Effect Of Angelica Sinensis And Danggui Wuji Granule On Blood Deficiency Syndrome And The Mechanism Based On Intestinal Flora

Objective:Blood deficiency syndrome is one of the traditional syndromes of traditional Chinese medicine,which refers to the pathological state of insufficient blood or decreased nourishing function of blood.It exists in many diseases,most of which are caused by excessive blood loss,spleen and stomach weakness,or insufficient blood biochemical source and blood stasis.Intestinal flora is a symbiotic microbial community living in the host,which plays a variety of roles in the physiological activities of human and animal bodies.It is influenced by many factors such as diet,environment,diseases and drugs.A large number of studies show that there is a close relationship between intestinal flora and the health of the host.In this study,we observed the effects of blood tonic drugs and products on the general condition,weight,blood routine,spleen index,thymus index and other indicators of blood deficiency mice,and analyzed the diversity,community composition,species differences,metabolic pathways and metabolites of intestinal contents of mice by using 16S rDNA diversity analysis,metagenomics and metabonomics methods.Combined with fecal bacteria transplantation,we studied the treatment of pseudo-sterile blood deficiency mice with intestinal contents to verify the effect of blood tonic drugs on blood deficiency.The purpose is to explore the explanation of the principle of traditional Chinese medicine with modern science,reveal the correlation between blood deficiency syndrome and intestinal flora,and the improvement effect of blood tonic drugs on intestinal flora.Methods and Results:1.Comparison of different models of blood deficiency syndromeICR mice were randomly divided into normal group,simple bloodletting group,acetylphenylhydrazinegroup,cyclophosphamidegroupand acetylphenylhydrazine+cyclophosphamide group.The results showed that the mice in the model group showed listlessness,decreased activity,hunched back,lethargy,eyes closed,sparse and dull fur,loose stool and thin body.Compared with the normal group,the blood routine indexes(RBC,WBC,HGB,MCV,RET)of mice in the simple bloodletting group decreased significantly(P<0.05 or P<0.01),and the spleen index increased significantly(P<0.01).The blood routine indexes(RBC,HCT,MCV,MCH,MCHC,RDW)of mice in phenylhydrazine group decreased significantly(P<0.01),and the spleen index increased significantly(P<0.01).The blood routine indexes(RBC,WBC,HCT,MCHC,RET),thymus index,spleen index and serum Ig G content in cyclophosphamide group were significantly decreased(P<0.05 or P<0.01).The weight,thymus index,serum Ig G content and blood routine indexes(RBC,WBC,HCT,MCH,MCHC,RET)of mice in the Acetylhydrazine+Cyclophosphamide group decreased significantly(P<0.05 or P<0.01),while the spleen index increased significantly(P<0.01).After comparative analysis,the models of blood deficiency syndrome caused by simple bloodletting method and cyclophosphamide method can simulate the symptoms of clinical blood deficiency syndrome,so the efficacy and mechanism of blood tonics were studied by using these two models.2.The influence of blood tonic drugs and products on the efficacy and intestinal flora of blood deficiency syndrome caused by cyclophosphamide.(1)The efficacy of blood tonic drugs and products on blood deficiency syndrome caused by cyclophosphamide.ICR mice were randomly divided into normal group,model group,Angelica sinensis water extract group,Angelica sinensis black-chicken granule high,middle and low dose groups,with 12 mice in each group.The mice were given corresponding drugs by gavage for 30 days,and from the 24th day after administration,except the normal group,CTX 40 mg/kg was injected intraperitoneally for 5 consecutive days.The results showed that compared with the normal group,the mice in the model group showed mental fatigue,decreased activity,arched back,lethargy,eyes closed,sparse and dull fur,loose stools,thin body,weight loss,and RBC,WBC,HGB,HCT,RDW,PLT and RET in blood routine decreased significantly(P<0.01).Compared with the model group,the water extract of Angelica sinensis and low-dose Angelica Wuji Granule group can significantly increase RBC,HGB,HCT and other levels in peripheral blood of mice(P<0.01),and significantly increase thymus index and spleen index of mice(P<0.01).