Résumé
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 mRNA:F =294.660,P =0.000 ; α-SMA protein:F =346.950,P =0.000 ; E-cad mRNA:F =264.250,P =0.000 ; E-cad protein:F =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-SMA:F =693.864,P =0.000 ;E-cad:F=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.
Résumé
Objective To discuss the effects of extract of Ginkgo BilobaLeaves(EGb) on expression of cytokine of renal interstitial fibrosis induced by transforming growth factor-?1 (TGF-?1) and extracellular matrix. Methods Cultured human kidney cells(HKC) were divided into three groups: control group,TGF-?1(8 ng/mL) group,and TGF-?1(8 ng/mL) added EGb(25,50,100,150 mg/L)group.After 72 h,expression of ?-SMA was detected by cell immunochemistry ABC,and collagen type I by Real-time PCR and Western blotting. Results Treated with TGF-?1(8 ng/mL) for 72 h,expression of ?-SMA and collagen type Ⅰ were up-regulated markedly compared with control group.Treated with EGb(25,50,100,150 mg/L)and TGF-?1(8 ng/mL)concomitantly for 72 h,expression of ?-SMA and collagen typeⅠ were down-regulated in dosage dependent manner compared with TGF-?1 group. Conclusion EGb can inhibit expression of ?-SMA and collagen type I in HKC induced by TGF-?1,and the possible mechanism might be related to the inhibition of EGb on renal tubular epithelial-myofibroblast transdifferentiation.
Résumé
Objective Rapamycin (RAPA) is an anti-proliferative immunosuppressant and has been used to suppress rejection of transplanted organs. In present study, we observed the effect of rapamycin on epithelial-myofibroblast transition (EMT)of cultured HKC cells in vitro. Methods Cultured human proximal tubular epithelial cells (HKCs) were divided into three groups: blank control, treated with TGF-?1 (1 ?g/L) and treated with TGF-?1 (1 ?g/L) plus rapamycin (0.1, 1, 10, 100 ?g/L). The protein and mRNA for ?-SMA and E-cadherin in HKC cells were determined by Western Blot and RT-PCR.The mRNA level of Snail in HKC was detected by RT-PCR. Results Rapamycin dramatically abrogated TGF-?1 induced ?-SMA expression and restored E-cadherin expressionin HKC cells in a dose-dependent manner. At a concentration of 100 ?g/L, rapamycin almost completely blocked ?-SMA mRNA and protein expression induced by TGF-?1(1 ?g/L). Rapamycin also suppressed expression of ?-SMA in HKC cells at both mRNA and protein level in a time dependent manner.We also found rapamycin dramatically abrogated TGF-?1 induced Snail mRNA expression in HKC cells in a dose-dependent manner. Conclusion Rapamycin may inhibit EMT of tubular cells in vitro. The downregulation of Snail expression might be one of the mechanisms of rapamycin blocking EMT.