| Background Pritoneal dialysis (PD) is one of efficient therapies for end stage renal disease(ESRD). The number of global patients treated with PD was in 5-10% annual growth rate since 2005.By 2009,the number reached 15 million.However, peritoneal ultrafiltration failure result in a large number of patient withdraw from PD, which seriously affected the survival and life of patients. Studies showed that during peritoneal dialysis, peritoneal mesothelial cell(MC) play an important role in peritoneal fibrosis and loss of function.Studies suggest that Transforming growth factor-β1 (TGF-β1)plays a key role in the pathogenesis of inducing epithelial-mesenchymal transdifferentiation(EMT) and promoting peritoneal fibrosis. It has been confirmed that classic TGF-β-smad pathway can mediate the EMT of peritoeal mesothelial cell. non-Smad-dependent pathways, downstream of TGF-β, have been recently found played important roles in the occurrence and development of EMT, in which PI3K/Akt signaling pathway is concerned about particularly. Phosphatidil inositol 3 kinase (PI3K) is one important intracellular kinase of creatures. PI3K/Akt signaling pathway plays an important role in cell metabolism, apoptosis, proliferation and differentiation.Recent studies suggest that PI3K/Akt signaling is required for TGF-β1-induced EMT in varied cells,such as renal tubular epithelial cells, mesangial cells, etc. Inhibting PI3K or knockout of Akt both relieve the degree of EMT. Currently there is little research on whether PI3K/Akt signalling pathway is involved in EMT of peritoneal mesothelial cells except Pranali Pate's report about EMT in peritoneal mesothelial cells of mice. Recently more and more extensive attention was paid on PI3K/Akt pathway as which is potential preventative and therapeutic target for EMT.But the mechanisms underlying EMT and fibrosis mediated by PI3K/Akt pathway is still not fully understood.In the past PI3K/Akt signalling pathway was considered had independent function from TGF-β/smad pathway. But recent studies suggested that they might interact through the ubiquitin-proteasome pathway. Studies show that TGF-β1 signaling is regulated by the ubiquitin-proteasome pathway degrading Smads. smad ubiquitin regulatory factor-1(Smurf2) is important E3 ubiquitin ligase among of them. In nephritis and renal fibrosis animal models, expression of Smurf2 were found increased and promote ubiquitin-dependent degradation of Smad2 and Smad7. N.Ohashi and colleages confirmed that TGF-β1 activates PI3K/Akt pathway and promote up regulation of smurf2 in tumor cells. Smad2 is considered as a propective mediator of EMT, while smad3 acts as a key mediator of EMT and fibrosis. So smurf2 might be key ligament linking PI3K/Akt signaling and EMT induced by TGF-β1 in peritoneal mesothelial cells. there is some significance to explore the interaction between PI3K/Akt signaling and smurf2 and mechanism in the process of EMT induced by TGF-β1 in peritoneal mesothelial cells. Objective To investigate the expression of PI3K/Akt and smurf2 and the relationship with EMT in mesothelial cells in a mice model of peritoneal EMT.Methods Male ICR mice were randomizely divided into normal control group(n=6), sham-operated groups(sham group) and model groups. sham groups were divided into NS 15d group(n=6) and NS 30d group(n=6); Model groups were divided into 4.25% PDS15d group(n=6) and 4.25% PDS 30d group(n=6). The mice in model groups received a daily infusion of 1.5ml 4.25%PDS (produced by Baxter,USA).The mice in sham groups were intraperitoneally injected of saline.The animals were sacrificed at day 15 or 30 according to groups.Visceral peritoneum were harvested to extract tissue protein and mRNA.Parietal peritoneum was collected for HE and Masson staining to observe the thickness of peritoneum.The expression of zo-l,vimentin, pAkt, smurf2,smad2 and smad3 were examined by immunofluorescence(IF), Westem-blot and Real Time-PCR.Results1. Compared with control, the thickness of peritoneum in PDS 15d group was markedly elevated (P<0.05) and in PDS 30d group it increased further(P<0.01); Compared with control, there was no significant difference in sham-operated group(sham 15d group and sham 30d group). 