(2)Effects of blood tonic drugs and products on intestinal flora of blood deficiency syndrome caused by cyclophosphamideThe intestinal flora of cecum contents in normal group,model group,Angelica sinensis water extract group and Angelica sinensis Wuji Granule low dose group was studied.The total DNA of cecum contents was extracted and the purity was detected by16S rDNA diversity analysis.PCR amplification,identification,purification and quantification were carried OTU.PE library and Illumina sequencing were constructed.The results were analyzed by data optimization,out clustering,αdiversity analysis,βdiversity analysis,principal coordinate analysis,community composition analysis and species difference.The results showed that,at the phylum level,compared with the normal group,the abundance of Bacteroidota,Patescibacteria,Actinobactota and Desulfobacteria in the model group decreased,while the abundance of Firmicutes increased.Compared with the model group,the abundance of Bacteroidota and Patescibacteria in Angelica sinensis water extract group and Angelica sinensis Wuji granule low dose group increased.At the genus level,the significance level of species abundance differences among the four groups was evaluated by Kruskal-Wallis rank sum test,and there were significant differences among Candidatus＿Saccharimonas,Lachnospiraceae,Enterorhabdus,Alloprevotella and Turicibacter.Compared with the normal group,the abundance of Muribaculaceae,Clostridia,Candidatus＿Saccharimonas,Lachnospiraceae in the model group decreased.Compared with the model group,the abundance of Muribaculaceae,Clostridia,Candidatus＿saccharomyces,Lachnospiraceae in Angelica sinensis aqueous extract group increased,and that of Muribaculaceae and Candidatus＿saccharomyces in Angelica sinensis Wuji Granule low dose group increased.(3)Experiment of fecal bacteria transplantationEffect of intestinal contents on drug efficacy and intestinal flora of pseudo-sterile mice with blood deficiency syndromeICR mice were selected.Except the normal group,other mice were given antibiotics by gavage for 7 consecutive days,and fecal smear was carried out to verify whether the pseudo-sterile mice were successfully prepared.Pseudo-sterile mice were randomly divided into FMT normal group,FMT model group,FMT Angelica sinensis water extract group and FMT Angelica Wuji Granule low-dose group according to their body weight.The feces of normal group,model group,Angelica sinensis water extract group and Angelica sinensis Wuji Granule low-dose group in blood deficiency syndrome experiment induced by cyclophosphamide method were taken,and the bacterial liquid was prepared,and the pseudo-sterile mice were given intragastric administration for 2 weeks.After one week’s administration,except the normal FMT group,the other groups were injected with CTX 40 mg/kg intraperitoneally for 5 days to make the model,and the diversity of 16S rDNA in the cecum of mice was analyzed.The results showed that compared with the normal FMT group,the mice in FMT model group showed listlessness,decreased activity,arched back,lethargy,eyes closed,sparse and dull fur,loose stools,thin body and weight loss.Compared with the normal FMT group,WBC,RBC,HGB,HCT,MCHC,thymus mass,thymus index and spleen mass in FMT model group decreased significantly(P<0.01 or P<0.05).Compared with the FMT model group,the RBC content in the low dose group of FMT Danggui Wuji Granule increased significantly(P<0.05).The results of intestinal flora showed that,at the portal level,compared with the normal FMT group,the abundance of Bacteroidota and Patescibacteria FMT model group decreased;Compared with the FMT model group,the abundance of Actinobactota in FMT Angelica sinensis water extract group increased,the abundance of Bacteroidota in FMT Angelica sinensis black-chicken granule low-dose group increased,and the abundance of Patescibacteria in FMT Angelica sinensis water extract group and FMT Angelica black-chicken granule low-dose group increased.At genus level,Kruskal-Wallis rank sum test showed that there were significant differences among four groups,such as Lactobacillus,Clostridia,Lachnospiraceae,Colidextribacter.Compared with normal FMT group,the abundance of Muribaculacea and Clostridia in FMT model group decreased;Compared with FMT model group,the abundance of Alloprevotella in FMT Angelica sinensis water extract group increased,and the abundance of Bacteroides and Alloprevotella in FMT Angelica sinensis Wuji Granule low dose group increased.3.The effects of blood tonic drugs and products on drug efficacy and intestinal flora of mice with blood deficiency syndrome caused by bloodletting(1)Effect of blood tonics and products on blood deficiency syndrome caused by simple bloodlettingICR mice were randomly divided into normal group,model