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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.
Objective: To investigate the role of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase dependent formation of reactive oxygen species (ROS) in the transforming growth factor β1 (TGF-β1) -induced epithelial-mesenchymal transition (EMT) in rat peritoneal mesothelial cells ), and the effect of Astragalus injection (AGI) intervention. Methods: Primary RPMCs were cultured to the second generation in vitro. AfterAfter 24 hours, 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 -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- 1 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 induced α-SMA mRNA and protein expression, and down-regulated E-cadherin mRNA and protein expression 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 .