Src-family tyrosine kinases regulate apoptosis and EMT stimulated by high glucose in the human lens epithelial cells

Abstract

Purpose: Formation of the opacification under the lens capsules is the typical changes of diabetic cataract. Our previous studies have showed that Src-family tyrosine kinases (SFK) may be activated by high glucose. We hypothesize that Src regulates the signaling pathway between the cell death and EMT of the lens epithelial cells (HLE) stimulated by high glucose, which involves in formation of cataract.

Methods: HLE-B3 cells were cultured in DMEM for 24 hours, which is normal glucose group (NG, 5.5 mmol/L GS). For high glucose group (HG), the cells were treated with 35.5 mmol/L glucose, and for PP1 group (PP1), the cells were treated with 35.5mmol /L glucose and 10umoL/L PP1, which is a specific inhibitor of SKF. At 3h, 6h, 12h and 24h, flow cytometry assay was employed to detect the apoptotic rate. Immunofluorescence assay was used to determine the expressions of the E-cadherin and a-SMA. Western-blot assay was employed to determine the expressions of p-Src⁴¹⁸, c-Src, BcL-xl, Survivin, Caspase-3, E-cadherin and α-SMA.

Results: The expression of p-Src⁴¹⁸ was higher in HG group than that in both NG and PP1 groups at 6h (P<0.05). The apoptosis rate was higher in PP1 group than that in both NG and HG groups at 6h and 24h (P<0.05). In PP1 group, the expressions of Bcl-xL and Survivin were lower and the active Caspase-3 was higher than those in NG and HG groups at 6h and 12h (P<0.05). Under the inverted microscope, the typical fibroid cells were observed in HG grope，but in PP1 group the cells remained irregularly polygonal which was similar to those in NG group. The expression of E-cadherin was lower, but α-SMA was higher in HG group than that in NG and PP1 groups at 6h (P<0.05) by Western blot and immunofluorescence staining.

Conclusions: High glucose may activate Src kinase, which may promote EMT and inhibit apoptosis in human lens epithelial cells. Inhibition of Src activity by PP1 may suppress EMT and induce cell apoptotic process, which may help to maintain the properties of the lens epithelial cells in the stimulation of high glucose.