Effect of a new ROCK inhibitor thiazovivin on the morphology and function of human corneal endothelial cells

Abstract

To evaluate the effect of thiazovivin, a novel ROCK inhibitor, on the morphology and function of human corneal endothelial cells(HCECs). The primary HCECs were identified by light microscopy and immunofluorescence staining of neuron-specific enolase. To screen the optimal concentration and action time of thiazovivin for maintaining the morphology and function of primary HCECs, Na (+)/K (+)-ATPase and N-cadherin were chosen as indicators, and the morphology and function of HCECs in various concentrations(0 μmol/L, 2 μmol/L, 4 μmol/L, and 6 μmol/L)for different durations(24 h and 48 h)were examined by immunofluorescence experiments. The effect of thiazovivin on the expression of ROCK was investigated by immunofluorescence and Western blot. The primary HCECs cultured were hexagonal, closely packed, homogeneously and obviously stained by neuron-specific enolase. The immunofluorescence staining of Na(+)/K(+)-ATPase showed that when the primary HCECs cultured with various concentrations of thiazovivin(0, 2, 4, 6 μmol/L)for 24 h, the fluorescence were obvious, and the average absorbance values(A)were 1.27±0.08, 3.72±0.17, 21.07±4.67, 3.69±0.34, respectively. And the immunofluorescence staining of N-cadherin revealed that when the primary HCECs treated with 4 μmol/L thiazovivin for 24 h, the cell boundary was clear and the structure of the cells was intact. While the treating time of thiazovivin(4 μmol/L)on HCECs extended to 48 h, the immunofluorescence staining of Na(+)/K(+)-ATPase and N-cadherin showed that compared to HCECs treated with thiazovivin(4 μmol/L)for 24 h, the fluorescence intensity did not change significantly, but the cells arranged slightly untidy. In addition, the immunofluorescence staining of ROCK was weakened and the expression of ROCK was reduced by thiazovivin. Thiazovivin was effective for protecting the morphology and function of HCECs. An optimal improvement in the morphology, connection and function of HCECs was found when the primary HCECs were cultured with 4 μmol/L thiazovivin for 24 h. Moreover, the expression of ROCK protein could be significantly inhibited by thiazovivin. (Chin J Ophthalmol, 2016, 52: 686-692).