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We have previously demonstrated that thioredoxin reductase (TrxR) inhibition attenuates hyperoxic lung injury in adult mice. Clara cells, non-ciliated columnar airway epithelial cells, protect the lung against oxidative injury and are a predominant site of thioredoxin and TrxR expressions. the present study tested the hypothesis that TrxR inhibition enhances Clara cell viability in hyperoxia via nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent antioxidant responses. Murine transformed Clara cells (mtCC) were treated with 1 µM auranofin (AFN), 15 µM dinitrochloro-benzene (DNCB), or vehicle for 1 h and exposed to room air (RA) or 85% O2(HO) for 24 h. TrxR1 activities were determined by insulin disulfide reductase assay. Viable cell numbers were determined using trypan blue exclusion. Nrf2 protein levels were determined by western blotting and transcripts of Nrf2-responsive genes were determined by qRT-PCR. for GSH depletion studies, buthionine sulphoximine (BSO) was added to the media during the hyperoxia exposure. Data were analyzed by either ANOVA with Bonferonni or Kruskal-Wallis with Dunn’s post hoc (n= 9-15, P< 0.05). AFN and DNCB treatment decreased TrxR1 activities by 90% and 61%, respectively, at 1 h. in RA, AFN pretreatment decreased viable cell numbers (control 100±5.9 vs. AFN 71.3±9.8) compared to vehicle-treated controls. HO exposure decreased cell viability by 71%(28.2±1.9) in vehicle-treated cells, and AFN-treated cells were not more susceptible to HO (24.8±3.4). AFN or DNCB treatment increased nuclear Nrf2 at 1 h, increased Nrf2-responsive gene transcripts at 1, 3, and 6 h, and increased total …