Gut microbiota affects the efficacy of Danggui Buxue Tang by affecting plasma concentration of active ingredients.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui Buxue Tang (DBT) is a traditional Chinese medicine, which has the function of supporting Qi and enriching blood. Antibiotics can cause Gut microbiota disorder and affect efficacy of DBT. AIM OF THE STUDY: Explore the manner in which Gut microbiota affects the efficacy of Danggui Buxue Tang. MATERIALS AND METHODS: In this study, antibiotics were used to destroy gut microbiota. The changes of DBT efficacy were detected to verify the effect of gut microbiota on DBT efficacy. The changes of gut microbiota was detected using 16S rRNA sequencing, and UPLC-MS/MS was used to analyze the plasma concentration of active ingredients. Correlation analysis was used to establish the relationship between gut microbiota, blood components and drug efficacy, and to explore the role of gut microbiota in the efficacy of DBT. RESULTS: The results showed that the efficacy in the DBT group was significantly improved compared with the control group (p<0.05). Compared with DBT group, the efficacy in antibiotic DBT treatment (ABXDBT) group was significantly reduced, 194 plasma metabolites and 18 DBT blood components were significantly altered in ABXDBT group, and 11 DBT blood components such as caffeic acid and formononetin were significantly decreased. Correlation analysis showed that 6 DBT blood components were related with the decrease of efficacy. Network pharmacology analysis showed that the above 6 DBT blood components participated in the hematopoietic regulation through PI3K-Akt and HIF-1 signaling pathways. Correlation analysis showed that Bacteroides and other intestinal bacteria were related to the absorption of DBT active ingredients. The drug metabolic pathway of gut microbiota was significantly decreased after antibiotic treatment (p = 0.033). CONCLUSIONS: Gut microbiota such as Bacteroides affects the efficacy of DBT by affecting the metabolism and absorption of DBT active ingredients such as caffeic acid and formononetin.

Danggui Buxue Tang restores antibiotic-induced metabolic disorders by remodeling the gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui Buxue Tang (DBT) has been used to promote hematopoiesis and relieve myelosuppression in China. Antibiotics can cause myelosuppression through gut microbiota disorders. AIM OF THE STUDY: This study aims to explore the way of DBT to alleviate the metabolic disorder caused by antibiotics. MATERIALS AND METHODS: In this study, 16S rRNA sequencing was used to detect the change of gut microbiota, metabolomics to analyze the change of metabolites. Correlation analysis was used to establishment the correlation between gut microbiota and metabolites. PICRUST 2 was used to predict the function of gut microbiota. RESULTS: Results showed that eighty-two genera of gut microbiota were affected by antibiotic, while twelve were significantly restored after DBT. Seventy-four potential metabolites were significantly different from the antibiotics and DBT. We found significant recovery by the Bacteroides and Rikenellaceae RC9 after DBT. The metabolic pathways influenced by the antibiotic treatment included primary and secondary bile biosynthesis, etc. The metabolic pathways that could be restored after DBT included the primary and secondary bile acid biosynthesis pathway, etc. Through correlation analysis, we found a correlation between the Bacteroides, Rikenellaceae_RC9_gut_group and other potential differential metabolisms such as those of taurodeoxycholic acid, N-phenylacetyl glycine, etc. The functional prediction showed that the biosynthesis of primary bile acid, secondary bile acid was significantly affected. CONCLUSIONS: DBT can restore the gut and reverse the metabolic disorder caused by antibiotics through Bacteroides, and it provides a new medical idea regarding the gut microbiota balance.

Metabolomic analysis of serum reveals the potential effective ingredients and pathways of Danggui Buxue Tang in promoting erythropoiesis.

Danggui Buxue Tang has been used for menopausal women in China for more than 800 years. However, the potential effective ingredients and pathways require further investigation. The main objective of this work was to explore the potential effective ingredients and pathways. The optimal administration time was optimized by detecting the changes of reticulocytes in peripheral blood. Drug-containing serum (DCS) was taken every 30 min after last administration. Because of the different concentration of effective ingredients absorbed into blood at different time, the pharmacodynamic effect is different. Therefore, bone marrow stromal cells as a member of hematopoietic microenvironment were used to evaluate the pharmacodynamics of DCS. Metabolomics was used to detect changes of metabolites (DBT and endogenous metabolites). The correlation of the metabolites and pharmacodynamics was used to identify the metabolites associated with erythropoiesis. After 14 days, the number of reticulocytes in peripheral blood, erythroid-related cells and erythroid progenitor cells in bone marrow in the DBT group were significantly increased. In vitro experiments showed that DCS at different time had different proliferation effects on BMSCs. Metabolomic analysis showed that the concentration of metabolites in DCS at different time was significantly different. The correlation analysis identified 7 DBT metabolites and 15 endogenous metabolites related to erythropoiesis. 15 endogenous metabolites were finally connected to different pathways. Glutamate is a node molecule. 7 potential effective ingredients of DBT were found. DBT promoted erythropoiesis via promoting the metabolism of glutamate and further affect other pathways.

A low-cost, accurate method for detecting reticulocytes at different maturation stages based on changes in the mitochondrial membrane potential.

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2-1 (MTGhigh/TMREhigh/FSbig), R2-2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.

Danggui Buxue Tang promotes the adhesion and migration of bone marrow stromal cells via the focal adhesion pathway in vitro.

ETHNOPHARMACOLOGY RELEVANCE: Danggui Buxue Tang has been used in China to treat clinical anemia for more than 800 years. However, there is no scientific report on its effect on bone marrow stromal cells. AIM OF THE STUDY: Here, we aimed to explore the effect of Danggui Buxue Tang on bone marrow stromal cell adhesion and migration. MATERIALS AND METHODS: Bone marrow stromal cells were used as a model to evaluate the effect of Danggui Buxue Tang on the adhesion and migration of bone marrow stromal cells. RNA-sequencing, quantitative polymerase chain reaction, and western blotting were used to detect and confirm the expression of genes related to the focal adhesion pathway before and after drug delivery. RESULTS: Danggui Buxue Tang significantly increased the number of bone marrow stromal cells. After 12 days of 16 mg/mL Danggui Buxue Tang treatment, bone marrow stromal cells were significantly increased (by 0.527 ±â€¯0.008 fold; p < 0.001) as compared to the control group (0.180 ±â€¯0.019). The effect was not due to enhanced cell proliferation, as there was no difference in the cell cycle (p > 0.05). The adhesion area of a single cell was doubled by Danggui Buxue Tang treatment (p < 0.001), and the time required for cell adhesion to a Petri dish was shortened. Thus, Danggui Buxue Tang increases the number of bone marrow stromal cells by promoting adhesion. Danggui Buxue Tang also significantly promoted bone marrow stromal cell migration (p <  0.001). Transcript analysis revealed that the focal adhesion and PI3K-Akt signaling pathways were activated. Expression analysis confirmed that the gene and protein expression of focal adhesion-related factors were upregulated. CONCLUSION: Danggui Buxue Tangaffects bone marrow stromal cell adhesion and migration by enhancing the focal adhesion pathway in vitro, and bone marrow stromal cells are a target of DBT-regulated hematopoiesis, and the active ingredients of DBT involved in the effects require further investigation.