Targeting the Gut-Kidney Axis in Diarrhea with Kidney-Yang Deficiency Syndrome: The Role of Sishen Pills in Regulating TMAO-Mediated Inflammatory Response.

BACKGROUND Sishen Pills (SSPs) are commonly used to treat diarrhea with kidney-yang deficiency syndrome. Trimethylamine-N-oxide (TMAO) is produced through the metabolism of gut microbiota and can participate in diarrhea in kidney-yang deficiency syndrome by mediating the "gut-kidney axis" to transmit inflammatory factors. This study combined network pharmacology with animal experiments to explore whether SSPs can treat diarrhea with kidney-yang deficiency syndrome by affecting the interaction between TMAO and gut microbiota. MATERIAL AND METHODS A mouse model of diarrhea with kidney-yang deficiency syndrome was constructed by using adenine and Folium sennae decoction, and SSP decoction was used for treatment. This study utilized network pharmacology to predict the potential mechanisms of SSPs in treating diarrhea with kidney-yang deficiency syndrome. 16S rRNA high-throughput sequencing was used to analyze gut mucosal microbial characteristics. ELISA was used to measure TMAO, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), interleukin-1ß (IL-1ß), and transforming growth factor-ß1 (TGF-ß1) levels. We performed Masson and immunohistochemical (Occludin, ZO-1) staining of kidney and small intestinal tissues. The fluorescein diacetate (FDA) hydrolysis spectrophotometric method was used to assess the microbial activity in contents of the small intestine. RESULTS Network pharmacology analysis revealed that SSPs can modulate 108 target points involved in the development of diarrhea, including IL-1ß and TNF. The experimental results demonstrated that SSP decoction significantly improved the general behavioral profiles of the mice, and also reduced TMAO, NLRP3, IL-1ß, and TGF-ß1 levels (P<0.05). Correlation analysis revealed significant positive correlations between TMAO concentrations and NLRP3, IL-1ß and TGF-ß1 levels (P<0.05). Pathological analysis revealed improvements in renal fibrosis and increased expression of the Occludin and ZO-1 proteins in intestinal tissue. In the SSP group, there was a significant increase in microbial activity (P<0.001). According to the sequencing results, the characteristic bacteria of the SSP and NR groups included Succinatimonas hippei, uncultured Solirubrobacter sp., and Clostridium tyrobutyricum. Furthermore, TMAO, NLRP3, IL-1ß, and TGF-ß1 were significantly positively correlated (P<0.05) with Succinatimonas hippei and Clostridium tyrobutyricum. By modulating Firmicutes, Succinatimonas hippei, and Clostridium tyrobutyricum, SSP decoction lowers TMAO levels to alleviate diarrhea with kidney-yang deficiency syndrome. CONCLUSIONS TMAO likely plays a significant role in the "gut-kidney axis" of diarrhea with kidney-yang deficiency syndrome. By adjusting gut microbiota to reduce the inflammatory response that is transmitted through the "gut-kidney axis" as a result of elevated TMAO levels, SSP decoction can alleviate diarrhea with kidney-yang deficiency syndrome.

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.

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.