The Anti-fibrotic Effect Of Combination XL413 With Pirfenidone Via TGF-?1/Smad Pathway

Purpose: To investigate the effect and mechanism of pirfenidone and XL413,a novel antifibrotic agent,on proliferation,migration,and myofibroblast differentiation of the mesenchymal stem C3H10T1/2 cells,which plays an important role on the origin of myofibroblasts.Methods: After treatment of C3H10T1/2 cells with transforming growth factor-?1(TGF-?1),pirfenidone and/or XL413,cell proliferation was measured by CCK8 assay.Cells were analyzed for DNA content by flow cytometry.Cell migration was investigated by transwell technique and scratch assay.Cell cytotoxity was determined by LDH assay.The expression of cell division cycle 7(Cdc7),?-SMA,Smad2,p-Smad2,Smad4 and cyclin D1 were estimated with real-time PCR,Western blot,or immunofluorescence analyses.Statistical analysis was performed using the software of GraphPad Prism version 5 Demo for Windows.All values are expressed as mean ± standard deviation(SD),and Student's t test(two tailed)was performed to assess the statistical significance of difference.P value <0.05 was considered statistically significant.Results: We recently found that pirfenidone had anti-proliferative activity via G1 phase arrest and Cdc7 kinase expression decrease in TGF-?1-stimulated murine mesenchymal stem C3H10T1/2 cells.Pirfenidone also had inhibiting effect on the migration and ?-SMA expression.Moreover,in this study we showed for the first time that Cdc7 inhibitor XL413 enhanced the anti-fibrotic activity of pirfenidone via depressed the expression of Smad2/4 proteins,and also prevented the nuclear accumulation and translocation of Smad2 protein.Conclusion: we demonstrated that pirfenidone inhibited proliferation,migration and differentiation of TGF-?1-stimulated C3H10T1/2 cells,which could be enhanced by Cdc7 inhibitor XL413,via Smad2/4.Combination with pirfenidone and XL413 might provide a potential candidate for the treatment of TGF-?1 associated fibrosis.