| Qi stagnation and blood stasis syndrome is a common clinical syndrome.Qi stagnation leads to the disorder of blood circulation,resulting in the coexistence of qi stagnation and blood stasis.Qi stagnation and blood stasis evidence leads to various clinical cardiovascular system diseases,gynecological diseases and mental system diseases,which endanger human health.Xuefu Zhuyu decoction(XFZYD)is a commonly used clinical prescription for the treatment of qi stagnation and blood stasis syndrome.XFZYD is composed of 11 drugs:peach kernel,safflower,raw land,red peony,angelica,Ligusticum chuanxiong,bupleurum,Fructus aurantii,Platycodon grandiflorum,Achyranthes bidentata and licorice.At present,there are many reports on the therapeutic mechanism of XFZYD,including the mechanism of treating myocardial ischemia,ischemic stroke,anti atherosclerosis and so on.However,based on the study of qi stagnation and blood stasis syndrome,the research on the biological and material basis of the efficacy of XFZYD is insufficient,especially the material basis.Based on the rat model of chronic qi stagnation and blood stasis induced by adrenaline,this study used metabonomics technology to explore the regulatory effect of XFZYD on qi stagnation and blood stasis syndrome,and studied the material basis of the active part of XFZYD.ObjectiveTo optimize the modeling dose of adrenaline induced chronic qi stagnation and blood stasis model in rats,based on metabonomics technology to study the biomarkers and metabolic pathways of qi stagnation and blood stasis syndrome and XFZYD regulating qi stagnation and blood stasis syndrome.On this basis,to screen the active parts of XFZYD regulating qi stagnation and blood stasis syndrome and clarify its biomarker group,metabolic pathway and material basis.MethodThis study is divided into three parts:the optimization of the rat model of chronic qi stagnation and blood stasis caused by adrenaline,the study of the biomarker group of the efficacy of XFZYD based on targeted metabolomics,and the material basis of the efficacy of XFZYD.Part Ⅰ:Model Study of chronic qi stagnation and blood stasis syndrome in rats.The rats were divided into normal group,low dose group(0.3mg/kg),medium dose group(0.6mg/kg)and high dose group(0.9mg/kg).Adrenaline was injected subcutaneously for modeling,and the best modeling dose was selected according to the general state,body weight,blood viscosity and coagulation of rats.Based on LC-MS/MS,the non targeted metabolomics of normal group and three different doses of adrenaline model groups were studied to explore the biomarkers and related metabolic pathways of qi stagnation and blood stasis syndrome.Part Ⅱ:the rats were divided into normal group,model group and XFZYD group.The model group and XFZYD group were subcutaneously injected with adrenaline at the dose of 0.6mg/kg,and the XFZYD group was gavaged with XFZYD at the dose of 1.92g/kg.Rat plasma was analyzed by targeted metabolomics to explore the biological basis of XFZYD in regulating qi stagnation and blood stasis syndrome.Part Ⅲ:investigate the effects of three kinds of macroporous adsorption resins on the separation of active parts of XFZYD,and select the best macroporous adsorption resin to prepare the active parts of XFZYD.XFZYD was divided into water and 30%ethanol elution part(30E),60%and 95%ethanol elution part(95E)and insoluble part(BR).Based on targeted metabonomics,the regulatory effects of different active parts of XFZYD on the rat model of qi stagnation and blood stasis were studied,and the active parts were screened and their chemical components were identified.ResultsPart Ⅰ:compared with the normal group,the body weight of rats in the model group of qi stagnation and blood stasis syndrome with different doses decreased significantly(P<0.05 or P<0.01).The whole blood viscosity increased significantly in low shear,medium shear and high shear rate.PT shortened,TT shortened and FIB increased in the four items of coagulation.In addition,on the third day of modeling,there was a difference in the body weight of the medium dose group and the high dose group.The experimental results showed that the optimal dose of adrenaline subcutaneously injected into the rat model of chronic qi stagnation and blood stasis was 0.6mg·kg-1·d-1.At this dose,the indexes of qi stagnation and blood stasis in rats were significantly different,the rat model was stable and the indexes fluctuated little.The results of non targeted metabolomics found 30 biomarkers of rat models with qi stagnation and blood stasis syndrome.They have dose-response relationship with adrenaline dose,including arachidonic acid,phenol sulfate,lactic acid,taurine,indoleacrylic acid and various long-chain fatty acids.They are mainly involved in taurine and taurine metabolism,arachidonic acid metabolism,bile acid metabolism,pyrimidine metabolism and fatty acid metabolism.Part Ⅱ:the results of targeted metabolomics showed that 45 endogenous metabolites in the plasma of rats with qi stagnation and blood stasis model had significant changes,while XFZYD significantly corrected 20 metabolites,including taurine,tryptophan,acyl carnitine,aspartic acid,glycocholic acid and 5-HT,mainly involving taurine and taurine metabolism,arginine metabolism Tryptophan metabolism and energy metabolism.Part Ⅲ:through screening,it is finally determined to separate XFZYD with D101 macroporous adsorption resin.The effects of different active parts of XFZYD on regulating the rat model of qi stagnation and blood stasis were analyzed by targeted metabolomics.The results showed that 19 metabolites in the plasma of the rat model group of qi stagnation and blood stasis were changed.Group 30E recalled 9 metabolites,group 95E recalled 15 metabolites and group BR recalled 17 metabolites.Three different active parts can regulate the metabolic disorder caused by the modeling of qi stagnation and blood stasis syndrome.These effects are reflected in different metabolic pathways and play a role in regulating qi stagnation and blood stasis syndrome.The active parts of XFZYD were analyzed qualitatively.The results showed that the main active components of part 30E of XFZYD were paeoniflorin,amygdalin,citric acid,gallic acid,hydroxysafflower yellow A and safflower yellow A,which mainly came from peach kernel,red peony and safflowe.The main active components of 95E are ferulic acid,paeoniflorin,hesperidin,neohesperidin,glycyrrhizin,glycyrrhizin,glycyrrhizic acid,rutin,naringin and kaempferol,which are derived from the medicinal flavors of licorice,fructus aurantii,Angelica sinensis,Ligusticum chuanxiong,red peony and so on.The insoluble part of XFZYD has a regulatory effect in the results of metabolomics,but the analysis has not been completed due to the limitation of method and time.ConclusionThe regulatory effects of different active parts of XFZYD on the rat model of chronic qi stagnation and blood stasis are reflected in different targets and metabolic pathways.The main active components of 30E are paeoniflorin,amygdalin,citric acid,gallic acid,hydroxysafflor yellow A and safflor yellow a.They mainly come from peach kernel,red peony,safflower and other medicinal flavors,and act on the metabolism of taurine and taurine,phenylalanine and the biosynthesis of phenylalanine,tyrosine and tryptophan;The main active components of 95E are ferulic acid,paeoniflorin,hesperidin,neohesperidin,glycyrrhizin,glycyrrhizin,glycyrrhizic acid,rutin,naringin and kaempferol.They come from the medicinal flavors of licorice,fructus aurantii,Angelica sinensis,chuanxiong,red peony and act on the metabolic pathways such as taurine and taurine metabolism. |
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