Regulation Mechanism Of AEE Treatment On Thrombosis In Rats From Cardiac Tissue Based On Transcriptome

AEE was a new kind of esters compound which was synthesized from molecular precursor aspirin and eugenol. Studies have shown that compared with aspirin, AEE has smaller side-effect and lower toxicity in aspects of the antithrombotic and anti-inflammatory effects, which plays an obvious role in the treatment and prevention of all kinds of thrombotic disease. However, these studies have focused on clinical research and drug dynamics and the studies on its target genes mechanism is not yet studied. In this study, intraperitoneal injection of k-carrageen is used to establish Wistar rat thrombosis model and experimental rats were divided into six groups(each group with seven rats) including 10, 15, 20, 25 and 30 mg/kg k-carageen dosage induced thrombus group and the control group with 2 m L saline. At 6, 12, 18 h and 24 h, the thrombosis black tail length were measured, then the close clinical and pathological study of thrombus model was established by pathology observation and blood cell morphology analysis as the influence of dosage and time of black tail length and the thrombus black tail ratio. Using AEE, aspirin, eugenol, sodium-carboxymethyl-cellulose, combination of aspirin and eugenol by oral dosing of thrombus model rats for 7 days, total RNA was extracted for gene expression profile chip for the organization anatomy analysis of the rat heart tissue. Statistics of differentially expressed genes in the heart tissue was used to analyze the difference between the different drugs/thrombus model group and normal group/solvent group in the Venn diagram analysis. Clustering analysis and signal pathways were analyzed after gene expression profile chip, screening the participation signaling pathways of drugs in heart tissue, and identifying the differentially expressed important genes in signaling pathways. In this study, followed results are obtained:1. Through the pathology and observation of the blood cells, drug induced thrombus model dose and time were studied. When the ambient temperature was at 18±2 ℃ after 6 h, the thrombus-induced rate was reached 100% by injecting k-carrageen in enterocoelia. Statistical analysis showed that the time impact on black tail length of thrombus was not significant under the same dose(P > 0.05), while the IV dose effect on it was significant at the same time(P < 0.05). Compared with the other dosage, the effect of k-carageen at the dosage of 20 mg/kg on the black tail length and ratio was more significant(P < 0.05) and the ratio was 78.45%, at which dosage the rats mesenteric generating no lesions, the abdominal cavity without water, while blood cells appeared relatively obvious aggregation. The results showed that 20 mg/kg k-carrageen can be rational to establish the clinical research thrombosis model, which will provide qualified animal pathology model for thrombus diseases and new drug research.2. The differences in gene number of each drug treatment group were statistically analyzed and multiple comparison analysis of the Venn diagram about genetic variations was carried out in gene expression profile chip. It was found that when compared with model group, differentially expressed genes number in AEE at medium dose and combination of aspirin and eugenol group was 7076 and 9988, respectively. When compared with normal group, their number of differentially expressed genes was 7593 and 8728, respectively, which was the highest of all the differences in number genes in drug group. The differentially expressed genes number of AEE was higher than that of aspirin and eugenol, respectively. Venn diagram showed that the common number of differentially expressed genes were 4610 in AEE at medium dose group and combination of aspirin and eugenol group when compared with model group, while there were 4266 differentially expressed genes when compared with normal group, which were the most differences in all drug treatment groups.3. Gene expression profile data of AEE and other drug treatment group were analyzed with cluster and KEGG pathway, through which signal pathway and target genes were selected and then tested with the q RT-PCR analysis. Through cluster analysis, it was found that AEE mainly affected the biological processes which were related with protein, lipid, inflammatory mediators and ionic reaction, as well as cell molecular components. KEGG Pathway analysis indicated that AEE mainly influenced the complement and blood coagulation cascade signaling pathways to play its antithrombotic effects. Through the KEGG Pathway analysis, 5 target genes were screened out and they were named Fga, Fgb, F2, Serpinc1 and Plaur. By the q RT-PCR analysis, it was found the genes Fga, Fgb, F2 and Serpinc1 were down regulated but Plaur was up. The results showed that AEE had not only antiplatelet effect of it precursors, but also specific thrombolysis. Cancer cells and tumor caused by thrombosis can be cured.