Establishment Of An In Vitro Model Of Renal Fibrosis Based On High-content Technology And Screening Of Traditional Chinese Medicine Compounds

Renal fibrosis is a common pathology of chronic kidney disease(CKD)to end-stage renal disease(ESRD).The main feature of renal fibrosis is that persistent chronic injury initiates the loss of innate cells and physiological structure of the kidney and replacement by the accumulation of extracellular matrix(ECM).It is a progressive,irreversible pathological process,accompanied by a progression of renal malfunctions.With a high prevalence,modern medicine is still lack of effective methods and drugs to chronic kidney disease and end-stage renal disease.Although effective therapy for renal fibrosis is still lacking,Chinese medicine showed better application prospects in the treatment of renal fibrosis in recent years.Objective:To establish an in vitro cell model for the screening of anti-renal fibrosis drugs based on the high-content screening technology platform,and to evaluate and validate the model with the compounds with good efficacy.On the basis of establishment and validation of the model,344 kinds of traditional Chinese medicine compounds were screened with the high-content screening systerm,and the drugs with anti-renal fibrosis activity were obtained,which laid the foundation for the further research on anti-renal fibrosis drug development and related mechanism.Methods:1.The establishment of an in vitro model for high content screening of anti-renal fibrosis drugs:In this study,normal rat kidney fibroblasts NRK49F was used,which was treated with transforming growth factor-?1(TGF-?1)as an inducer.As the main marker of myofibroblasts,the expression of a-SMA in NRK49F cells was detected by high-content analysis system.The conditions including cell density,induction time and serum concentration in induction medium were optimized.2.Model validation:To verify the reliability of the model,curcumin,emodin and TGF-?1 receptor blocker SB525334,which are reported as anti-renal fibrosis compounds,were used as positive drugs,and the expression of a-SMA in NRK49F cells was detected by high-content analysis system.3.The high content screening of Chinese medicine compounds with anti-renal fibrosis efficacy:On the basis of establishment of cell model,344 kinds of traditional Chinese medicine compounds(10?mol/L)were screened by testing the expressiong of a-SMA.The compounds(72?mol/L,24?mol/L,8?mol/L,2.6?mol/L,0.89?mol/L)with anti-renal fibrosis activity were added to the renal fibrosis in vitro model.The expression of a-SMA and type ? collagen were tested.Results:The model of renal fibrosis in vitro based on high-content screening syeterm was established as follows with largest up-regulated expression of a-SMA:cell density of 6,000 cells/well cultured in 96-well plate,TGF-?1(10?g/L)by adding 5%fetal bovine serum in DMEM medium,incubated for 72 hours.Curcumin,emodin and TGF-?1 receptor blocker SB525334,could inhibit the expression of a-SMA,which were consistent with the literatures.16 compounds of the 344 kinds of traditional Chinese medicine compounds showed anti-renal fibrosis efficacy.Rosmarinic acid had no effect on the expression of a-SMA and type ?collagen.Peiminine,Imperatorin,Ginsenoside F2,and Neohesperidin may downregulate the expression of a-SMA but have no significant effect on the expression of type ? collagen.Salvianolic acid B,Danshensu and Taurine inhibited the expression of type ? collagen,but failed to inhibit the expression of a-SMA.Curcumin,Emodin,Glycyrrhizic acid,Ligustrazine,Hydroxysafflor yellow A,Astragaloside,Gypenosides and Crocin downregulate both the expression of ?-SMA and type ? collagen.Conclusion:We explored the conditions for the establishment of an in vitro model of renal fibrosis based high-content system.The effectiveness and stability of the model were verified.On this basis,more than ten kinds of Chinese medicine compounds were obtained with anti-renal fibrosis efficacy.Some of them,including glycyrrhizic acid and hydroxysafflor yellow A,crocin,gypenosides,have not been reported against renal fibrosis.These compounds can be used as candidate drugs for the study of animal in vivo.