Effects of rapamycin on expression of Bcl-2 and Bax in human lens epithelial cells and cell cycle in rats

Abstract

The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells (LECs) and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract. The cultured LECs of second and third passages were collected and treated with rapamycin. The LECs were transferred into 96-well culture plates and divided into 6 groups, and each group was set to have 8 duplicate wells. In the negative control group, the LECs were given culture medium only, and in the blank control group, only culture medium was given. In the four rapamycin-treated groups, different concentrations (20, 40, 60 and 80 ng/mL) of rapamycin were given. After treatment for 24, 48 and 72 h, the absorbance (A) values in each well were determined by MTT assay. The cell cycles of all groups were detected by using flow cytometry. Real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR) and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively. MTT assay showed that rapamycin could inhibit proliferation of LECs in a time- and dose-dependent manner. Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase, resulting in the increase of cells in G₁ phase and the decrease of the cells in S phase. RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA, but up-regulate the expression of Bax mRNA, suggesting it could induce apoptosis of LECs. Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein, but promote the expression of Bax protein. It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle, but also by promoting the induction of apoptosis.

Keywords

rapamycin / human lens epithelial cell / cell cycle / apoptosis

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