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

BACKGROUND: 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. PURPOSE: 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. METHODS: Firstly, the content determination methods of QXLZY were established with calycosin-7-O-ß-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. 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 metabolism and lipid metabolism, analyzed by network pharmacology. Disorders of amino acid metabolism did occur in db/db mice. QXLZY could revert the levels of metabolites, such as quinolinic acid, arginine, and asparagine. CONCLUSION: This study was the first time to demonstrate that QXLZY alleviated diabetes-induced pathological changes in the kidneys of db/db mice by correcting disturbances in amino acid metabolism. This work could provide a new experimental basis and theoretical guidance for the rational application of QXLZY on DN, exploring the new pharmacological effect of traditional Chinese medicine, and promoting in-depth research and development.

The integrated study on the chemical profiling and in vivo course to explore the bioactive constituents and potential targets of Chinese classical formula Qingxin Lianzi Yin Decoction by UHPLC-MS and network pharmacology approaches.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingxin Lianzi Yin Decoction (QXLZY), a Chinese classical formula, has been widely used in the treatment of various chronic kidney diseases over 1,000 years. However, the current studies on QXLZY were mostly focused on its clinical efficacy, lacking systematic material basis research on constituents. AIM OF THE STUDY: This work aims to elucidate and quantify the chemical constituents, clarify the blood-absorbed components and excretion pathways, predict major bioactive constituents and discover potential therapeutic targets. MATERIALS AND METHODS: UHPLC-LTQ-Orbitrap HRMS was employed to clarify the chemical constituents and metabolites of QXLZY. The extraction of diagnostic ion and neutral loss fragment was aimed for searching specific type of constituents. The plasma, urine, bile and feces samples of rats after oral administration of QXLZY were systematically studied. UHPLC-QQQ-MS/MS was employed to simultaneously detect different types of constitutes. Based on the analysis of ingredients in vivo, the bioactive constituents and potential therapeutic targets in the treatment of diabetic nephropathy (DN) was investigated by using network pharmacological analysis. RESULTS: Totally, 220 compounds were identified or tentatively characterized by UHPLC-LTQ-Orbitrap HRMS. Among them, 59 compounds were confirmed by reference standards. Meanwhile, 21 representative components were simultaneously determined within 15 min by UHPLC-QQQ-MS/MS. 123 components (74 prototypes as well as 49 metabolites) were identified or tentatively characterized. By using network pharmacological analysis, baicalein, liquiritigenin, succinic acid, formononetin, wogonin might be the major effective constituents in QXLZY during the treatment of DN. CONCLUSIONS: Flavonoids, saponins and organic acids were the major chemical ingredients of QXLZY. Flavonoids were the main components absorbed into blood, followed by organic acids. Phase II conjugation reaction was the major metabolic type. The pathways that QXLZY in the treatment of DN were probably related to glucose and lipid metabolism, oxidative stress and inflammation.

Historical Evolution and Modern Clinical Application of Qingxin Lianzi Yin / 中国实验方剂学杂志

Qingxin Lianzi Yin （QXLZY）， as an ancient classical formula for clearing the heart and nourishing the Yin， was composed of nine herbs （Scutellariae Radix， Ophiopogonis Radix， Lycii Cortex， Plantaginis Semen， Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma）， coming from Prescriptions of the Bureau of Taiping People's Welfare Pharmacy. It could clear away the heart-fire， promote the interaction of the heart and kidney， replenish Qi and Yin， and stop strangury-turbidity. It was used to treat gonorrhea of urination， seminal emission， restlessness， wasting-thirst and so on. At present， the usage and dosage of QXLZY and its addition and subtraction are different in clinical practice. Most of the studies just focus on its clinical efficacy， and there is few review literature reflecting its historical evolution. Based on this， this paper systematically clarified the historical evolution， composition， preparation， interpretation， function， and modern clinical application of QXLZY. This work has been explained the historical evolution of QXLZY， and found that it was wildly used in modern clinical， especially suitable for the treatment of chronic urinary system diseases. At the same time， QXLZY also had significant therapeutic effects on neurasthenia， stomatitis， diabetic nephropathy and other aspects. Through the comprehensive analysis of ancient and modern literature， this work explores the true connotation of QXLZY from the perspective of traditional Chinese medicine theory， which can point out the direction of the clinical application and positioning of this famous classical formula after it comes into the market， and also can provide reference basis for its subsequent in-depth research and development.