【作者】 张根水；

【导师】 王汝俊；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 背景与目的:和厚朴酚（Honokiol）为中药厚朴中的提取化合物,具有钙通道阻滞、抗炎、抗氧化等作用。钙拮抗作用提示其在心血管疾病治疗中可能价值,抗氧化与抗炎作用提示其在高血压疾病的防治中还可能具有其它重要意义。高血压是心脑血管病死亡的主要因素之一,严重危害人们的身心健康。本研究旨在观察和厚朴酚的降压作用及其可能机制。利用大鼠离体胸主动脉环,观察和厚朴酚对SD大鼠内皮完整及去内皮离体胸主动脉环的作用,以及其作用与钙通道、NO、PGI₂的关系。用Langendorff离体心脏模型,观察和厚朴酚对离体心脏的直接作用。用培养新生SD大鼠心肌细胞,观察和厚朴酚对心肌细胞钙瞬变的作用,探讨其对心脏作用与心肌细胞内钙离子动态变化的关系。采用原发性高血压大鼠模型,观察和厚朴酚口服7周高血压大鼠血压、血管反应性、氧化应激水平、炎症水平以及心脏与血管组织学变化。方法:①采用大鼠胸主动脉环离体灌流技术。观察和厚朴酚对内皮完整及内皮去除的血管环在基础状态（2.5 g张力）、苯肾上腺素或氯化钾预收缩状态下的作用。并用左旋硝基精氨酸甲酯、吲哚美辛、硝苯地平处理血管环,观测和厚朴酚影响血管张力的具体机制。②健康成年大鼠,雄性,取离体心脏,建立Langendorff模型,观察和厚朴酚对左室发展压（LVDP）、心率（HR）、左室压最大变化速率（+/-dp/dt max）以及冠脉流量（CFR）的影响。③原代培养乳鼠心肌细胞,记录心肌细胞自发性收缩细胞内钙瞬变,观察1、3μmol/L和厚朴酚对心肌细胞钙瞬变的影响,并观察3μmol/L和厚朴酚对异丙肾上腺素增强心肌细胞钙瞬变的作用。④利用原发性高血压大鼠模型,观察和厚朴酚口服7周给药过程中高血压大鼠血压、心率、体重变化,末次给药后,观察SHRs胸主动脉对乙酰胆碱、硝普钠的反应性,比较大鼠肝脏丙二醛、血清NO₂^-/NO₃^-与TNF-α水平,观察心脏冠状动脉与胸主动脉组织学变化。结果:①和厚朴酚对血管基础张力无明显影响,能剂量依赖的缓慢舒张高钾、PE引起去内皮血管环收缩,最大舒张分别为96.82%±3.043%、96.80%±2.166%。对PE引起内皮完整血管呈快速（2 min）、缓慢（15 min）双相舒张作用;最大快速、缓慢舒张分别达45.35%±3.004%、97.77%±1.466%,效应呈明显剂量依赖现象。其快速舒张作用不受吲哚美辛影响、而可被NOS抑制剂L-NAME完全阻断;缓慢舒张作用不能被硝苯地平完全消除。②和厚朴酚可以降低大鼠离体心脏的LVDP、HR、+/-dp/dt max、CF。100μmol/L和厚朴酚明显降低大鼠离体心脏心率至144±12 beats/min（P＜0.05）。终浓度10μmol/L、30μmol/L、100μmol/L和厚朴酚组大鼠左室发展压（LVDP）均逐渐降低,与DMSO对照组差异具有显著性（P＜0.05）,100μmol/L和厚朴酚引起左室最大收缩率（+dp/dtmax）明显下降（1250±92 mmHg/s vs 1959±320 mmHg/s,P＜0.05）;左室最大舒张率（-dp/dt max）也明显低于DMSO对照组（995±268 mmHg/s,913±101 mmHg/s vs1477±124 mmHg/s,P＜0.05）,终浓度30μmol/L、100μmol/L和厚朴酚明显降低冠脉流量（CFR）（10.7±3.1 mL/min,4.0±1.2 mL/min vs 21.5±1.0 mL/min,P＜0.05）。去除和厚朴酚后,再次用正常K-H液灌注,60 min心脏各指标无明显变化,说明和厚朴酚对心脏的抑制不可逆。③1、3μmol/L和厚朴酚明显降低自发性收缩乳鼠心肌细胞钙信号,基线（Baseline）、峰值/基线比（Peak height/baseline%）明显降低,收缩50%时程（Timeto peak 50%）、舒张50%时程（Time to baseline 50%）明显延长,与DMSO组比较差异有显著性（P＜0.05）。3μmol/L和厚朴酚引起基线（Baseline）降低至98.1%±1.0%、峰值/基线比（Peak height/baseline%）降低至43.2%±11.0%。3μmol/L和厚朴酚分别延长收缩50%时程（Time to peak 50%）、舒张50%时程（Time to baseline 50%）131.7%±27.0%、132.9%±11.2%。当和厚朴酚终浓度增加至10μmol/L时,乳鼠心肌细胞自发必收缩几乎停止,同时也记录不到细胞内钙瞬变。给予3μmol/L和厚朴酚预孵育后再给予1μmol/L异丙肾上腺素,钙瞬变基线（Baseline）增高为104.8%±49.2%、峰值/基线比（Peak height/baseline%）增强114.6%±7.1%,与单给异丙肾上腺素组比较明显降低（P＜0.05）,收缩50%时程（Time to peak 50%）、舒张50%时程（Time tobaseline 50%）分别缩短为95.6%±8.7%、89.5%±8.7%,与单给异丙肾上腺素组比较明显延长（P＜0.05）。④治疗7周时间过程中,各组大鼠体重、心率无明显差异（P＞0.05）,大剂量和厚朴酚（400 mg/kg·d）长期给药能明显降低高血压大鼠收缩压（P＜0.05）,收缩压约下降25-30 mmHg。SHRs模型大鼠胸主动脉对乙酰胆碱舒张反应较差,最大舒张为47.2%±5.7%,口服和厚朴酚小剂量（200 mg/kg·d）与大剂量（400 mg/kg·d）均明显改善胸主动脉对乙酰胆碱的舒张反应,最大舒张分别达到54.8%±1.8%、59.0%±3.0%。与溶媒对照组比较差异具有显著性（P＜0.05）。各组SHRs胸主动脉对硝普钠舒张反应无明显差异（P＞0.05）。溶媒对照组SHRs血清NO₂^-/NO₃^-水平为6.52±3.03μmol/L,口服和厚朴酚低、高两个剂量组（200 mg/kg·d,400 mg/kg·d）7周后,血清NO₂^-/NO₃^-水平明显高于溶媒对照组（P＜0.05）,分别为15.35±2.95μmol/L,55.78±4.49μmol/L。溶媒对照组SHRs血清TNF-α水平为97.81±7.82 pg/mL,口服和厚朴酚低、高两个剂量组（200 mg/kg·d,400 mg/kg·d）7周后,血清TNF-α水平明显低于溶媒对照组（P＜0.05）,分别为84.07±6.91 pg/mL、81.69±7.15 pg/mL。