NADPH Oxidase-Dependent Formation of Reactive Oxygen Species Contributes to Transforming Growth Factor β1-Induced Epithelial-Mesenchymal Transition in Rat Peritoneal Mesothelial Cells,and the Role of Astragalus Intervention

Objective:To investigate the role of nicotinamide-adenine dinucleotide phosphate(NADPH)oxidasedependent formation of reactive oxygen species(ROS)in the transforming growth factorβ1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in rat peritoneal mesothelial cells(RPMCs),and the effect of Astragalus injection(AGI)intervention.Methods:Primary RPMCs were cultured to the second generation in vitro.After synchronization for 24 h,the cells were randomly assigned to the following groups:control(Group A),AGI(2 g/mL;Group B),TGF-β1(10 ng/mL;Group C),TGF-β1(10 ng/mL)+AGI(2 g/mL;Group D;pretreated for 1 h with AGI before TGF-β1 stimulation).Reverse transcription-polymerase chain reaction(RT-PCR)and Western blot analysis were employed to evaluate the mRNA and protein expression of the NADPH oxidase subunit p67phox,α-smooth muscle actin(α-SMA)and E-cadherin.The dichlorofluorescein-sensitive cellular ROS levels were measured by a fluorometiic assay and confocal microscopy.Results:TGF-β1 significantly induced NADPH oxidase subunit p67phox mRNA and protein expression in RPMCs,as well as inducing the production of intracellular ROS.AGI inhibited this TGF-β1-induced up-regulation by 39.3%and 47.8%,respectively(P<0.05),as well as inhibiting the TGF-β1-induced ROS generation by 56.3%(P<0.05).TGF-β1 also inducedα-SMA mRNA and protein expression,and down-regulated E-cadherin mRNA and protein expression(P<0.05).This effect was suppressed by AGI(P<0.05).Conclusions:NADPH oxidase-dependent formation of ROS may mediate the TGF-β1-dependent EMT in RPMCs.AGI could inhibit this process,providing a theoretical basis for AGI in the prevention of peritoneal fibrosis.