大黄、黄连及其提取物对实热证模型大鼠肝基因表达谱的影响

发布时间：2019-04-12 13:41

【摘要】：目的：利用基因芯片技术检测实热证模型大鼠肝全基因表达谱的改变，经典寒性中药大黄、黄连及其提取物对实热证模型大鼠肝全基因表达谱的影响，从功能基因组角度探讨寒性中药的物质基础及其性效发生机制，探讨寒性中药属性界定的可能依据和中药药性理论研究的思路和方法。方法：SPF级Wistar大鼠96只。实验分2批完成，每批48只。第一批动物分为空白对照组，模型对照组，大黄水煎液、大黄乙酸乙酯萃取物，大黄正丁醇萃取物、大黄水萃取物组。其中大黄水煎液治疗组、各萃取物组按照10ml/Kg灌胃相应液体，空白对照组给予等量蒸馏水。第二批动物分为空白对照组，模型对照组，黄连水煎液、黄连乙酸乙酯萃取物，黄连正丁醇萃取物、黄连水萃取物组。其中黄连水煎液治疗组、各萃取物组按照10ml/Kg灌胃相应液体，空白对照组给予等量蒸馏水。大鼠背部皮下注射2,4-二硝基苯酚（DNP）生理盐水溶液复制实热证模型，造模后0.5h给药治疗，2h、4h、6h测肛温。之后提取肝组织总RNA，应用基因芯片检测各组大鼠肝脏基因表达，筛选差异表达基因，进行基因聚类分析和功能分类注释。选择部分差异基因进行荧光实时定量PCR实验验证芯片结果的准确性。结果：实热模型对照组与空白对照组比较有167条差异表达基因；大黄乙酸乙酯组、大黄正丁醇组、大黄水提物组和大黄水煎液组与实热模型对照组比较分别有177、139、287和177条差异表达基因；黄连乙酸乙酯组、黄连正丁醇组、黄连水提物组和黄连水煎液组与实热模型对照组比较有336、362、417和218条差异表达基因；对差异基因进行基因功能分类注释，实热模型对照组与空白对照组相比，查询到15项显著性基因功能.。大黄乙酸乙酯组、大黄正丁醇组、大黄水提物组、大黄水煎液组与实热模型对照组相比分别查询到34、31、53和29项显著性基因功能，主要为代谢过程基因功能。黄连乙酸乙酯组、黄连正丁醇组、黄连水提物组、黄连水煎液组与实热模型对照组相比分别查询到59、78、72、32项显著性基因功能，主要为代谢过程功能，催化活性功能项。结论：实热证大鼠主要通过调节代谢过程、钙离子平衡和异生物质刺激应答相关基因实现对机体活动的调节。大黄水煎液主要通过调节代谢过程功能基因，尤其是糖代谢相关基因表达，降低机体过高的能量代谢过程。黄连水煎液主要通过调节代谢过程功能基因，尤其是氨基酸代谢相关基因表达，发挥其对机体的调节作用。寒性中药大黄、黄连及其提取物均能通过上调Gclc、Adh1、Rpl6、Nqo1、RGD1562920_predicted基因表达，下调Ubd、Hamp、Sds、LOC683385基因表达发挥作用。以上基因改变可能是寒性中药发挥其清热、泻火、解毒作用的分子机制之一。
[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.
【学位授予单位】：山东中医药大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R-332;R285.5

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