Gene Regulation Analysis For Xuefu Zhuyu Decoction Influenced On Angiogenesis With Rats Hind-limb Ischemic Model

1ObjectiveWe purposed to observe and compare differences of pathological angiogenesis among high-dose,regular-dose XFZYD group and control goup to choose optimum angiogenic group tissues with model of hind-limb ischemic rats.Then taking advantage of microarray analysis technology to ascertain angiogenesis related key function targets and overall rules that XFZYD regulated.2Methods2.1The model of hind-limb ischemic rats were made with Bletilla microsphere embolization agent and then water solution of XFZY capsules were given by gavage on the first day after operation. Low ischemic limb done with amputation on third day after modeling with Bletilla embolization.Draw materials of granulation tissue in ischemic and necrotic areas on36th and48th hour after amputation. By routine pathological section of granulation tissue, we observed pathological repair status and counted number of blood vessels from different drug doses and groups at both two time points to select the best group for microarray analysis.2.2Taking advantage of microarray analysis, granulation tissues from optimal drug group were analyzed through differentially expressed genes, GO function classification and pathway analysis.2.3Real-time PCR was used to validate main factors which got from microarray analysis.3Results3.1Through microscopic observation of granulation tissue after HE stain,we found both36th and48th hour materials were characterized by fresh granulation,and tissue form the48th hour showed tendency of fibrosis,and as time being72th hour, granulations transformed into scar tissues. Comparison among groups of the36th hour had statistical significance and P values that high dose XFZYD compared respectively with regular dose and saline group both were less than0.001. Regular drug compared with saline group,the P value was less than0.05. There was no statistical significance among groups at the48th hour (P>0.05),and the vessels number at36th hour was all more than48th. Consequently, we choose granulation tissues of high-dose XFZYD at36th hour for microarray analysis.3.22,085differentially expressed genes got through microarray analysis, which included860up-regulated genes and1,225down-regulated. And also got4BP terms,which respectively were angiogenesis, regulation of angiogenesis, positive regulation of angiogenesis and positive regulation of VEGF receptor signaling pathway(P value<0.05),and the4BP terms enriched25DEGS directly related with angiogenesis by the function of GO classification. Microarray analysis result also showed49significant pathways including11pathways related with angiogenesis(P value<0.05). Among the11pathways, ECM-receptor interaction, Hippo and Hedgehog signaling pathway, Cell adhesion molecules, Adherens junction and TGF-beta signaling pathway were up-regulated, and Cytokine-cytokine receptor interaction, Chemokine, Toll-like receptor,Jak-STAT and Hifl signaling pathway were down regulated.3.3Genetic expression from Real-time PCR including VEGFb, TGFβr2, Klf5and Cyr61were all more than2fold changes,among which VEGFb、TGFPr2、Klf5were up regulated and Cyr61was down regulated,which were mainly consistent with results of microarray analysis.4.Conclusions4.1Compared with regular group and saline control group, high-dose XFZYD can promote angiogenesis better in short time for rats hind-limb ischemic necrosis granulation4.2Angiogenic process regulated by XFZYD was really complicated.This experiment result showed4BP terms,25DEGS and11pathways directly related with angiogenesis,which further proved mechanism of angiogenesis regulated by XFZYD relied on multi-gene target and multi-pathway.4.3Validated results from real-time PCR were generally consistent with chip results,which also proved the accuracy of microarray analysis.