毛莨总苷心血管药理作用研究
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摘要
本论文正文由三部分组成,第一部分主要介绍了毛茛总苷对去甲肾上腺素诱导的大鼠主动脉血管平滑肌细胞增值的影响。第二部分介绍了毛茛总苷对多种因素诱发的大鼠离体胸主动脉环收缩的影响以及相关机制的探讨。第三部分介绍了毛茛总苷对垂体后叶素所致小鼠心肌缺血的保护作用。
第一部分毛茛总苷对去甲肾上腺素诱导的大鼠主动脉平滑肌细胞增殖的影响
本实验采用噻唑蓝(MTT)染色法,以去甲肾上腺素(NE)诱导体外培养的成年SD大鼠胸主动脉血管平滑肌细胞(Vascular Smooth Muscle Cells,VSMC)作为细胞增殖模型,以酚妥拉明等作为阳性对照药物,观察毛茛总苷对大鼠主动脉平滑肌细胞增殖的影响。结果显示:200μg/ml或以上浓度的毛茛总苷对体外正常培养的血管平滑肌细胞的增殖有显著的抑制作用(p<0.05),12.5μg/ml～100μg/ml毛茛总苷对体外正常培养的血管平滑肌细胞的增殖没有明显的影响(p>0.05),而对由去甲肾上腺素诱导血管平滑肌细胞的增殖具有一定的抑制作用(p<0.05),且这种抑制作用呈现一定的剂量和时间依赖性。
第二部分毛茛总苷对大鼠离体胸主动脉环的作用研究及其机制探讨
本实验采用离体胸主动脉环张力记录法,观察毛茛总苷对各种因素引起的离体大鼠胸主动脉环收缩的影响,并探讨其作用部位及可能的作用机制。结果显示:毛茛总苷对由去甲肾上腺素或者氯化钾及氯化钙溶液引起的胸主动脉环收缩均有抑制作用(p<0.05),且其作用呈现一定的剂量依赖性而不具有内皮依赖性。结果提示其作用机制可能与拮抗血管平滑肌上的肾上腺素α1受体和阻断钙离子通道,减少内钙释放和外钙内流,降低细胞浆钙浓度有关。
第三部分毛茛总苷对垂体后叶素所致小鼠心肌缺血的保护作用研究
本实验采用尾静脉注射垂体后叶素(PIT)制备小鼠心肌缺血模型,监测标准Ⅱ导联心电图变化(心率、T波)。观察毛茛总苷对垂体后叶素所致实验小鼠心肌缺血的影响。结果显示:毛茛总苷能够降低小鼠心肌缺血心脏的心电图T波的抬高幅度,在一定程度上抑制心肌缺血所致的心率减慢,与生理盐水组相比存在显著性差异(p<0.05),表明毛茛总苷对垂体后叶素所致小鼠的实验性心肌缺血有保护作用,其中高剂量组的效果稍优于低剂量组的,而灌胃口服给药和腹腔注射给药的两种给药方式的效果无显著性差异。
This thesis is composed of three parts. PartⅠmainly introduced inhibitory effect of total glycosides of Ranunculus japonicus(TGRJ) on proliferation of thoracic aortic vascular smooth muscle cells induced by norepinephrine. PartⅡcovered my study on the influence of TGRJ on multiple factors induced contraction of rats’thoracic aorta and my discussion related to possible mechanisms. PartⅢreported protective effect of TGRJ on myocardial ischemia in mice caused by pituitrin.
PartⅠInhibitory effect of TGRJ to norepinephrine-induced proliferation of thoracic aortic vascular smooth muscle cells
In this thesis, norepinephrine-induced thoracic aortic vascular smooth muscle cells cultured in vitro as cell proliferation model, phentolamine and nimodipine as positive control drugs, methyl thiazolyl tetrazolium colorimetric assay was applied to observe the inhibitory effect of TGRJ to norepinephrine-induced proliferation of thoracic aortic vascular smooth muscle cells. Result showed that TGRJ at and over concentration of 200μg/ml had significantly inhibitory effect on proliferation of thoracic aortic vascular smooth muscle cells in general cultivation (p<0.05) while 12.5μg/ml～100μg/ml TGRJ had no significant effect on it but had significantly inhibitory effect on proliferation induced by norepinephrine of thoracic aortic vascular smooth muscle cells (p<0.05) with dose and time dependent manner.
