大黃、黃連及其提取物對實(shí)熱證模型大鼠肝基因表達(dá)譜的影響
發(fā)布時間：2019-04-12 13:41
【摘要】：目的：利用基因芯片技術(shù)檢測實(shí)熱證模型大鼠肝全基因表達(dá)譜的改變，經(jīng)典寒性中藥大黃、黃連及其提取物對實(shí)熱證模型大鼠肝全基因表達(dá)譜的影響，從功能基因組角度探討寒性中藥的物質(zhì)基礎(chǔ)及其性效發(fā)生機(jī)制，探討寒性中藥屬性界定的可能依據(jù)和中藥藥性理論研究的思路和方法。 方法：SPF級Wistar大鼠96只。實(shí)驗(yàn)分2批完成，每批48只。第一批動物分為空白對照組，模型對照組，大黃水煎液、大黃乙酸乙酯萃取物，大黃正丁醇萃取物、大黃水萃取物組。其中大黃水煎液治療組、各萃取物組按照10ml/Kg灌胃相應(yīng)液體，空白對照組給予等量蒸餾水。第二批動物分為空白對照組，模型對照組，黃連水煎液、黃連乙酸乙酯萃取物，黃連正丁醇萃取物、黃連水萃取物組。其中黃連水煎液治療組、各萃取物組按照10ml/Kg灌胃相應(yīng)液體，空白對照組給予等量蒸餾水。 大鼠背部皮下注射2,4-二硝基苯酚（DNP）生理鹽水溶液復(fù)制實(shí)熱證模型，造模后0.5h給藥治療，2h、4h、6h測肛溫。之后提取肝組織總RNA，應(yīng)用基因芯片檢測各組大鼠肝臟基因表達(dá)，篩選差異表達(dá)基因，進(jìn)行基因聚類分析和功能分類注釋。選擇部分差異基因進(jìn)行熒光實(shí)時定量PCR實(shí)驗(yàn)驗(yàn)證芯片結(jié)果的準(zhǔn)確性。 結(jié)果：實(shí)熱模型對照組與空白對照組比較有167條差異表達(dá)基因；大黃乙酸乙酯組、大黃正丁醇組、大黃水提物組和大黃水煎液組與實(shí)熱模型對照組比較分別有177、139、287和177條差異表達(dá)基因；黃連乙酸乙酯組、黃連正丁醇組、黃連水提物組和黃連水煎液組與實(shí)熱模型對照組比較有336、362、417和218條差異表達(dá)基因； 對差異基因進(jìn)行基因功能分類注釋，實(shí)熱模型對照組與空白對照組相比，查詢到15項(xiàng)顯著性基因功能.。大黃乙酸乙酯組、大黃正丁醇組、大黃水提物組、大黃水煎液組與實(shí)熱模型對照組相比分別查詢到34、31、53和29項(xiàng)顯著性基因功能，主要為代謝過程基因功能。黃連乙酸乙酯組、黃連正丁醇組、黃連水提物組、黃連水煎液組與實(shí)熱模型對照組相比分別查詢到59、78、72、32項(xiàng)顯著性基因功能，主要為代謝過程功能，催化活性功能項(xiàng)。 結(jié)論：實(shí)熱證大鼠主要通過調(diào)節(jié)代謝過程、鈣離子平衡和異生物質(zhì)刺激應(yīng)答相關(guān)基因?qū)崿F(xiàn)對機(jī)體活動的調(diào)節(jié)。大黃水煎液主要通過調(diào)節(jié)代謝過程功能基因，尤其是糖代謝相關(guān)基因表達(dá)，降低機(jī)體過高的能量代謝過程。黃連水煎液主要通過調(diào)節(jié)代謝過程功能基因，尤其是氨基酸代謝相關(guān)基因表達(dá)，發(fā)揮其對機(jī)體的調(diào)節(jié)作用。 寒性中藥大黃、黃連及其提取物均能通過上調(diào)Gclc、Adh1、Rpl6、Nqo1、RGD1562920_predicted基因表達(dá)，下調(diào)Ubd、Hamp、Sds、LOC683385基因表達(dá)發(fā)揮作用。以上基因改變可能是寒性中藥發(fā)揮其清熱、瀉火、解毒作用的分子機(jī)制之一。
[Abstract]:Objective: to detect the gene expression profile of liver in rats with excess heat syndrome by gene chip technique, and to study the effects of traditional Chinese medicine rhubarb, Rhizoma Coptidis and its extracts on liver gene expression profile in rats with heat deficiency syndrome. From the point of view of functional genome, this paper discusses the material basis and mechanism of cold Chinese medicine, and discusses the possible basis of defining the attribute of cold Chinese medicine and the thought and method of theoretical research on the properties of cold Chinese medicine. Methods: 96 SPF grade Wistar rats were used. The experiment was completed in two batches with 48 rats in each batch. The first batch of animals were divided into blank control group, model control group, rhubarb decoction, ethyl acetate extract of rhubarb, rhubarb n-butanol extract and rhubarb water extract group. Among them, rhubarb decoction group, extract group according to 10ml/Kg gavage corresponding liquid, blank control group was given the same amount of distilled water. The second batch of animals were divided into blank control group, model control group, Rhizoma Coptidis decoction, ethyl acetate extract of Coptis chinensis, n-butanol extract of Coptis chinensis and Coptis chinensis water extract group. In the treatment group, the extract group was given the corresponding liquid according to 10ml/Kg, and the blank control group was given the same amount of distilled water. The rats were subcutaneously injected with 2,4-dinitrophenol (DNP) saline solution to reproduce the model of solid heat syndrome. The rats were treated with medicine 0.5 h, 2 h, 4 h and 6 h after the establishment of the model, and the anal temperature was measured at 6 h. Then the total RNA, of liver tissue was extracted and gene microarray was used to detect the gene expression in liver of each group. The differentially expressed genes were screened for gene cluster analysis and functional classification annotation. Partial differential genes were selected for real-time PCR assay to verify the accuracy of the chip results. Results: there were 167 differentially expressed genes between the control group and the control group. There were 177139287 and 177 differentially expressed genes in rhubarb ethyl acetate group, rhubarb n-butanol group, rhubarb water extract group and rhubarb decoction group compared with the real-heat model control group. There were 336362417 and 218 differentially expressed genes in the ethyl acetate group, n-butanol group, water extract group and decoction group of Coptis chinensis compared with the control group. Compared with the blank control group, 15 significant gene functions were queried in the real-heat model control group according to the classification and annotation of the gene functions of the differentially expressed genes. In rhubarb acetate group, rhubarb n-butanol group, rhubarb water extract group and real heat model control group, 34, 31, 53 and 29 significant gene functions were queried, mainly in metabolic process gene function. In the ethyl acetate group, n-butanol group, water extract group and decoction group of Coptis chinensis, there were 59, 78, 72, 32 significant gene functions, mainly metabolic process function and catalytic activity function, compared with the real-heat model control group. Conclusion: the regulation of metabolism, calcium balance and allogenic substance stimulation response related genes are mainly involved in the regulation of body activity in rats with excess heat syndrome. Rhubarb decoction can reduce the excessive energy metabolism by regulating the functional genes of metabolic process, especially the expression of genes related to glucose metabolism. Rhizoma Coptidis decoction plays an important role in the regulation of metabolic processes by regulating the expression of functional genes, especially genes related to amino acid metabolism. The cold Chinese medicine rhubarb, Coptis chinensis and its extracts could up-regulate the expression of Gclc,Adh1,Rpl6,Nqo1,RGD1562920_predicted gene and down-regulate the expression of Ubd,Hamp,Sds,LOC683385 gene. The above gene changes may be one of the molecular mechanisms of cold Chinese herbs in clearing heat, purging fire and detoxification.