2. Comparede with control, IF, Western Blot and Real Time-PCR showed that accompanied with prolonged intraperitoneal injection of dialysis fluid, expression level of zo-1 in MCs of model group mice gradually declined, while vimentin expression up regulated gradually. But zo-1 and vimentin protein and mRNA expression were not statistically different.3. In the visceral peritoneal tissue of mice model, real Time-PCR showed that the expression of TGF-β1 mRNA increased accompanied with time of the injection of PDS extended; IF, Western Blot and Real Time-PCR showed that pAkt and smurf2 protein and mRNA expression all elvated gradually.4. Western Blot and Real Time-PCR showed that smad2 mRNA level in peritoneaum of model mice gradually rose while smad2 protein simultaneously decreased; smad3 protein and mRNA expression were both elevated.5. Linear correlation analysis showed that in model group, pAkt and smurf2 protein expression were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.Conclusion Successfully set up the mice model of peritoneal EMT.During the process pAkt and smurf2 expression were significantly increased in the peritoneum of mice peritoneal EMT model and both positively correlated with EMT; the expression of smurf2 was positvely correlated with pAkt, which suggests that P3K/Akt signaling and smurf2 might play important roles in EMT of mice peritoneal mesothelial cells. Objective To observe changes of PI3K/Akt and smurf2 expression in EMT induced by TGF-β1 in human peritoneal mesothelail cells (HPMCs);to investigate the relationship of PI3K/Akt with smurf2 and them with EMTMethods Human peritoneal mesothelial cell line were exposed respectively in different concentrations of TGF-β1 and harvest mRNA and protein of the cells.The expression of PI3K, pAkt, smurf2, smad2, smad3,etc and markers of EMT in HPMCs(zo-1, vimentin) were examined by Westem-blot, Real Time-PCR and IF.Results1. IF, Western Blot and Real Time-PCR showed that expression of vimentin protein and mRNA were up regulated in a time and dose-dependent manner in HPMCs stimulated with TGF-β1,meanwhile, the expression of zo-1 protein and mRNA were down regulated2. Western Blot showed that PI3K protein expression was elevated gradually in HPMCs stimulated with TGF-β1;IF and Western Blot showed that the expression level of pAkt protein,smurf2 protein and mRNA were increased in a time and dose-dependent manner in HPMCs stimulated with TGF-β1.3. Western Blot and Real Time PCR showed that the level of smad2 mRNA was elevated in HPMCs stimulated with TGF-β1, on the contrary, smad2 protein was down-regulated; smad3 and their mRNA were decreased in a time and dose-dependent manner in HPMCs stimulated with TGF-β1 The expression level of psmad2 and psmad3 were increase to the peak at 30min with the stimulation of TGF-β1,then declined gradually.4. Linear correlation showed that in EMT induced by TGF-β1 in HPMCs, pAkt and smurf2 protein were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.Conclusion It was demonstrated firstly that Smurf2 was constitutively exprsessed in HPMCs; pAkt and smurf2 expression were significantly correlated with EMT and elevated in a time-and dose-dependent way induced by TGF-β1 in HPMCs;.Simultaneously, TGF-β1 promoted the degradation of smad2 but increased smad3 expression in EMT induced by TGF-β1. Objective To investigate the role and mechanism of PI3K/Akt and smurf2 in HPMCs during the process of EMT through observing the effect on signaling molecule(smurf2, etc.)in EMT induced by TGF-β1 in HPMCs using PI3K inhibitor(LY294002) and Domain Negtive-Akt.Methods We treated normal HPMCs with TGF-β1 in the presence or absence of LY294002 and one kind of plasmid (pUSEamp+, domain-negtive Akt, activated Akt and wild type Akt) respectively. The tag myc protein was examined by Westem-blot to judge the plasmids transfection efficiency. The expression of pAkt, smurf2, smad2, smad3, psmad2, psmad3 and markers of EMT in HPMCs(zo-1, vimentin) and and relation of them were examined by Westem-blot, Real Time-PCR and IF.Results1. IF, Western Blot and Real Time PCR all showed that the expression of vimentin protein were partly inhibited by LY294002 or DN-Akt, meanwhile, decrease of zo-1 protein expression was increased by them.2. Western blot, IF and Real Time PCR all showed that induction of pAkt protein, smurf2 protein and mRNA were decreased in HPMCs stimulated with TGF-β1 in the presence of LY294002 or DN-Akt compared with HPMCs stimulated with TGF-β1.3. Real Time PCR and Western Blot showed that both LY294002 and DN-Akt attenuated smad2 mRNA in HPMCs stimulated with TGF-β1, but elevated smad2 protein expression; expression of smad3 protein and mRNA were both inhibited by them. Compared with HPMCs stimulated with TGF-β1, DN-Akt did not significantly affect the expression of psmad2 and psmad3 protein.4. Linear correlation showed that in HPMCs treated by TGF-β1 using LY294002 or DN-Akt, smurf2 protein were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.Conclusion Blocking PI3K/Akt signaling pathway can lessen the extent of EMT induced by TGFβ1 in HPMCs and inhibit the induction of smurf2; suggest that The EMT in HPMCs induced by TGF-β1 is partly positively regulated by PI3K/Akt signalling pathway,smurf2 and smad2 also play important roles in it. |
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