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Uterine leiomyomas are most frequent benign tumors in genital organs. At present, Neither Western medicine nor TCM has get satisfied effect. So operation is the main method to cure uterine leiomyomas , however, it brings many harmful effects to female. Eligible animal model is the key of drug candidates screening before clinic stage and investigative pathogenesy. However, it takes 4 months to establish the animal model of uterine leiomyoma, which prolonged the new drug’s preliminary screening mostly and cost lots of fund and time. So it is very important to study the earlier biomarker of Uterine leiomyomas model.Recently, people ;have initiated to utilize metabonomics to study drug screening, but mostly on toxicology. It is rare to apply it in animal model. Metabonomics based on NMR can reflect the whole physiologic and pathologic state of organism. It can analysis, detect, definite, quantitate and systematize the phenotype of metabolism, and discover the abnormal changes and correlated biological tag promptly, so as to provide a technique platform.In this study, we adopted exogenous hormone to duplicate animal model of uterine leiomyomas , collected rats’ urine and blood at different time points of constructing model process. Then determined the NMR spectrum, found the PC and biomarker by pattern recognition. At the same time, we made use of IHC to assay the expression of ER,PR, and PCNA in muscular tumor, and caugnt out histopathological examination to check the result of metabonomics, so as to find the biomarkers related with animal model of uterine leiomyomas.At the same time, we also observed the ciliate bugle ’s intervention to biomarkers.1 objectiveTo search the earlier biomarker which is related with rat model of uterine leiomyomas and explore the dynamic and non-invasi index which indicate the success of model and estimate the drug.2 MethodWe duplicated animal model of uterine leiomyomas by administrating female rat exogenous estrogen and progestogen , collected rats’ urine and blood at different time points, determined the NMR spectrum, and assaied the expression of ER、PR and PCNA in muscular tumor ; examed pathological changes in rat uterus by two different staining method.3 Results3．1 Study of metabonomicsFrom the ~1H-NMR spectrum ,We can see the endogenous metabolism in rats’ urine and blood varied in the process of building model. We use pattern recognition to analysis the ~1H-NMR spectrum of biological body fluid which was collected at different time points, The results showed that the PC scores between control group and model group all can be distinct at different time points, and in oval scatterplot, presented as clustering distribution in two areas. According to the reciprocal factor load gram, the chemical shift in distant place are principal constituent which resulted in the differences between control group and model group. In urine , they are 1．94(acetic ester), 2．7(DMA), 3．42(taurine),and 3．26(TMAO); In blood, they 3．52(carbohydrate), 1．24(3-hydroxybutyric acid), 1．36(threonine), 1．40(lactate), 1．32(lipide), 0．92(VLDL) and 2．28(acetone), these compound were correlated mostly with model. At the same time, we used PLS-DA to analyze the ~1H-NMR spectrum of urine and blood, the results suggested that the PC score of metabolite collected at 4,8,12,16 week distributed in four different areas, and all model groups didn’t intersect with control group, which manifeseted that at various stage, the metabolism phenotype of mode and control was different.The metabolism phenotype of model in the forth week was different between with control group, but the change was most obvious in the eighth week, in the twelfth and sixteenth week, the change of metabolism phenotype was in metastable state. After administrating drug, the model, control and medication administration team can be separated obviously in PCA score, and the medication administration team was in the middle; According to the factor load gram, the change of compound in medication administration team was near the control group.3．2 Study of IHCApply SABC to detect the expressions of ER, PR and PCNA in rats’ uterus, we found that the expression all step up in the process of building model. In the sixteenth week, the expressions of ER, PR and PCNA were significantly different with control group. After administrating ciliate bugle, the expression of ER, PR and PCNA declined at different degrees, and at the eighteenth week, the expression of medication administration group had significant difference with model group.3．3 Check of pathologyApply HE and VG in dyeing rats’uterus muscular tissues, from the spectacle, we can see the smooth muscle of uterus in control group was comparatively thin, the contractile fiber cells were slender, and line up in order. In the forth and eighteenth week, the smooth muscle of uterus in model group hadn’t accrementitioned yet, and in the twelfth and sixteenth week, the contractile fiber cells in uterus increased, muscular coat thickened significantly, muscular tissues arrayed anomaly ,some had whirlpool structure, which lost the normal order. The muscle cells were spawn or rod shape, the karyotin was comparatively deep, and the kytoplasm reduced significantly. After administrating ciliate bugle, the increase of thickness and disorder of muscle cell were softened significantly.4 ConclusionsAt the eighteenth week of constrcuting model, the change of metabolism which has related with model can be taken as the eariler biomarkers of Uterine leiomyomas model. In this experiment, the biomarkers will contribute to pre-clinic drug screening and assessment of drug action, at the same time , it has deeply significance to clinical diagnosis and study of new drug.
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