Protective Role of Transduced Tat-Thioredoxin1 (Trx1) against Oxidative Stress-Induced Neuronal Cell Death via ASK1-MAPK Signal Pathway

Abstract

Oxidative stress plays a crucial role in the development of neuronal disorders including brain ischemic injury. Thioredoxin 1 (Trx1), a 12 kDa oxidoreductase, has anti-oxidant and anti-apoptotic functions in various cells. It has been highly implicated in brain ischemic injury. However, the protective mechanism of Trx1 against hippocampal neuronal cell death is not identified yet. Using a cell permeable Tat-Trx1 protein, protective mechanism of Trx1 against hydrogen peroxide-induced cell death was examined using HT-22 cells and an ischemic animal model. Transduced Tat-Trx1 markedly inhibited intracellular ROS levels, DNA fragmentation, and cell death in H₂O₂-treatment HT-22 cells. Tat-Trx1 also significantly inhibited phosphorylation of ASK1 and MAPKs in signaling pathways of HT-22 cells. In addition, Tat-Trx1 regulated expression levels of Akt, NF-κB, and apoptosis related proteins. In an ischemia animal model, Tat-Trx1 markedly protected hippocampal neuronal cell death and reduced astrocytes and microglia activation. These findings indicate that transduced Tat-Trx1 might be a potential therapeutic agent for treating ischemic injury.

Keywords: ASK1; Apoptosis; Ischemia; Protein therapy; ROS; Tat-Trx1.

Fig. 1

Purification and transduction of Tat-Trx1 protein into HT-22 cells. (A) Diagrams of the expressed Tat-Trx1 proteins. (B) Purified Tat-Trx1 and Trx1 proteins were identified by 15% SDS-PAGE and detected by Western blot analysis using an anti-histidine antibody. (C) Transduction of Tat-Trx1 proteins into HT-22 cells. HT-22 cell culture media were treated with Tat-Trx1 protein at different doses (0.5-5 μM) or with the Trx1 protein for 1 h. (D) The cell culture media were treated with Tat-Trx1 protein (5 μM) or Trx1 protein for different time periods (10-100 min). (E) Intracellular stability of transduced Tat-Trx1 protein. HT-22 cell culture media were incubated for 24 h after transduction of Tat-Trx1 protein for 1 h. Transduction of Tat-Trx1 protein was measured by Western blotting and the intensity of the bands was measured by a densitometer. (F) The localization of transduced Tat-Trx1 protein was examined by confocal fluorescence microscopy. Scale bar=20 μm. The bars in the figure represent the mean ± SEM obtained from 3 independent experiments.

Fig. 2

Effects of transduced Tat-Trx1 protein on cell viability in response to oxidative stress. (A) Effect of transduced Tat-Trx1 protein on cell viability. HT-22 cells were pretreated with Tat-Trx1 protein (1-5 μM) for 1 h and exposed to H₂O₂ (700 μM) for 1 h 30 min. Cell viabilities were estimated using a colorimetric assay using WST-1. Effects of Tat-Trx1 protein on H₂O₂-induced ROS production and DNA damage. Treatment with Tat-Trx1 protein (5 μM) and Trx1 protein was followed by 1 h treatment with H₂O₂ (700 μM). Intracellular ROS levels were measured by (B) DCF-DA staining and (C) DNA fragmentation was detected by TUNEL staining. The fluorescence intensity was measured by ELISA plate reader; scale bar=50 μm. The bars in the figure represent the mean ± SEM obtained from 3 independent experiments. *p<0.05 compared with H₂O₂-treated cells.

Fig. 3

Effect of Tat-Trx1 protein against H₂O₂-induced cellular signaling pathways in HT-22 cells. One-hour pretreatment of HT-22 cells with Tat-Trx1 protein (5 μM) or Trx1 protein was followed by treatment with H₂O₂ (700 μM). Then, the expression levels of (A) ASK1, (B) MAPK, (C) Akt, (D) p65 and (E) Bcl-2, Bax, Caspase-3 and cleaved Caspase-3 were determined by Western blotting. Band intensity was measured by densitometer. The bars in the figure represent the mean ± SEM obtained from 3 independent experiments. *p<0.05 compared with H₂O₂-treated cells.

Fig. 4

Protective effects of transduced Tat-Trx1 protein on ischemic injury. Gerbils were treated with single injections Tat-Trx1 (2 mg/kg) proteins and killed after 7 days (n=10 per groups). Then, the effects of transduced Tat-Trx1 protein on neuronal cell viability after ischemic insults using immunostaining. (A) The hippocampus was stained with CV and (B) Iba-1, GFAP, F-JB in sham-, vehicle-, Tat-Trx1-, Trx1- and Tat peptide-treated animals 7 days after I/R. Relative numeric analysis of CV-, Iba-1-, GFAP- and F-JB-positive neurons in CA1 region. Scale bar=400 and 50 μm. The brains from each group were harvested. (C) The intracellular ROS level was measured using an ROS assay kit. (D, E) Analysis of HNE and endogenous Trx1 protein levels. The levels of 4-HNE and endogenous Trx1 protein in the brain were analyzed by Western blot analysis using a 4-HNE and Trx1 antibody. *p<0.05, significantly different from the vehicle group.