抑制剂羟胺(Hydroxylamine,HA)及不同的干预试剂；采用左心室和股动脉插管法及四道生理记录仪记录平均动脉血压(mean arterial pressure,MABP)、心率(heart rate,HR)、左室收缩压(Left ventricle systolic pressure,LVSP)、左室舒张末压(left ventricle end-diastolic pressure,LVEDP)及左室最大压力上升及下降心室内压的变化率(rate of the rise of LV ventricular pressure,±p/dt);采用气管插管法测呼吸深度和呼吸频率；采用免疫印迹法检测3-巯基丙酮酸硫基转移酶和胱硫醚-p-合成酶蛋白浓度；采用亚甲基蓝法测定组织硫化氢表达水平；采用生物化学方法测定心肌和血管组织Na+-K+-ATPase活性；采用放射免疫法测定血浆洋地黄含量。结果：(1)免疫印迹实验证实,中枢神经系统存在高水平的H2S生成酶CBS和MPST。CBS依赖的H2S生成前体半胱氨酸,下丘脑和皮质分别生成62.5±5.3和63.8±10.3nmol/min/g组织的H2S。给予3-巯基丙酮酸,H2S生成大约是给予半胱氨酸的2倍即125.7±16.8和134.6±17.7nmol/min/g组织,各自与半胱氨酸生成H2S比较,两者的差异有显著意义(a11P0.01)。而预先给予CBS的抑制剂羟胺(HA),再给予半胱氨酸和MPST, HA能够显著抑制半胱氨酸生成H2S,但不影响MPST生成H2S的作用。 (2)侧脑室(ICV)给予不同浓度H2S饱和缓冲液可剂量依赖性的导致大鼠平均动脉血压急剧下降,而后迅速上升。其最大降低幅度分别是7.8±2.3、11.4±2.6和16.1±3.6mmHg,最大上升幅度分别是9.2±3.4、12.2±2.1和20.8±2.5mmHg,各组变化最显著的对应时间点统计学均有显著差异(p0.001)。 (3)ICV连续缓慢注射H2S饱和缓冲液大鼠平均动脉血压先降低迅速升高而后缓慢升高,在110min时达到最高值即38±8.6mmHg,峰值出现在100-110min之间,观察期内血压没有下降。与对照组比较,各时间点差异均有统计学意义(P0.01),而心率和呼吸没有显著的变化。 (4)ICV分别注射H2S生成前体L-半胱氨酸(L-Cys)、H2S生成酶3-巯基丙酮酸硫基转移酶(MPST)导致快速的升高平均动脉血压。ICV注射H2S生成酶胱硫醚-β-合成酶抑制剂羟胺(HA),平均动脉血压快速下降而后缓慢的回升。 (5)ICV注射L-Cys在观察期内心率缓慢的降低,其最大降低值大约是100次/分,与正常对照比较统计学有显著差异(P0.01)；ICV注射HA或注射H2S气体饱和缓冲液(2.8μmol/kg)对心率没有影响；预先ICV给予HA阻断CBS活性,10min后再ICV注射L-Cys,显著的减弱L-Cys减慢心率的作用(P0.01)。 (6)ICV微量注射泵持续注射不同浓度的H2S饱和溶液,剂量依赖性的增加左心室发展压和心室发展压最大变化率,但对左心室舒张末压没有显著影响；ICV注射H2S生成前体L-Cys增加左心室发展压和心室发展压变化率；相反,H2S生成酶CBS抑制剂羟胺能够短暂的降低左心室发展压和心室发展压最大变化率。 (7)外周静脉给与α受体阻断剂酚妥拉明,几乎完全阻断了中枢给予外源性H2S和内源性H2S的升压效应,与对照组比较统计学有显著差异(P0.01)。 (8)ICV不同浓度的H2S饱和缓冲液可剂量依赖性的显著增加大鼠的呼吸深度减慢呼吸频率,在前1分钟内变化最显著,而后呼吸逐渐恢复正常,各组间均有统计学意义(P均0.001)。 (9)ICV给予H2S饱和缓冲液和KATP通道开放剂吡那地尔,能够显著的降低平均动脉血压。而预先给与KATP通道阻滞剂glibenclimad,能够显著的阻断H2S饱和缓冲液和KATP通道开放剂吡那地尔的降压效应(P0.01),但不影响H2S的升压效应。 (10)β受体阻断剂美托洛尔显著的抑制了中枢内源性H2S对心脏的正性肌力作用,而glibenclamide却没有影响。 (11)与生理盐水组比较,I.C.V半胱氨酸组血浆内洋地黄类因子水平增加了大约1倍(P0.01)。而羟胺(HA)轻微的降低了洋地黄类因子水平,与生理盐水组比较没有显著差异(P0.05)。 (12)与对照组比较,内源性H2S显著抑制了心脏和腹主动脉检测心肌和腹主动脉Na+-K+-ATP酶活性(P0.001)；而羟胺显著增加了它们的Na+-K+-ATP酶活性(P0.001) 结论：中枢神经系统由CBS和MPST催化产生高水平的内源性H2S。中枢给予外源性和内源性H2S均能够增加大鼠平均动脉血压,减慢心率,增强心功能。中枢神经系统H2S通过激活外周交感神经活性、增加呼吸深度减慢呼吸频率、激活中枢KATP通道、而调节动脉血压；通过激活外周交感神经活性、增加心肌内源性洋地黄类因子的释放和抑制心肌和腹主动脉Na-K+-ATP酶活性增强心功能。
