天麻、钩藤归经理论的基因表达研究
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摘要
本研究以治疗肝阳上亢证的经典药对天麻和钩藤为对象,利用全基因表达谱芯片用于中药归经理论的研究,在基因表达水平上,定性定量地反映中药药理作用机制的全貌。
天麻、钩藤的配伍应用来源于治疗高血压病肝阳上亢证的经典代表方天麻钩藤饮(出自《中医内科杂病证治新义》)。
目的：
1.明确“归肝经”药物的主要效应器官,寻找中医“脏腑”与解剖学器官的联系；
2.辨析天麻、钩藤两者在配伍中的相互作用方式及地位；
3.探讨“归肝经”药物主要作用的生物路径；
4.阐明天麻“归肝经”的主要作用机理。
方法：
1.理论研究
通过整理肝阳上亢模型建立的相关文献,最终确立最接近理想病症且造模方式对药物研究无干扰的SHR大鼠作为肝阳上亢症的模型。
通过学习生物信息学,掌握基因差异表达谱分析方法,整理基因芯片在中药现代研究中应用的相关文献,建立基因芯片分析技术与中药研究相结合的模式——“疾病-给药”差异表达谱。
2.实验研究
设立空白、天麻粗粉、钩藤煎液、天麻钩藤合煎剂灌胃给药四个平行对照组别(SHR大鼠连续灌胃给药10天,末次给药30分钟后处死),制作四组动物的心脏、肝脏、胰腺、肺、肾脏、大脑、大血管七个组织的病理切片,观察药物是否对组织的病变有效逆转；用液氮研磨法提取四组动物肝脏和肾脏的总RNA,共8个样本,Cy3单色荧光标记后分别和安捷伦大鼠全基因组表达芯片(Agilent whole mouse genome)进行杂交并进行数据处理,建立“疾病-给药”差异表达谱,筛选目标基因,并进行聚类分析、生物功能(GO)分析以及生物通路(Pathway)分析。
结果：
1.天麻组、钩藤组、天麻钩藤组SHR大鼠较空白对照组的肾小球玻璃样变有明显好转,肾小球动脉血管管壁和肝脏血管增厚情况有逆转,钩藤组肝脏细胞出现明显空泡变性且组内个别大鼠肝脏出现囊泡。
2.8个样本的基因差异表达谱如散点图(附图2-1～附图2-6)。筛选出天麻对肾脏的作用相关的基因30个,钩藤对肾脏的作用相关的基因26个,天麻对肝脏的作用相关的基因16个,钩藤对肝脏的作用相关的基因15个。
3.双向分层聚类分析结果如图5-5。
结论：
1.肾脏、肝脏是天麻、钩藤的主要效应器官；
2.天麻和钩藤配伍时,天麻为君药,钩藤为佐药,天麻和钩藤的配伍使得药理作用更专注于肝脏,符合“引经配对”理论。
3.肝阳上亢与血脂代谢异常,氨基酸代谢异常,G-蛋白偶联受体介导的信号通路紊乱以及体液失衡有关,会出现全身性的炎症反应。
4.对与肝阳上亢的病患而言,天麻可以调节机体血脂代谢,影响高密度脂肪酸代谢,参与体内胆固醇逆转运,起到抗动脉粥样硬化作用；能够抑制肝脏炎症反应；能够调节氨基酸代谢以保护肝脏；能够有效调控G-蛋白偶联受体介导的信号传导通路——主要是环磷酸腺苷(AC)通路,降低患者的血压；能够降低多巴胺(DA)和去甲肾上腺素(NE)水平,改善胰岛素抵抗,抑制甲状腺功能亢进以平息肝阳；能够降低血清总胆汁酸的含量,控制血压保护靶器官。
5.相较于天麻单药,合煎剂对肾脏的一些药理作用削弱了,而保肝护肝的作用更加全面更加显著,使得作用更靶向定位于肝脏。配伍后一些与治疗肝阳上亢无关的药效也不再发挥。
6.有机阳离子转运蛋白参与了天麻有效成分的转运,而天麻中有效成分并非以金属离子络合物的状态存在。
7.钩藤单独作用于SHR大鼠,对肝脏有一定的毒性,可能引起肝脏的病变。SHR大鼠给予钩藤后成纤维细胞生长因子FGF21表达下降,天冬酰胺合成酶ASNS表达下降,可溶性半乳糖凝集素结合蛋白LGALS3BP的表达显著下调,是造成肝功能下降或癌变的主要原因。而药对的配伍能够使这些基因的表达有效逆转,对钩藤有“解毒”的作用。
This study used whole gene chips for the study of meridian theory of traditional Chinese medicine, focusing on Gastrodia and Uncaria which is the classic therapy for Liver-Yang. Gene expression level can qualitatively and quantitatively reflect the full view of pharmacological mechanism.
