【作者】 任永生；

【导师】 欧阳静萍；

【作者基本信息】 武汉大学 ， 病理和病理生理学， 2012， 博士

【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(a11P<0.01)。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(p<0.001)。(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(P<0.01),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(P<0.01)；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(P<0.01)。(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(P<0.01)。(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(P<0.01),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(P<0.01)。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(P>0.05)。(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(P<0.001)；而羟胺显著增加了它们的Na+-K+-ATP酶活性(P<0.001)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。更多还原

【Abstract】 AIM:Endogenous hydrogen sulfide is recongnized as a new neuromodulator which take part in the regulation of central neurous system phyisology and diseases. To explore the effect of endogenous hydrogen sulfide on hemodynamic effects and its probable mechanism.Method:Rats were given H2S gas saturation buffer and H2S different precursor-L-Cys or MP or CBS inhibitor-HA and different intervention agent by lateral cerebral ventricle(I.C.V.);the mean arterial pressure (MAP) and heart rate (HR) were recorded by Powerlab/4S instruments. To measure left ventricular pressure, the arterial catheters were inserted into the left-ventricle by right carotid arteries, and the left ventricular developed pressure (LVDP), left-ventricle maximal rate of systolic and diastolic pressure development (±LVdp/dtmax) were recorded; respiration were monitored by tracheal intubation. Western blot for MPST and CBS protein concentration; Measurement of H2S production by methylene; Measurement of the Na+-K+-ATPase activity by biological chemistry; Radioimmunoassay for endogenous digitalis like factors。Result:(1) Western blotting data showed, that both CBS and MPST protein expressed in thalamus and cortex; using L-cys as precursor (CBS dependent), the H2S releasing from thalamus and cortex were62.5±5.3and63.8±10.3nmol/min/g tissues; while administrated MP as precursor (MPST dependent), the endogenous H2S generation were approximate to two-fold (125.7±16.8,134.6±17.7nmol/min/g tissues respectively; all P<0.01) in the thalamus and cortex, comparison to L-cys as precursor. Pre-incubated with HA-an inhibitor of CBS, significantly blocked the endogenous H2S production from L-Cys; but not affected H2S production form MP。(2) The exogenous H2S injection by I.C.V. induced a dose-dependent transient hypotension (within in10-240seconds), the time of hypotensive effect was longer in low concentration H2S. After transient hypotension, dramatic MAP increased rapidly in dose-dependent manner were observed and maintained long times (5-20min).(3) Continuously injected H2S gradually increased MAP during2hours. At the end of injection, the MAP increased about30mmHg compared with physiological saline injection, peaked between90min and100min of infusion (P<0.01versus baseline).(4) Bonus injection L-Cys, induced quickly dramatic elevation of MAP, the peak appeared at10-20min, the maximum changes of MAP was about43mmHg and the pulse pressure also increased approximately to1fold. As following, the MAP and pulse pressure recovered gradually. Bonus injection MP, the MAP slowly gradually increased, the maximum changes of MAP was approximate to24mmHg, which continued to60min (the end of observation) without attenuation. Administration with CBS inhibitor-HA block endogenous H2S, lowered the MAP for20min, the maximum reduction of MAP was about20mmHg.(5) I.C.V. administrated the L-Cys induced the heart rate decreased during60min by bonus injection L-Cys, the maximum changes of heart rate was approximate to100bpm (P<0.01versus baseline). Injection HA or intravenous injection H2S did not affect heart rate. Pretreartment with HA blocked CBS activity significantly attenuated the heart rate inhibition by L-Cys (P<0.01). Whereas pre-intravenous injection phentolamine did not affect (p>0.05). Pretreatment with HA inhibition CBS activity in part blocked the effects of L-Cys, The phentolamine also partly blocked the hypertensive effect of L-Cys.(6) Continuously injected different concentration of H2S by I.C.V. and found that increasing CNS endogenous H2S production induced dose-dependently increases left ventricular develop-pressure (LVDP) and±LVdp/dtmax, without affecting left ventricular end of diastolic pressure. Bonus injection H2S precursor L-Cys also increased LVDP and±LVdp/dtmax; on the contrary, HA injection induced a transient decreases of LVDP and±LVdp/dtmax.(7) Intravenous injection phenolamine alpha receptor blocker largely blocked the hypertensive effects of H2S (P<0.01).(8)The I.C.V.injection H2S induced a dose-dependent deeper and slower respiration.(9) Pre-injection KATP channel blocker-glibenclamide by I.C.V), significantly blocked the transient hypotension effects of H2S (P<0.01); whereas did not block the following hypertension effects;(10) The beta receptor blocker metoprolol in part lowered the positive inotropic effect of L-Cys, but not glibenclamide (P<0.05).(11) L-Cys I.C.V. quickly elevated the plasma EDLF level approximately to1fold compared with physiological saline injection control group (P<0.01). HA injection slightly lowered the EDLF level in plasma but no statistical difference.(12) The L-Cys I.C.V. inhibited cardiac and aortic membrane Na+-K+-ATPase, otherwise, HA increased it.Conclusion:Central nervous system endogenous hydrogen sulfide up-regulated mean arterial pressure and cardiac systolic function by activation sympathetic nervous, increase the depth of respiration to slow down the respiratory rate, the activation of central KATP channels, inhibition of the cardiac and abdominal aortic micro-vesicles the Na+-K+-ATPase activity and increase endogenous digitalis like factorrelease.更多还原