Research On The Pharmacodynamic Substances And Mechanism Of Tangniaokang Capsules In The Treatment Of Diabetic Kidney Disease Based On AGE-RAGE/NF-κB/TGF-β1 Signaling Pathway

Objective: Explain the mechanism of action of Tangniaokang capsules and its active ingredients in the treatment of diabetic kidney disease,provide data support for Tangniaokang capsules in the treatment of diabetic kidney diseasee.Method:1.Establish the chromatographic and mass spectrometric detection methods of Tangniaokang capsules,and analyze the chemical composition of Tangniaokang capsules through the self-built compound library,accurate molecular weight and secondary fragment information of Tangniaokang capsules.2.The chemical composition of Tangniaokang capsules was proofread on the Huayuan website,and the Swiss Target Prediction database was used to predict the component targets.Gene Cards,TTD,CTD and other databases were used to obtain DKD disease targets;STRING database was used to construct PPI network,GO analysis and KEGG pathway enrichment analysis were performed through DAVID database,and Cytoscape 3.8.2software was used to display the network diagram of "active ingredients-key targetspathways-diseases".Finally,the HMC cell model was used to validate the network pharmacology study results.3.MTT assay was used to observe the effect of experimental drugs on AGE-induced HMC cell inflammation model,and the optimal administration concentrations of Tangniaokang capsules and 4-Hydroxybenzoic acid,Echinacoside,Acteoside and Safflor yellow(A)were selected;ELISA assay was used to observe the effect of experimental drugs on the expression of IL-6,IL-1β,and TNF-α secreted by AGE-induced HMC cells;Western blot was used to detect the expression of RAGE,p65,JUN,and TGF-β1 protein,respectively,so as to investigate the regulation of AGE-RAGE/NF-κB/TGF-β1 signaling pathway by experimental drugs.Results:1.A total of 58 chemical constituents were identified from Tangniaokang capsules,including phenylethanoid glycosides,flavonoid,organic acids,alkaloids,and others.2.It was found that the active ingredients of Tangniaokang capsules in the treatment of diabetic kidney disease are mainly 4-Hydroxybenzoic acid,Echinacoside,Acteoside and Safflor yellow(A),etc;the core targets are mainly ALB,TNF,GAPDH,AKT1,VEGFA,IL-1β,TP53,PPARG,EGFR,PTGS2,etc;the signaling pathways involved include AGERAGE,HIF-1,IL-17,TNF,MAPK,NF-κB,etc.3.(1)Compared with the model group,Tangniaokang capsules and its active ingredients echinacoside,p-hydroxybenzoic acid,ergosteroid glycoside,and safflower yellow A could significantly inhibit AGE-induced proliferation of HMC cells,with statistical significance(P < 0.05),and the optimal concentration of Tangniaokang capsules concentration was 120μg/m L;the optimal concentration of 4-Hydroxybenzoic acid,Echinacoside,Acteoside and Safflor yellow(A)was 60 μg/m L.(2)ELISA experiment: After successful membrane creation with 200 μg/m L AGE,the expression of IL-6,IL-1β,and TNF-α secreted by HMC cells was significantly increased,while Tangniaokang capsules and four active ingredients could effectively reduce the levels of IL-6,IL-1β,and TNF-α in HMC cells;Safflor yellow(A)contained in Tangniaokang capsules was the main component inhibiting IL-6 levels(P< 0.0001),Echinacoside was the main component inhibiting TNF-α levels(P < 0.0001),while Acteoside was the main component reducing IL-1β levels(P < 0.001).(3)Western blot test: Compared with the model group,the RAGE protein level tended to be significantly down-regulated after intervention with Tangniaokang capsules and four active ingredients,with statistical significance(P < 0.01,P < 0.001,P < 0.0001).Safflor yellow(A)in Tangniaokang capsules inhibited NF-κB protein level with statistical significance(P < 0.01),while Tangniaokang capsules,Echinacoside,Acteoside,and 4-Hydroxybenzoic acid had no statistical difference in NF-κB protein level.The four active ingredients of Tangniaokang capsules inhibited JUN protein level,with statistical significance(P < 0.001,P < 0.0001).Tangniaokang capsules and active ingredients could down-regulate TGF-β1 protein levels,with statistical significance(P < 0.001,P < 0.0001).Conclusion: The main components contained in Tangniaokang capsules include phenylethanoid glycosides,flavonoid,organic acids.Its pharmacodynamic substances for the treatment of diabetic kidney disease are 4-hydroxybenzoic acid,echinacoside,acteoside,safflor yellow(A)and so on.Tangniaokang capsules and its active ingredients may regulate the release of IL-6,IL-1β,TNF-α in the treatment of diabetic kidney disease by acting on AGE-RAGE/NF-κB/TGF-β1 signaling pathway.In this study,the pharmacodynamic substances and mechanism of Tangniaokang capsules in the treatment of diabetic kidney disease were clarified,and the results will effectively promote the application of Tangniaokang capsules in the treatment of diabetic kidney disease.