The Compatibility of Gastrodia and Uncaria is referred to the clinical application of Tianma Gouteng Decoctiont.
Objective:
1. To definite the tissue which the herbal attributing to liver meridian mainly effect on, and to look for relationship of Chinese "organs" and the anatomy of organs;
2. To analyze the interaction manner between Gastrodia and Uncaria during the compatibility process;
3. To investigate the important biological pathways of the herbal attributing to liver meridian;
4. To clarify the main mechanism of "Liver attribution of Gastrodia".
Methods:
1. Theoretic research
The relevant literatures about modeling of Liver-Yang were reviewed. SHR rats were determined as the model of "Liver-Yang" which is the closest to the ideal condition. What's more, this modeling approach had no interference for herbal study.
Through the study of bio-informatics, the differential gene expression spectrum analysis method was set up., Relevant literatures about the application of gene chip in Modernization of Traditional Chinese Medicine were reviewed to established the Union of gene chip analysis techniques and traditional Chinese medicine research- "disease-drug treatment" differential expression profile.
2. Experiment research
Four parallel control groups were set up including blank group, Gastrodia treatment group, Uncaria treatment group, Gastrodia and Uncaria combined decoction treatment group. SHR rats were continuously administered intragastrically for 10 days then were killed 30 minutes after the last administration. Histopathological slides of four rats groups were produced, including heart, liver, pancreas, lung, kidney, brain, and major blood vessel, to observe whether drugs reversed tissue lesions effectively; Total RNAs from liver and kidney of four rats groups were extracted by liquid nitrogen grinding,8 samples in total were hybridized with Agilent whole mouse genome chip after Cy3 labeling. Final data were analyzed to establish "disease-drug treatment" differential expression profile, filterting target genes, carrying on cluster analyze, biological function (GO) analyze and biological pathway analyze.
Results:
1. Compared with blank group, SHR rats from the glomerular hyalinization of Gastrodia treatment group, Uncaria treatment group, Gastrodia and Uncaria combined decoction treatment group were improved significantly. Renal and hepatic artery vascular wall thickening situation were reversed. The liver cells of Uncaria treatment group rats vacuolar degenerated significantly, and vesicles appeared individually in the liver of this group.
2. The genes were differentially expressed in 8 samples as shown in Scatter (Appendix Figure 2-1-Appendix Figure 2-1). Totally,30 Gastrodia-kidney related genes,26 Uncaria-kidney related genes,16 Gastrodia-liver related and 15 Uncaria-liver related genes were selected.
3. Cluster analysis results were shown in Figure 5-5.
Conclusions:
1. Liver and Kidney were the certain tissues that Gastrodia and Uncaria mainly effect on.
2. During the compatibility process, Gastrodia acted as master, meanwhile, Uncaria acted as slave. The compatibility makes the pharmacological effects more focused on the liver, consistent with " Guiding compatibility" theory.
3. Liver-yang was related to lipid metabolism, amino acid metabolism, G protein-coupled receptor-mediated signaling pathway disorder and imbalance in the body fluids. There also exists systemic inflammatory response.
4. For patients with liver yang, Gastrodia can adjust lipid metabolism, affect high-density lipoprotein (HDL) metabolism, get involved in cholesterol ester transport, have the efficacy of anti-atherosclerosis. Gastrodia can inhibit liver inflammation; is able to adjust amino acid metabolism in order to protect the liver; is able to regulate G-protein coupled receptor mediated signal transduction pathway effectively-especially the cyclic adenosine monophosphate (AC) pathway, then the blood pressure could be decreased; can reduce dopamine (DA) and to Norepinephrine (NE) levels, improve insulin resistance, inhibit hyperthyroidism in order to calm the Liver-yang; can reduce serum total bile acid content, control of blood pressure, protect the target organs.
5. Compared to single Gastrodia, the pharmacological effects on kidney of combined decoction were weaker, while the protecting for liver was more comprehensive and more significant, which means it is specially localized on liver.
6. Organic cation transporter protein was involved in the transport of Gastrodia active ingredients, but the active ingredients of Gastrodia were not existed within metal ion complexes.
7. Single Uncaria may have certain toxicity to the SHR rat's liver, which might cause liver disease. After administered intragastrically with Uncaria, three particular genes of SHR rats were differentially expressed:fibroblast growth factor(FGF21) expression was decreased, asparagine synthetase (ASNS) expression was decreased, soluble galectin-binding protein (LGALS3BP) expression was significantly reduced. These may be the main reasons which cause the liver function decreasing or liver cancer. Meanwhile the combined decoction can make an effective reversal of the expression of the three particular genes, which could relieve the toxicity of Uncaria.
