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 ... -  《-》

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 ... -  《-》

Integrated gas chromatography-mass spectrometry and ultra-high-performance liquid chromatography-mass spectrometry renal metabolomics and lipidomics deciphered the metabolic regulation mechanism of Gushudan on kidney-yang-deficiency-syndrome rats.

Kidney-yang-deficiency-syndrome is a neuroendocrine disease caused by the dysfunction of the adrenal-pituitary-target gland axis. Gushudan is a traditional Chinese medicine prescription with the functions of tonifying the kidney and strengthening bone, and its bone-strengthening effect has been confirmed by previous anti-osteoporosis research. However, its kidney-tonifying mechanism has not been clear so far. In this study, renal metabolomics and lipidomics based on gas chromatography-mass spectrometry and ultra-high-performance liquid chromatography-high resolution mass spectrometry were integrated to find the metabolic disorders in kidney-yang-deficiency-syndrome rats. Protein precipitation and liquid-liquid extraction were used to extract metabolome and lipidome from the kidney. Gushudan regulated abnormal levels of amino acids, lipids, purines, and carbohydrates, such as L-arginine, hypoxanine, stearic acid, and phosphatidylethanolamine (P-18:1/20:4), which had effects on many metabolic pathways, such as glycerophospholipid metabolism, sphingolipid metabolism, glycine, serine and threonine metabolism and purine metabolism, and so forth. By integrating metabolomics and lipidomics, this study comprehensively revealed the abnormal metabolic activities of amino acids, lipids, and nucleotides in kidney-yang-deficiency-syndrome, and the metabolic regulation mechanism of Gushudan in preventing kidney-yang-deficiency-syndrome, as well as the improvement of Gushudan in maintaining renal cell structure, mitochondrial function, and energy supply, which also provided some new evidence and connotation for "kidney-bone" axis.

Lu Q ，Zhang J ，Xin L ，Lou Y ，Qin F ，Zhao L ，Xiong Z ... -  《-》

(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 ... -  《-》

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 ... -  《-》