Metabonomic study of syndrome of liver qi stagnation and spleen deficiency in rats

Abstract

To determine the changes of the plasma metabolic phenotype in rats with chronic restraint stress (rats with syndrome of liver qi stagnation and spleen deficiency), so as to reveal the biological features of the syndrome of liver qi stagnation and spleen deficiency, and to examine potential application of metabonomic analysis in studies of syndromes in traditional Chinese medicine. Twenty-four male Sprague-Dawley rats were randomly divided into four groups: group A, 7 d normal control group; group B, 21 d normal control group; group C, 7 d stress group; and group D, 21 d stress group, with 6 rats in each group. Chronic restraint was used to induce stress in rats. Blood was collected from the cardio-ventricle under anesthesia on the 8th (groups A and C) or 22nd day (groups B and D) and detected by using the Fourier variable superconducting nuclear magnetic resonance (NMR) spectrometer (Varian UnityInova 600 M). Free induction decay signals were transferred into one-dimensional NMR spectrogram via 32 k Fourier transformation. Segmental integral calculus (0.04 ppm per segment) was performed from 4.5-0.5 ppm (Carr-Purcell-Meiboom-Gill, CPMG) or 6.0-0 ppm (longitudinal eddy-delay, LED) as defaulted 1H spectra values by using the VNMR software. Data were saved as text or excel files after normalization and then used for pattern recognition analyses. All the data were analyzed by principal component analysis (PCA) using the SIMCA-P 10.0 software (Umetrics AB, Umea, Sweden). The PCA analysis of rat plasma (1)H NMR spectra revealed different metabolic spectra between stress and control groups, which were consistent with alterations of in vivo metabolisms in rats under stress stimuli. Compared with the normal control group, rats with repeated stress displayed significant changes in spectral peak shapes of acetate, lactate, tyrosine, low-density lipoprotein, and unknown compounds (3.44 ppm). These altered metabolites can be used as biomarkers of syndrome of liver qi stagnation and spleen deficiency for further studies. The (1)H NMR spectra of metabolites in the rat blood are differentially changed after chronic stress. Specific, characteristic metabolic products can be identified by analyses of metabonomics, which lead to interpretation of biological feature of Chinese medicine syndromes. Therefore, metabonomic analysis is an approach with good development prospects to studies of TCM syndromes.