天麻、钩藤的配伍应用来源于治疗高血压病肝阳上亢证的经典代表方天麻钩藤饮(出自《中医内科杂病证治新义》)。
目的：
1.明确“归肝经”药物的主要效应器官,寻找中医“脏腑”与解剖学器官的联系；
2.辨析天麻、钩藤两者在配伍中的相互作用方式及地位；
3.探讨“归肝经”药物主要作用的生物路径；
4.阐明天麻“归肝经”的主要作用机理。
方法：
1.理论研究
通过整理肝阳上亢模型建立的相关文献,最终确立最接近理想病症且造模方式对药物研究无干扰的SHR大鼠作为肝阳上亢症的模型。
通过学习生物信息学,掌握基因差异表达谱分析方法,整理基因芯片在中药现代研究中应用的相关文献,建立基因芯片分析技术与中药研究相结合的模式——“疾病-给药”差异表达谱。
2.实验研究
设立空白、天麻粗粉、钩藤煎液、天麻钩藤合煎剂灌胃给药四个平行对照组别(SHR大鼠连续灌胃给药10天,末次给药30分钟后处死),制作四组动物的心脏、肝脏、胰腺、肺、肾脏、大脑、大血管七个组织的病理切片,观察药物是否对组织的病变有效逆转；用液氮研磨法提取四组动物肝脏和肾脏的总RNA,共8个样本,Cy3单色荧光标记后分别和安捷伦大鼠全基因组表达芯片(Agilent whole mouse genome)进行杂交并进行数据处理,建立“疾病-给药”差异表达谱,筛选目标基因,并进行聚类分析、生物功能(GO)分析以及生物通路(Pathway)分析。
结果：
1.天麻组、钩藤组、天麻钩藤组SHR大鼠较空白对照组的肾小球玻璃样变有明显好转,肾小球动脉血管管壁和肝脏血管增厚情况有逆转,钩藤组肝脏细胞出现明显空泡变性且组内个别大鼠肝脏出现囊泡。
2.8个样本的基因差异表达谱如散点图(附图2-1～附图2-6)。筛选出天麻对肾脏的作用相关的基因30个,钩藤对肾脏的作用相关的基因26个,天麻对肝脏的作用相关的基因16个,钩藤对肝脏的作用相关的基因15个。
3.双向分层聚类分析结果如图5-5。
结论：
1.肾脏、肝脏是天麻、钩藤的主要效应器官；
2.天麻和钩藤配伍时,天麻为君药,钩藤为佐药,天麻和钩藤的配伍使得药理作用更专注于肝脏,符合“引经配对”理论。
3.肝阳上亢与血脂代谢异常,氨基酸代谢异常,G-蛋白偶联受体介导的信号通路紊乱以及体液失衡有关,会出现全身性的炎症反应。
4.对与肝阳上亢的病患而言,天麻可以调节机体血脂代谢,影响高密度脂肪酸代谢,参与体内胆固醇逆转运,起到抗动脉粥样硬化作用；能够抑制肝脏炎症反应；能够调节氨基酸代谢以保护肝脏；能够有效调控G-蛋白偶联受体介导的信号传导通路——主要是环磷酸腺苷(AC)通路,降低患者的血压；能够降低多巴胺(DA)和去甲肾上腺素(NE)水平,改善胰岛素抵抗,抑制甲状腺功能亢进以平息肝阳；能够降低血清总胆汁酸的含量,控制血压保护靶器官。
5.相较于天麻单药,合煎剂对肾脏的一些药理作用削弱了,而保肝护肝的作用更加全面更加显著,使得作用更靶向定位于肝脏。配伍后一些与治疗肝阳上亢无关的药效也不再发挥。
6.有机阳离子转运蛋白参与了天麻有效成分的转运,而天麻中有效成分并非以金属离子络合物的状态存在。
7.钩藤单独作用于SHR大鼠,对肝脏有一定的毒性,可能引起肝脏的病变。SHR大鼠给予钩藤后成纤维细胞生长因子FGF21表达下降,天冬酰胺合成酶ASNS表达下降,可溶性半乳糖凝集素结合蛋白LGALS3BP的表达显著下调,是造成肝功能下降或癌变的主要原因。而药对的配伍能够使这些基因的表达有效逆转,对钩藤有“解毒”的作用。
This study used whole gene chips for the study of meridian theory of traditional Chinese medicine, focusing on Gastrodia and Uncaria which is the classic therapy for Liver-Yang. Gene expression level can qualitatively and quantitatively reflect the full view of pharmacological mechanism.
