The Role Of TGF-?1 And Twist1 Molecule In Epithelial-Mesenchymal-Transition Of Peritoneal Mesothelial Cells Induced By Indoxyl Sulfate

Peritoneal dialysis(Peritoneal dialysis,PD)is a way of renal replacement therapy.Because of its unique protection advantage for residual renal function,it has become the first choice of treatment for End-stage renal disease(ESRD).Peritoneal fibrosis(peritoneal fibrosis PF)is an important reason why patients withdrew from peritoneal dialysis.This pathological change is caused by multiple factors.The peritoneal epithelial cells to mesenchymal transitions(epithelial-mesenchymal--transitions EMT)plays a key role in the pathogenesis of peritoneal fibrosis.Previous studies have shown that high concentration of Indoxyl sulfate(IS)can induce EMT in peritoneal mesothelial cells,and the involvement of transforming growth factor beta 1(transforming growth factor ?1,TGF-beta 1)may play an important role.But it's no clear whether the main signs of epithelial cells E-cadherin changes in the EMT of IS induced,and TGFbeta1 and Twist,Slug,ZEBl protein,ZEB2 and E12 / E47 and other transcription factors are likely to exhibit transcription inhibition of E-cadherin.This topic is mainly about the further research on the EMT of HPMCs process caused by IS.[Objective](1)Human peritoneal mesothelial cell immortalized cell line(HMr SV5)was induced by indoxyl sulfate to detect changes in cell morphology,E-cadherin and to study cell transdifferentiation.(2)To study the expression changes of transforming growth factor ?1(TGF-?1)and Twist1 in the above process and to explore the role of both in epithelial-mesenchymal-transitions(EMT)of peritoneal mesothelial cells and the relationship between these three factors.[Methods]Through cell culture in vitro,1000umol/L concentration of IS and cultivation of liquid culture for IS group;add 4.25% Peritoneal dialysis fluid co cultured with culture medium for PD group F;TGF-beta1 receptor inhibitor(Transforming growth factor-R1 TRI beta inhibitor)group: with 2umol/L LY364947 + 4.25% peritoneal dialys fluid and with 2umol/L LY364947+1000umol/L concentration of IS;The morphological changes of cells in 0h,4h,24 h,48h and 72 h were observed by immunofluorescence.The relative expression of TGF-beta 1,E-cadherin and Twist1 genes at each time point was measured by q RT-PCR.[Results](1)After IS stimulated cells,the cells became thinner and longer,they looked like paving stone and changed in spindle shape.The expression level of E-cadherin gene increased first and then decreased,peaked at 24 hours after cell culture,then gradually decreased,and the difference was statistically significant(P<0.05).(2)The expression of TGF-?1 increased most significantly after stimulation with IS/PDF for 48 hours,which was significantly different from the corresponding TRI group at time points of 24 h,48h,and 72h(P<0.05).(3)The expression of Twist1 increased most significantly after 48 hours of stimulation with IS/PDF,which was significantly different from the corresponding TRI group at time points of 24 h,48h,and 72h(P<0.05).There were negative correlation between E-cadherin and Twist1 gene expression in IS experimental group(R2=0.3948).[Conclusion](1)The expression of E-cadherin in peritoneal mesothelial cells of 48 h group was significantly reduced by IS stimulation.Combined with previous studies,IS could stimulate the expression of alpha-SMA and the secretion of FN(Fibronectin)and LN5(laminin5)in peritoneal mesothelial cells,and further clarify that IS can induce EMT in peritoneal cells.(2)Insulin sulfate induced TGF-?1 gene expression during the induction of EMT in peritoneal mesothelial cells,and the TGF-?1 inhibited E-cadherin gene expression.Combined with previous studies on the promotion of IS through TGF-?1 on the promotion of ?-SMA,it is suggested that IS can promote peritoneal mesothelial cell EMT by increasing TGF-?1 gene expression.(3)Insulin sulfate induces peritoneal mesothelial cells to undergo EMT,which promotes gene expression of twist1.The expression level of TGF-?1 can regulate the expression of Twis-t1 gene and bind to the effect of Twist1 on cell transdifferentiation,suggesting that Twist1 is involved in IS-induced EMT of peritoneal mesothelial cells.