Study The "Warm Meridian And Channel" Mechanism Of Danggui Sini Decoction Based On Metabolomics

OBJECTIVES:1.The "cold coagulation and blood stasis syndrome"(BSS)is one of the common clinical syndromes of traditional Chinese medicine,traditional Chinese medicine believes that this syndrome is due to the cold of the meridians and veins,resulting in poor circulation of Qi and blood,and then form blood stasis.Or attribute to body is often in a cold and humid environment,the Yang Qi of body is damaged and deficiency.The BSS is a common basic and pathological condition in the development of many chronic diseases.In this study,a method of continuous cold water bath combined with subcutaneous injection of epinephrine hydrochloride was used to construct a BSS rat model.A metabolomics-based method was used to study the serum metabolic network of BSS rats to reveal the pathogenesis of BSS.2.To study the intervention effect of different compatibility groups of Danggui Sini Decoction(DSD)prescription on the occurrence and development of BSS rats based on metabolomics.Then,reveal the "warm meridian and channel" mechanism and the compatibility principle of DSD prescription,and provide new ideas and evidence to explain the scientific compatibility of Traditional Chinese Medicine formulas.METHODS:1.Forty-eight female Sprague-Dawley rats were randomly divided into control group,model group,Danggui Sini Decoction(DSD)intervention group,and the compatibility groups,including the removal of the monarch medicine group(NJ),minister medicine group(NC),and assistant medicine group(NZ).The rats in the model,DSD,NJ,NC,and NZ groups were soaked with a 14-day continuous cold water bath combined with subcutaneous injection of epinephrine hydrochloride(on day 10 of the experiment)to construct a BSS rat model.Rats in the DSD group,NJ group,NC group and NZ group were intragastrically administered with DSD,NJ,NC and NZ drug extracts at a dose of 1.8 mg·g-1;the control group and the model group were given the same volume of distilled water.Oral gavage was administered for all of the animals once daily for 14 days.2.BSS rat model construction was evaluated using,overall index,including chills,diet,weight increment,hair condition,volume of stool and venous blood stasis,and blood rheology.3.Metabolomics approach based on ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry(UPLC-Q-TOF/MS)was used to obtain the metabolic spectrum of serum of rats in each group,and then principal component analysis(PCA),partial least-squares discriminant analysis(PLS-DA)and orthogonal partial-squares discriminant analysis(OPLS-DA)were used to find the differences in serum metabolic components between the model group and the control group,to analyze the possible relationship between different substances and BSS,and to reveal the pathogenesis of BSS.In addition,this study further characterized thechanges of serum endogenous metabolites in each intervention groups,and looked for potential biomarkers related to the efficacy of "warm meridian and channel".Then to reveal the "warm meridian and channel" mechanism and the compatibility law of DSD prescription from the whole network adjustment point of view.RESULTS:1.During the modeling period,according to the overall index evaluation of the success of BSS rat construction,the total score of the model group rats was 16 points indicated as severe BSS performance.The score of the DSD group was 3 points indicated as mild BSS performance,while the NJ group,NC group and NZ group scored 10 points,9 points and 12 points respectively which indicated as moderate BSS performance.2.The body weight of the control and DSD rats showed an increasing trend during the modeling time.The body weight fluctuations of the model group,NJ group,NC group and NZ group were obvious.On the sixth day of modeling,the body weight of these groups showed a decreasing trend.After the sixth day of modeling,the body weight showed a slow growth trend,but the weight of rats in the NZ group showed a reduction trend generally.The order of average weight gain of each group of rats was DSD>control group>NC group>model group>NJ group>NZ group.In addition,after 14 consecutive days of modeling,the model group showed a significant increase in whole blood viscosity(WBV)and high erythrocyte aggregation index(P<0.01).Compared with the model group,the WBV and erythrocyte aggregation index of the whole blood of the DSD group were significantly regulated(P<0.01).Medium shear rate(50 s-1,30 s-1),low shear rate(5 s-1,1 s-1)and erythrocyte aggregation index in whole blood of NJ,NC,and NZ groups compared with the model group were regulated(P<0.01),however,WBV at high shear rate(200 s-1)had a reversal trend but was not statistically significant.3.PCA and PLS-DA score plots showed that the metabolic profile of serum samples of model rats were deviated from the control rats,indicating that BSS caused changes in the metabolic profile of the rat.A total of 20 potential biomarkers were identified,13 of which were initially identified as related to arachidonic acid metabolism,glycerophospholipid metabolism,linoleic acid metabolism,biosynthesis of unsaturated fatty acids,pyruvate metabolism,and biosynthetic pathways of bile acids.Among them,pathway importance analysis discovered that the arachidonic acid metabolic pathway is the most interfered(pathway analysis,impact=0.33).4.Nine of the potential biomarkers may be associated with the role of DSD in nourishing blood to warm meridians.Moreover,each functional unit(monarch,minister,and assistant)involved 5 different potential biomarkers respectively in the effect on dispelling blood stasis.CONCLUSIONS:1.This study successfully constructed a rat BSS model,which can provide a reference basis for clinical research of BSS.2.DSD and its compatibility groups can significantly improve the abnormal hemorheology of BSS rats,while the DSD group has the best improvement effect.3.The serum metabolic profile of BSS rats was significantly changed.BSS significantly disturbed arachidonic acid metabolism,glycerophospholipid metabolism,linoleic acid metabolism,biosynthesis of unsaturated fatty acids,pyruvate metabolism and biosynthesis of bile acids.DSD and its compatibility groups performed different degrees of callback on the disturbed metabolic pathway,thus preventing the occurrence and development of BSS.The preventive effect of DSD group was the best,which might be related to the regulation of multiple metabolic pathways by multi-functional units.4.The research method based on metabolomics can reveal the inherent law of compatibility of traditional Chinese medicine prescription from the perspective of metabolic network regulation.