PartⅡStudy on the influence of TGRJ to multiple factors induced contraction of rats’thoracic aorta and investigation of possible mechanisms
In this thesis, isolated aortic ring tension recording method was applied to further observe the effects of TGRJ on multiple factors-induced vasoconstriction of isolated thoracic aortic ring, also investigation on its action sites and possible action mechanisms. Results showed that, TGRJ had markedly inhibitory effect on vasoconstriction induced by norepinephrine, potassium chloride or calcium chloride solution (p<0.05), and the effect of which was with dose dependent manner and without endothelium dependent manner. And the results concluded that its possible mechanisms have relation withα1 adrenoceptor antagonist and calcium channels block which lead to decrease intracellular calcium release and calcium influx so as to decrease calcium concentration in cytoplasm.
PartⅢProtective effect of TGRJ to pituitrin caused myocardial ischemia on mice In this thesis, the electrocardiographs (ECGs) leadⅡwere monitored in myocardial ischemia rats caused by pituitrin through tail vein in order to observe the effect of TGRJ on myocardial ischemic in mice induced by pituitrin. Results showed that total glycosides of ranunculus japonicus have effect on reducing the rise amplitude of T-wave and inhibiting decent of heart rates to a certain degree compared with NS groups(p<0.05) in electrocardiogram. The effects of high dose groups were superior to low dose groups’. No significant difference of the effects was showed between intragastric administration and intraperitoneal administration. These results demonstrated that total glycosides of Ranunculus japonicus possesses protective effects on myocardial ischemia caused by pituitrin.
第一部分毛茛总苷对去甲肾上腺素诱导的大鼠主动脉平滑肌细胞增殖的影响
本实验采用噻唑蓝(MTT)染色法,以去甲肾上腺素(NE)诱导体外培养的成年SD大鼠胸主动脉血管平滑肌细胞(Vascular Smooth Muscle Cells,VSMC)作为细胞增殖模型,以酚妥拉明等作为阳性对照药物,观察毛茛总苷对大鼠主动脉平滑肌细胞增殖的影响。结果显示:200μg/ml或以上浓度的毛茛总苷对体外正常培养的血管平滑肌细胞的增殖有显著的抑制作用(p<0.05),12.5μg/ml～100μg/ml毛茛总苷对体外正常培养的血管平滑肌细胞的增殖没有明显的影响(p>0.05),而对由去甲肾上腺素诱导血管平滑肌细胞的增殖具有一定的抑制作用(p<0.05),且这种抑制作用呈现一定的剂量和时间依赖性。
第二部分毛茛总苷对大鼠离体胸主动脉环的作用研究及其机制探讨
本实验采用离体胸主动脉环张力记录法,观察毛茛总苷对各种因素引起的离体大鼠胸主动脉环收缩的影响,并探讨其作用部位及可能的作用机制。结果显示:毛茛总苷对由去甲肾上腺素或者氯化钾及氯化钙溶液引起的胸主动脉环收缩均有抑制作用(p<0.05),且其作用呈现一定的剂量依赖性而不具有内皮依赖性。结果提示其作用机制可能与拮抗血管平滑肌上的肾上腺素α1受体和阻断钙离子通道,减少内钙释放和外钙内流,降低细胞浆钙浓度有关。
第三部分毛茛总苷对垂体后叶素所致小鼠心肌缺血的保护作用研究
本实验采用尾静脉注射垂体后叶素(PIT)制备小鼠心肌缺血模型,监测标准Ⅱ导联心电图变化(心率、T波)。观察毛茛总苷对垂体后叶素所致实验小鼠心肌缺血的影响。结果显示:毛茛总苷能够降低小鼠心肌缺血心脏的心电图T波的抬高幅度,在一定程度上抑制心肌缺血所致的心率减慢,与生理盐水组相比存在显著性差异(p<0.05),表明毛茛总苷对垂体后叶素所致小鼠的实验性心肌缺血有保护作用,其中高剂量组的效果稍优于低剂量组的,而灌胃口服给药和腹腔注射给药的两种给药方式的效果无显著性差异。
This thesis is composed of three parts. PartⅠmainly introduced inhibitory effect of total glycosides of Ranunculus japonicus(TGRJ) on proliferation of thoracic aortic vascular smooth muscle cells induced by norepinephrine. PartⅡcovered my study on the influence of TGRJ on multiple factors induced contraction of rats’thoracic aorta and my discussion related to possible mechanisms. PartⅢreported protective effect of TGRJ on myocardial ischemia in mice caused by pituitrin.