group,water extract group of Angelica sinensis,high,middle and low dose groups of Angelica black-chicken granules,with 16 mice in the model group and 12 mice in each group.Each group was administered orally for 35 consecutive days.From the 21st day of administration,except the normal group,0.3 m L of blood was bled to the orbital venous plexus of other mice once every other day for 3 weeks.The results showed that compared with the normal group,the mice in the model group showed mental burnout,decreased activity,arched back,lethargy,eyes closed,sparse and dull fur,loose stools,weak body shape and weight loss.In the female model group,RBC,HGB,HCT,RDW,thymus index and Na~+-K~+-ATPase activity of erythrocyte membrane decreased significantly(P<0.01 or P<0.05).Compared with the model group,RBC,WBC of Angelica sinensis water extract group,RBC,HGB,HCT of Angelica sinensis Wuji Granule high-dose group and HCT of Angelica sinensis Wuji Granule low-dose group were significantly increased(P<0.05),and the thymus index of each administration group showed an upward trend.The activity of Na+-K+-ATPase in erythrocyte membrane of Angelica sinensis water extract group and Angelica sinensis Wuji Granule high and low dose groups increased significantly(P<0.05 or P<0.01).Compared with the normal group,RBC,HGB,MCV and RDW in the male model group decreased significantly(P<0.05).Compared with the model group,the peripheral hemogram and thymus index of each administration group showed an upward trend.(2)Study on intestinal flora of intestinal contents in mice with blood deficiency syndrome caused by simple bloodletting.16S rDNA diversity analysis and metagenome analysis were carried out on cecal contents of mice in normal group,model group,angelica sinensis water extract group and angelica wuji granule high dose group.Genome analysis:After total DNA extraction and Illumina sequencing of cecum contents,the sequencing results were subjected to data quality control,splicing and assembling,gene prediction,construction of non-redundant genes,gene abundance calculation,and species annotation and analysis at NR,COG and KEGG levels.16S rDNA diversity analysis showed that at the portal level,the abundance of Firmicutes,Bacteroidota and Actinobactota in female mouse model group decreased compared with that in normal group,but the abundance of Bacteroidota in Angelica sinensis water extract group and Angelica sinensis Wuji Granule high dose group increased compared with that in model group.Compared with the normal group,the abundance of Firmicutes,Bacteroidota and Desulfobacterota in the male model group increased,while the abundance of Actinobacteriota and Patescibacteria decreased.Compared with the model group,the abundance of Bacteroidota,Desulfobacterota,Actinobacteriota and Patescibacter in Angelica sinensis water extract group and Angelica sinensis Wuji Granule high dose group increased,but the abundance of Firmicutes decreased.At genus level,the abundance of Lactobacillus,Muribaculaceae and Clostridia in female mouse model group decreased compared with that in normal group,and the abundance of Alistipes increased.Compared with the model group,The abundance of Lactobacillus,Muribaculaceae and Alloprevotella in Angelica sinensis water extract group increased,while that of Muribaculaceae and Alloprevotella in Angelica sinensis Wuji Granule high-dose group increased,while the abundance of harmful bacteria such as Erysipelotrichaceae and Enterorhabdus decreased.The abundance of Muribaculaceae,Lachnospiraceae and unclassified＿f＿Lachnospiraceae in male model group increased compared with that in normal group,while the abundance of Lactobacillus and Desulfovibrio decreased.Compared with the model group,the abundance of Muribaculaceae,Lactobacillus and Desulfovibrio in Angelica sinensis water extract group increased,but the abundance of Lachnospiraceae and Unclassified＿F＿Lachnospiraceae decreased.The abundance of Muribaculaceae and Desulfovibrio increased,but the abundance of Lachnospiraceae,Unclassified＿F＿Lachnospiraceae and Lactobacillus decreased in the high dose group of Angelica sinensis Granule.Correlation analysis showed that RBC,HGB and HCT in female mice were significantly positively correlated with Dubosiella and Turicibacter(P<0.05 or P<0.01),and the abundance of Lachnoclostridium was positively correlated with RBC and HCT(P<0.05 or P<0.01),Gem Male Clostridium innocuum and Bacillus were positively correlated with RBC,HGB and HCT(P<0.05 or P<0.01),but negatively correlated with WBC(P<0.05).Erysipelatoclostridium was positively correlated with HGB and