溶媒对照组肝脏MDA水平为61.52±6.17 nmol/g,口服和厚朴酚低、高两个剂量组（200mg/kg·d,400 mg/kg·d）7周后,肝脏MDA水平明显低于溶媒对照组（P＜0.05）,分别为46.12±5.46 nmol/g、34.75±9.61 nmol/g。此外口服和厚朴酚低、高两个剂量组7周后,胸主动脉弹性膜层数明显低于溶媒对照组（P＜0.05）,且大剂量和厚朴酚（400 mg/kg·d）与卡托普利能明显降低SHRs胸主动脉血管中膜厚度（P＜0.05）;但对SHRs心脏冠状动脉壁厚度无明显影响（P＞0.05）。结论:和厚朴酚对离体血管环具有舒张作用;机制与电压依赖性钙通道及受体操控性钙通道有关,并增强NO作用而不影响PGI₂;和厚朴酚对大鼠离体心脏有明显的负性肌力、负性频率作用,降低心肌细胞内钙离子是和厚朴酚抑制大鼠心脏导致负性肌力、负性频率的主要机制之一;和厚朴酚对原发性高血压大鼠具有治疗作用,其血管扩张作用以及抗炎抗氧化特性有助于抑制血压的升高。更多还原

【Abstract】 BACKGROUND AND AIM:Honokiol,is the major phenolic compound purified from the medical plant Cortex magnoliae officinalis,which has activity of calcium channel blocking,anti-inflammatory effects and antioxidative effects.The main aim of this paper is to probe the antihypertensive potential and mechanism of effects of honokiol on rats.Isolated thoracic aorta with or without the endothelium of Sprague-Dawley rat were used to verify the effect of honokiol on aortic rings.And the mechanism of the effects of honokiol involved of calcium channel,NO and PGI₂ were investigated.In Langendorff isolated hearts model,the direct effects of honokiol on cardiac function in isolated rat hearts were observed.The direct effects of honokiol on calcium transients in cardiomyocytes from neonate rats were investigated to probe the mechanism of the negative inotropic and chronotropic effects of honokiol on heart.To study the effects of chronic treatment with 200 and 400 mg/kg honokiol oral administration on spontaneously hypertensive rats,the effects of honokiol were investigated by determination of the level of blood pressure,vascular reactivity,oxidative parameters,the level of inflammation and histological change of aorta and coronary artery.METHODS:（1） Isometric tension recordings were applied to evaluate the effects of honokiol on resting,phenylephrine（PE）-preconstricting or KCl-preconstricting aortic rings with or without the endothelium.Using the corresponding agents such as L-N（ω）-nitro-arginine methyl ester（L-NAME）, indomethacin and nifedipine,the underlying mechanism of vasorelaxation-induced honokiol was determined.（2） Langendorff model were established from adult male rats of and were subjected to continuous perfusion of K-H solution,honokiol,K-H solution in turn.Left ventricular developed pressure（LVDP）,heart rate（HR）,the maximal rate of left ventricular pressure rise and fall（+/-dp/dt max）,and coronary flow rate（CFR） were measured to observe the direct effect of honokiol on isolated rat heart.