He-gu Luo1, Jia-xu Chen, Guang-xin Yue. 1. Department of Chinese Medicinal Diagnosis, Beijing University of Chinese Medicine, Beijing.
Abstract
OBJECTIVE: To provide a scientific basis for systematic research on the mechanism of chronic immobilization stress (CIS) induced metabolic network change in rats, through detecting the changes of endogenous metabolites in rats with CIS, treated or un-treated with Xiaoyao Powder (XYP), for determining the small molecule marker compound that closely associated with the metabonomical specificity of CIS and acting mechanism of XYP. METHODS: Thirty-six experimental male SD rats were divided into 3 groups, the normal control group, the model group and the XYP group. And all the three groups were subdivided into two subgroups respectively on day 7 and day 21 of the experiment. The stress rat model of CIS was made by chronic restraining method for 3 h every day. Starting from the first day of modeling, XYP 3.854 g/kg in volume of 1 mL/100 g body weight was administered 1 h before restraining via gastrogavage to rats in the XYP group, while equal volume of distilled water was given to rats in the other two groups instead. Blood samples were collected on the 8 th day and 22 th day for detection in the following procedure: at 27 degrees C, 300 microL supernate of blood plasma was taken, calling the Carr-Purcell-Meiboom-Gill (CPMG) and longitudinal eddy-delay (LED) sequence respectively on a Fourier variable nuclear magnetic resonance (NMR) spectrometer, pre-saturated inhibition of the water peak was performed; free induction decay (FID) signals were transferred via 32 k Fourier transformation to gain one-dimensional NMR spectrogram; by taking TSP as the chemical migration reference peak, the segmental integral calculus (0.04 ppm per segment) was performed from 4.5 - 0.5 ppm (CPMG) and 6.0 - 0 ppm (LED) within the peak ranges in 1H spectra using the VNMR software; after normalization, centering and scaling were conducted on data, then used for pattern recognition of principal component analysis (PCA) using the SIMCA-P 10.0 software, or if necessary, the partial least squares discriminate analysis (PLS-DA) was performed. RESULTS: (1) The metabolites in the model group were significantly different from those in the control group, suggesting that the animal model was successfully established with the metabolic network different to that of control. The model group and the XYP group could be differentiated from the control group by the differences of metabolites and metabolic networks between groups; XYP could intervene the metabolites or the metabolic path to cause changes in final metabolites. (2) The serum contents of lactic acid (1.4, 4.16), choline (3.24), N-acetylgalactosamine (NAC) and saturated fatty acids (1-3) increased, but unsaturated fatty acids (1.99,4-5), blood sugar (34), HDL (0.83), etc. reduced in the CIS rats. XYP showed obvious regulatory effects on final metabolites, causing decrease of lactic acid, choline, NAC, saturated fatty acids and blood sugar, and increase of unsaturated fatty acids, blood sugar, HDL, 3.44 ppm compound, etc. CONCLUSIONS: The metabolic phenotype in CIS rats includes the increase of lactic acid, choline, NAC, saturated fatty acid, and the decrease of blood sugar contents, unsaturated fatty acid, HDL, 3.44 ppm compound, etc., these may be the markers of the metabolites. The final metabolites changes induced by CIS are primarily the lipid substances. XYP markedly regulates the contents of final metabolites, showing the regulatory effects on final metabolites, but what is the metabolite or metabolic pathways it interferes to alter the final metabolites should be confirmed by further studies.
OBJECTIVE: To provide a scientific basis for systematic research on the mechanism of chronic immobilization stress (CIS) induced metabolic network change in rats, through detecting the changes of endogenous metabolites in rats with CIS, treated or un-treated with Xiaoyao Powder (XYP), for determining the small molecule marker compound that closely associated with the metabonomical specificity of CIS and acting mechanism of XYP. METHODS: Thirty-six experimental male SD rats were divided into 3 groups, the normal control group, the model group and the XYP group. And all the three groups were subdivided into two subgroups respectively on day 7 and day 21 of the experiment. The stress rat model of CIS was made by chronic restraining method for 3 h every day. Starting from the first day of modeling, XYP 3.854 g/kg in volume of 1 mL/100 g body weight was administered 1 h before restraining via gastrogavage to rats in the XYP group, while equal volume of distilled water was given to rats in the other two groups instead. Blood samples were collected on the 8 th day and 22 th day for detection in the following procedure: at 27 degrees C, 300 microL supernate of blood plasma was taken, calling the Carr-Purcell-Meiboom-Gill (CPMG) and longitudinal eddy-delay (LED) sequence respectively on a Fourier variable nuclear magnetic resonance (NMR) spectrometer, pre-saturated inhibition of the water peak was performed; free induction decay (FID) signals were transferred via 32 k Fourier transformation to gain one-dimensional NMR spectrogram; by taking TSP as the chemical migration reference peak, the segmental integral calculus (0.04 ppm per segment) was performed from 4.5 - 0.5 ppm (CPMG) and 6.0 - 0 ppm (LED) within the peak ranges in 1H spectra using the VNMR software; after normalization, centering and scaling were conducted on data, then used for pattern recognition of principal component analysis (PCA) using the SIMCA-P 10.0 software, or if necessary, the partial least squares discriminate analysis (PLS-DA) was performed. RESULTS: (1) The metabolites in the model group were significantly different from those in the control group, suggesting that the animal model was successfully established with the metabolic network different to that of control. The model group and the XYP group could be differentiated from the control group by the differences of metabolites and metabolic networks between groups; XYP could intervene the metabolites or the metabolic path to cause changes in final metabolites. (2) The serum contents of lactic acid (1.4, 4.16), choline (3.24), N-acetylgalactosamine (NAC) and saturated fatty acids (1-3) increased, but unsaturated fatty acids (1.99,4-5), blood sugar (34), HDL (0.83), etc. reduced in the CIS rats. XYP showed obvious regulatory effects on final metabolites, causing decrease of lactic acid, choline, NAC, saturated fatty acids and blood sugar, and increase of unsaturated fatty acids, blood sugar, HDL, 3.44 ppm compound, etc. CONCLUSIONS: The metabolic phenotype in CIS rats includes the increase of lactic acid, choline, NAC, saturated fatty acid, and the decrease of blood sugar contents, unsaturated fatty acid, HDL, 3.44 ppm compound, etc., these may be the markers of the metabolites. The final metabolites changes induced by CIS are primarily the lipid substances. XYP markedly regulates the contents of final metabolites, showing the regulatory effects on final metabolites, but what is the metabolite or metabolic pathways it interferes to alter the final metabolites should be confirmed by further studies.