Protective Effect And Mechanism Study Of Flavanomarein On Renal Tubular Epithelial Cell Injury Induced By High Glucose

Objective: Diabetic nephropathy(DN)is one of the main causes of end-stage renal failure,which seriously threatens the life and quality of life of patients.DN is characterized by the accumulation of extracellular matrix(ECM)protein.the deposition of ECM subsequently leads to renal fibrosis-tubulointerstitial fibrosis,glomerulosclerosis,infiltration of inflammatory mediators,and activates α-SMA positive myofibroblasts.Epithelial mesenchymal transition(EMT)occurs in some renal tubular epithelial cells under pathological stimulation such as high glucose.A large number of literatures have shown that EMT is the main source of myofibroblasts in diabetic nephropathy.Our previous study confirmed that the flavonoids of Coreopsis tinctoria Nutt have protective effect on early diabetic nephropathy.Flavanomarein(FM)is the main active component in Coreopsis tinctoria Nutt.the results of pharmacokinetic study showed that the concentration of FM in renal tissue changed most significantly,reached the highest value first,and the concentration was the highest and has the longest duration,absorbed into the blood by prototype.At present,there is no targeted and specific drug for the treatment of DN.In this study,we studied the anti-EMT effect of FM on human renal tubular epithelial cells(HK-2)stimulated by high glucose and its mechanism.Including exploring the mechanism of its direct target,downstream signal-related proteins and its anti-EMT effect through the cellular communication and carrier role of exosomes,and the effect of FM on human renal fibroblasts(KFB)through paracrine.It is hoped to provide a theoretical basis for the further development and utilization of Coreopsis tinctoria Nutt,and the research and development of new drugs for DN.Methods: 1)The potential target proteins of FM were predicted by DS 2017R2 ligand-protein reverse docking software.2)According to the selected 15 proteins,the protein-protein interaction was constructed by usingBisoGenet software of Cytoscape platform,and the relationship between each target protein was analyzed.3)Through the gene ontology(GO)of Cytoscape platform,the biological process of 15 proteins in the network was enriched.4)The specific binding of FM to the target protein was studied by molecular dynamics and surface plasmon resonance(SPR).5)MTS method,combined with cell morphology and Western blot method,were used to determine the high glucose modeling concentration of EMT and the effective anti-EMT concentration of FM.FN,E-cadherin,α-SMA and Vimentin were used as marker proteins of EMT.6)The effects of different concentrations of FM on TGF-β1,Syk,Smad2/p-Smad2 and Smad4 proteins in HK-2 cells were detected by Western blot combined with gene silencing technique,and the pathway of FM was studied.7)The marker proteins CD9,CD63,Calnexin and TSG101 were detected by Western blot,the morphology of exosome body was observed by electron microscope,and the particle size of exosome was detected by NTA.8)Western blot assay was used to detect the anti-EMT effect of FM,Syk inhibitor(predicted target inhibitor of FM)and losartan(positive control)on HK-2 cells induced by high glucose.9)Proteomics method was used to study the anti-EMT mechanism of FM,Syk inhibitor and losartan intervented exosomes on HK-2cells.10)Western blot method combined with Gene silencing technique was used to verify the proteomics results and to study the pathway.11)Cell immunofluorescence technique,scratch test and Western blot method to explore the effect of FM,Syk inhibitor,losartan intervented exosomes on anti-fibrosis of KFB cells treated with high glucose.Results: 1)Spleen tyrosine kinase(Syk)is a direct target of FM related to DN.2)FM can promote the proliferation of HK-2 cells and inhibit the EMT process of HK-2 cells through Syk/TGF-β1/Smads signaling pathway,and Syk inhibitor has synergistic anti-EMT effect.3)The exosomes secreted by HK-2 cells can be Internalized by itself and KFB.The exosomes(secreted from HK-2 cells)intervened by FM and Syk inhibitor can produce anti-EMT effect on HK-2 cells induced by high glucose through c-Kit/Syk/TGF-β1signaling pathway.4)The exosome proteomics,bioinformatics analysis and Western blot method were used to predict and verify one of the main targets of exosomes intervention of high glucose,flavanomarein,Syk inhibitor BAY61-3606 Syk against EMT induced by high glucose in HK-2 cells is c-KIT.5)The exosomes(secreted from HK-2 cells)interfered with high glucose can promote the proliferation,migration and transformation of KFB cells to myofibroblasts.6)The exosomes(secreted from HK-2 cells)treated with FM,Syk inhibitor and losartan could inhibit the proliferation,migration and transformation of KFB cells into myofibroblasts.Conclusion: FM can produce anti-EMT effect on HK-2 cellsinduced by high glucose through Syk/TGF-β1/Smads signaling pathway and paracrine effect of exosomes.The exosomes intervened by FM can inhibit the fibrosis of KFB cells.Through the above researchs,we hope to provide significance for the development of effective DN treatment drugs.