Combined (1)H NMR fecal metabolomics and 16S rRNA gene sequencing to reveal the protective effects of Gushudan on kidney-yang-deficiency-syndrome rats via gut-kidney axis.

Based on traditional Chinese medicine (TCM) theory, kidney is regarded as governing the bones and dominating the storage of essence ('jing' in Chinese). Gushudan (GSD) is a traditional Chinese medicine prescription with the effects of strengthening bone and nourishing kidney, which has been used to treat osteoporosis for years. Several anti-osteoporosis effects of GSD have been investigated based on metabolomics in previous studies. However, the specific mechanism of GSD on kidney tonifying and its alterations in gut microbiota are still unclear. In this study, 1H NMR fecal metabolomics and 16 S rRNA gene sequencing technology were integrated to comprehensively explore the microbiota and metabolic changes in kidney-yang-deficiency-syndrome (KYDS) rats and to elucidate the protective mechanism of GSD through the gut-kidney axis. GSD significantly regulated the levels of 12 out of 31 potential metabolites and the abundance of 11 out of 16 potential microbial biomarkers related to KYDS, respectively. Fecal metabolomics showed that GSD could reserve the abnormal levels of gut microbial-mediated metabolites of KYDS rats, such as tryptophan, lysine, dimethylamine, creatinine, acetate and butyrate, which mainly involved in amino acid metabolism, methylamine metabolism, energy metabolism and short-chain fatty acid metabolism. Specifically, GSD could promote butyrate-producing bacteria g_Lachnospiraceae_NK4A136_group and lactate-producing bacteria g_Lactobacillus. Interestingly, there was a strong relationship between altered fecal metabolites and perturbed intestinal microflora in genus. For example,lysine was negatively correlated with g_Lactobacillus, while acetate was positively correlated with g_Barnesiella. In conclusion, the study showed that the gut-kidney axis had scientific implications, which not only offered new insights into the in-depth understanding of the pathogenesis of KYDS, but also provided further evidence for the efficacy evaluation of GSD.

Tong L ，Feng Q ，Lu Q ，Zhang J ，Xiong Z ... -  《-》

Integrated UHPLC-MS untargeted metabolomics and gut microbe metabolism pathway-targeted metabolomics to reveal the prevention mechanism of Gushudan on kidney-yang-deficiency-syndrome rats.

Gushudan (GSD) was a traditional Chinese prescription with the remarkable effect of kidney-tonifying and bone-strengthening. However, the potential prevention mechanisms of the GSD on kidney-yang-deficiency-syndrome (KYDS) and its regulation on gut microbe metabolism still need to be further systematically investigated. This study established untargeted urinary metabolomics based on RP/HILIC-UHPLC-Q-Orbitrap HRMS and combined with multivariate statistical analysis to discover differential metabolites and key metabolic pathways. And the gut microbe metabolism pathway-targeted metabolomic based on HILIC-UHPLC-MS/MS was developed and validated to simultaneously determine 15 gut microbe-mediated metabolites in urine samples from the control group (CON), KYDS model group (MOD), GSD-treatment group (GSD) and positive group (POS). The results showed that a total of 36 differential metabolites were discovered in untargeted metabolomics. These differential metabolites included proline, cytosine, butyric acid and nicotinic acid, which were primarily involved in the gut microbe metabolism, amino acid metabolism, energy metabolism and nucleotide metabolism. And GSD played a role in preventing KYDS by regulating these metabolic pathways. The targeted metabolomics found that the levels of 10 gut microbe-mediated metabolites had significant differences in different groups. Among them, compared with the CON group, the levels of lysine, tryptophan, phenylacetylglycine and hippuric acid were increased in the MOD group, while the levels of threonine, leucine, dimethylamine, trimethylamine, succinic acid and butyric acid were decreased, which verified the disorders of gut microbe metabolism in the KYDS rats and GSD had a significant regulatory effect on this disorder. As well as by comparing analysis, it was found that the experimental results were consistent with previous metabolomics and microbiomics of fecal samples. Therefore, this integrated strategy of untargeted and targeted metabolomics not only elucidated the potential prevention mechanism of GSD on KYDS, but also provided a scientific basis for GSD preventing KYDS via the "gut-kidney" axis.

Xin L ，Ren M ，Lou Y ，Yin H ，Qin F ，Xiong Z ... -  《-》

Fecal metabonomics combined with 16S rRNA gene sequencing to analyze the changes of gut microbiota in rats with kidney-yang deficiency syndrome and the intervention effect of You-gui pill.

