| Blood stasis syndrome is a common clinical symptom,which is related to coronary heart disease,hypertension,hyperlipidemia,tumor and other diseases.The main physiological and biochemical basis of blood stasis is abnormal hemorheology,blood circulation disorder and abnormal hemodynamics.A large number of basic and clinical studies have shown that the bitterness exerts the effect of "expel stagnation".One of the key problems in the research of bitter herbs is the flavour-effect relationship.It is found that bitter receptors are distributed in the heart and blood vessels besides the tongue tissue.With the further research of bitter receptor,different bitter receptors have become the potential drug targets for the treatment of many diseases.Based on the results of the virtual screening,bitter herbs(such as Salvia miltiorrhiza,Rheum officinale and Coptis chinensis)can activate different bitter receptors.Due to the exact effect of expel stagnation of bitter herbs,bitter receptors may be the breakthrough point to reveal the difference of biological characterization of different substances of "expel stagnation".In this paper,we examined behaviors,hemorheology,hemodynamics,microcirculation and other indicators for evaluating the apparent differences in indicators in three kinds of blood stasis syndrome model.The blood stasis syndrome was established by high fat diet combining with CUMS.Furthermore,different genes involved in the pathogenesis,related biological processes and pathways and the significant changes in the bitter receptor gene by comparing the changes of bitter receptor gene in blood stasis model group and normal group,were found by gene chip technique.The blood type of blood activating and stasis eliminating drug Salvia miltiorrhiza with a bitter taste,exhibited effects of promoting blood circulation to restore menstrual flow,removing stasis and relieving pain,cooling blood and eliminating carbuncle,clearing away the heart-fire and relieving restlessness and nourishing the blood and tranquilization.The diarrhea drug Rheum officinale with a bitter taste,showed effects of purging heat and promoting intestine,removing pattogenic heat from the blood and toxic material from the body,removing blood stasis and stimulating the menstrual flow.The bitter drug of non blood activating and stasis eliminating Coptis chinensis with a bitter taste,exerted effects of heat dampn and purging fire detoxification.By analyzing the pharmacodynamic action spectrums of three bitter traditional Chinese medicine carriers based on different chemical materials,which can be a good foundation to reveal that bitter receptors may be the breakthrough points to reveal the difference of biological characterization of different substances of "expel stagnation".1.The establishment and evaluation of three different kinds of animal models of blood stasis syndromeAim To establish phlegm and blood stasis,qi-stagnation and blood stasis,phlegm turbid+qi-stagnation and blood stasis model in rats and to study the characteristics of animal models with different blood stasis.Methods SD rats were randomly divided into normal group,high fat diet group,chronic unpredictable mild stress group(CUMS)and high fat diet+chronic unpredictable mild stress group.Different state of blood stasis rat model were established by different stress factors for 6 weeks.Indexes of weight,open field behavior,serum lipids and corticosterone were monitored dynamically at the 2nd,4th,6th weeks.At the end of the experiment(6th week),the heart function was detected by small animal ultrasound and the left ventricular intubation.The blood rheology indexes were detected by the viscosity tester and red blood cell deformation/aggregation test instrument.Results Compared with the normal group,blood stasis could be induced by high fat diet and chronic unpredictable mild stress,introducing the influence of different degree on animal behavior,blood lipids,heart function and blood viscosity.When the two factors were superimposed,the changes of the indexes about blood stasis were the most significant.Performance as:compared with normal control group,a significant reduction was observed in body weight(P<0.01).Horizontal movement,vertical movement and movement time were reduced(P<0.01 or P<0.05)at the 2nd week.At the 2nd and 4th weeks,serum corticosterone was increased(P<0.01 or P<0.05)as well as TG at the 4th and 6th weeks(P<0.01).At the 6th week,velocity of blood was slowddown(P<0.01);left ventricular anterior wall and posterior wall thickness at end-systolic was increased(P<0.01 or P<0.05);left ventricular diastolic index was increased(P<0.01);the maximum rate of myocardial contraction was decreased(P<0.05);the whole blood viscosity was increased(P<0.01).Conclusion In the high fat diet group,the positive indexes were 19.35%.The number of positive indexes in the CUMS group was about 35.48%,while the number of positive indexes in the high fat diet+ CUMS group was about 32.26%.Although the number of changed indexes was more in the CUMS group,among which the number of behavioral indicators was more,while the proportion of the number of indicators related to blood stasis was less.However,in the high fat diet+CUMS group,the changes of heart function,blood lipid level and whole blood viscosity were obvious.Therefore,the animal model of high fat diet+CUMS group was selected as the later research object.2.Alteration of whole genome gene expression of tongue tissue in blood stasis syndrome rats by gene chip techniqueAim To study the changes of gene expression profile of tongue tissues in rats with blood stasis