16S rDNA sequencing combined with metabolomics profiling with multi-index scoring method reveals the mechanism of salt-processed Semen Cuscuta in Bushen Antai mixture on kidney yang deficiency syndrome.

Kidney yang deficiency syndrome (KYDS) is a classic syndrome of traditional Chinese medicine (TCM). The salt-processed product of Semen Cuscuta (YP) is the monarch drug in Bushen Antai Mixture (BAM), can improve the reproductive dysfunction caused by KYDS, and the effect is better than that of raw products of Semen Cuscuta (SP). However, its mechanism is not completely clear yet. In this study, an integrated strategy combining untargeted metabolomics with microbiology was used to explore the mechanism of YP in the BAM improving KYDS. 16S rDNA gene sequencing showed that BAM containing YP (Y-BAM) had a significantly better regulatory effect on Desulfobacterota and Desulfovibrionaceae_unclassified than BAM containing SP (S-BAM). Untargeted metabolomics studies showed that Y-BAM significantly regulated 4 metabolites and 4 metabolic pathways. In addition, multi-index analysis showed that the effect of Y-BAM on arachidonic acid metabolism, tyrosine metabolism, purine metabolism, fructose and mannose metabolism and total metabolism was closer to that of the control group compared to S-BAM. The analysis of serum biochemical indexes showed that Y-BAM had more significant regulating effect on the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) and superoxide dismutase (SOD) in serum of KYDS rats compared to S-BAM. Spearman correlation analysis showed that there was a significant correlation between intestinal microorganisms and metabolites and serum biochemical indexes. For example, Desulfovibrionaceae_unclassified was positively correlated with arachidonic acid, and negatively correlated with SOD and LH. This study suggests that YP may enhance the regulation of intestinal flora and endogenous metabolism of KYDS, so that BAM shows a better therapeutic effect on KYDS, which also reasonably explains why BAM uses Semen Cuscuta stir-baked with salt solution.

Metabolomic Analysis of Stephania tetrandra-Astragalus membranaceus Herbal Pair-Improving Nephrotic Syndrome Identifies Activation of IL-13/STAT6 Signaling Pathway.

The Stephania tetrandra−Astragalus membranaceus herbal pair (FH) is a classic herbal pair widely used in the treatment of nephrotic syndrome (NS). The effects of Stephania tetrandra (FJ) and Astragalus membranaceus (HQ) on NS have been reported, but the mechanism of their combination on the improvement of NS are still unclear. The NS model was established by injecting adriamycin into the tail vein. FH intervention reduced the levels of serum triglyceride, total cholesterol, interleukin-6 (IL-6), blood urea nitrogen (BUN), urinary protein, and the gene expression levels of aquaporin 2 (AQP2) and arginine vasopressin (AVP) in NS rats. In addition, FH improved kidney injury in NS rats by inhibiting the expression of interleukin 13 (IL-13), phospho-signal transducers, and activators of transcription 6 (p-STAT6), Bax, cleaved-caspase3, while promoting the expression of Bcl-2. By comprehensive comparison of multiple indexes, the effects of FH on lipid metabolism, glomerular filtration rate, and inflammation were superior to that of FJ and HQ. Metabonomic studies showed that, compared with FJ and HQ, FH intervention significantly regulated tricarboxylic acid (TCA) cycle, cysteine and methionine metabolism, and alanine, aspartic acid and glutamic acid metabolism. Pearson correlation analysis showed that succinic acid and L-aspartic acid were negatively correlated with urinary protein, cystatin C (Cys C) and BUN (p < 0.05). In summary, FH could reduce renal injury and improve NS through inhibiting the IL-13/STAT6 signal pathway, regulating endogenous metabolic pathways, such as TCA cycle, and inhibiting the expression of AQP2 and AVP genes. This study provides a comprehensive strategy to reveal the mechanism of FH on the treatment of NS, and also provides a reasonable way to clarify the compatibility of traditional Chinese medicine.

Comprehensive evaluation of the mechanism of Gastrodia elata Blume in ameliorating cerebral ischemia-reperfusion injury based on integrating fecal metabonomics and 16S rDNA sequencing.

Gastrodia elata Blume was used to treat stroke and headaches caused by "Feng" for thousands of years. The present study has shown a significant effect of G. elata Blume in improving cerebral ischemia-reperfusion injury (CIRI). However, the mechanism of G. elata Blume in improving CIRI by regulating the intestinal flora has not been reported until now. This research aimed to comprehensively evaluate the mechanism of G. elata Blume in CIRI based on fecal metabolomics and 16S rDNA sequencing. The rat model with CIRI was created based on the Zea Longa method. Enzyme-linked immunosorbent assay (ELISA) was used to monitor the inflammatory factors in rat serum. Damages of brain tissues were observed using hematoxylin and eosin (H&E) staining. Cerebral infarction was observed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The balance of intestinal flora in cecal contents of rats was evaluated by high-throughput sequencing. Changes of metabolites in the intestinal flora were evaluated by fecal metabolomics through Ultra high performance liquid chromatography-orbitrap exploris-mass spectrometer (UHPLC-OE-MS). The area of brain necrosis, cerebral infarction volume, and the contents of inflammatory factors in CIRI rats can be effectively reduced after oral administration of G. elata Blume. CIRI can cause disturbances in the intestinal flora and its associated metabolites. G. elata Blume can significantly regulate the composition of the intestinal microflora. It reversed CIRI-induced changes in the levels of multiple intestinal bacteria, including Prevotellaceae, Coriobacteriaceae; Prevotella, Gamma proteobacteria unclassified, Barnesiella, Escherichia, Shigella; uncultured Shigella sp., Flavonifractor sp., Escherichia sp. enrichment culture clone NBAR004, Veillonella sp. R-32, and Lactobacillus intestinalis. The levels of metabolites in cecal contents were disturbed in rats with CIRI, including amino acid, purine, and sphingolipid metabolism. The changes in the level of biomarkers in amino acid metabolism induced by CIRI were significantly reversed after treatment with G. elata Blume. Correlation studies show that Prevotellaceae was significantly positively correlated with interleukin (IL)-6, and L. intestinalis and L-phenylalanine were negatively interrelated to IL-1ß. Beta-glycerophosphoric acid was significantly negatively interrelated to high-sensitivity C-reactive protein (hs-CRP). There were significantly negative correlations between L-phenylalanine and L. intestinalis, beta-glycerophosphoric acid and Prevotellaceae. G. elata Blume protected against CIRI, which may be related to improved intestinal microflora composition and metabolism, resulting in decreased inflammation.