Deficiency, Hot State Of The Liver Tissue Level Of Dna Methylation

The study is a part of "Essential study of the property of TCM base on three factors theory", which is supported by the Major State Basic Research Development Program of China (National Project 973) (No.2007CB512605). The property theory of TCM is the basic and guideline of TCM applications in clinic. The study focuses on investigating the factors of the formation and expression of the property of TCM. A hypothesis is proposed that the property of TCM derives from its bioactivity, which is related to the status of organism and chemical constituents. Therefore, the status of organism, chemical constituents and bioactivity are considered to be three factors of the property of TCM. The organism plays a role in expression of bioactivity of drugs. This study is searching for physiological indexes characterizing the status of organism and mechanisms of bioactivity of drugs in different organism status. The study will provide some foundations to understand the property of TCM.Previous research by our group showed that the activity levels of liver metabolic enzyme were reducing both in Deficiency-Cold rat and Deficiency-Heat rat. The correct expression of genetic information carried by DNA is a prerequisite for synthesis of metabolic enzymes. DNA methylation is the content of epigenetics; it's able to change gene function through influencing gene expression.DNA methylation refers to transfer the methyl from S-adenosylmethionine to 5'position of carbon atoms of Cytosine, which catalyzed by methyltransferase. Main sites of DNA methylation in mammalian is Cytosine of CPG dinucleotide pairs. In the human genome,70%-80% Cytosine of CPG dinucleotide pairs is unmethylated. Recent researches show that DNA methylation is closely related to cancer, genetic diseases, autoimmune diseases and aging.The relationship between DNA methylation and body state has not been reported. This study intends to explore the relationship between DNA methylation and body state by detecting the DNA methylation level of liver tissue in Deficiency-Cold rat and Deficiency-Heat rat. Experimental part of this paper is divided into four sections:First part, the extraction and hydrolysis methods of DNA were established. DNA was extracted from rat liver by phenol chloroform extracting method, and then hydrolyzed by Nuclease P1 and CIAP.Second part, established the detection method of global DNA methylation level by HPLC. The methodological study showed that this method had a good linear relationship, accuracy and repeatability, can be used to detect the DNA methylation level of rat liver.Third part, established Deficiency-Cold model rats by gavage aconite, dried ginger, cinnamon (1:1:1). Detect the DNA methylation level of liver tissues. The results showed that the DNA methylation level of liver tissue in Deficiency-Cold rats were lower than that in normal rats (P＜0.05)Forth part, established Deficiency-Cold model rats by injected hydrocortisone for two weeks and Deficiency-Heat model rats by injected dexamethasone sodium phosphate for two weeks. Part of the Deficiency-Cold and Deficiency-Heat model rats were treated with Rhizoma curculiginis. Detect the DNA methylation level of liver tissues. The results showed that the DNA methylation level of liver tissue had no difference among the groups(P> 0.05).This is the first study of the relationship between DNA methylation and body state. The results of experiments showed that hydrocortisone and dexamethasone sodium phosphate had no effect on the level of global DNA methylation. Aconite, dried ginger, cinnamon (1:1:1) was able to decrease the level of global DNA methylation, the reasons were worthy to study.