TGF-β1-induced Epithelial-Mesenchymal Transition Of Rat Peritoneal Mesothelial Cells Via RhoA/ROCK Signaling Pathway

Objective:To investigate the role of RhoA/ROCK signaling pathway in the process of RPMCs Epithelial-Mesenchymal Transition (EMT)induced by TGF-β1.Methods:(1)Primary rat peritoneal mesothelial cells were harvested from the peritoneum of male Sprague-Dawley rats,the cultured cells were identified by invert microscope and immunohistochemistry.(2)The second generation RPMCs were divided into the control group and TGF-β1 stimulated group.Growth arrested and synchronized cells were stimulated by 10ng/ml TGF-β1 for different time.Semi-quantification RT-PCR,Western blot and immunofluorescence assay were used to detect the mRNA and/or protein expression of E-cadherin,α-smooth muscle actin(α-SMA),CollagenⅠ,Vimentin and RhoA(included active RhoA and total RhoA).(3)After synchronization for 24 hours,the second generation RPMCs were randomly assigned to 4 groups:group A (control),group B(TGF-β1,10ng/ml),group C(TGF-β1,10ng/ml +Y-27632,an inhibitor of ROCK,10μM),group D(Y-27632 alone, 10μM).The mRNA and protein expression levels of E-cadherin,α-SMA and CollagenⅠwere measured by RT-PCR,Western blot and fluorometric assay and confocal microscopy,respectively.The protein expression levels of Vimentin was measured by Western blot.Results:(1)95%of the cells were proved to be RPMCs by identification.(2)The results of semi-quantification RT-PCR showed that the mRNA levels of intracellular E-cadherin in the TGF-β1 stimulated groups compared with that in the control group were decreased in a time-dependent manner(12h,24h,48h P＜0.05),and the mRNA levels ofα-SMA and CollagenⅠin the TGF-β1 stimulated groups compared with those in the control group were increased from 6h and 12h, respectively(P＜0.05),peaking at 24h and dropping at 48h,and increased in a time-dependent manner.The results of Western blot amd IF showed that the protein levels of E-cadherin in the TGF-β1 stimulated groups compared with that in the control group were obviously decreased from 24h(P＜0.05),and the protein levels ofα-SMA,Vimentin and CollagenⅠ in the TGF-β1 stimulated groups compared with those in the control group were upregulated in a time-dependent manner(24h,48h P＜0.05). TGF-β1 stimulation elicited a robust increase in RhoA activity in a 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,it was(4.35±0.41)folds compared with control group(P＜0.05). (3)The results of semi-quantification RT-PCR,Western blot amd IF respectively showed that the mRNA and protein levels of intracellular E-cadherin in TGF-β1+Y-27632 groups compared with the TGF-β1 stimulated groups had no increase(P＞0.05),the mRNA levels ofα-SMA and Collagen I decreased 25.4%and 55.7%,and the protein levels ofα-SMA,Vimentin and Collagen I decreased 30.0%,60.3%and 58.1% compared with TGF-β1 stimulated groups(P＜0.05).Conclusions:(1)TGF-β1 can induce EMT and upregulate the expression of active RhoA simultaneously in RPMCs.(2)The ROCK inhibitor Y-27632 effectively revered TGF-β1-induced expression ofα-SMA,Collagen I and Vimentin,but had no effect on loss of E-cadherin. (3)RhoA/ROCK signaling pathway may mediates EMT induced by TGF-β1 in rat peritoneal mesothelial cells.RhoA/ROCK pathway may be the potential therapeutic targets in the progress of peritoneal fibrosis.