（3） Recording the calcium transients of spontaneous contraction in primary culture cardiomyocytes from neonate rats.The effects of 1,3μmol/L honokiol on calcium transients were observed.And the effects of 3μmol/L honokiol on the potentiation of calcium transients induced by isoproterenol were observed. （4） The spontaneously hypertensive rats were used to investigate the effects of long-term honokiol administration on hypertension.Systolic blood pressure, heart rate and body weight were observed through 7 weeks drug administration. After the last drug administration,the vascular reactivity to acetylcholine and sodium nitroprusside were observed.The plasma level of NO₂^-/NO₃^- and TNF-αand the level of malondialdehyde in SHRs were tested.And histological change of aorta and hearts were observed too.RESULTS:（1） Honokiol exhibited negligible vasomotor actions on aortic rings with or without endothelium at resting tension.Honokiol had dose-dependent relaxation on endothelium-denuded rings stimulated by KCl or PE.Honokiol caused two-phases（fast in 2min,slow in 15min） relaxation on endothelium-intact rings stimulated by PE.These two relaxations were concentration-dependent.The fast relaxation was completely antagonized by L-NAME,but not by indomethacin.（2） The LVDP,CF,+/-dp/dt max and CFR were markedly decreased irreversibly while perfusing the isolated heart with honokiol.（3） The baseline and the peak height/baseline（%） were markedly decreased,the time to peak 50%and time to baseline 50%were markedly prolonged after honokiol were added in solution directly.And 3μmol/L honokiol inhibited the potentiation of calcium transients induced by isoproterenol significantly.（4） In all groups,HR and BW were no different compared with vehicle-treated group（P＞0.05）.Long-term administration with honokiol （400mg/kg） decreased systolic blood pressure significantly（P＜0.05）.In SHRs, honokiol enhanced the aortic relaxation to acetylcholine after 49 d treatment （P＜0.05）,but has no significant effects on the relaxation to sodium nitroprusside（P＞0.05）.Oral administration of honokiol significantly increased the plasma level of NO₂^-/NO₃^-,but decreased the plasma level of TNF-αand the level of MDA in liver compare to the control vehicle（P＜0.05）. In addition,honokiol showed significant reduction in the elastin bands and media thickness in the rat aorta（P＜0.05）,but has no significant effects on coronary arterial wall-thickness（P＞0.05）.CONCLUSION:Honokiol has relaxing effects on vascular smooth muscle.The mechanisms involved voltage dependent calcium channel（VDCC）,receptor operating calcium channel（ROCC） and NO bioavaibility.Honokiol possesses negative inotropic and chronotropic effects in hearts isolated from rats. Decreasing the intracellular calcium concentration is the one of the mechanism of the negative inotropic and chronotropic effects of honokiol on heart of rats.There results suggest that chronic treatment with honokiol experts an antihypertensive effect in SHRs,and its vscorelaxant action,and anti-oxidant, anti-inflammatory properties may contribute to reduce the elevated blood pressure.更多还原