Modern Medical Characterization Of Qi Deficiency And Blood Stasis Syndrome And Its Preliminary Molecular Basis

As the root of treatment based on syndrome differentiation, treating principle and prescription, syndromes has been considered as a breakthrough for the modernization of TCM research, in which the essence of syndrome is of uttermost important. Within numerous syndromes in Chinese medicine, Qi deficiency and blood stasis syndrome is very important and universal. Although many researches have been reported, its essence is still unclear and it is not as simply as Qi deficiency or blood stasis. After the completion of Human Genome Project, the arrival of post genome era provides an opportunity to study the syndrome of TCM thoroughly. Due to the complexity of syndrome and the limitation of individual gene, it is very necessary to explore the essence of syndrome from the genome level. Astragalus and Angelica Compatibility (AAC) is one of typical couples with widely theoretical and clinical uses. The formation of this herb couple is guided under relationship of Qi and blood, and they can be used singly or with other herbs. But so far, the molecular biology mechanism about the effect of AAC on tonifying Qi and controlling blood circulation has not been reported. This study was designed to explore the dynamic evolution and molecular basis of Qi deficiency and blood stasis through gene expression profile on the animal model. Mechanism about the effect of AAC on tonifying Qi and controlling blood circulation was explained through modulation the functional genes.Methods1. The animal model of Qi deficiency and blood stasis was established through hungered, fatigued, and cold stimulated in rats. The whole experimental period was four weeks.2. The changes of hemorheological, T-lymphocyte transformation, T cell subtypes count, adrenal gland weight and adrenal gland weight index were observed to characterize Qi deficiency and blood stasis syndrome at 5th, 14th and 28th day during the development of this syndrome. 3. Gene expression profiles of syndrome development were detected using Affymetrix's GeneChip Rat Genome 230 Arrays and analyzed with Gene Ontology and GenMAPP for the biology function and the signal pathway.5. Effects of AAC on hemorheology, T-lymphocyte transformation, T cell subtypes count and adrenal gland weight were determined at 5th, 14th and 28th day.6. Effect of AAC on mRNA expressions of IL-1β, TNF-α, HSP-70, NF-κB, p38MAPK and JNK in blood cells of rats with Qi deficiency and blood stasis were measured with real-time fluorescent quantitative-PCR at 5th, 14th and 28th day. And that in artery wall were determined at the 28th day.8. Effect of AAC on the pathologic alteration in rat artery wall with Qi deficiency and blood stasis were observed.9. Effect of AAC on NF-κB/p65 and p-c-jun protein expressions in rat artery wall with Qi deficiency and blood stasis were detected by western blotting.Result1. Animal model characterization was in line with clinical performance of Qi deficiency and blood stasis syndrome.2. In model group, whole blood viscosity at high shear rate and middle shear rate had no obvious difference compared with that of control group. Whole blood viscosity at low shear and whole blood reduced viscosity increased significantly at 28th day (P<0.01); plasma viscosity and fibrinogen content increased at 14th, and 28th day (P<0.01); RBC count, hematocrit and RBC rigid index were no obviously changed. RBC aggregation index and electrophoresis time were all increased at 14th and 28th day compared with that of control group(P<0.01).3. Compared with control group, CD4+T count decreased significantly at 28th day (P<0.05); CD8+T and CD3+T count decreased significantly at 5th day (P<0.05), 14th and 28th day (P<0.01).4. 3H-TdR incorporation in T lymphocytes in model group decreased markly compared with control group (P<0.01).5. Adrenal gland weight in control group was always increasing. Adrenal gland weight in model group increased significantly at 5th day (P<0.05), but that decreased significantly at 28th day compared with control group (P<0.05). 