Investigation Of The Mechanism Of Tamoxifen Ameliorates Peritoneal Epithelial To Mesenchymal Transition In Encapsulating Peritoneal Sclerosis

Aims:To define the effect and illuminate the underlying signal transduction mechanism of Tamoxifen treatment with high glucose(HG)-induced peritoneal epithelial-mesenchymal transition(EMT).Methods:Rat peritoneal mesothelium cells (RPMCs) were isolated from normal rat’ peritoneal and 2-3 generation of the subconfluent RPMCs were chosen to study. HG intervention:RPMCs were stimulated with high glucose (30,60,120mM) to extract RNA for 24h and proteins for 72h. Drug intervention:(1) Some RPMCs were stimulated with 60mM HG, while others were treated with 60mM HG in the presence of different doses of Tamoxifen (0.5,2μM) for 72h;(2) Cells co-cultured with 60mM HG and 2μM tamoxifen were simultaneously treated with or without 2μM pure ER-a antagonist to extract RNA for 6h and proteins for 1h. (3) Cells co-cultured with 60mM HG and 2μM tamoxifen were pretreated with or without 1μM proteasome inhibitor for 1h. Expression of various proteins were evaluated by western blot analysis and mRNA level of related genes were evaluated by quantitative real-time PCR.Results:We found that high concentration of glucose(30,60,120mM) could induce the EMT of RPMC shown as an increased expression of a-SMA and decreased expression of E-cadherin at 72h in a dosage-dependent manner. Tamoxifen blocked the EMT induced by 60mM HG, as it preserved the expression of E-cadherin and reduced the expression of a-SMA. Tamoxifen treatment reduced the 60mM HG-induced increase of rapid phosphorylation level of Smad2 called phospho-Smad2 and mRNA level of CTGF and PAI-1, but this inhibition could be blocked by ER-a antagonist. All kinds of proteins could not be influenced by Tamoxifen alone. Eventually, the reduction of Tamoxifen on HG-induced increase of phospho-Smad2 could be reversed by proteasome inhibitor.Conclusions:Tamoxifen showed an effect on preventing EMT in a dosage-dependent manner. Tamoxifen suppressed TGF-β1/Smad signal pathway though reducing the level of rapid phosphorylation of Smad2 and ER-a played an important role in the process. The reduction of Tamoxifen on phospho-Smad2 might be attributed to degradation by proteasome approach.