Combined systems pharmacology and fecal metabonomics to study the biomarkers and therapeutic mechanism of type 2 diabetic nephropathy treated with and Leech.

In our study, systems pharmacology was used to predict the molecular targets of and Leech, and explore the therapeutic mechanism of type 2 diabetic nephropathy (T2DN) treated with and Leech. Simultaneously, to reveal the systemic metabolic changes and biomarkers associated with T2DN, we performed H NMR-based metabonomics and multivariate analysis to analyze fecal samples obtained from model T2DN rats. In addition, ELISA kits and histopathological studies were used to examine biochemical parameters and kidney tissue, respectively. Striking differences in the Pearson's correlation of 22 biomarkers and 9 biochemical parameters were also observed among control, T2DN and treated rats. Results of systems pharmacology analysis revealed that 9 active compounds (3,9-di--methylnissolin; (6aR,11aR)-9,10-dimethoxy-6,11-dihydro-6-benzofurano[3,2-]chromen-3-ol; hirudin; l-isoleucine; phenylalanine; valine; hirudinoidine A-C) and 9 target proteins (l-serine dehydratase; 3-hydroxyacyl-CoA dehydrogenase; tyrosyl-tRNA synthetase; tryptophanyl-tRNA synthetase; branched-chain amino acid aminotransferase; acetyl-CoA C-acetyltransferase; isovaleryl-CoA dehydrogenase; pyruvate dehydrogenase E1 component alpha subunit; hydroxyacylglutathione hydrolase) of and Leech were closely associated with the treatment of T2DN. Using fecal metabonomics analysis, 22 biomarkers were eventually found to be closely associated with the occurrence of T2DN. Combined with systems pharmacology and fecal metabonomics, these biomarkers were found to be mainly associated with 6 pathways, involving amino acid metabolism (leucine, valine, isoleucine, alanine, lysine, glutamate, taurine, phenylalanine, tryptophan); energy metabolism (lactate, succinate, creatinine, α-glucose, glycerol); ketone body and fatty acid metabolism (3-hydroxybutyrate, acetate, -butyrate, propionate); methylamine metabolism (dimethylamine, trimethylamine); and secondary bile acid metabolism and urea cycle (deoxycholate, citrulline). The underlying mechanisms of action included protection of the liver and kidney, enhancement of insulin sensitivity and antioxidant activity, and improvement of mitochondrial function. To the best of our knowledge, this is the first time that systems pharmacology combined with fecal metabonomics has been used to study T2DN. 6 metabolites (-butyrate, deoxycholate, propionate, tryptophan, taurine and glycerol) associated with T2DN were newly discovered in fecal samples. These 6 metabolites were mainly derived from the intestinal flora, and related to amino acid metabolism, fatty acid metabolism, and secondary bile acid metabolism. We hope the results of this study could be inspirational and helpful for further exploration of T2DN treatment. Meanwhile, our results highlighted that exploring the biomarkers of T2DN and therapeutic mechanisms of Traditional Chinese Medicine (TCM) formulas on T2DN by combining systems pharmacology and fecal metabonomics methods was a promising strategy.