Résumé
Objective To investigate the role of RhoA-Rock signaling pathway in the process of rat peritoneal mesothelial cells (RPMCs) epithelial-mesenchymal transition (EMT) induced by transforming growth factor β1 (TGF-β1). Methods Primary RPMCs were cultured in vitro. After synchronization for 24 hours, RPMCs were randomly assigned to 4 groups: group A (control), group B (TGF-β1, 10 μg/L), group C (10 μg/L TGF-β1+10 μmol/L Y-27632, an inhibitor of Rock, pretreated for 2 hours with Y-27632 before TGF-β1 stimulation), group D (Y-27632 alone, 10 μmol/L). Growth arrested and synchronized RPMCs were stimulated by 10 μg/L TGF-β1 for different time. The mRNA and protein expression levels of E-eadherin, α-SMA and collagen Ⅰ were measured by RT-PCR and Western blotting respectively. The protein expression level of vimentin was measured by Western blotting. Active RhoA was extracted by Plasma Membrane Protein Extraction Kit, then it was assessed by Western blotting. Results (1) TGF-β1 stimulation elicited a robust increase in RhoA activity in time-dependent manner, which was (2.57±0.52) folds compared with control group (P<0.05) after 10 min stimulation. RhoA activity peaked at 1 hour, which was (4.35±0.41) folds compared with control group (P<0.05). (2) TGF-β1 up-regulated mRNA and/or protein expression of α-SMA, vimentin and collagen Ⅰ , and down-regnlated mRNA and protein expression of E-cadherin in RPMCs. (3) The Rock inhibitor Y-27632 effectively revered TGF-β1-induced expression of α-SMA, collagen Ⅰ and vimentin. The mRNA levels of α-SMA and collagen Ⅰ decreased by 53.8% and 55.7%, and the protein levels of α-SMA, vimentin and collagen Ⅰ decreased by 42.6%, 60.1% and 58.1% compared with TGF-β1-stimulated groups (P< 0.05). But Y-27632 had no effect on the level of E-cadherin. Conclusions RhoA-Bock signaling pathway may mediate EMT induced by TGF-β1 in rat peritoneal mesothelial cells. RhoA-Rock pathway may be the potential therapeutic target in the progress of peritoneal fibrosis.
Résumé
Objective To evaluate the inhibiting effect of antisense vascular smooth muscle alpha adenovirus vector on tubular epithelial myofibroblast transdifferentiation induced by TGF-?1.Methods The pAdanti-?-SMA was constructed and identified.The ?-SMA expression in tubular epithelial cells(TEC) was detected by immunofluorescence and Western blot.Five groups were designed as control(A),TEC transfected by 2 ?l pAdanti-?-SMA(B),TEC induced by TGF-?1(C),TEC induced by TGF-?1 and transfected by 2 ?l pAdanti-?-SMA(D),TEC induced by TGF-?1 and transfected by 10 ?l pAdanti-?-SMA(E).Results The pAdanti-?-SMA was successfully constructed.The expression of ?-SMA in TEC induced by TGF-?1 was inhibited by pAdanti-?-SMA.Conclusion Tubular epithelial myofibroblast transdifferentiation can be inhibited by pAdanti-?-SMA.