6. Rat Gene Chip was used for examination of gene expression profiles in rats with Qi deficiency and blood stasis syndrome and normal rat. Differential expression genes focused on cellular process, metabolic process and biological regulation according to biological process. Differential expression genes focused on cell, organelle, and organelle part according to cell component, which focused on binding, catalytic activity according to cell function. The number of differential expression gene was more at 14th day.7. There were 212 differential expression genes associated with immune function, which mostly focused on immune response. Analyzed by GenMAPP software indicated that the change of cytokines and inflammation response was obvious.8. In AAC high dose group, weight increased at 28th day (P<0.01), swimming time extended (P<0.05), and tongue texture score decreased significantly at 28th day (P<0.01); whole blood viscosity low shear, whole blood reduced viscosity and fibrinogen content all decreased significantly at 28th day(P<0.05), plasma viscosity decreased obviously at the beginning of 14th day(P<0.01), RBC aggregation index always decreased significantly (P<0.05), RBC electrophoresis time shorten (P<0.05 or 0.01); CD8+T count is increased obviously (P<0.05); 3H-TdR incorporation in T lymphocytes increased significantly (P<0.05 or 0.01); adrenal gland weight decreased obviously at 5th day (P<0.05); however there was no significant difference between AAC high dose group and model group in CD3+T, CD4+T countand CD4+T/CD+8T.9. In stragalus group,RBC electrophoresis time shorten at 5th and 28th day (P<0.05); 3H-TdR incorporation in T lymphocytes increased significantly at 14th and 28th day (P<0.05).10. Compared with model group, rat weight, swimming time, tongue texture score, hemorheoligy, T subtypes counts, 3H-TdR incorporation and adrenal gland weight in AAC middle and low dose group all did not change significantly; in stragalus group, there was only RBC electrophoresis time shorten obviously at 5, 28th day (P<0.05).11. In model group, TNF-α, IL1-β, NF-κB, HSP-70, p38MAPK and JNK mRNA expression in blood cell increased significantly compared with control group(P<0.05 or 0.01). In stragalus group, mRNA expression of IL1-βand JNK decreased significantly (P< 0.05 or 0.01); TNF-αdecreased at 5th, 14th day (P<0.05 or 0.01); p38MAPK decreased at 14th, 28th day (P<0.05); HSP-70 and NF-κB decreased at 28th day (P<0.01) compared with control group. In AAC high dose group, mRNA expression of IL1-βand JNK decreased significantly (P< 0.01); TNF-αdecreased at 5th, 14th day (P< 0.01); HSP-70, p38MAPK decreased at 14th, 28th day (P<0.01); NF-κB decreased at 28th day (P<0.01). Moreover, IL1-βand JNK mRNA expression in AAC high dose group decreased significantly compared with stragalus group(P<0.05 or 0.01).12. Pathological changes of artery in model group showed that vascular endothelium was injuryed and neointimal hyperplasia. Preventment and treatment of AAC high dose group was more obvious than stragalus group.13. In model group, TNF-α, IL1-β, NF-κB, p38MAPK and JNK mRNA expression in artery increased compared with control group, excluded HSP-70 (P< 0.01), and that in AAC high dose group and stragalus group decreased significantly (P<0.05 or 0.01). Moreover, IL1-βand JNK mRNA expression in AAC high dose group decreased significantly compared with stragalus group(P<0.05 or 0.01).14. In model group, NF-κB/p65 and p-c-jun protein expression in artery increased compared with control group (P< 0.01), and that in AAC hig hdose group and stragalus group decreased significantly (P<0.05 or 0.01) compared with model group. Compared with stragalus group, the effects of AAC high dose group was more obvious (P<0.05 or 0.01).Conclusion1. Hungered, fatigued, and cold stimulated in rats could induced Qi deficiency, then blood stasis coulde be resulted from and resulted in Qi deficiency.2. Changes of T-lymphocyte subtypes counts, T-lymphocyte transformation and modulation of PHA axis were involved in Qi deficiency and blood stasis. Immune system disorder was run through whole process of Qi deficiency and blood stasis.3. Rats gene profile indicated differentially expressed genes categories included different bioglogy pathway, molecular function and molecular component. Qi deficiency and blood stasis was abnormal expression of functional gene groups.4. The immunological profile of Qi deficiency and blood stasis syndrome was closely related to immune network disorder, including depression of T amd B cell activation, abnormal expression of complement system and cytokine genes, and inflammation activation.5. AAC could obviously prevent and treat Qi deficiency and blood stasis syndrome through improving symptoms,hemorheology, T-lymphocyte subtypes counts, T-lymphocyte transformation and modulation of PHA axis.6. The effects of AAC on Qi deficiency and blood stasis syndrome was brought from the regulating of cytokine network at multi-link and multi-target, NF-κB, p38MAPK and JNK signal transduction pathways included. Through which the immune system and whole body reached to a functional balance status. Supported by the adminicular evidence of"syndrome differentiation through drug effects", the relationship between immune disorder of Qi deficiency and blood stasis syndrome and cytokine disorder was reasonable and scientific.