HCT(P<0.05),and was related to WBC.Lachnospiraceae＿NK4A136 and Desulfovibrio were negatively correlated with HGB and HCT(P<0.05),but positively correlated with WBC(P<0.05).Metagenome results:NR functional annotation(species level)showed that the abundance of Candidatus＿Amulumruptor in Angelica sinensis water extract group was significantly higher than that in model group(P<0.05);The abundance of unclassified＿f＿muribaculaceae and Alistipes in the high dose group of Danggui Chicken Granule increased significantly(P<0.05).COG functional annotation shows(amino acid sequence of gene set)that there are significant differences in cell wall/membrane/envelope biogenesis,transcription,coenzyme transportation and metabolism,lipid transportation and metabolism,cell cycle control,cell division,chromosome allocation and other functions between the administration group and the model group.The annotation of KEGG metabolic pathway shows that the metabolic pathways in the administration group and the model group mainly focus on secondary metabolite biosynthesis,ABC transporter,amino sugar and nucleotide sugar metabolism,pyrimidine metabolism,glycolysis/gluconeogenesis,aminoacyl-trna biosynthesis,peptidoglycan biosynthesis,starch and sucrose metabolism,pyruvate metabolism,pentose phosphate pathway,etc.(3)Metabonomics study of colon contents in mice with blood deficiency syndrome caused by simple bloodletting.Based on LC-MS technique,the contents of colon in normal group,model group,Angelica sinensis water extract group and Angelica sinensis Wuji Granule high dose group were metabonomics analyzed.The results showed that the structure of metabolites in colon contents of mice with blood deficiency syndrome caused by simple bloodletting changed,and the high dose of Angelica sinensis water extract and Angelica sinensis Wuji Granule had certain intervention effect on it.The identification results of different metabolites in each group showed that there were 94 different metabolites in normal group and model group,30 different metabolites in model group and Angelica sinensis water extract group,and 121 different metabolites in model group and Angelica sinensis Wuji Granule high-dose group.According to KEGG annotation analysis,the different metabolites mainly concentrated in thyroid hormone synthesis,α-linolenic acid metabolism,galactose metabolism,lysine degradation,unsaturated fatty acid biosynthesis,tyrosine metabolism,amino acid biosynthesis and metabolic pathways.Conclusion:1.Compare the models of blood deficiency syndrome caused by different modeling methods,and evaluate the efficacy of two models of blood deficiency syndrome:cyclophosphamide method and simple bloodletting method.Angelica sinensis water extract and Angelica sinensis Wuji granules have therapeutic effects on both models,which can improve the general situation of blood deficiency syndrome,increase blood routine,spleen index,thymus index and erythrocyte membrane Na~+-K~+-ATPase,and verify it by fecal bacteria transplantation experiment.2.The study of intestinal flora,16S rDNA diversity analysis showed that Angelica sinensis water extract and Angelica sinensis black-bone chicken granules increased the relative abundance of Bacteroidota、Patescibacteria、Muribaculacea、Alistipes、Alloprevotella.Correlation analysis showed that RBC,WBC,HGB,HCT of blood deficiency mice had significant positive correlation with intestinal flora such as Bacteroides,Dubosiella,Turicibacter,etc.Genome analysis shows that,The administration group and the model group were treated in M-cell wall/membrane/envelope biology,K-Transcription,H-coenzyme transport and metabolism,I-Lipid transport and metabolism,The functions of D-cell cycle control,cell division,chromosomome partitioning,etc.are significantly different;Metabolic pathways mainly focus on secondary metabolite biosynthesis,ABC transporter,amino sugar and nucleotide sugar metabolism,pyrimidine metabolism,glycolysis/gluconeogenesis,aminoacyl-trna biosynthesis,peptidoglycan biosynthesis,starch and sucrose metabolism,pyruvate metabolism,pentose phosphate pathway,etc.3.Metabonomics analysis showed that the structural outline of metabolites in colon of blood deficiency mice was changed by simple bloodletting method.Angelica sinensis water extract and Angelica sinensis Wuji granules could improve the metabolic disorder of colon of blood deficiency mice,and the pathways involved were mainly metabolic pathways,amino acid biosynthesis and metabolism,unsaturated fatty acid metabolism,galactose metabolism and so on,which were closely related to immune level and energy metabolism.