A Metabolomics And Network Pharmacology-based Approach To Study The Material Basis And Mechanism Of Action Of Qingrekasen Granule For Treating Nephrotic Syndrome

Objective: To investigate the material basis and mechanism of action of Qingrekasen Granule in the treatment of nephrotic syndrome through experimental studies,so as to provide scientific and theoretical guidance for the clinical use of Qingrekasen Granule.Methods: Based on ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）,the active ingredients in the pellets were identified and analyzed.48 Sprague Dawley（SD）male rats were randomly divided into 6 groups: control,model,Qingrekasen Granule low dose（0.81g/kg/d）,medium dose（1.62 g/kg/d）,high dose（3.24 g/kg/d）and Benazepril（0.9mg/kg/d）positive control groups,and rats in each group were administered by gavage.The rat model of nephrotic syndrome was established by tail vein injection of Adriamycin（7 mg/kg）.The physiological status and behavior of the rats were observed;the urine of the rats was taken and the urine protein content was measured;the pathological changes of the rats in each group were observed by HE（hematoxylin-eosin）staining of the kidney sections;the changes of the glomerular foot cells in each group were observed under the electron microscope after the kidney tissue was fixed;the serum biochemical indexes of TG（total triglycerides）,BUN（urea nitrogen）,TC（total cholesterol）,Cr（creatinine）and ALB（albumin）were analyzed by automatic biochemical analyzer.Metabolomic analysis of the main differential metabolites and metabolic pathways in the treatment of nephrotic syndrome in rats with Qingrekasen Granule.Network pharmacology was used to establish the compound-target-pathway network and protein interaction network for the treatment of nephrotic syndrome with Qingrekasen Granule,and to analyze the targets and mechanisms of Qingrekasen Granule in the treatment of nephrotic syndrome.We also analyzed the core targets and mechanisms of action of Qingrekasen Granule in the treatment of nephrotic syndrome by combining network pharmacology and metabolomics.The expression levels of PI3K/Akt/m TOR signaling pathway-related proteins and apoptosis-related proteins CASP3 and BCL2L1 were detected by Western Blot（WB）assay;the affinity of docking validated compounds to the targets was analyzed to clarify the pathways and material basis involved in the treatment of nephrotic syndrome with Qingrekasen Granule.Results: Based on retention time and mass spectrometry analysis,we identified that Qingrekasen Granule contained 30 active ingredients.The results of in vivo experiments in animals showed that Qingrekasen Granule could reduce the levels of TG,BUN,TC and Cr in serum,while increasing the levels of ALB.At the same time,Qingrekasen Granule could reduce the pathological damage caused by Adriamycin-induced nephrotic syndrome in the kidney of rats.It indicated that Qingrekasen Granule had therapeutic effects on rats with nephrotic syndrome.The metabolomics analysis showed "D-glutamine and Dglutamate metabolism" and "alanine,aspartate and glutamate metabolism" as the main targeted metabolic pathways for treatmenting of nephrotic syndrome in rats.The results of network pharmacology analysis showed that PI3K-Akt signaling pathway and AGE-RAGE signaling pathway were the main pathways of action,and AKT1,BCL2L1,MTOR,STAT3,EGFR,HSP90AA1,CASP3 and other targets might be the core targets for the treatment of nephrotic syndrome with Qingrekasen Granule.The results of combined metabolomics and network pharmacology analysis showed that AKT1,BCL2L1,CASP3 and MTOR were the core targets of Qingrekasen Granule in the treatment of nephrotic syndrome.WB experimental results verified that the expression levels of p-PI3 K,p-AKT1,p-m TOR and CASP3 significantly decreased in the middle dose group of Qingrekasen Granule,while BCL2L1 increased compared to the model group.Molecular docking results showed that the core targets had strong affinity with flavonoids,terpenoids and phenolic acids.Conclusion: 1.Qingrekasen Granule have certain protective effects on rats with nephrotic syndrome;2.Qingrekasen Granule play a role in kidney protection by reducing urinary protein,alleviating renal pathological damage in rats,lowering serum levels of TG,BUN,TC and Cr,and increasing levels of ALB;3.Qingrekasen Granule function to protect podocytes and maintain renal tubular function by inhibiting the PI3K/Akt/m TOR signaling pathway,affecting the expression of proteins related to apoptosis BCL2L1,CASP3,and promoting autophagy and anti-apoptosis.