syndrome,to analyze the biological processes and pathways related to different genes and to compare the changes of bitter receptors in blood stasis syndrome,providing a scientific basis for the study of whether bitter Chinese medicine can improve the blood stasis syndrome through bitter receptors.Methods Blood stasis model in rats were established by high fat diet and CUMS.The changes of the whole gene expression profiles in the normal rats and the model rats were analyzed by gene chip technique to compare the variation of bitter receptors and find the significant difference of bitter receptor genes.Biological processes and pathways associated with differential genes were analyzed by GO and Pathway.Results Compared with the normal rats,the blood stasis rats showed 277 differential expression genes,including 68 up-regulated genes and 209 down regulated genes.The expression of Tas2R110 was decreased(P<0.01).GO and Pathway analysis showed that the blood stasis syndrome was closely related to inflammation,lipid metabolism,immune response and other biological process as well as the complement and coagulation cascade,PPAR signaling pathway,metabolism of xenobiotics by cytochrome P450 and other 4 pathways.Conclusion There was a significant change in the expression of bitter receptors and a relationship with the complement and coagulation cascade,PPAR signaling pathway,metabolism of xenobiotics by cytochrome P450 and other related pathways in the blood stasis syndrome,which could provide a theoretical basis for the study of bitter herbs improving blood stasis syndrome by bitter taste receptors.3.Study on pharmacodynamics of three kinds of bitter herbs of Salvia miltiorrhiza,Rheum officinale and Coptis chinensis on rats with blood stasis syndromeAim To establish the pharmacodynamic spectrums on the effect of promoting blood circulation and removing blood stasis of three kinds of bitter herbs of Salvia miltiorrhiza,Rheum officinale and Coptis chinensis on rats with blood stasis syndrome,To clarify the differences of efficacy of bitter drugs based on the same efficacy and different substances in promoting blood circulation and removing blood stasis.Methods The animal model of blood stasis was induced by the combination of high fat diet and chronic unpredictable stimulation.the animal successfully were randomly divided into model group,Salvia miltiorrhiza group(1.75g/kg),Rheum officinale group(1.75g/kg),Coptis chinensis group(0.58g/kg)by continuous intragastric administration for 8 weeks.During the course of administration,the stress regimen lasted for 12 weeks.Body weight,food intake and sucrose percentage were monitored dynamically during the whole experimental period.At the end of the experiment,the open field behavior index,blood lipid levels were measured by biochemical methods,CORT and ATCH were examined by radioimmunoassay,the heart function were determined by small animal ultrasound,the average perfusion of hind limbs by microcirculation detector and coagulation indices was analyzed by blood coagulation analyzer.Results At the 4th week of modeling,compared with normal control group,a significant reduction was observed in body weight and food intake(P<0.01).Sucrose consumption percentage was significantly reduced(P<0.05).Horizontal movement,vertical movement and movement time were reduced(P<0.05).Serum TG and TC were significantly increased(P<0.01)as well as serum corticosterone(P<0.05).After 8 weeks of continuous administration,compared with normal control rats,the body weight,food intake,sucrose consumption percentage were decreased significantly(P<0.01)as well as horizontal movement,vertical movement and movement time(P<0.01)in the model group.Serum TG,TC,CORT and ATCH levels were significantly increased(P<0.01)and cardiac function,microcirculation,coagulation system indicators were significantly abnormal(P<0.01).Compared with the model group,food intake was significantly increased as well as horizontal movement,vertical movement and movement time(P<0.05 or P<0.01)in Salvia miltiorrhiza group.Serum TG,TC,CORT and ATCH were significantly decreased(P<0.05 or P<0.01).All indices of cardiac function were improved except LVIPs(P<0.05 or P<0.01).The average perfusion of right hind limb was significantly increased(P<0.05)as well as coagulation indexes TT,FIB,APTT and PT(P<0.05 or P<0.01)in Salvia miltiorrhiza group.Food intake was improved(P<0.01)and serum TG was significantly decreased(P<0.01).Meanwhile,except EF and FS,cardiac function indicators were improved(P<0.05 or P<0.01).Blood coagulation index TT and APTT were significantly increased(P<0.01)in Rheum officinale group.Food intake was improved(P<0.05).Serum TG,CORT and ATCH were significantly reduced(P<0.05).Cardiac function indexes were significantly improved except LVPWd and LVPWs(P<0.01)as well as blood coagulation index TT,FIB and APTT(P<0.01).Conclusion Salvia miltiorrhiza,Rheum officinale and Coptis chinensis can improve the hemorheology and the hemodynamics of blood stasis indexes.In addition,Salvia miltiorrhiza,the drug for invigorating blood circulation and eliminating stasis,can also improve behavior and microcirculation disorders.Three bitter Chinese medicine carriers can achieve the effect of"expel stagnation",but due to the different chemical substances,the apparent indicators of"expel stagnation" are different,which can be a good foundation to reveal that bitter receptors may be the breakthrough points to reveal the difference of biological characterization of different substances of "expel stagnation". |
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