PartⅠInhibitory effect of TGRJ to norepinephrine-induced proliferation of thoracic aortic vascular smooth muscle cells
In this thesis, norepinephrine-induced thoracic aortic vascular smooth muscle cells cultured in vitro as cell proliferation model, phentolamine and nimodipine as positive control drugs, methyl thiazolyl tetrazolium colorimetric assay was applied to observe the inhibitory effect of TGRJ to norepinephrine-induced proliferation of thoracic aortic vascular smooth muscle cells. Result showed that TGRJ at and over concentration of 200μg/ml had significantly inhibitory effect on proliferation of thoracic aortic vascular smooth muscle cells in general cultivation (p<0.05) while 12.5μg/ml～100μg/ml TGRJ had no significant effect on it but had significantly inhibitory effect on proliferation induced by norepinephrine of thoracic aortic vascular smooth muscle cells (p<0.05) with dose and time dependent manner.
PartⅡStudy on the influence of TGRJ to multiple factors induced contraction of rats’thoracic aorta and investigation of possible mechanisms
In this thesis, isolated aortic ring tension recording method was applied to further observe the effects of TGRJ on multiple factors-induced vasoconstriction of isolated thoracic aortic ring, also investigation on its action sites and possible action mechanisms. Results showed that, TGRJ had markedly inhibitory effect on vasoconstriction induced by norepinephrine, potassium chloride or calcium chloride solution (p<0.05), and the effect of which was with dose dependent manner and without endothelium dependent manner. And the results concluded that its possible mechanisms have relation withα1 adrenoceptor antagonist and calcium channels block which lead to decrease intracellular calcium release and calcium influx so as to decrease calcium concentration in cytoplasm.
PartⅢProtective effect of TGRJ to pituitrin caused myocardial ischemia on mice In this thesis, the electrocardiographs (ECGs) leadⅡwere monitored in myocardial ischemia rats caused by pituitrin through tail vein in order to observe the effect of TGRJ on myocardial ischemic in mice induced by pituitrin. Results showed that total glycosides of ranunculus japonicus have effect on reducing the rise amplitude of T-wave and inhibiting decent of heart rates to a certain degree compared with NS groups(p<0.05) in electrocardiogram. The effects of high dose groups were superior to low dose groups’. No significant difference of the effects was showed between intragastric administration and intraperitoneal administration. These results demonstrated that total glycosides of Ranunculus japonicus possesses protective effects on myocardial ischemia caused by pituitrin.
引文
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2.郑 威,周长新,张水利等.毛茛化学成分的研究[J]. 中国中药杂志,2006,31(11):892-4
3.熊辅信编著.中药现代研究荟萃[M]. 昆明:云南科技出版社,2002,5:158
4.蔡飒,谭毓治等.毛茛提取液对离体子宫平滑肌作用及其机制的研究[J]. 广东药学院学报,2004,20(1):37-9
5.蔡飒,江涛等.毛茛提取液含药血清对家兔子宫平滑肌细胞的影响[J]. 中草药,2005,36(4):566-8
6.蔡飒,李校坤.用血清药理学方法观察毛茛提取液对家兔主动脉平滑肌细胞内钙的影响[J]. 中药材,2004,27(10):741-4
7.李鸿燕,黄春英,叶朝钧.乌头乙醇提取物的抗炎镇痛作用[J]. 中国现代实用医学杂志,2006,5(6):27-9
8.王榕乐,谭毓治.毛茛总苷对血管紧张素Ⅱ致心肌肥大的影响[J]. 广东药学院学报,2008,24(2):154-6
9.周滢等.毛茛中白头翁素的制备工艺研究[J]. 中药材, 2004,27(10):762-4
10.王宏镛,王淑芬,翁福海.白头翁素对小鼠腹腔巨噬细胞释放IL-6及TNF的影响[J].中国中西医结合外科杂志,1997,3(3):191-2
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13.刘洪瑞,李智,王晓红等. 滨蒿内酯对豚鼠气管平滑肌细胞细胞内钙离子浓度的影响[J]. 中国医科大学学报. 2002,31(4):249-51
14.李智,刘洪瑞,杨海玲等. 滨蒿内酯对离体大鼠主动脉平滑肌作用的研究.中国医科大学学报[J]. 2002, 31(4):244-6
15.Yamahara J, Kobayashi G, Matsuda H, et al. Vascular dilatory action of the Chinese crude drug. II. Effects of scoparone on calcium mobilization. Chem Pharm Bull [J], 1989, 37(2):485-9
16.Huang H C, Lee C R, Weng Y I, et al. Vasodilator effect of scoparone(6, 7-dimethoxycoumarin) from a Chinese herb[J]. Eur J Pharmacol.1992, 218(1):123-8
17. Sourivonq P, Schronerova K, Babincova M. Scoparone inhibits ultraviolet radiation-induced lipid peroxidation [J]. Z Naturfosch.2007, 62(2):61-4
18. Eun Kyung K, Kang Beom K, Ju Hyung L,et al. Inhibition of cytokine-mediated nitric oxide synthase expression in rat insulinoma cells by scoparone [J]. Biol Pharm Bull. 2007, 30(2): 242-6
1.徐正云,任国珍,马爱群.大鼠胸主动脉平滑肌细胞的培养与鉴定[J]. 岭南心血管病杂志, 2005, 11(3):202-204.