【學(xué)位授予單位】：山東中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R-332;R285.5
本文編號：2457070
[Abstract]:Objective: to detect the gene expression profile of liver in rats with excess heat syndrome by gene chip technique, and to study the effects of traditional Chinese medicine rhubarb, Rhizoma Coptidis and its extracts on liver gene expression profile in rats with heat deficiency syndrome. From the point of view of functional genome, this paper discusses the material basis and mechanism of cold Chinese medicine, and discusses the possible basis of defining the attribute of cold Chinese medicine and the thought and method of theoretical research on the properties of cold Chinese medicine. Methods: 96 SPF grade Wistar rats were used. The experiment was completed in two batches with 48 rats in each batch. The first batch of animals were divided into blank control group, model control group, rhubarb decoction, ethyl acetate extract of rhubarb, rhubarb n-butanol extract and rhubarb water extract group. Among them, rhubarb decoction group, extract group according to 10ml/Kg gavage corresponding liquid, blank control group was given the same amount of distilled water. The second batch of animals were divided into blank control group, model control group, Rhizoma Coptidis decoction, ethyl acetate extract of Coptis chinensis, n-butanol extract of Coptis chinensis and Coptis chinensis water extract group. In the treatment group, the extract group was given the corresponding liquid according to 10ml/Kg, and the blank control group was given the same amount of distilled water. The rats were subcutaneously injected with 2,4-dinitrophenol (DNP) saline solution to reproduce the model of solid heat syndrome. The rats were treated with medicine 0.5 h, 2 h, 4 h and 6 h after the establishment of the model, and the anal temperature was measured at 6 h. Then the total RNA, of liver tissue was extracted and gene microarray was used to detect the gene expression in liver of each group. The differentially expressed genes were screened for gene cluster analysis and functional classification annotation. Partial differential genes were selected for real-time PCR assay to verify the accuracy of the chip results. Results: there were 167 differentially expressed genes between the control group and the control group. There were 177139287 and 177 differentially expressed genes in rhubarb ethyl acetate group, rhubarb n-butanol group, rhubarb water extract group and rhubarb decoction group compared with the real-heat model control group. There were 336362417 and 218 differentially expressed genes in the ethyl acetate group, n-butanol group, water extract group and decoction group of Coptis chinensis compared with the control group. Compared with the blank control group, 15 significant gene functions were queried in the real-heat model control group according to the classification and annotation of the gene functions of the differentially expressed genes. In rhubarb acetate group, rhubarb n-butanol group, rhubarb water extract group and real heat model control group, 34, 31, 53 and 29 significant gene functions were queried, mainly in metabolic process gene function. In the ethyl acetate group, n-butanol group, water extract group and decoction group of Coptis chinensis, there were 59, 78, 72, 32 significant gene functions, mainly metabolic process function and catalytic activity function, compared with the real-heat model control group. Conclusion: the regulation of metabolism, calcium balance and allogenic substance stimulation response related genes are mainly involved in the regulation of body activity in rats with excess heat syndrome. Rhubarb decoction can reduce the excessive energy metabolism by regulating the functional genes of metabolic process, especially the expression of genes related to glucose metabolism. Rhizoma Coptidis decoction plays an important role in the regulation of metabolic processes by regulating the expression of functional genes, especially genes related to amino acid metabolism. The cold Chinese medicine rhubarb, Coptis chinensis and its extracts could up-regulate the expression of Gclc,Adh1,Rpl6,Nqo1,RGD1562920_predicted gene and down-regulate the expression of Ubd,Hamp,Sds,LOC683385 gene. The above gene changes may be one of the molecular mechanisms of cold Chinese herbs in clearing heat, purging fire and detoxification.
【學(xué)位授予單位】：山東中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R-332;R285.5
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