本文關(guān)鍵詞： 中樞神經(jīng)系統(tǒng) 硫化氫 平均動(dòng)脈血壓 心率 心功能 洋地黃類因子 K_(ATP)通道 Na~+-K~+-ATP酶活性　出處：《武漢大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：目的：內(nèi)源性硫化氫(Hydrogen Sulfide,H2S)作為一個(gè)新的神經(jīng)調(diào)質(zhì),參與了中樞神經(jīng)系統(tǒng)的生理功能的調(diào)節(jié)和疾病過(guò)程。本課題的目的是探討中樞神經(jīng)系統(tǒng)內(nèi)源性H2S對(duì)正常大鼠血流動(dòng)力學(xué)的調(diào)節(jié)作用及其機(jī)制。 方法：側(cè)腦室給予外源性H2S飽和溶液和H2S生成酶胱硫醚-p-合成酶(cystathionine β-synthase, CBS)或3-巰基丙酮酸(3-mercaptopyruvate, MP)或H2S前體半胱氨酸氨或胱硫醚-p-合成酶抑制劑羥胺(Hydroxylamine,HA)及不同的干預(yù)試劑；采用左心室和股動(dòng)脈插管法及四道生理記錄儀記錄平均動(dòng)脈血壓(mean arterial pressure,MABP)、心率(heart rate,HR)、左室收縮壓(Left ventricle systolic pressure,LVSP)、左室舒張末壓(left ventricle end-diastolic pressure,LVEDP)及左室最大壓力上升及下降心室內(nèi)壓的變化率(rate of the rise of LV ventricular pressure,±p/dt);采用氣管插管法測(cè)呼吸深度和呼吸頻率；采用免疫印跡法檢測(cè)3-巰基丙酮酸硫基轉(zhuǎn)移酶和胱硫醚-p-合成酶蛋白濃度；采用亞甲基藍(lán)法測(cè)定組織硫化氫表達(dá)水平；采用生物化學(xué)方法測(cè)定心肌和血管組織Na+-K+-ATPase活性；采用放射免疫法測(cè)定血漿洋地黃含量。 結(jié)果：(1)免疫印跡實(shí)驗(yàn)證實(shí),中樞神經(jīng)系統(tǒng)存在高水平的H2S生成酶CBS和MPST。CBS依賴的H2S生成前體半胱氨酸,下丘腦和皮質(zhì)分別生成62.5±5.3和63.8±10.3nmol/min/g組織的H2S。給予3-巰基丙酮酸,H2S生成大約是給予半胱氨酸的2倍即125.7±16.8和134.6±17.7nmol/min/g組織,各自與半胱氨酸生成H2S比較,兩者的差異有顯著意義(a11P0.01)。而預(yù)先給予CBS的抑制劑羥胺(HA),再給予半胱氨酸和MPST, HA能夠顯著抑制半胱氨酸生成H2S,但不影響MPST生成H2S的作用。 (2)側(cè)腦室(ICV)給予不同濃度H2S飽和緩沖液可劑量依賴性的導(dǎo)致大鼠平均動(dòng)脈血壓急劇下降,而后迅速上升。其最大降低幅度分別是7.8±2.3、11.4±2.6和16.1±3.6mmHg,最大上升幅度分別是9.2±3.4、12.2±2.1和20.8±2.5mmHg,各組變化最顯著的對(duì)應(yīng)時(shí)間點(diǎn)統(tǒng)計(jì)學(xué)均有顯著差異(p0.001)。 (3)ICV連續(xù)緩慢注射H2S飽和緩沖液大鼠平均動(dòng)脈血壓先降低迅速升高而后緩慢升高,在110min時(shí)達(dá)到最高值即38±8.6mmHg,峰值出現(xiàn)在100-110min之間,觀察期內(nèi)血壓沒(méi)有下降。與對(duì)照組比較,各時(shí)間點(diǎn)差異均有統(tǒng)計(jì)學(xué)意義(P0.01),而心率和呼吸沒(méi)有顯著的變化。 (4)ICV分別注射H2S生成前體L-半胱氨酸(L-Cys)、H2S生成酶3-巰基丙酮酸硫基轉(zhuǎn)移酶(MPST)導(dǎo)致快速的升高平均動(dòng)脈血壓。ICV注射H2S生成酶胱硫醚-β-合成酶抑制劑羥胺(HA),平均動(dòng)脈血壓快速下降而后緩慢的回升。 (5)ICV注射L-Cys在觀察期內(nèi)心率緩慢的降低,其最大降低值大約是100次/分,與正常對(duì)照比較統(tǒng)計(jì)學(xué)有顯著差異(P0.01)；ICV注射HA或注射H2S氣體飽和緩沖液(2.8μmol/kg)對(duì)心率沒(méi)有影響；預(yù)先ICV給予HA阻斷CBS活性,10min后再I(mǎi)CV注射L-Cys,顯著的減弱L-Cys減慢心率的作用(P0.01)。 (6)ICV微量注射泵持續(xù)注射不同濃度的H2S飽和溶液,劑量依賴性的增加左心室發(fā)展壓和心室發(fā)展壓最大變化率,但對(duì)左心室舒張末壓沒(méi)有顯著影響；ICV注射H2S生成前體L-Cys增加左心室發(fā)展壓和心室發(fā)展壓變化率；相反,H2S生成酶CBS抑制劑羥胺能夠短暫的降低左心室發(fā)展壓和心室發(fā)展壓最大變化率。 (7)外周靜脈給與α受體阻斷劑酚妥拉明,幾乎完全阻斷了中樞給予外源性H2S和內(nèi)源性H2S的升壓效應(yīng),與對(duì)照組比較統(tǒng)計(jì)學(xué)有顯著差異(P0.01)。 (8)ICV不同濃度的H2S飽和緩沖液可劑量依賴性的顯著增加大鼠的呼吸深度減慢呼吸頻率,在前1分鐘內(nèi)變化最顯著,而后呼吸逐漸恢復(fù)正常,各組間均有統(tǒng)計(jì)學(xué)意義(P均0.001)。 (9)ICV給予H2S飽和緩沖液和KATP通道開(kāi)放劑吡那地爾,能夠顯著的降低平均動(dòng)脈血壓。而預(yù)先給與KATP通道阻滯劑glibenclimad,能夠顯著的阻斷H2S飽和緩沖液和KATP通道開(kāi)放劑吡那地爾的降壓效應(yīng)(P0.01),但不影響H2S的升壓效應(yīng)。 (10)β受體阻斷劑美托洛爾顯著的抑制了中樞內(nèi)源性H2S對(duì)心臟的正性肌力作用,而glibenclamide卻沒(méi)有影響。 (11)與生理鹽水組比較,I.C.V半胱氨酸組血漿內(nèi)洋地黃類因子水平增加了大約1倍(P0.01)。而羥胺(HA)輕微的降低了洋地黃類因子水平,與生理鹽水組比較沒(méi)有顯著差異(P0.05)。 (12)與對(duì)照組比較,內(nèi)源性H2S顯著抑制了心臟和腹主動(dòng)脈檢測(cè)心肌和腹主動(dòng)脈Na+-K+-ATP酶活性(P0.001)；而羥胺顯著增加了它們的Na+-K+-ATP酶活性(P0.001) 結(jié)論：中樞神經(jīng)系統(tǒng)由CBS和MPST催化產(chǎn)生高水平的內(nèi)源性H2S。中樞給予外源性和內(nèi)源性H2S均能夠增加大鼠平均動(dòng)脈血壓,減慢心率,增強(qiáng)心功能。中樞神經(jīng)系統(tǒng)H2S通過(guò)激活外周交感神經(jīng)活性、增加呼吸深度減慢呼吸頻率、激活中樞KATP通道、而調(diào)節(jié)動(dòng)脈血壓；通過(guò)激活外周交感神經(jīng)活性、增加心肌內(nèi)源性洋地黃類因子的釋放和抑制心肌和腹主動(dòng)脈Na-K+-ATP酶活性增強(qiáng)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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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