| BackgroundSyndrome differentiation and treatment are the advantage of the Traditional Chinese Medicine. Being one of features of TCM theory, syndrome was based on TCM basic theory, has been paid attention in studying of Traditional Chinese Medicine in recent years. As basic syndrome of liver diseases of Chinese medicine is concerned, liver-qi stagnation syndrome is one of common syndromes in clinical practice and highly valuable for profound research. The nature of liver-qi stagnation syndrome is the focus of TCM theory study today. Although lots of achievement was got during the pasted 50 years, they still can't evaluation the essence of liver depression syndrome integrity, more exploration was needed.Gene chip, also called DNA chip or DNA array, is one of advanced molecular biological technology which was accompanied by development of study of human genome and is applied to many medical and biological science research areas already. As an important method in the post-genome era, gene chip gives a chance to study of liver-qi stagnation syndrome deeply. Both genomics and the study of traditional Chinese medicine syndrome integrity and dynamic generated the syndrome-genomics accordingly. ObjectiveTo set up the effective rat model of liver depression syndrome from chronic constraint stress to find differentially expressed genes in hippocampus between the rat model and the normal control, and between the rat model and rat treated by Xiaoyao Powder through gene chip.MethodsSet up the rat model of liver depression syndrome from chronic constraint stress, compared with normal control group and treatment group of Xiaoyao Powder, every group 6 rats. The changes in body weight and food intake in the animals were observed. By open-field test and fluid consumption test, the behaviors of the animals were determined every week. We harvested the serum and separated hippocampus tissues of rats respectively after 3 weeks. Corticotropin releasing factor (CRF, also CRH) in serum was detected after the animals were executed.Gene chip is used in this study. Method of one step of Trizol reagent was applied to extract the total RNA of hippocampus of every group. The total RNA was reverse transcribed to cDNA which was then transcribed to cRNA marked by cy3 and cy5 in vitro. The cRNA was hybridized with 27K Rat Genome Array, and the gene chip was scanned. After analyze the gene chip image, we screened the differentially expressed genes, and then searched the NCBI genomic databases to defined the gene by means of CapitalBio(?) Molecule Annotation System (CB-MAS). Comparing and analyzing the differentially expressed genes between the rat model and normal control, we speculated the pathogenesis of liver depression syndrome, and the differentially expressed genes between the rat treated by Xiaoyao Powder and the rat model to search for the mechanisms of Xiaoyao Powder to liver depression syndrome at the level of genome and prove reliability of liver depression syndrome model on the other hand. Some differentially expressed genes of hippocampus of rats in different groups are verified through real-time PCR.ResultsThe rat model of liver depression syndrome has been established successfully at 21st day. The model rats displayed the typical appearances such as low activity, slow reaction, taper cry, reduces food and drink, like adherence, and so on. In the model group, the rats showed decreased body weight and food intake, reduced squares crossing and rearing in open-field test, a significantly reduced consumption of preference for sucrose solutions and an increased pure water consumption as compared with control group. The serum level of CRH was also increased in rat model, showing the function disorder of HPA axis.We found that a total of 48 genes expressed differentially in hippocampus between rat model and normal control, among which 15 were up-regulated and 33 were down-regulated. After analyzing the function of genes relate to common disease, we knew the differential expression genes of liver-qi stagnation syndrome involved in three categories: (1) neurologic and psychiatric diseases genes, such as Bdnf, Tph1, Gsk3b etc; (2) tumor genes, including Midkine, Folr1, Aqp1, Igfbp2 and so on; (3) genes related to other diseases, such as Ace, Clic, Phex.It was found that Xiaoyao Powder can improve the typical symptoms of liver depression syndrome, increasing the food intake, consumption of and preference for sucrose solutions and squares crossing and rearing in open-field test. It can also reduced the serum level of CRH in rat model. In addition, a total of 22 genes expressed differentially in hippocampus between rat treated by Xiaoyao Powder and rat model, among which 7 were up-regulated and 15 were down-regulated. We analyzed these genes closely related to diseases such as Ncs-1 and Pomc and found that Xiaoyao Powder probably can regulate the neuroendocrinology and function of HPA axis. Expressions of Bdnf, Tph1 and Midkine of hippocampus in different groups tsested through real-time PCR are consistent with results of the gene chips. The results of real-time PCR prove part of chip datas are credible.ConclusionThe rat model of liver depression syndrome from chronic constraint stress is reliable and credible. The abnormal expressed genes in hippocampus of rat model suggest that liver depression syndrome is probably related to disorder of neuroendocrinology and neurotransmitters, and have tumor susceptibility at level of genome, witch may be regulated by Xiaoyao Powder. The results of real-time PCR of some differentially expressed genes of hippocampus of rats in different groups prove the reliability of the datas of gene chips.The study of gene differential expression in Rat Modal of liver depression by gene chip established a foundation from theory and technique for the deeper study of liver-qi stagnation syndrome. |
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