Integrated untargeted and targeted testicular metabolomics to reveal the regulated mechanism of Gushudan on the hypothalamic-pituitary-gonadal axis of kidney-yang-deficiency-syndrome rats.

Modern studies have shown that neuroendocrine disorders caused by the dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis are one of the important pathogenetic mechanisms of kidney-yang-deficiency-syndrome (KYDS). The preventive effect of Gushudan on KYDS has been reported, but its regulatory mechanisms on the HPG axis have not been elucidated. In this study, we developed an integrated untargeted and targeted metabolomics analysis strategy to investigate the regulatory mechanism of Gushudan on the HPG axis in rats with KYDS. In untargeted metabolomics, we screened 14 potential biomarkers such as glycine, lysine, and glycerol that were significantly associated with the HPG axis. To explore the effect of changes in the levels of potential biomarkers on KYDS, all of them were quantified in targeted metabolomics. With the quantitative results, correlations between potential biomarkers and testosterone, a functional indicator of the HPG axis, were explored. The results showed that oxidative stress, inflammatory response, and energy depletion, induced by metabolic disorders in rats, were responsible for the decrease in testosterone levels. Gushudan improves metabolic disorders and restores testosterone levels, thus restoring HPG axis dysfunction. This finding elucidates the special metabolic characteristics of KYDS and the therapeutic mechanism of Gushudan from a new perspective.

Lou Y ，Liang Q ，Xin L ，Ren M ，Hang Q ，Qin F ，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 ... -  《-》

(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 ... -  《-》

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 ... -  《-》

Integrated adrenal and testicular metabolomics revealed the protective effects of Guilingji on the Kidney-Yang deficiency syndrome rats.

Guilingji (GLJ) is a well-known traditional Chinese medicine (TCM) prescription for the treatment of Kidney-Yang deficiency syndrome (KYDS). This study aimed to address the protective effects of GLJ against KYDS in rats with pharmacodynamic indicators and target tissues (adrenal gland and testis) metabolomics. The rats were injected intraperitoneally (i.p) hydrocortisone to simulate KYDS and administered orally of GLJ for 30 days. Traditional pharmacodynamic indicators (body weight, behavioral indicators, biochemical parameters and histological examination) were performed to evaluate the efficacy of GLJ. Furthermore, adrenal gland and testis metabolic profiles obtained by UHPLC-Q Exactive Orbitrap-MS coupled with multivariate analysis were conducted to explore the metabolic regulation mechanism of GLJ. After administration of GLJ, the weight, levels of behavioral indicators and biochemical parameters of rats were increased compared with those of the model group, and the abnormalities of morphology in adrenal and testicular tissues were improved. Furthermore, GLJ had recovering effects via the adjustment of vitamins metabolism, which was accompanied by lipids metabolism, amino acid metabolism and nucleotides metabolism. The study firstly integrated the target tissues metabolic profiles, which were complementary, and GLJ had protective effects on KYDS rats via the regulation of steroid hormone biosynthesis, oxidant-antioxidant balance and energy acquisition.

Du K ，Gao XX ，Feng Y ，Li J ，Wang H ，Lv SL ，Wang PY ，Zhang B ，Qin XM ... -  《-》