Effect Of Sonic Hedgehog Signaling Pathway On Epithelial-mesenchymal Transition Of Peritoneal Mesothelial Cells Induced By High Glucose

Background:Peritoneal dialysis(PD)is one of the integrated treatments for end-stage renal disease,It has gradually become the first choice of most end-stage renal disease(ESRD)patients because of easyily operate,light economic burden,flexible treatment time,and effective protection of residual renal function.Peritoneal mesothelial cells(PMCs)is the main cell population that play the role of abdominal penetration.But peritoneum has long-term contact with non-physiological high glucose dialysate,High glucose leads to damage of PMCs,and cause the abnormalities of peritoneal structural and functional,make the inflammatory cells to proliferate.Finally,through a series of pathof physiological changes,causes a continuous decrease in filtration function to the point of exhaustion.It is the main reason that many patients with peritoneal dialysis to withdraw from peritoneal dialysis,greatly reduce the quality of life of patients with end-stage renal disease.Sonic hedgehog(Shh)signal transduction pathway is a group of genes widely expressed during normal growth and development to regulate cell growth.Control the origin and development of organs.Gli is located at the end of the Hedgehog signaling pathway,it is the key step of transcriptional activation and the final common pathway of different expressions.The expression level of Gli1 indicates the activity of the Hedgehog signaling pathway.In the physiological condition,Shh signaling pathway is in a low activity state,but under pathological conditions,itcan be reactivated.Most of the existing studies focus on the role of Shh pathway in pulmonary and renal fibrosis,the expression of Shh pathway on peritoneum was reported a little.This case study aims to establish a rat model,explorethe potential role of Shh signalingpathways in the epitheliummesenchymal transdifferentiation of PMCs,and to explore blocking the activation of Shh signal transduction pathway whether can avoid peritoneal injury,and improve the efficacy of long-term peritoneal dialysis.Hoping thenew ideas may reduce EMT and prevent peritoneal fibrosis in clinic.Methods1.Primary culture of RPMCs was performed by trypsin digestion,and identifing the cultured RPMCs by immunohistochemistry.2.Using Western blot test(Western blot)and reverse transcription polymerase chain reaction(reverse transcription PCR,RT-PCR)determination of Shh in cultured cells and the expression of transcription factor GLi1,observing the effect of high glucose stimulation on the expression of Shh and GLi1 in RPMCs.3.Given different doses of Shh signaling pathway antagonists-cyclopamine(CPN)after the intervention,and detecting the expressions of Shh、GLi1、α-SMA、snail1 and E-cadherin in rat peritoneal mesothelial cells were by Western blot and RT-PCR.To observe the effect of blocking Shh signaling pathway expression in rat peritoneal mesothelial fibrosis induced by high glucose.Results:1.Culture and identification of RPMCs:under an inverted light microscope,it can be seen that the cells are irregular polygons,It’s like the shape of a pebble;And expression of cytokeratin and wavy protein,cannot express Ⅷ factor related antigen and leucocyte antigen of CD45 after the immunohistochemical,in accordance with documented PMCs phenotype Identified of RPMCs.2.The expression of Shh and GLi1 in RPMCs was increased in a time anddosedependentmanner in the high concentration of glucose:RPMCs were stimulated by glucose medium with a concentration of 2.5% for different periods of time,then detected by RT-PCR and Western blot,Founded that compared with the control group,The expression of Shh and Gli1 mRNA and protein were increased in a time-dependent manner in RPMCs on high glucose group,the longer the time,the greater the increase(P<0.05).Apply the same amount of time(24hours),Stimulating RPMCs with a glucose concentration of5.4mmol/L、83.3mmol/L(1.5%)、150.0mmol/L(2.5%)、236.1mmol/L(4.25%)culture medium respectively,the expressions of Shh and Gli1 mRNA and protein of on high glucose group were increased in a dose-dependent manner in RPMCs,the higherthe glucose concentration used,the more significant the increase(P < 0.05).3.CPN inhibited the Shh signaling pathway of RPMCs induced by high glucose,and accompanied by decreased expression of PMCs fibrosis factors:pretreating the PMCs with different concentrations of CPN(2uM,5uM),then incubate with 2.5% glucose for 24 hours.Detecting by RT-PCR and Western blot,that CPN could not inhibit the mRNA and protein expression of Shh,but the expression of mRNA and protein on Gli1 were significantly decreased.It shows that CPN is very effective in blocking Shh signal transduction(P<0.05).After blocking Shh signaling,comparing with the high sugar group,the expression of E-cadherin in RPMCs was increased,the expression of related fibrosis factor like α-SMA was decreased.and this effect shows a dose-dependent trend.Conclusion:1.High glucose can stimulate the activation of Shh signal transduction pathway in peritoneal mesothelial cells in a time-dependent and dose-dependent manner;2:The Shh signaling pathway inhibitor cyclopamine inhibits the Shh signaling pathway activation induced by high glucose in a concentration-dependent manner,3:Cyclopamine also inhibited the transdifferentiation of peritoneal mesenchymal cells induced by high glucose.