| Blood deficiency, which includes many diseases correlated to anaemia and immune response decreased etc, is a kind of symptom in Chinese traditional medicine. Three mice models of blood deficiency have been established respectively by using 60Co irradiation, injection of CTX and combination bleeding, and the development mechanisms were investigated by using genomic and proteomic techniques. Siwutang decotion, which has been wildly used to cure blood deficiency in clinic for hundreds of years in China, consists of paeoniae alba, angelica root, prepared rehmannia root and szechwan lovage rhizome. We have found its major effective components, which are composed of peaoniflorin, ligustrazine, fructose and ferulaic acid. In the same time the effect of peaoniflorin on increasing the blood was discovered. Based on the above researches, we will further launch two aspects of studies respectively.To clarify the characteristic changes of blood deficiency, we take advantage of the metabonomic techniques to learn these three models. Metabonomics involves in the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Three methods of 3.5Gy 60 Co irradiation, injection of CTX and combination bleeding were used to establish mice models of blood deficiency. Blood was obtained from femoral artery on the 7th day after irradiation and injection of CTX. The samples of marrow, thymus and spleen were also collected at same time. In the group of combination bleeding, blood and the other samples were gotten on the 10th day after the beginning of bleeding. All these were detected by using the Fourier variable superconducting nuclear magnetic resonance spectrometer. Data were analyzed by principal component analysis using the SIMCA-P 10.0 software. There were obvious changes between three model groups and normal including lactic acid, choline, taurine, long-chain fatty acid, HDL, sugar, unsaturated fatty acid and some unidentified substances. Although, the changes of this material were different in three mice models, the decrease of lactic acid was observed in all samples and all models. This suggested that suppression of energy metabolism played some roles in blood deficiency of three mice models. At same time, the change of choline in serum of three models was confirmed, and this phenomena was also discovered in thymus and spleen of irradiation and CTX. All these changes were explained by involvements of neurotransmitters. Obviously, there are different extent changes respectively in different models indicating that some unknown mechanisms were also involved.Secondly, the molecular mechanisms of paeoniflorin on increasing expression of erythropoietin were investigated. In the process, RT-PCR was used to investigate influences of paeoniflorin, PI-3 kinase inhibitor (LY294002 30μmol·L-1), antagonist of adenosine A1 receptors(DPCPX 10μmol·L-1), agonist of A1 receptors(CPA 1μmol·L-1) and antagonist of adenosine A2a receptors(SCH58261 0.2μmol·L-1) on mRNA expression of erythropoietin, adenosine A1, A2a, A2b receptors in hypoxia and normoxia. The method of western blotting was used to observe the changes in erythropoietin, AKT and phosphorylation AKT in different oxygen levels. We discovered that paeoniflorin decreased expressions of erythropoietin in normoxia. However, paeoniflorin(2μmol·L-1 ) significantly increased expression of erythropoietin mRNA after exposure hypoxia environment from 16 to 24 hours in HepG2 cells. And this phenomenon was inhibited by LY294002, DPCPX and SCH58261. In the same time, increasing expressions of A2a mRNA through longing exposure in hypoxia and of phosphorylation AKT by dealing with paeoniflorin were also observed. From above results, we suggested that paeoniflorin increases erythropoietin mRNA levels by activating PI-3 kinase pathway and then making AKT phosphorylation through adenosine A1 and A2a receptors in hypoxia. |
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