Reversion Of High Glucose-induced EMT In HPMC By The SARA/SBD Fusion Protein Containing Protein Transduction Domain

BackgroundPeritoneal fibrosis is a common complication of peritoneal dialysis which will make malfunction of peritoneum and ultrafiltration failure. More seriously, People with a peritoneal fibrosis have to stop the peritoneal dialysis because of peritoneal failure. Today, there are not any good prevention and treatment for peritoneal fibrosis. It is confirmed that epithelial-to-mesenchymal transition of human peritoneal mesothelial cells is an early mechanism of peritoneal fibrosis. It plays a key role in the occurrence of peritoneal fibrosis and progression of peritoneal failure. SARA/SBD is a negatively regulatory factor in the process of fibrosis and is expected to become an effectual prevention drug of peritoneal fibrosis.ObjectiveTo express and purify the TAT PTD-SARA/SBD fusion protein in prokaryotic cells and determine its effect on the epithelial-to-mesenchymal transition of human peritoneal mesothelial cells treated with high glucose and possible mechanisms.MethodsTAT PTD-SARA/SBD gene was cloned into prokaryotic expression vector pET-44a(+) with His tag. Then the constructed recombinant plasmid was transformed to E.coli BL21 for expression under induction of IPTG. The expressed product was purified by Ni²⁺-NTA affinity chromatography and identified by SDS-PAGE and Western blot; Immunocytochemistry methods was used to test the transmembrane efficiency and colocalization of the fusion protein with Smad2 which was one of factors in TGF-β1 signal transduction pathway in HPMC. Western Blot was used to test the level of E-cadherin,α-SMA, Smad2/3 and pSmad2/3 protein in HPMC to confirm whether the recombinant fusion protein can inhibit the epithelial-to-mesenchymal transition of HPMC treated with high glucose and its possible mechanisms.ResultsThe TAT PTD-SARA/SBD fusion protein was expressed and purified using the methods of genetic engineering. It was indicated that the expressed product contained 20% of total somatic protein and existed in a soluble form. The purity quotient was beyond 95% after being purified by Ni²⁺-NTA affinity chromatography. The results of function experiments indicated that the TAT PTD-SARA/SBD fusion protein could transduce into HPMC efficiently which mainly located in nucleolus and could colocalize with Smad2.The fusion protein could increase the level of E-cadherin protein and decrease the level ofα-SMA protein in HPMC treated with high glucose. It could decrease the level of pSmad2 protein, which had no effect on the Smad3 phosphorylation level. ConclusionTAT PTD-SARA/SBD fusion protein was successfully expressed which could translocate across the plasma membrance of HPMC efficiently and locate with Smad2 in nuclei.The fusion protein could prevent the epithelial-to-mesenchymal transition of HPMC treated with high glucose.The possible mechanism was that the fusion protein could prevent the phosphorylation of Smad2 and had no effect on the phosphorylation level of Smad3.