Study on the potential mechanism of Qingxin Lianzi Yin Decoction on renoprotection in db/db mice via network pharmacology and metabolomics

•This study established content determination method of QXLZY index components.•This study is the first time to elucidate that amino acid metabolism was disturbed in the kidney of db/db mice, which could be callback by QXLZY.•Untargeted metabolomics studies were performed to investigate by UHPLC-LTQ-Orbitrap MS on urine, serum, and kidney samples.•The potential therapeutic targets of QXLZY in the treatment of DN were investigated by network pharmacological analysis.•Targeted metabolomics by UHPLC-QQQ-MS/MS was performed on urine, serum, and kidney samples for validation. Diabetic nephropathy (DN) was one of the most popular and most significant microvascular complications of diabetes mellitus. Qingxin Lianzi Yin Decoction (QXLZY) was a traditional Chinese classical formula, suitable for chronic urinary system diseases. QXLZY had good clinical efficacy in early DN, but the underlying molecular mechanism remained unrevealed. This study aimed to establish the content determination method of QXLZY index components and explore the mechanism of QXLZY on DN by network pharmacology and metabolomics studies. Firstly, the content determination methods of QXLZY were established with calycosin-7-O-β-d-glucoside, acteoside, baicalin and glycyrrhizic acid as index components. Secondly, pharmacological experiments of QXLZY were evaluated using db/db mice. UHPLC-LTQ-Orbitrap MS was used to carry out untargeted urine metabolomics, serum metabolomics, and kidney metabolomics studies. Thirdly, employing network pharmacology, key components and targets were analyzed. Finally, targeted metabolomics studies were performed on the endogenous constituents in biological samples for validation based on untargeted metabolomics results. A method for the simultaneous determination of multiple index components in QXLZY was established, which passed the comprehensive methodological verification. It was simple, feasible, and scientific. The QXLZY treatment alleviated kidney injury of db/db mice, included the degree of histopathological damage and the level of urinary microalbumin/creatinine ratio. Untargeted metabolomics studies had identified metabolic dysfunction in pathways associated with amino acid metabolism in db/db mice. Treatment with QXLZY could reverse metabolite abnormalities and influence the pathways related to energy metabolism and amino acid metabolism. It had been found that pathways with a high degree were involved in signal transduction, prominently on amino acids metab