Inhibitory effect of Tanshinone ⅡA on the proliferation of human retinal pigment epithelial cells in hypoxia and its mechanism

Objective

To investigate the effect of Tanshinone ⅡA on the proliferation and the signaling pathway of human retinal pigment epithelial (RPE) cells in hypoxia.

Methods

CoCl₂ (150 μmol/L) was used to simulate hypoxic condition and the ARPE-19 cells cultured in vitrowere divided into blank control group, hypoxia control group, Tanshinone ⅡA group and hypoxia-inducible factor-1α (HIF-1α) inhibitor group.The different doses of Tanshinone ⅡA were used to treat ARPE-19 for 24, 48 and 72 hours, respectively.The inhibitory rate of cell proliferation of different groups were detected by MTT after 24, 48 and 72 hours of administration cultured, and the apoptosis rate and the cell cycle distribution of cells in the hypoxia were analyzed by flow cytometry.Real-time PCR and Western blot were used to detect the expressions of mRNA and protein of HIF-1α and vascular endothelial growth factor (VEGF).

Results

MTT assay showed that Tanshinone ⅡA could inhibit the proliferation of ARPE-19 cells in a dose- and time-dependent manner, and the proliferation inhibitory rate gradually increased in the 1, 5 and 10 mg/L Tanshinone ⅡA groups, with significant differences between any two groups (all at P<0.05). Flow cytometry showed that the apoptosis rate of ARPE-19 in 1, 5 and 10 mg/L Tanshinone ⅡA groups gradually increased with the elevation of Tanshinone ⅡA dosage, with significant differences between any two groups (all at P<0.05). The cell proportion in the G₀/G₁ phase gradually increased, while the cell proportion in the S phase gradually decreased along with the elevation of Tanshinone ⅡA concentration, significant differences were obtained among the hypoxia control group, 1, 5 and 10 mg/L Tanshinone ⅡA groups (all at P<0.05). RT-PCR and Western blot showed that the relative expression of VEGF mRNA, HIF-1α and VEGF protein in the the blank control group, hypoxia control group, 1, 5 and 10 mg/L Tanshinone ⅡA groups and HIF-1α inhibitor group were significantly different (all at P<0.05). The expression of VEGF mRNA, HIF-1α and VEGF protein decreased successively in the 1, 5 and 10 mg/L Tanshinone ⅡA groups, with significant differences between them (all at P<0.05). There were no significant differences between 10 mg/L Tanshinone ⅡA and the HIF-1α inhibitor group of all the test indexes(all at P>0.05).

Key words: Tanshinone ⅡA; Retinal pigment epithelial cells; Hypoxia-inducible factor-1α; Vascular endothelial growth factor; Proliferation