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. 2016 Mar;13(3):2864-70.
doi: 10.3892/mmr.2016.4855. Epub 2016 Feb 3.

Thioredoxin 1 protects astrocytes from oxidative stress by maintaining peroxiredoxin activity

Mengfei Wang  1 , Kunting Zhu  1 , Luyu Zhang  2 , Lingyu Li  1 , Jing Zhao  1
Affiliations

Thioredoxin 1 protects astrocytes from oxidative stress by maintaining peroxiredoxin activity

Mengfei Wang et al. Mol Med Rep. 2016 Mar.

Abstract

Previous studies have demonstrated that thioredoxin 1 (Trx1) exerts neuroprotective effects against cerebral ischemia/reperfusion injury caused by oxidative stress. While Trx1 is known to maintain the anti‑oxidant activity of 2‑Cys peroxiredoxins (Prdxs), the underlying mechanisms of its protective effects have remained to be elucidated, which was the aim of the present study. For this, an in vitro ischemic model of hypoxemia lasting for 4 h, followed by 24 h of reperfusion was used. Primary astrocytes from neonatal rats were pre‑treated with small interfering RNA targeting Trx1 prior to oxygen glucose deprivation/reperfusion (OGD/R). MTS and lactate dehydrogenase assays were performed to evaluate cell viability. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis were employed to assess the mRNA and protein expression levels of Prdx1‑4 and Prdx‑SO3. Furthermore, a dual luciferase reporter assay was used to assess the interaction between activator protein‑1 (AP‑1) and Trx1. The present study demonstrated that OGD/R decreased the cell viability and increased cellular damage, which was more marked following Trx1 knockdown. The expression of Prdx1‑4 and Prdx‑SO3 protein was higher in the cells subjected to OGD/R. Knockdown of Trx1 markedly decreased the levels of Prdx1‑4 but increased Prdx‑SO3 mRNA and protein levels. The results of the present study also suggested that AP‑1 directly activated the expression of Trx1. The present study demonstrated that Trx1 exerts its neuroprotective effects by preventing oxidative stress in astrocytes via maintaining Prdx expression.

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Figures

Figure 1
Figure 1
Trx1 mRNA expression levels following knockdown or OGD/R. (A) The expression of Trx1 was significantly knocked down by LV3-288. (B) Trx1 mRNA expression demonstrated a significant decrease following Trx1 knockdown but a significant increase following OGD/R. Values are expressed as the mean ± standard error of the mean. **P<0.01 vs. the controls, ##P<0.01 vs. si-Trx1 group. RTC, relative to control; Trx1, thioredoxin 1; LV, lentivirus; si, small interfering; OGD/R, oxygen glucose deprivation/reperfusion.
Figure 2
Figure 2
Effect of Trx1 on cell viability and cell damage following exposure to OGD/R. (A) Cell viability was measured using the MTS assay. (B) Cell damage was evaluated with LDH levels. Values are expressed as the mean ± standard error of the mean (n=6 from 3 independent experiments). *P<0.05, **P<0.01 vs. the control; #P<0.05, ##P<0.01 vs. the si-Trx1 group. Trx1, thioredoxin 1; LDH, lactate dehydrogenase; si, small interfering; OGD/R, oxygen glucose deprivation/reperfusion.
Figure 3
Figure 3
Trx1 knockdown suppresses 2-Cys Prdxs expression. (A) Prdx1, (B) Prdx2, (C) Prdx3 and (D) Prdx4 were determined by reverse-transcription quantitative polymerase chain reaction. Prdx1-4 mRNA expression increased following OGD/R, which was inhibited by Trx1 knockdown. Values are expressed as the mean ± standard error of the mean (n=4). *P<0.05, **P<0.01 vs. the controls; #P<0.05, ##P<0.01 vs. the si-Trx1 group. Trx1, thioredoxin 1; si, small interfering; OGD/R, oxygen glucose deprivation/reperfusion; Prdx, peroxiredoxin.
Figure 4
Figure 4
Trx1 knockdown attenuates 2-Cys Prdxs protein expression and promotes Prdx-SO3 formation. (A) Representative western blot of Prdx1-4 and Prdx-SO3 in astrocytes following Trx1 knockdown. Quantification of (B) Prdx1, (C) Prdx2, (D) Prdx3, (E) Prdx4 and (F) Prdx-SO3 was performed by densitometric analysis. Following Trx1 knockdown, expression levels of Prdx1-4 were decreased, while PRDX-SO3 was elevated. In addition, PRDXs1-4 and PRDX-SO3 protein expression was increased following OGD/R treatment. Values are expressed as the mean ± standard error of the mean (n=4). *P<0.05, **P<0.01 vs. the control; #P<0.05, ##P<0.01 vs. the si-Trx1 group. Trx1, thioredoxin 1; si, small interfering; OGD/R, oxygen glucose deprivation/reperfusion; Prdx, peroxiredoxin.
Figure 5
Figure 5
Expression of Trx1 is partly dependent on AP-1. (A) Three tandem AP-1 binding sites are present in the Trx1 promoter. (B) The combining capacity of AP-1 and Trx1 was detected by dual luciferase activity assay. Astrocytes were transfected with luciferase reporter constructs driven by a Wt or TRE-mutated fragment from the Trx1 promoter as well as an AP-1 plasmid. At 24 h post-transfection, luciferase activity of the Wt plasmid was shown to be increased by AP-1, while the mutant vector was not affected by Ap-1. Values are expressed as the mean ± standard error of the mean (n=6). *P<0.05 vs. the control. Trx1, thioredoxin 1; si, small interfering; AP-1, activator protein-1; Luc, luciferase; Wt, wild-type; MtTRE, TRE mutant; TRE, 12-O-tetradecanoylphorbol-13-acetate response element.
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