[Abstract]:Objective: endogenous hydrogen sulfide (Hydrogen Sulfide, H2S) as a new neurotransmitter involved in the regulation of physiological function and disease of the central nervous system. The purpose of this study is to investigate the central nervous system regulation of endogenous H2S on hemodynamics in normal rats were used and its mechanism.
Methods: intracerebroventricular administration of exogenous H2S solution and H2S generating enzyme cystathionine synthase -p- (cystathionine beta -synthase, CBS) or 3- mercaptopyruvate (3-mercaptopyruvate, MP) or H2S precursor cysteine ammonia or cystathionine synthase inhibitor -p- hydroxylamine (Hydroxylamine, HA) and different intervention by the left ventricle and reagents; femoral artery intubation and four channel physiological recorder to record the mean arterial blood pressure (mean arterial, pressure, MABP), heart rate (heart rate, HR), left ventricular systolic pressure (Left ventricle systolic pressure, LVSP), left at the end of Shi Shuzhang (left ventricle end-diastolic pressure, pressure LVEDP) changes and left ventricular maximum pressure rise and decline of ventricular the internal pressure ratio (rate of the rise of LV ventricular pressure + p/dt); method of measuring the depth of breathing with tracheal intubation and respiratory frequency detection; 3- mercaptopyruvate Sbased metastasis by Western blot Enzyme and cystyl sulfide -p- synthetase protein concentration; methylene blue method was used to detect tissue hydrogen sulfide expression level; biochemical activity was used to detect Na+-K+-ATPase activity in myocardium and vascular tissue; plasma digitalis content was measured by radioimmunoassay.
Results: (1) confirmed by Western blot, H2S generating enzyme with high levels of CBS and MPST.CBS on the formation of H2S precursor cysteine in the central nervous system, hypothalamus and cortex were 62.5 + 5.3 and 63.8 + 10.3nmol/min/g H2S. given 3- mercaptopyruvate, H2S generation is about 2 times that of 125.7 cysteine to + 16.8 and 134.6 + 17.7nmol/min/g, respectively with cysteine H2S generation, there are significant differences (a11P0.01). The pretreatment with CBS inhibitor hydroxylamine (HA), and then give the cysteine and MPST, HA can significantly inhibit the formation of cysteine H2S, but does not affect the formation of MPST H2S.
