川西獐牙菜毒性及其机制研究

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	川西獐牙菜毒性及其机制研究
	杨红霞
学位类型	博士
导师	魏立新 ; 杜玉枝
	2014-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	植物学
关键词	川西獐牙菜毒性机制
摘要	藏药清肝热处方中，多以藏茵陈为主药。青藏高原及毗邻地区民间肝炎患者，多以藏茵陈泡水饮用来治疗各种肝炎，疗效确切。我们课题组在对藏茵陈代表植物川西獐牙菜保肝活性及机制研究过程中，发现其提取物给药小鼠时出现短期昏迷、长期昏迷、直至死亡现象。文献中也发现有獐牙菜属植物提取物或单成分副作用或毒性的相关报道。因此，本论文在实验室发现和文献查阅基础上，先进行川西獐牙菜提取物致小鼠机体损伤或死亡等毒副作用观察，再结合小鼠死亡前心律加快现象和“迷走神经心脏效应或心肌细胞收缩—心脏功能”这一反应途径，从细胞、基因和蛋白三个方面对其死亡原因进行探讨。主要研究结果：毒性观察：(1) 不同提取物给药小鼠，正丁醇提取物(SMBE) 28.00 g/Kg·WB致死率为85%，75 %乙醇提取物(SME) 44.00 g/Kg·WB致死率为95 %；(2) SMBE的LD₅₀为21.81 g/Kg·WB，不同剂量给药小鼠预防CCl4致急性肝损伤实验显示，随SMBE给药剂量增加，血清生化指标谷丙转氨酶(ALT)和总胆汁酸(TBA)呈现肝损伤加重趋势，肝切片中肝细胞浸润、细胞核扩大等损伤加剧，肝脏系数逐渐增大；(3) SME的LD₅₀为27.50 g/Kg·WB，不同剂量给药小鼠预防CCl4致急性肝损伤实验显示，随SME给药剂量的增加，肝脏系数呈先降低（恢复）后增加（损伤）趋势，两高剂量组肝切片中肝细胞肿胀和凋亡损伤加重；30 d 低剂量SME(0.02g/Kg·WB)给药小鼠，肝脏、肾脏、胃和脑切片出现不同程度损伤。毒性机制探讨：(1) 小鼠小肠推进率随SME给药剂量(1.25-10.00 g/Kg·BW)增加逐渐降低(30.12-18.79 %)，氯化乙酰胆碱(ACh)能促进由SME抑制的小肠推进率，证实SME和ACh之间的拮抗作用；同时，SME和ACh联合给药小鼠达LD₅₀的时间由SME单独给药时的9 h左右显著缩短到0.4 h之内；(2) 獐牙菜苦苷，龙胆苦苷和芒果苷三种成分对小鼠小肠平滑肌细胞(MUS-M1)的增殖抑制率比三种成分混合对其增殖抑制率明显；三种成分能降低正常细胞中游离钙离子浓度，并能显著性抑制由ACh所引起的细胞中游离钙离子浓度升高，有剂量依赖关系；(3) 三种成分能显著降低细胞中M₂R，M₃R的mRNA表达水平，有引起细胞收缩异常的作用；(4) 獐牙菜苦苷和龙胆苦苷能显著增加心肌细胞中钙调蛋白依赖性蛋白激酶II(CaMKII)的蛋白表达量，增加心率失常发生的可能。由实验结果可知，上述川西獐牙菜不同溶剂提取物能够导致小鼠脏器损伤和急性死亡；其单成分獐牙菜苦苷，龙胆苦苷或芒果苷可以降低心肌细胞中游离钙离子浓度，明显抑制心肌细胞中胆碱受体M₂和M₃的mRNA表达水平，增加CaMKII蛋白表达量（芒果苷除外），提示川西獐牙菜提取物致小鼠死亡原因与“抑制胆碱受体表达→导致心肌细胞收缩异常或减弱迷走神经对心脏跳动的抑制→从而引起心律失常”这一过程有关。
其他摘要	The medicinal plant of Swertia L. is used extensively in the Tibetan traditional system of medicine as a hepatoprotective agent, and is widely noted for its notable curative effect. So far, there are many researchers have approved its other more than 10 pharmacological actions besides the hepatoprotective. Our research group found the death of mice when they were treated with Swertia mussotii Franch. extract during observing the relationship between efficacy and mechanism of SME, and found many documents about the side effects of parts of those medicinal plants extracts and their own compositions. Therefor, this thesis is to approve the toxcity first, then to explore the cause of death through different experiments combine the phenomenon before mice death and the reaction path of “cardiac effects of vagus nerve or myocardial contraction - cardiac function”. The following are the main research results. Toxicity observations after mice were treated with extracts. (1) When the mice were treated with different extracts, the mice mortality rate of N-buty alcohol extract(SMBE) is 85 % in the dose of 28.00 g/Kg·WB, and 75% ethanol extracts(SME) is 95 % in the dose of 44.00 g/Kg·WB; (2) The LD50 of SMBE is 21.81 g/Kg·WB, the experiment of SMBE hepatoprotective efficacy on CCl4 induced the acute liver injury shows, with the increase of dose, alanine aminotransferase (ALT) and total bile acids(TBA) show the injury aggravate, the liver injury such as liver cellular infiltration, nuclear expansion, cellular swelling become more serious and relative liver weight also increase; (3) LD₅₀ of SME is 27.28 g/Kg·WB; the experiment of SME hepatoprotective efficacy on CCl4 induced the acute liver injury shows, with the increase of dose, relative liver weight show the trend of reduce (recovery) frist then increase (damage), relative liver weight increase at the higher two dose groups is same to the cellular swelling and apoptosis at these two groups; Mice were treated with SME one month with dose of 0.02g/Kg·WB have different drgree damage to liver, kindey, stomach and brain. Toxicity mechanisms: (1) The experiment of small intestine drive rate of mice indicate SME has obvise effect on restraining small intestine drive rate (30.12 - 18.79 %), and has positive correlation with dose (1.25-10.00 g/Kg·BW), ACh promote drive rate of mice that were treated with SME; the time reach to the LD₅₀ from 9 h that die induced by SME single down to 0.4 h that induced by SME and ACh. (2) Three components of Swertiamarin, Gentiamarin and Mangiferin that dose MUS-M1 show more toxicity than combinations on it; the components can reduce Ca ²⁺ concentration in nomal cells, obviously reduce the Ca ²⁺ concentration that was raised by ACh and there is a dose-dependent relationship; (3) Three components decrease M2R and M3RmRNA expression levels, which induce cell contract abnormality; (4) Swertiamarin and Gentiamarin increase CaMKII expression levels, which can increase the chance of arrhythmia occurred. Experimental results show different extracts of Swertia mussotii Franch. can damage the mice organ and induce mice acute death; the constituents of Swertiamarin, Gentiamarin and Mangiferin can decrease Ca ²⁺ concentration in nomal cells, obviousely restrain M2R and M3RmRNA expression levels, increase CaMKII expression levels (except Mangiferin), all these hint the relationship between cause of death and the process of “restrain MR expression - myocardial contraction abnormal or weaken restrain of vagus on heartbeat - arrhythmia”.
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/4007
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	杨红霞. 川西獐牙菜毒性及其机制研究[D]. 北京. 中国科学院研究生院,2014.

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