There is some evidence that oxidative stress plays a crucial role in neuronal disorders including brain ischemic injury. Production of large amounts of reactive oxygen species (ROS) can trigger oxidative stress and lead to dysfunction of cells due to DNA and protein damage. Finally, overproduction of ROS could lead to cell death. Although ROS have beneficial roles in regulating cellular signaling pathways, overproduction of ROS is involved in brain ischemic injury (Floyd, 1990; Li
Thioredoxin 1 (Trx1), a small (12 kDa) protein, is one of cellular redox enzymes ubiquitously expressed in mammalian cells. Trx1 has a variety of biological functions, including regulating cell growth and apoptosis as an antioxidant protein (Haendeler
Mitogen activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-Jun
Protein transduction domain (PTD) is known as an effective tool for delivering proteins into cells. Thus, PTDs including Tat PTD have been used to delivery therapeutic proteins into cells and tissues (Schwarze
Ni2+-nitrilotriacetic acid Sepharose Superflow was purchased from Qiagen (Valencia, CA, USA). PD-10 columns were purchased from Amersham (Brauncschweig, Germany). Fetal bovine serum (FBS) and antibiotics (streptomycin and penicillin) were obtained from Gibco BRL (Grand Island, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Lonza/BioWhittaker (Walkersville, MD, USA). 2′,7′-Dichlorofluorescein diacetate (DCF-DA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Histidine antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The indicated antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Unless otherwise stated, all other agents were of the highest grade available.
Mouse hippocampal HT-22 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics (100 μg/mL streptomycin, 100 μg /mL penicillin) at 37°C in a humidity chamber with 5% CO2 and 95% air.
Preparation of the Tat expression vector has been described in a previous study (Shin
To examine Tat-Trx1 protein transduction efficiency, HT-22 cells were cultured in a 60 mm dish plate and exposed to different concentrations of Tat-Trx1 and Trx1 protein (0.5-5 μM) for 1 h or over various time periods (10-100 min) of Tat-Trx1 and Trx1 protein (5 μM). The cells were treated with trypsin-EDTA (Gibco BRL) and washed twice with phosphate-buffered saline (PBS). To determine the intracellular stability of transduced Tat-Trx1 protein, cells were cultured over various time periods (1-24 h) after Tat-Trx1 protein transduction. We confirmed the transduced levels of Tat-Trx1 protein which were measured by Western blot analysis and fluorescence microscopy analysis using an anti-His antibody.
After transduction of Tat-Trx1 protein, protein extraction was performed using cell lysis buffer (RIPA; ELPIS BIOTECH, Daejeon, Korea) according to the manufacturer’s instructions. Then, equal amount of proteins were loaded into 15% SDS-PAGE and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with TBS-T (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5) buffer containing 5% non-fat dry milk or BSA for 1 h. After being washed with TBS-T buffer, the membrane was incubated with the indicated primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Then the membranes were washed with TBS-T buffer three times and the protein bands were identified using chemiluminescent reagents as recommended by the manufacturer (Amersham, Franklin Lakes, NJ, USA) (Shin
To determine the intracellular distribution of transduced Tat-Trx1 protein in HT-22 cells, we performed confocal fluorescence microscopy as described previously (Shin
A cell viability assay was performed using a water-soluble tetrazolium salt-1 (WST-1) cytotoxicity assay (EZ-Cytox cell viability assay kit, Daeil Lab service Co., Seoul, Korea) according to the manufacturer’s protocols (Shin
Intracellular ROS levels were determined using 2′,7′-Dichlorofluorescein diacetate (DCF-DA) as described previously (Shin
To examine whether transduced Tat-Trx1 protein protects against H2O2-induced DNA damage in cells, HT-22 cells were pretreated with 5 μM Tat-Trx1 protein for 1 h and exposed to 700 μM H2O2 for 6 h. DNA fragmentation was determined using a Cell Death Detection Kit (Roche Applied Science, Basel, Switzerland) according to the manufacturer’s instructions. Fluorescent images were obtained by fluorescence microscopy (Eclipse 80i, Nikon) and the fluorescence intensity was detected with excitation at 485 nm and emission at 538 nm using a Fluoroskan ELISA plate reader (Thermo Labsystems) (Shin
HT-22 cells were incubated in the absence or presence of Tat-Trx1 (5 μM) for 1 h, and then treated with H2O2 for various times. The expression of Akt, MAPKs and apoptotic protein expression levels were determined by Western blotting using indicated specific antibodies. The bands were quantified by Image J software (NIH, Bethesda, MD, USA) (Shin
Male gerbils (65-75 g; 6 months old) obtained from the Experimental Animal Center, at Hallym University (Chuncheon, Korea) were housed at a temperature of 23ºC, with humidity of 60%, and exposed to 12 hour periods of light and dark with free access to food and water. All experimental procedures involving animals and their care conformed to the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research & Quarantine Service of Korea and were approved by the Institutional Animal Care and Use Committee of Soonchunhyang University (Cheonan, Korea) [SCH 15-0002-3].
