The Role Of TGF-β1 And Snail1 In Epithelial-mesenchymal-transition Of Peritoneal Mesothelial Cells Induced By Indoxyl And Sulfate

Peritoneal dialysis(PD)as one of the alternative methods for the treatment of end-stage renal disease has been more and more widely used clinically.Peritoneal dialysis(PF)is the main cause of failure of peritoneal dialysis patients.Many factors can lead to PF,and epithelial-to-mesenchymal transition(EMT)of peritoneal mesothelial cells is a key factor in peritoneal fibrosis.Indoxyl Sulfate(IS)is an endogenous toxin,and its relationship with kidney fibrosis has been widely recognized.However,there are few studies related to IS and peritoneal dialysis at home and abroad.Our previous studies found that IS can induce EMT in peritoneal mesothelial cells(PMC),and transforming growth factor beta 1(TGF-β1)may be involved in cell transdifferentiation.The process,but IS is the path through which PMC causes EMT is not clear.In the development of EMT,Snail1 is a transcription factor that studies more E-cadherin,which can inhibit the expression of E-cadherin and promote the expression of α-SMA.It is not clear whether IS can promote EMT of peritoneal mesothelial cells through the expression of TGF-β1 and Snail1.Based on the above analysis,this project intends to study the mechanism of TGF-β1 and Snail1 in IS-induced EMT of peritoneal mesothelial cells through in vitro experiments.[Objective](1)Determine whether IS can promote PMC transdifferentiation through cell morphology and protein expression of α-SMA.(2)To study the promoting effect of IS on the expression of TGF-β1 gene and the change of cell morphology and α-SMA protein expression after using TGF-β1 inhibitor,and to clarify the role of TGF-β1 in IS-induced PMC transdifferentiation.(3)To investigate the expression of Snail1 gene after stimulation with PMC and using TGF-β1 inhibitors,and to infer whether it participates in IS-induced EMT of PMCs.[Methods]1.Human peritoneal mesothelial cell line(HMrSV5)was cultured in vitro and resuscitated and passaged.2.The cells were divided into 5 groups: 1 control group(RPMI1640+10%FBS medium,no intervention during the culture),2IS group(IS culture with a concentration of 1000umol/L and RPMI1640+10%FBS medium),3IS+ LY group(containing 2umol/L LY364947+1000umol/Ls co-cultured with RPMI1640+10%FBS medium),4PDF group(4.25% peritoneal fluid co-cultured with RPMI1640+10%FBS medium),5 PDF+ LY(co-culture of LY364947+4.25% PD fluid with RPMI1640+10%FBS medium);the cells were intervened when the cells grew to approximately 70-80% confluence,and they were cultured at 0h,4h,24 h and 48 h respectively.At 72 hours,the morphological changes of cells in each group were observed,and the expression of relevant markers was detected.3.The protein expression of α-SMA in each group was detected by Western blot.The relative expression of TGF-β1 and Snail1 gene was detected by qRT-PCR at each time point.[Results](1)After IS stimulated PMC,the cells gradually taper and lengthen from the typical pebbles and paving stones,showing a change in the shape of the shuttle and a time-dependent manner.(2)After stimulation of PMCs with IS,the expression of α-SMA protein was most obvious at 72 h.Under the action of TGF-β receptor inhibitors,the expression of α-SMA protein was significantly reduced at 24 h,48 h,and 72 h compared with the control group.Statistically significant(P<0.05).(3)After stimulation with PMC in the IS group,the gene expression of TGF-β1 increased most significantly at 48 h.Under the effect of TGF-β receptor inhibitor,the total expression of TGF-β1 was not obvious.After 48 h,the expression of TGF-β1 was higher than that of the IS group.Significantly reduced,the difference was statistically significant(P<0.05).(4)After stimulation of PMCs in IS group,the expression of Snail1 gene increased most significantly at 48 h.Under the effect of TGF-β receptor inhibitor,Snail1 expression was not obvious at all.After 48 h,Snail1 expression was significantly decreased compared with IS group.Statistical significance(P<0.05).[Conclusion] 1.IS stimulation of peritoneal mesothelial cells can cause morphological changes in cells,consistent with the morphological characteristics of EMT,while the increased expression of α-SMA protein,and further confirm that IS can induce transdifferentiation of peritoneal mesothelial cells.2.IS can induce TGF-β1 gene expression during the process of EMT induced by IS in peritoneal mesothelial cells,while inhibiting TGF-β1 can significantly inhibit EMT morphological changes and α-SMA protein expression after IS-stimulated PMC,indicating that TGF-β1 Β1 participates in this EMT process.3.IS induced peritoneal mesothelial cells in the process of EMT,IS can promote Snail1 gene expression,and inhibition of TGF-β1 expression can inhibit Snail1 gene expression,combined with the role of Snail1 in cell transdifferentiation,prompted Snail1 involved IS induces EMT in peritoneal mesothelial cells.