Inhibition of Rho-kinase inhibitor fasudil on the growth and migration of human LECs and extracellular matrix synthesis

Background

Biological behavior changes of human lens epithelial cells (LECs) and extracellular matrix (ECM) synthesis are major events in the pathogenesis of posterior capsule opacification (PCO). Rho-kinase inhibitor fasudil can inhibit the cytoskeleton remodeling, migration and fibrosis, however, the mechanism of its effect in LECs is below understood.

Objective

This study was to investigate the role of Rho-kinase inhibitor fasudil in proliferation, migration of human LECs and ECM synthesis in vitro.

Methods

Human LECs line HLEB-3 cells were regularly cultured and assigned to the control group and the drug treatment groups. Different doses of fasudil were added into the medium to make the final concentrations 10, 20, 40 and 60 μmol/L, respectively. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay in 12, 24 and 48 hours after addition of drug to calculate the growth inhibitory rate. Transwell test was employed to detect the migrating number of the cells in 12, 24, 36 and 48 hours after action of drug. The concentrations of collagen type Ⅰ (COL-Ⅰ), fibronectin (FN) in cell supernatant and α-smooth muscle actin (α-SMA) content in the cells were measured by ELISA.

Results

The cell growth inhibitory rates in different doses of fasudil groups were higher than that in the control group. The cell growth inhibitory rates were gradually elevated as the increases of fasudil concentrations and prolong of action time (F_{concentration}=966.727, P=0.000; F_time=280.428, P=0.000). Transwell migration assay showed that the number of migration cells across polycarbonate membrane was 29.20±1.28 in the control group after 48 hours of incubation, which was much more than 24.40±1.33, 17.00±1.10, 14.60±0.68, 6.60±1.29 in the 10, 20, 40 and 60 μmol/L fasudil groups, respectively, with a significant difference among the five groups (F=57.34, P<0.05). COL-Ⅰ and FN protein contents in supernatant were gradually declined with the increase of drug concentrations and lapse of time (COL-Ⅰ: F _{concentration}=143.992, P=0.000; F_time=113.745, P=0.000.FN: F_{concentration}=81.761, P=0.000; F_time=69.602, P=0.000), and the α-SMA levels in the cells were significantly reduced with the increase of drug concentrations and lapse of time (F_{concentration}=78.156, P=0.000; F_time=65.162, P=0.000).

Conclusions

Fasudil can inhibit the proliferation, migration and ECM synthesis of human LECs in vitro in a dose- and time-dependent manner.

Rho-associated kinases/antagonists and inhibitors; Epithelial cells, lens/drug effect; Humans; Cell migration; Cell proliferation; Collagen type I; Fibronectins; Extracellular matrix; Fasudil /pharmacology