| Objectives:This study aims to establish a model of DEHP exposure in pregnant and lactation to explore whether maternal DEHP exposture dutring pregnancy and lactation induces kidney fibrosis in adult rats.A rat renal proximal tubular cell line was used to explore the role of Wnt/β-catenin signaling pathwy in ep ithelia-mesenchymal(EMT)of the kidney.Methods:In tais studyg healthy female Wistar rats aged 8-10 weeks and weighing 230-250g were fed adaptively for a period of time.The pregnant rats were ra:ndomLy divided into Control gf Oup,30,300 and 750 mg/kg/d dose group by weight In PN21 of the pups,the pups were weaned and raised in separate cages.In PN28 of the pups,the renal tissues for each group were chosen and Masson’s trichroome stain was used to observe p athological changes of kidney.Western Blot was used to detect the process of renal EMT.IN vitro,A DEHP exposure model was established by Using rat renal proximal tubular cell line NRK5 2E as the experimental object.CCK-8 was used to detect cell viability.Westernblot was used to detect the expression of α-Smooth Muscle Actin(a-SMA)and E-cadherin(E-cad)wich are typical marker of EMT process,as well as phosphorylition of β-Catenin and p-GSK-3 β in Wnt/β-cateninpathway.Wnt/β-catenin Agonist 1 and IWR-1 were used as the activator and inhibitor of Wnt/β-catenin signaling pathway in each group to explore the EMT level of NRK52E cell and the protein about Wnt/β-catenini pathway in each group.Results:(1)Masson staining showed that there was no significant difference between the exposed group and the control group.The renal tubules atrophy was observed,interstitial space was enlaoged,extracellular matrix accumulation was increased obviously in 750mg/k g/d dose groups.(2)There was no significant difference in renal protein of female offspring(P>0.05).Compared with the control grotup,the expression of E-cad in kidney of male offspring were decreased significantly in each exposure group(P<0.05),the expression of a-SMA in the kidney was significantly increased in mate offspring in 300mg/kg/d and 750mg/kg/d group.(3)Compared with the control group and 50μM DEHP exposure group,the expression of E-cad in 200μM DEHP exposure group was decreased significantly(P<0.05);the expression of α-SMA in 125μM and 200μM DEHP exposure group were increased significantly(P<0.05).(4)Compared with the control group,the expression of β-catenin in each DEHP exposure group was increased significantly(P<0.05;the expression of p-GSK-3β in 125μM and 200μMDEHP exposure group were increased significantly(P<0.05),while the total expression levels of GSK-3βin each group were not significantly different(P>0.05),(5)Wnt/β-catenin Agonist 1 aggravated the EMT of DEHP-induced NRK52E cells by activating the Wnt/β-catenin signaling pathway.Compared with DEHP alone treatment group and Wnt/β-catenin Agonist 1 alone treatment group,there were signifieant change in protein expression in Wnt/β-catenin Agonist 1 and DEHP both treatment group(E-Cad down-regulation,p<0.05;α-SMA up-regulation,p<0.05;β-Catenian p-GSK-3β up-regulation,p<0.05).(6)IWR-1 alleviated EMT of NRK52E cells by itnhibitiing Wnt/β-catetnin siginaling pathway.There were significant change in protein expression levels in DEHP alone treatment group(E-Cad down-regulation,p<0.05;α-SMA,β-Catenin and p-GSK-3p up-regulation,p<0.05).Compared with DEHP alone treatment group,the expression level of E-Cad down-regulated by DEHP was increased,the expression of α-SMA,β-Catenin and p-GSK-3β up-regulated by DEHP were decreased in IWR-1 and DEHP both treatment group(p<0.05).Concludons:(1)Exposure to DEHP during pregnancy and lactation induced renal EMT in offspring rats and renal tubular epithelial cells.(2)Maternal exposure to DEHP during pregnancy and lactation induced EMT in renal tubular epithelial cells by activating the Wnt/β-catenin signaling pathway. |
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