| For a long time, the study on Qi-deficiency and blood-stasis syndrome's biological foundation is in a rough stage due to many limiting factors including methodology and technology. The difference between Chinese medical clinic and modern research mode is one of main reasons, which leads to disjointed biological markers of the syndrome. Based on the experience of prior related studies, we put forward a research plan of etiology-clinical findings-syndrome-treatment to make an animal model and a concept of exterior signs to evaluate the model. This study was to discuss the methods of objective evaluation of Qi-deficiency and blood-stasis syndrome's biological foundation via some macroscopical signs and biological findings.Objectives:1. To discuss the methods of establishing an animal model of Qi-deficiency and blood-stasis syndrome directed by the theory of Chinese medicine.2. To explore the methods of study macroscopical exterior signs of the syndrome's animal model.3. To reveal partly microcosmic biological foundation of Qi-deficiency and blood-stasis syndrome.4. To explore the methods of evaluating the research on Qi-deficiency and blood-stasis syndrome's biological foundation.Methods:1. Based on the Chinese medicine theory of overwork depleting Qi, we established a rat model on the assumption of swimming→strength exhausting→Qi deficiency→blood stasis. At different time point after swimming (1w, 2w and4w), data were gathered, including weight, food intake, dejecta, swimming time, vigilance, behavior examinations, multiple electrophysiological detections, grades for tongue characters, and tongue picture, to generate an dynamic macroscopical picture of Qi-deficiency and blood-stasis syndrome and select the most sensitive exterior signs. 2. Concurrent with external observations, metabolic and hemorheological parameters were also tested with respect to blood glucose (GLU),blood lipids, blood viscosity (VIS), erythrocyte deformation and aggregation (AGGRE) index, and coagulation related parameters.3. Superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO), endothelin-1 (ET-1), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) in tissues of heart and brain were tested to discuss the possible biological mechanism of Qi deficiency and blood stasis syndrome.4.Canonical correlation analysis was used to analyze the relation between external signs and biological markers in anticipation of finding sensitive signs and markers to establish the evaluation system for Qi deficiency and blood stasis.Results:1. The model rats after exhausting swimming showed low spirits, decreased activity, narrow eye, dim coat and faint-colored nails. 2w and 4w group had less weight than control group (P<0.05) while model groups had increased food intake. Compared with 1w group, 2w and 4w group had shorter swimming time. Model groups also showed higher grades in spirit evaluation as the swimming continued (P<0.05 in 2w and 4w group). In broad field analyses, 2w and 4w group showed longer staying in middle square than control group (P<0.05). There was no significant difference either in Morris water maze or in multiple electrophysiological detections. The tongue color of model rats turned from light red to dark purple 1week after exhausting swimming and the grades increased as the swimming continued(P<0.05).2. For metabolic parameters, rats in model groups (1w, 2w and 4w) showed an increase in GLU (P<0.05), and increase in total cholesterol (TC) or triglyceride (TG) was only found in 4w group (P<0.05). There is no significant difference between groups in high density lipoprotein (HDL) or low density lipoprotein (LDL). For hemorheological parameters, compared with that in control group, model rats showed an increased plasm viscosity in 2w group (P<0.05), and a decreased erythrocyte aggregation index in 1w and 2w group (P<0.05). There was shorter prothrombin time (PT) in 1w (P<0.01), 2w (P<0.01) and 4w (P<0.05) group, and shorter activated partial thromboplastin time (APTT) and decreased fibrinogen (Fbg) in 4w group (P<0.05). There is no significant difference between groups in the other hemorheological parameters.3. Model groups showed an increase in MDA and ET in heart tissue (P<0.05). There is no significant difference between groups in the other biological parameters. 4.In canonical correlation analysis on sensitive exterior signs and macroscopical signs, exterior signs of Qi deficiency showed significant correlations, indicating consistence between every two aspects of external signs. While negative correlations were found between swimming time and staying in middle square time, indicating consistence between habit changes and activity decrease. Tongue characters, the most representative exterior sign of blood stasis, showed correlation with spirit, one of Qi deficiency exterior signs, indicating correlation between exterior signs of blood stasis and Qi deficiency. The sensitive exterior signs showed little correlation with other signs, indicating the characteristic of sensitive exterior signs representing Qi deficiency and blood stasis.In canonical correlation analysis on sensitive exterior signs and microcosmic parameters, the parameters significantly (P<0.05) correlated with exterior signs of Qi deficiency were as follows. 1) Metabolic parameters: GLU and TG. 2) Horheological parameters: VIS10, VIS60 and AGGRE. 3) Oxidation stress related parameters: SOD2. 4) Blood vessel controlled substances: ET1, ET2 and NO1. 5) IL-1β. The parameters significantly (P<0.05) correlated with exterior signs of blood stasis were PT, APTT, LDL and TNF.Based on above results, discriminant analysis was used to analyze the relationship between sensitive exterior signs of Qi deficiency and blood stasis and related microcosmic parameters to discuss the contributions from related signs and parameters to the model of Qi deficiency and blood stasis. The result showed that PT and APTT had more contributions to the model, and the nonstandard discriminant function of correlation variable was y = -23.979+1.287PT +0.327APTT.Conclusions:1. The exterior signs of model rats such as low spirits, less weight gain, shorter swimming time, longer staying in middle square, and transitory more dejecta etc., are consistent respectively with the character signs of Qi deficiency such as tired, thin, worn-out, slothful, and loose stool etc. So they could be the sensitive exterior signs of Qi deficiency. And the dark purple tongue could be the sensitive exterior signs of blood stasis. Four weeks of exhausting swimming are found to be the time period when the exterior signs were most significant. The results showed that the exhausting swimming was an effective manipulation to induce syndrome of Qi deficiency and blood stasis.2. It could be concluded that metabolic and hemorheological changes are partly the biological foundation of Qi deficiency and blood stasis. Lipid peroxidation in heart tissue and dysfunctions in blood vessel control but no evident inflammation indicated that damage to tissues and organs is a relative long-term process.3. PT and APTT are sensitive microcosmic parameters of Qi deficiency and blood stasis. This study confirmed that it was feasible to evaluate Qi-deficiency and blood-stasis syndrome's biological foundation objectively via multiple analyses on macroscopical exterior signs and microcosmic biological parameters. |
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