Transforming growth factor β1-induced epithellal-mesenchymal transition of rat peritoneal mesothelial cells via RhoA-Rock signaling pathway / 中华肾脏病杂志

ABSTRACT

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.