Effects of rapamycin on transforming growth factor-β2-induced epithelial-myofibroblast transition of human lens epithelial cells / 中华实验眼科杂志

ABSTRACT

Background Epithelial-myofibroblast transition (EMT) of human lens epithelial cells (LECs) induced by transforming growth factor-β2 (TGF-β2) is the main mechanism in the pathogenesis of posterior capsular opacification(PCO).Seeking an effective drug capable of inhibiting this process is important for the prevention and treatment of PCO.Objective The purpose of this study was to investigate the inhibitory effect of rapamycin (RAPA)on the proliferation of human LECs and TGF-β2-induced EMT.Methods Human LEC strain(SRA01/04)was cultured in DMEM with high glucose and 10％ fetal bovine serum.The cells were consequently cultured in serumfree DMEM with 5 mg/L TGF-β2,TGF-β2+10 mg/L RAPA,TGF-β2 + 100 mg/L RAPA,TGF-β2 + 1000 mg/L RAPA or TGF-β2 +10 000 mg/L RAPA for 72 hours,and SRA01/04 cultured in serum-free DMEM were used as control.The proliferation rate(A490)of SRA01/04 in the different groups was detected using the MTT assay and the rate of inhibition of RAPA was calculated.The expressions of the α-smooth muscle actin(α-SMA) and E-cadherin(E-cad)mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The changes in the expression of α-SMA and E-cad in SRA01/04 were evaluated by Western blot 24,48 and 72 hours after TGF-β2 +400 mg/L RAPA treatment.Results The A490 value of SRA01/04 was 0.680±0.020,0.550±0.013,0.480±0.014,0.400±0.011 and 0.200±0.019 in the control group,TGF-β2 group,TGF-β2 + 10 mg/LRAPA group,TGF-β2 + 100 mg/L RAPA group,TGF-β2 + 1000 mg/L RAPA and TGF-β2 + 10 000 mg/L RAPA group,respectively,showing a gradually declining trend in SRA01/04 rate of proliferation with increasing RAPA concentrations (F =101.920,P =0.000).RT-PCR and Western blot assay showed that the relative expression levels of α-SMA mRNA (α-SMA mRNA/β-actin mRNA)and protein (α-SMA/β-actin)in the cells were significantly increased in the TGF-β2 treatment group.However,with exposure to RAPA,the relative expression levels of α-SMA mRNA and protein were significantly lowered with increasing RAPA concentrations,but the expression levels of E-cad mRNA and protein were raised (α-SMA mRNAF =294.660,P =0.000 ; α-SMA proteinF =346.950,P =0.000 ; E-cad mRNAF =264.250,P =0.000 ; E-cad proteinF =317.327,P =0.000).In addition,after exposure to 400 mg/L RAPA,the expression levels of α-SMA protein gradually reduced and those of E-cad protein gradually increased with increasing treatment durations,showing significant differences among the different time points (α-SMAF =693.864,P =0.000 ;E-cadF=369.286,P =0.000).Conclusions RAPA can inhibit the proliferation of SRA01/04 in vitro and arrest EMT of SRA01/04 induced by TGF-β2 in a dose-and time-dependent manner.