(2) the lateral ventricle (ICV) with different concentrations of H2S buffer saturated dose dependently resulted in mean arterial blood pressure of rats decreased rapidly, then increased rapidly. The maximum decreases were 7.8 + 2.3,11.4 + 2.6 and 16.1 + 3.6mmHg, the largest increases were 9.2 + 3.4,12.2 + 2.1 and 20.8 + 2.5mmHg. Each time point corresponds to a change of the most significant differences were statistically significant (p0.001).
(3) ICV continuous slow injection of H2S saturated mean arterial blood pressure of rats decreased buffer increased rapidly and then slowly increased, when 110min reached the highest value is 38 + 8.6mmHg, the peak at 100-110min, the observation period of blood pressure did not drop. Compared with the control group at each time point, the differences were statistically significant (P0.01). There are no significant changes in heart rate and respiration.
(4) ICV were injected with H2S generated precursor L- cysteine (L-Cys), H2S synthase 3- mercaptopyruvate sulfurtransferase (MPST) led to the rapid increase of mean arterial pressure.ICV injection of H2S generating enzyme cystathionine beta synthase inhibitor hydroxylamine (HA), mean arterial blood pressure decreased rapidly and then slow recovery.
(5) ICV injection of L-Cys during the observation period of heart rate decreased slowly, the maximum reduction is about 100 beats per minute, compared with normal control showed a statistically significant difference (P0.01); ICV injection of HA or injection of H2S gas saturated buffer (2.8 mol/kg) had no effect on heart rate; pre ICV HA blocking CBS activity. After 10min ICV injection of L-Cys, L-Cys significantly attenuated the effect of slowing heart rate (P0.01).
(6) ICV micro injection pump continuous injection of different concentrations of H2S solution, dose dependent increase in left ventricular developed pressure and ventricular developed pressure maximum rate of change, but has no significant effect on left ventricular end diastolic pressure; ICV injection increased generation of H2S precursor L-Cys left ventricular developed pressure and ventricular developed pressure change rate; on the contrary, H2S synthase CBS inhibitor hydroxylamine can reduce left ventricular developed pressure and ventricular developed pressure maximum rate of change short.
(7) peripheral vein administration of alpha blocker, phentolamine, almost completely blocked the pressor effect of exogenous H2S and endogenous H2S in the central nervous system, which was significantly different from that in the control group (P0.01).
(8) ICV H2S buffer at different concentrations increased significantly in a dose-dependent manner, breathing depth and respiratory rate in rats, and the most significant change in the first 1 minutes, and then returned to normal after breathing. There was statistical significance among all groups (P = 0.001).
(9) ICV H2S saturated buffer and KATP channel opener pinacidil, can significantly reduce the mean arterial blood pressure. But given the KATP channel blocker glibenclimad can block the hypotensive effect of H2S saturated buffer and KATP channel was opener pinacidil (P0.01), but does not affect the pressor effect of H2S.
(10) metoprolol, a beta blocker, significantly inhibited the positive inotropic action of the central endogenous H2S on the heart, while glibenclamide did not affect it.
(11) compared with the saline group, the plasma level of digitalis like factor increased by about 1 times (P0.01) in the I.C.V cysteine group, while hydroxylamine (HA) slightly reduced digitalis like factor level, which was not significantly different from that in the saline group (P0.05).
(12) compared with the control group, endogenous H2S significantly inhibited the Na+-K+-ATP activity of myocardium and abdominal aorta (P0.001) in heart and abdominal aorta, while hydroxylamine significantly increased their Na+-K+-ATP enzyme activity (P0.001).
Conclusion: the central nervous system by CBS and MPST catalyzed by endogenous H2S. central high levels of exogenous and endogenous H2S can increase mean arterial blood pressure of rats, decrease the heart rate and improve cardiac function. Central nervous system H2S through activation of peripheral sympathetic nerve activity, increase the depth of breathing slowed breathing frequency, activation of Central KATP channel. The regulation of blood pressure; through the activation of peripheral sympathetic nerve activity, increased myocardial endogenous digitalis factor release and inhibition of myocardial and aortic Na-K+-ATP activity enhanced heart function.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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