The Molecular Mechanisms Of Endothelial Protection By "Fufang Danshen" And It's Active Compounds By Applying Oligonucleotide Microarrays

Backgrounds and Aims: Traditional Chinese Medicines(TCM) have been proved to be a reliable and effective therapeusis after thousands years of traditional medical practices. To elucidate the mechanisms of TCM at the molecular level is one of important tasks in the study of TCM theory. The characteristics of TCM effects are multi-components, multi-target and multi-passage. And organisms undertake a multi-level and multi-crosstalk network in order to regulate themselves. So, we can apply the principles of systematical biology and study the gene expression regulation of TCM in organisms to further explore its molecular mechanisms. DNA microarray, a powerful means to query the relative transcript aboundance of many genes in parallel, has many advantages when it is applied in the study of the gene expression regulation of TCM. It is very important for the theory modernization to construct a study platform based on the DNA microarray technology. In present study, a study platform based on cardiovascular disease related genes oligonuleotide microarray was constructed, which was applied in the study of effects of gene expression profile in injured endothelial cellsby ginsenoside Rgl, Danshensu, ginsenoside Rel and fuang Danshen in order to further explore the molecular mechanisms of endothelial protection by those drugs. And cluster analysis of regulated genes in injured endothelial cells by Danshensu, ginsenoside Re 1 and fuang Danshen provides a useful method for TCM study.Materials and Methods: (1) Part I : search the Cardio database and obtain approximately 500 cardiovascular disease related genes; find the mRNA sequence of those genes from Genebank; obtain the most special seqence by online BLAST; design probes for positive control gene, negative control genes and housekeeping genes for quality control; prepare the oligonuleotide microarray; extract total RNA of HUVEC; reverse-transcript the same RNA to Cy3-cDNA or Cy5-cDNA; analyze the qualities of prepared oligonucleotide microarray. (2)Part II: stimulate HUVEC by TNF-a and detect the NO production of HUVEC to set up an in vitro model of endothelial dysfunction; screen the effective dose of ginsenoside Rgl; detect the alteration of the gene expression profile by ginsenoside Rgl in TNF- a stimulated HUVEC by using prepared oligonuleotide microarray; employ RT-PCR and Western blot to validate the expression level change of eNOS. (3 )PartIII: stimulate HUVEC by oxLDL and detect the MDA production of HUVEC to set up an in vitro model of endothelial oxidized injury; screen the effective dose of Fufang Danshen, Danshensu and ginsenoside Rel; detect the alteration of the gene expression profile by Fufang Danshen, Danshensu and ginsenoside Rel in oxLDL injured HUVEC by using prepared oligonuleotide microarray; cluster analyze the regulated genes in injured endothelial cells by Danshensu, ginsenoside Rel and Fuang Danshen.Results: (1) Part I : Background signals of printing solution pointsand negative control points indicated good speciality of self-prepared microarray; strong singnals of positive control points and housekeeping genes indicated good sensitivity of self-prepared microarray. CV values between some random selected points from the left part with its counterparts in the right part are all less than 15%, which indicated repeatable detection. All detected genes are less than 1.5-fold expression changes when two same RNA samples were detected for the alteraion of gene expression profile, which indicated good authenticity of self-prepared microarray. (2) Part II: NO production in HUVEC was decreased significantly after TNF- a treatment, whereas pretreatment of ginsenoside Rgl enhanced NO production in TNF- a stimulated HUVEC. Ginsenoside Rgl affected the expression levels of genes involved in vascular constriction, cell adherence, coagulation ,cell growth and signal transduction in TNF- a stimulated HUVEC. The validation of eNOS by RT-PCR and Western blot showed consistent results with microarray. Ginsenoside Rgl increased the expression level of eNOS mRNA and protein. (3) PartHI: MDA production was increased by oxLDL, and Fufang Danshen, Danshensu and ginsenosed Rel can decreased the MDA production in oxLDL injured HUVEC. Fufang Danshen can regulated 33 genes, and Danshensu can regulated 44 genes, and ginsenose Rel can regulated 27 genes. Among those regulated genes, there are many important genes with great function. Three group of genes were obtained by cluster analysis.Conclusions: (1 )Part I : Self-prepared cardiovascular diseases related genes detected oiigonucleotide microarray is a powerful and reliable study tool, which can be applied to the study of molecular mechanism of FufangDanshen and its active compounds. (2) Part II: Ginsenoside Rgl was able to enhance NO production and the expression of eNOS mRNA and protein in TNF- a stimulated HUVEC. Ginsenoside Rgl regulated sets of genes in endothelial cells and protected endothelial cells from TNF- a activation. Microarray analysis provided us valuable insights into the atheroprotective mechanism by gingsenoside Rgl. (3) Partlll: Danshemu, ginsenoside Re 1 and Fuang Danshen can protected HUVEC from oxidized injury of oxLDL. Those drugs can regulated different genes in different passway.