Astragalus Inhibits Epithelial-to-Mesenchymal Transition Of Peritoneal Mesothelial Cells By Down-Regulating ?-Catenin

Background/AimThe overwhelming majority of patients with end-stage renal disease require maintenance dialysis,includes peritoneal dialysis(PD),which is an effective renal replacement therapy.Due to improved clinical prescription,biomaterials,medical care and education,PD becomes the most widely used around the world.Unfortunately,continuous PD results in decreased efficiency and changeable clearance,even ultrafiltration failure.Peritoneum lesions is responsible for peritoneal fibrosis(PF)and ultimately leads to disruption of PD.Hence,approaches and solutions to challenges in prevention and treatment of PF becomes more important.Epithelial-mesenchymal transition(EMT)of peritoneal mesothelial cells(PMCs)has been deemed to antagonize PF,which is beneficial to preserve peritoneal structure and function.However,this molecular mechanism remains unknown.The active Wnt/?-catenin signaling is characterized with ?-catenin increment and nuclear translocation,as well as the crosstalk with TGF-?1/Smads signaling,which is associated to PMCs EMT.Previously,we indicated that Chinese herbal Astragalus can improve efficiency of PD and preserve PMCs lesions.This article hopes to illustrate the anti-EMT mechanism of Astragalus based on Wnt/?-catenin signaling,as well as the crosstalk between Wnt/?-catenmn and TGF-?1/Smads pathways.MethodsIn viro:?HMrSV5 cells were treated with various doses of Astragalus(0?20?100?200?400?800mg/mL)for 24h or 800mg/mL Astragalus for various times(0?12?24?36?48?72h).Cell viability was detected in a 96-well plate using CCK8 assay.?HMrSV5 EMT model made by 1 Ong/mL TGF-?1 for 24h and treated with various Astragalus(200?400?800mg/mL)for 24h.EMT markers E-cadherin,Vimentin and a-SMA were detected using Western blot and immunofluorescence.?Detect the effect of ?-catenin siRNA and Snail siRNA on HMrSV5 EMT respectively.?Detect ?-catenin,Snail c-myc andCyclinDl after various dose Astragalus-treated HMrSV5 EMT.?Detect the effect of Astragalus on GSK-3??p-GSK-3? in cytoplasm,as well as ?-catenin in cytoplasm and nucleus.?Confirm the binding between P-catenin and Smads protein by immunoprecipitation.Knowdown Smad7 gene in HMrSV5 EMT model,detect the change of EMT treated with Astragalus.In vivo:?The PF rat model was developed by daily intraperitoneal injections with standard PD solution(4.25%dextrose)for 35 days.Rats in treatment group were treated with 40mg/mL Astragalus PD solutions(4g/kg/day)for 35 days at the same time.Negative control rats were treated with 40mg/mL Astragalus saline.Observe peritoneal structure and PMCs of rats by HE staining.Observe peritoneal thickness and deposition of collagen fibers by Masson staining.Detect Collagen Is Snail by immunohistochemistry.Detect E-cadherinx Vimentin?a-SMA and P-catenin mRNA by quantitative real-time PCR.?The specific knockdown and overexpression Smad7 in peritoneum of rat model was developed by tail intravenous injection of Adeno-associated virus.Astragalus treatment was added on the 22th day after transfection.Detect EMT markersN ?-catenin and Smad7 by Western blot and immunohistochemistry.ResultsIn vitro:?Various dose of Astragalus(0?20?100?200?400?800mg/mL)for 24h or 800mg/mL Astragalus for various times(0?12?24?36?48?72h)neither enhanced cell proliferation nor apoptosis.?HMrSV5 EMT model was succeeded in TGF-?1,accompanied by decreased E-cadherin,and increased Vimentin and a-SMA.Various doses of Astragalus partial inhibited HMrSV5 EMT,especially with 800mg/mL.?Both knockdown ?-catenin and Snail inhibited EMT.?Wnt/?-catenin signaling was activated by TGF-?1,accompanied by induction of ?-catenin?Snail?c-myc and CyclinDl.Various doses of Astragalus effected on(3-catenin degradation and reduction of Snail c-myc and CyclinD1.?Astragalus treatment can inhibited phospho-GSK-3? and increased phospho-?-catenin in cytoplasm.Also,expression of ?-catenin in nucleus was decreased by Astragalus.??-catenin bond with Smad7,rather than Smad2/3/4.Binding between Smad7 and ?-catenin HMrSV5 cells was reduced by TGF-?1.Astragalus inhibited P-catenin by enhancing Smad7 expression.Silencing Smad7 antagonized the EMT-inhibitory effect ofAstragalus.In vivo:?Standard PD solutions induced PF rats.In PD group,HE and Masson staining showed mesothelial layer denudation,inflammation,fibrous capsule formation and collagen proliferation around the peritoneum.As well,Collagen I,Snail and EMT expression were increased.Astragalus treatment was beneficial to alleviate peritoneal lesions,PMCs EMT and P-catenin.?The effect of Astragalus on the downregulated expression of EMT and ?-catenin in PF rats was partially reversed by knockdown of Smad7.In addition,overexpression of Smad7 alleviated PMCs EMT and ?-catenin in PF rats.Conclusion? Astragalus inhibits TGF-?1-induced HPMCs EMT.?Astragalus inhibits PMCs EMT in PF rat.?Astragalus inhibits PMCs EMT and via regulating Wnt/?-catenin signaling,as well as the crosstalk between Wnt/?-catenin and TGF-?1/Smads pathways.? Astragalus down-regulates ?-catenin has two molecular mechanism,one is aimed to stabilize GSK-3?/?-catenin complex,which further promotes the degradation of P-catenin,another is due to enhance Smad7 and reduce ?-catenin.