The Compatibility of Gastrodia and Uncaria is referred to the clinical application of Tianma Gouteng Decoctiont.
Objective:
1. To definite the tissue which the herbal attributing to liver meridian mainly effect on, and to look for relationship of Chinese "organs" and the anatomy of organs;
2. To analyze the interaction manner between Gastrodia and Uncaria during the compatibility process;
3. To investigate the important biological pathways of the herbal attributing to liver meridian;
4. To clarify the main mechanism of "Liver attribution of Gastrodia".
Methods:
1. Theoretic research
The relevant literatures about modeling of Liver-Yang were reviewed. SHR rats were determined as the model of "Liver-Yang" which is the closest to the ideal condition. What's more, this modeling approach had no interference for herbal study.
Through the study of bio-informatics, the differential gene expression spectrum analysis method was set up., Relevant literatures about the application of gene chip in Modernization of Traditional Chinese Medicine were reviewed to established the Union of gene chip analysis techniques and traditional Chinese medicine research- "disease-drug treatment" differential expression profile.
2. Experiment research
Four parallel control groups were set up including blank group, Gastrodia treatment group, Uncaria treatment group, Gastrodia and Uncaria combined decoction treatment group. SHR rats were continuously administered intragastrically for 10 days then were killed 30 minutes after the last administration. Histopathological slides of four rats groups were produced, including heart, liver, pancreas, lung, kidney, brain, and major blood vessel, to observe whether drugs reversed tissue lesions effectively; Total RNAs from liver and kidney of four rats groups were extracted by liquid nitrogen grinding,8 samples in total were hybridized with Agilent whole mouse genome chip after Cy3 labeling. Final data were analyzed to establish "disease-drug treatment" differential expression profile, filterting target genes, carrying on cluster analyze, biological function (GO) analyze and biological pathway analyze.
Results:
1. Compared with blank group, SHR rats from the glomerular hyalinization of Gastrodia treatment group, Uncaria treatment group, Gastrodia and Uncaria combined decoction treatment group were improved significantly. Renal and hepatic artery vascular wall thickening situation were reversed. The liver cells of Uncaria treatment group rats vacuolar degenerated significantly, and vesicles appeared individually in the liver of this group.
2. The genes were differentially expressed in 8 samples as shown in Scatter (Appendix Figure 2-1-Appendix Figure 2-1). Totally,30 Gastrodia-kidney related genes,26 Uncaria-kidney related genes,16 Gastrodia-liver related and 15 Uncaria-liver related genes were selected.
3. Cluster analysis results were shown in Figure 5-5.
Conclusions:
1. Liver and Kidney were the certain tissues that Gastrodia and Uncaria mainly effect on.
2. During the compatibility process, Gastrodia acted as master, meanwhile, Uncaria acted as slave. The compatibility makes the pharmacological effects more focused on the liver, consistent with " Guiding compatibility" theory.
3. Liver-yang was related to lipid metabolism, amino acid metabolism, G protein-coupled receptor-mediated signaling pathway disorder and imbalance in the body fluids. There also exists systemic inflammatory response.
4. For patients with liver yang, Gastrodia can adjust lipid metabolism, affect high-density lipoprotein (HDL) metabolism, get involved in cholesterol ester transport, have the efficacy of anti-atherosclerosis. Gastrodia can inhibit liver inflammation; is able to adjust amino acid metabolism in order to protect the liver; is able to regulate G-protein coupled receptor mediated signal transduction pathway effectively-especially the cyclic adenosine monophosphate (AC) pathway, then the blood pressure could be decreased; can reduce dopamine (DA) and to Norepinephrine (NE) levels, improve insulin resistance, inhibit hyperthyroidism in order to calm the Liver-yang; can reduce serum total bile acid content, control of blood pressure, protect the target organs.
5. Compared to single Gastrodia, the pharmacological effects on kidney of combined decoction were weaker, while the protecting for liver was more comprehensive and more significant, which means it is specially localized on liver.
6. Organic cation transporter protein was involved in the transport of Gastrodia active ingredients, but the active ingredients of Gastrodia were not existed within metal ion complexes.
7. Single Uncaria may have certain toxicity to the SHR rat's liver, which might cause liver disease. After administered intragastrically with Uncaria, three particular genes of SHR rats were differentially expressed:fibroblast growth factor(FGF21) expression was decreased, asparagine synthetase (ASNS) expression was decreased, soluble galectin-binding protein (LGALS3BP) expression was significantly reduced. These may be the main reasons which cause the liver function decreasing or liver cancer. Meanwhile the combined decoction can make an effective reversal of the expression of the three particular genes, which could relieve the toxicity of Uncaria.
引文
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