Astragalus Inhibits EMT Of Peritoneal Mesothelial Cells Via Wnt/β-catenin Signaling Pathway

Aim/BackgroundPeritoneal fibrosis(PF)reduces efficiency of peritoneal dialysis(PD),resulting in ultrafiltration failure and discontinuation of PD therapy.The epithelial-mesenchymal transition(EMT)of peritoneal mesothelial cells(PMCs)is crucial process of PF with several signaling pathways participate.Thus,the first part of this research aims to detect the expression of TGF-β1 and β-catenin in effluent of PD patients,searching for clinical evidence of β-catenin activation.The second part intends to confirm Wnt/β-catenin signaling regulates PMCs EMT,and identify the anti-EMT mechanism of Astragalus,which would provide a new method for PF therapy.MethodsPart 1:Collect PD effluent from patients with different dialysis age(6 months≤PD age≤2years and PD age≥2years,16 patients per group).Detect the expression of TGF-β1 and β-catenin in effluent,as well as mRNA level of β-catenin in effluent-derived cells.Part 2:In vivo:The PF model in rats was developed by daily intraperitoneal injections with standard dialysate(4.25%dextrose)for 35 days.Astragalus treatment was added on the 15th day after the former 2-week PD fluid injection.Rats were divided into 8 groups(15 rats per group):normal control;negative control(equal volume of NS injection for 35 days);Astragalus alone(injected intraperitoneally with 4g/kg/day Astragalus for 35 days);peritoneal dialysis(PD group);different doses of Astragalus(PD rats injected intraperitoneally with 1/2/4(g/kg/day)Astragalus for 21 days);pretreatment with Astragalus(4g/kg/day Astragalus + dialysate for 35 days).Observe pathomorphological changes in rats’ peritoneum.Detect expression of collagen I,EMT markers(a-SMA,E-cadherin),β-catenin and its downstream factors(Axin,LEF1).In vitro:①Use TGF-β1 to induce EMT of HMrSV5 cells by dose and time-dependent manner,observe expression of EMT markers and β-catenin downstream protein(Cyclin D、c-Myc).② Detect the effect of β-catenin siRNA on HMrSV5 EMT.③Treat HMrSV5 with TGF-β1 for 6-72h and interfere with Astragalus in different dose(20-800 mg/mL),analyze nuclear level of β-catenin.Observe the effect of Astragalus on P-catenin nuclear translocation by immunofluorescence.Treat the cells by TGF-β1 with or without Astragalus,observe the influence of Astragalus on EMT markers,β-catenin signal factors and cell morphology.Use siRNA to knockdown β-catenin,further observe the regulation of Astragalus on EMT.④Confirm the binding between β-catenin and GSK-3β by immunoprecipitation,detect the change of interaction after treating with Astragalus.The level of p-β-catenin、GSK-3β、p-GSK-3β were also tested.ResultsPart 1:Compared with the patients undergoing PD for less than 2 years,the effluent of patients who received PD for more than 2 years express a higher level of TGF-β1 and β-catenin.Similar result shows in effluent-derived cells.Part 2:In vivo:It showed that mesothelial layer denudation,submesothelial fibrosis,collagen proliferation and inflammation in PD rat’s peritoneum.The rats treated with the middle/high dose of Astragalus exhibited significantly alleviative mesothelial injury and fibrosis of the submesothelial zone.Astragalus inhibited EMT of rat PMCs and restrained activation of β-catenin signaling.In vitro:①EMT marker was highly increased or decreased after treatment with 10 ng/mL TGF-β1 for 24 h,as well as β-catenin signal factors.②β-catenin knockdown inhibited TGF-β1 induced HmrSV5 EMT.③Because the duration of TGF-β1 treatment was extended,theβ-catenin level in the nucleus was increased and reached the highest at 24h but declined subsequently.Astragalus treatment reduced the nuclear protein of β-catenin in a dose-dependent manner,which were confirmed in immunofluorescence.The expression of EMT markers and β-catenin downstream signaling were also decreased.β-catenin knockdown cannot reverse anti-EMT function of Astragalus.④Binding between GSK-3β and β-catenin in live HMrSV5 cells was reduced by TGF-β1,as TGF-β1 could phosphorylate GSK-β1 and decrease phosphorylation of β－catenin.Astragalus,to some extent,suppressed this alteration.Conclusion①β-catenin and TGF-β1 are activated in effluent of PD patients,which is related with PD age.②Wnt/β-catenin signaling pathway is activated in high glucose-induced PF in rats,while Astragalus could inhibit EMT and PF in rats via inactivating β-catenin signaling.③Wnt/β-catenin signaling pathway regulates TGF-β1 induced PMCs EMT,while Astragalus inhibits PMCs EMT via blocking β-catenin signaling.④ Astragalus inhibits phosphorylation of GSK-3β,enhances the binding between GSK-3β and β-catenin.It also induces phosphorylation of β-catenin,leading to decrease of cytoplasmic β-catenin level.