Effects Of Fasudil On Proliferation And Migration In Urethral Fibroblast Cells After RNA Interference LIMK1

Purpose:LIMK1 is one of the important molecular regulation of the cytoskeleton, previous research has shown that Rho kinase inhibitor fasudil can decrease the expression of LIM kinase 1 which is central regulator of cytoskeletal dynamics thus inhibiting fibroblast proliferation and migration,This study was performed to determine the effiacy of ROCK inhibitor fasudil in blocking the development of TGF?1-induced urethral fibroblast cells migration after RNA Interference LIMK1, to clarify that LIMK1 is an important molecules in the migration process of fibroblast?Materials and methods: The research object was the original generation of rabbit urethral fibroblasts, using RNAi technology to inhibition the expression of LIMK1, RT-PCR and Western blot to test interference efficiency. The research was set into two group: RNA interference group(LIMK1 inhibiting group) and no interference group(LIMK1 normal expression), the culture medium were added TGF-beta 1(10 ng/m L) and fasudil(50 ?mol/L), thus divided into eight subgroups. Using cell scratch experiment and cell migration assay to detect cell migration ability; Using flow cytometry to detect cell apoptosis rate; Western blot test the expression of alpha SMA, p-Cofilin, p-MLC, LIMK-1 and the collagen I and III.Results: Western blot show that compared with normal control group, the expression of LIMK1 protein respectively reduced by 52%, 70%, 90% in R1-R3 si RNA, R3 of si RNA has the highest inhibition rate(P < 0.05). RT-PCR results also shows that R3 of si RNA significantly decreased(P < 0.05). So, use the R3 group of si RNA to inhibition LIMK1. Cell scratches and cell migration assay showed that the cell migration distance of RNA interference group was significantly less than control group(P < 0.05). Flow cytometry detection showed that RNA interference group has a higher cells apoptosis rate than control group(P < 0.05). Western blot showed that the expression of alpha SMA, LIMK-1, p-Cofilin, and I and III collagen is much less than control group(p < 0.05),but the expression of p-MLC has no difference in two group?Conclusion: RNA interference LIMK1 can derease the p-Cofilin?alpha SMA ? type I and III collagen protein to down regulation of Rho/ROCK pathway signaling, thus inhibit the fibroblast proliferation and migration, LIMKI interference can also increase the inhibition effect of fasudil on migration in urethra fibroblast cell, therefore, LIMK1 can be used as a potential new targets molecules for the treatment of urethra scar.