2.张量,隋璐,孙成林等. 组织块贴壁法培养大鼠主动脉血管平滑肌细胞的特征[J]. 中国临床康复,2005,9(19):108-110.
3. OUYANG Ping, Peng Wen-lie, Lai Wen-yan, XU An-long. Green tea polyphenols inhibit low-density lipoprotein-induced proliferation of rat vascular smooth muscle cells [J]. JourNEl of the Forth Military Medical University, 2004, 24(9): 975-979.
4.Shaw S, Wang X, Redd H, et al. High glucose augments the angiotensinⅡ induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway[J]. J Biol Chem, 2003, 278:30634230641.
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2.郑 威,周长新,张水利等.毛茛化学成分的研究[J]. 中国中药杂志,2006,31(11):892-4
3.熊辅信编著.中药现代研究荟萃[M]. 昆明:云南科技出版社,2002,5:158
4.蔡飒,谭毓治等.毛茛提取液对离体子宫平滑肌作用及其机制的研究[J]. 广东药学院学报,2004,20(1):37-9
5.蔡飒,江涛等.毛茛提取液含药血清对家兔子宫平滑肌细胞的影响[J]. 中草药,2005,36(4):566-8
6.蔡飒,李校坤.用血清药理学方法观察毛茛提取液对家兔主动脉平滑肌细胞内钙的影响[J]. 中药材,2004,27(10):741-4
7.李鸿燕,黄春英,叶朝钧.乌头乙醇提取物的抗炎镇痛作用[J]. 中国现代实用医学杂志,2006,5(6):27-9
8.王榕乐,谭毓治.毛茛总苷对血管紧张素Ⅱ致心肌肥大的影响[J]. 广东药学院学报,2008,24(2):154-6
9.周滢等.毛茛中白头翁素的制备工艺研究[J]. 中药材, 2004,27(10):762-4
10.王宏镛,王淑芬,翁福海.白头翁素对小鼠腹腔巨噬细胞释放IL-6及TNF的影响[J].中国中西医结合外科杂志,1997,3(3):191-2
11.国家药典委员会.中华人民共和国药典(2000年版,一部)[M]. 北京:化学工业出版社,2000:260
12.詹 莉 , 戴华成, 杨治平,等.小毛茛内酯影响耐药结核患者外周血淋巴细胞SHSP和GLS表达的研究[J]. 中国中药杂志,2002,27(9):677-9
13.刘洪瑞,李智,王晓红等. 滨蒿内酯对豚鼠气管平滑肌细胞细胞内钙离子浓度的影响[J]. 中国医科大学学报. 2002,31(4):249-51
14.李智,刘洪瑞,杨海玲等. 滨蒿内酯对离体大鼠主动脉平滑肌作用的研究.中国医科大学学报[J]. 2002, 31(4):244-6
15.Yamahara J, Kobayashi G, Matsuda H, et al. Vascular dilatory action of the Chinese crude drug. II. Effects of scoparone on calcium mobilization. Chem Pharm Bull [J], 1989, 37(2):485-9
16.Huang H C, Lee C R, Weng Y I, et al. Vasodilator effect of scoparone(6, 7-dimethoxycoumarin) from a Chinese herb[J]. Eur J Pharmacol.1992, 218(1):123-8
17. Sourivonq P, Schronerova K, Babincova M. Scoparone inhibits ultraviolet radiation-induced lipid peroxidation [J]. Z Naturfosch.2007, 62(2):61-4
18. Eun Kyung K, Kang Beom K, Ju Hyung L,et al. Inhibition of cytokine-mediated nitric oxide synthase expression in rat insulinoma cells by scoparone [J]. Biol Pharm Bull. 2007, 30(2): 242-6
1.徐正云,任国珍,马爱群.大鼠胸主动脉平滑肌细胞的培养与鉴定[J]. 岭南心血管病杂志, 2005, 11(3):202-204.
2.张量,隋璐,孙成林等. 组织块贴壁法培养大鼠主动脉血管平滑肌细胞的特征[J]. 中国临床康复,2005,9(19):108-110.
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