Integrated bioinformatics and large-scale clinical urine proteomics reveal Huangkui Capsules extract alleviating diabetic kidney disease.

Abstract

Background: Diabetic kidney disease (DKD) is a critical microvascular complication of diabetes mellitus. As a clinical therapeutic drug for DKD, the treatment mechanism of Huangkui Capsules (HKC) has not been clarified, which affects its individualized medication for patients. The multi-target characteristics of traditional Chinese medicine make its mechanism research rather difficult.

Purpose: This study adopted the research method of combining network pharmacology with proteomics to accurately explore the potential protein targets of HKC in treatment of DKD. Moreover, a large-scale prospective clinical trial was utilized to conduct urine proteome detection on DKD patients before and after HKC treatment, further verifying the protein targets of HKC in treatment of DKD and discovering protein markers that characterize the therapeutic effect.

Methods: Firstly, the active ingredients of HKC were extracted with 70 % ethanol and analyzed by mass spectrometry. Meanwhile, the drug-containing serum of HKC was also analyzed by mass spectrometry to verify the active ingredients and their metabolites in the blood. By comparing the protein content changes of high-glucose-stimulated HEK293T cells before and after HKC administration, the potential targets of HKC in treating DKD were explored in combination with network pharmacology. At the same time, A prospective clinical trial was designed to recruit 80 DKD patients, who received standard hypoglycemic treatment and oral administration of HKC for 6 months. The urine proteomics of these patients was used to further verify these targets and search for protein markers reflecting the treatment.

Results: There were 22 major active compounds in HKC extract, in which quercetin and palmitic acid are the main components, and the metabolites of these two main components were also found in the drug-containing serum of HKC. According to the cellular experiments, the extracellular matrix accumulation phenomenon of HEK293T cells stimulated by high glucose was alleviated after HKC administration. The proteins that were upregulated under high glucose stimulation and downregulated after HKC administration were selected and combined with network pharmacology analysis. Then a total of 4 known targets and 86 potential unknown targets of HKC were identified. Furthermore, we verified the above targets using the urine proteomics of DKD patients before and after HKC treatment. And combined with molecular docking, we found that quercetin and palmitic acid in HKC could improve renal injury in DKD patients through VEGF-A through RAP1 signaling and tight junction pathways.

Conclusion: This study provides an integrated bioinformatics combined with large-scale clinical urine proteomics to verified the protein targets of HKC in the treatment of DKD.