A myriad of evidence have shown that kidney-yang deficiency syndrome (KYDS) is associated with metabolic disorders of the intestinal microbiota, while TCMs can treat KYDS by regulating gut microbiota metabolism. However, the specific interplay between KYDS and intestinal microbiota, and the intrinsic regulation mechanism of You-gui pill (YGP) on KYDS' gut microbiota remains largely unknown so far. In the present study, fecal metabonomics combined with 16S rRNA gene sequencing analysis were used to explore the mutual effect between KYDS and intestinal flora, and the intrinsic regulation mechanism of YGP on KYDS's gut microbiota. Rats' feces from control (CON) group, KYDS group and YGP group were collected, and metabolomic analysis was performed using 1H NMR technique combined with multivariate statistical analysis to obtain differential metabolites. Simultaneously, 16S rRNA gene sequencing analysis based on the Illumina HiSeq sequencing platform and ANOVA analysis were used to analyze the composition of the intestinal microbiota in the stool samples and to screen for the significant altered microbiota at the genus level. After that, MetaboAnalyst database and PICRUSt software were apply to conduct metabolic pathway analysis and functional prediction analysis of the screened differential metabolites and intestinal microbiota, respectively. What's more, Pearson correlation analysis was performed on these differential metabolites and gut microbiota. Using fecal metabonomics, KYDS was found to be associated with 21 differential metabolites and seven potential metabolic pathways. These metabolites and metabolic pathways were mainly involved in amino acid metabolism, energy metabolism, methylamine metabolism, bile acid metabolism and urea cycle, and short-chain fatty acid metabolism. Through 16S rRNA gene sequencing analysis, we found that KYDS was related to eleven different intestinal microbiotas. These gut microbiota were mostly involved in amino acid metabolism, energy metabolism, nervous, endocrine, immune and digestive system, lipid metabolism, and carbohydrate metabolism. Combined fecal metabonomics and 16S rRNA gene sequencing analysis, we further discovered that KYDS was primarily linked to three gut microbiotas (i.e. Bacteroides, Desulfovibrio and [Eubacterium]_coprostanoligenes_group) and eleven related metabolites (i.e. deoxycholate, n-butyrate, valine, isoleucine, acetate, taurine, glycine, α-gluconse, β-glucose, glycerol and tryptophan) mediated various metabolic disorders (amino acid metabolism, energy metabolism, especially methylamine metabolism, bile acid metabolism and urea cycle, short-chain fatty acid metabolism. nervous, endocrine, immune and digestive system, lipid metabolism, and carbohydrate metabolism). YGP, however, had the ability to mediate four kinds of microbes (i.e. Ruminiclostridium_9, Ruminococcaceae_UCG-007, Ruminococcaceae_UCG-010, and uncultured_bacterium_f_Bacteroidales_S24-7_group) and ten related metabolites (i.e. deoxycholate, valine, isoleucine, alanine, citrulline, acetate, DMA, TMA, phenylalanine and tryptophan) mediated amino acid metabolism, especially methylamine metabolism, bile acid metabolism and urea cycle, short-chain fatty acid metabolism, endocrine, immune and digestive system, and lipid metabolism, thereby exerting a therapeutic effect on KYDS rats. Overall, our findings have preliminary confirmed that KYDS is closely related to metabolic and microbial dysbiosis, whereas YGP can improve the metabolic disorder of KYDS by acting on intestinal microbiota. Meanwhile, this will lay the foundation for the further KYDS's metagenomic research and the use of intestinal microbiotas as drug targets to treat KYDS.

Chen R ，Wang J ，Zhan R ，Zhang L ，Wang X ... -  《-》

(1)H NMR serum metabolomics and its endogenous network pharmacological analysis of Gushudan on kidney-yang-deficiency-syndrome rats.

The pharmacodynamics, 1H NMR metabolomics and endogenous network pharmacology strategy approaches were integrated to investigate the preventive mechanism of Gushudan (GSD) on kidney-yang-deficiency-syndrome (KYDS) rats in this study. Firstly, the KYDS rat model was achieved by hydrocortisone induction, and the efficacy of GSD on KYDS model rats was assessed by the pharmacodynamic indicators. Next, the comprehensive untargeted serum metabolic profile of rats was obtained in 1H NMR metabolomics study, 29 potential biomarkers closely associated with KYDS were identified, which were mainly involved in carbohydrate metabolism, amino acid metabolism and intestinal flora metabolism. In addition, the potential biomarkers-targets-pathways-disease metabolic network was further investigated for deeper understanding the preventive effects of GSD on KYDS rats and its mechanism, which was further obtained for the important targets related to biomarkers and diseases such as NOS3, PTGS2 and CXCL8, and important metabolic pathways such as glyoxylate and dicarboxylate metabolism, arginine and proline metabolism, and microbial metabolism in diverse environments. Finally, compared with our previous anti-osteoporosis study of GSD, it suggested that some similar metabolic pathways, which would provide some scientific reference of the existence of the kidney-bone axis under the traditional Chinese medicine (TCM) theory of "kidney dominates bone".

Feng Q ，Tong L ，Lu Q ，Liu S ，Zhao L ，Xiong Z ... -  《-》

Metabolomics and serum pharmacochemistry revealed the preventive mechanism of Gushudan in kidney-yang-deficiency-syndrome rats.

Kidney-yang-deficiency-syndrome (KYDS) is a metabolic disease caused by neuroendocrine disorder. Gushudan (GSD) is a traditional Chinese medicine prescription with the effect of nourishing kidney and strengthening bones. In this study, the mechanism of preventive effect of GSD on KYDS was explored by integrating metabolomics and serum pharmacochemistry. Reversed-phase/hydrophilic interaction chromatography-ultra-high-performance liquid chromatography-Quadrupole-Orbitrap high-resolution mass spectrometry (RP/HILIC-UHPLC-Q-Orbitrap HRMS)-based serum metabolomics indicated metabolic disturbances of KYDS rats, and 50 potential biomarkers including l-threonine, succinic acid and phytosphingosine were obtained, which were mainly involved in alanine, aspartate and glutamate metabolism, citrate cycle (tricarboxylic acid cycle) and glycerophospholipid metabolism, among others. Serum pharmacochemistry identified 29 prototypical ingredients and 9 metabolites of GSD after administration, such as icaritin and xanthotoxol. The combination of 10 serum migration ingredients in GSD, including icaritin and osthole, with 7 important targets, including AKT serine/threonine kinase 1 (AKT1) and MAPK14, was found to be key for GSD to prevent KYDS in the network pharmacology study. This study provided a new idea for the research of pathogenesis of diseases and the pharmacodynamic mechanism of traditional Chinese medicine.

Lu Q ，Feng Q ，Yu J ，Tong L ，Zhang J ，Sun J ，Zhao J ，Xiong Z ... -  《-》