The transient forebrain ischemia model was performed as described previously (Shin
To explore the protective effects of Tat-Trx1 protein against ischemic damage, the animals were divided into 5 groups (each n=10): control sham group, vehicle-treated group, Tat-Trx1-treated group, Trx1-treated group and Tat peptide-treated group. The Tat-Trx1 proteins, Trx1 proteins and Tat peptide (2 mg/kg) were administered intraperitoneally 30 min before ischemia-reperfusion. The brains from each group were harvested and the levels of 4-hydroxynonenal (4-HNE) and endogenous Trx1 proteins were determined by Western blot analysis using 4-HNE (Santa Cruz, CA, USA) and Trx1 (Cell Signaling Technology) antibodies. Also, intracellular ROS level was determined using a ROS assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
Immunohistochemistry was performed as described in previous studies (Shin
The positive neuronal cell number and intensity of immunoreactivity were calculated using an image analyzing system equipped with a computer based CCD camera (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA). The staining intensity of the immunoreactive structures was evaluated as the relative optical density (ROD). A ratio of the ROD was calibrated as % (Shin
Data are expressed as the mean ± SEM of three experiments. Differences between groups were analyzed by ANOVA followed by a Bonferroni’s post-hoc test. Statistical significance was considered at
We constructed a Tat-Trx1 fusion protein for Trx1 protein transduction by fusing human Trx1 gene in frame with Tat-peptide and His-tag. As a negative control, Trx1 gene was fused to His-tag alone (Fig. 1A). After the protein was overexpressed by IPTG indcution, Tat-Trx1 and Trx1 proteins were purified by Ni-NTA and PD-10 chromatography. By SDS-PAGE, one single band was detected for purified Tat-Trx1 or Trx1 protein at their expected molecular weights. Furthermore, both purified Tat-Trx1 and Trx1 proteins were identified by Western blotting using an anti-His antibody (Fig. 1B).
Transduction of Tat-Trx1 protein was examined in HT-22 cells. These cells were treated with Tat-Trx1 protein (0.5-5 μM) for 1 h or with Tat-Trx1 protein (5 μM) at different time intervals (10-100 min). Then, transduced proteins were determined by Western blotting. As shown in Fig. 1C and 1D, a dose- and time-dependent increase in the amount of transduced Tat-Trx1 protein was detected in HT-22 cells. However, Trx1 protein without a Tat-peptide was not transduced in HT-22 cells.
We further conﬁrmed the stability and distribution of transduced Tat-Trx1 protein by Western blotting and confocal ﬂuorescence microscopy. As shown in Fig. 1E, transduced Tat-Trx1 protein levels were signiﬁcantly increased in HT-22 cells at 12 h compared to those in controls. Immunofluorescence staining showed that transduced Tat-Trx1 protein was distributed in the cytoplasm and nucleus of cells (Fig. 1F). These results indicate that Tat-Trx1 protein transduced into HT-22 cells and persisted for 12 h.
After pretreatment with Tat-Trx1 protein at different doses for 1 h, HT-22 cells were exposed to 700 μM of H2O2 and their viability was determined using a WST-1 assay. As shown in Fig. 2A, only 51% of cells survived in H2O2 only exposed cells. However, cells pretreated with Trx1 protein and Tat peptide did not show significant decrease of cell viability after exposure to H2O2. Tat-Trx1 protein significantly increased the survival of HT-22 cells up to 92% compared to H2O2 only exposed cells.
To determine effects of Tat-Trx1 protein on oxidative stress, ROS generation and DNA fragmentation in H2O2 exposed HT-22 cells were assessed. As shown in Fig. 2B and 2C, levels of ROS generation and DNA fragmentation were markedly reduced in Tat-Trx1 protein treated cells as compared with H2O2 only exposed cells. However, they showed no significant difference between H2O2 alone treated cells and Trx1 protein or Tat peptide treated cells. These results indicate that transduced Tat-Trx1 protein can inhibit cell death caused by oxidative stress by decreasing ROS generation and DNA fragmentation.
The cascade of ASK1 and MAPKs has emerged as a key cell death pathway in response to oxidative stress (Ichijo
To explore cellular mechanisms underlying the protective effect of Tat-Trx1 protein, we investigated expression levels of Akt, p65, and apoptotic related proteins in H2O2 exposed HT-22 cells. Phosphorylation levels of Akt and p65 were reduced in Tat-Trx1 protein treated cells. However, their levels were unchanged in Trx1 protein or Tat peptide treated cells (Fig. 3C, 3D). Tat-Trx1 protein increased expression levels of Bcl-2 and Caspase-3 in H2O2 treated HT-22 cells. In contrast, expression levels of Bax and cleaved Caspase-3 showed opposite patterns compared to Bcl-2 and Caspase-3 expression (Fig. 3E). These results indicate that Tat-Trx1 protein can inhibit HT-22 cell death by modulating the expression of ASK1, MAPKs, and apoptotic proteins.
To investigate effects of Tat-Trx1 protein against ischemic injury in an animal model, we performed immunohistochemistry. As shown in Fig. 4A, Tat-Trx1 protein significantly protected neuronal cell death in the hippocampal CA1 region. However, both Trx1 protein and Tat peptide treated groups showed a similar pattern compared with vehicle treated group. We also examined whether Tat-Trx1 protein inhibited the activation of microglia and astrocytes using F-JB, Iba-1, and GFAP staining, respectively (Fig. 4B). In the vehicle-, Trx1 protein, and Tat peptide protein-treated groups, F-JB, Iba-1, and GFAP fluorescence signals were intensively detected in the hippocampal CA1 region. In contrast, intensive fluorescence signals were markedly reduced in Tat-Trx1 protein treated group. These results indicate that Tat-Trx1 protein could protect against neuronal cell damage resulting from ischemic injury by decreasing microglia and astrocyte activation.
To examine whether Tat-Trx1 protein could inhibit ischemia-induced oxidative stress, we performed DHE and 4-HNE to determine ROS generation and lipid peroxidation levels in an ischemic injury animal model. As shown in Fig. 4C and 4D, levels of ROS and lipid peroxidation were significantly increased in vehicle-, Trx1 protein-, and Tat peptide-treated groups compared with the sham control group. In contrast, Tat-Trx1 protein treated group reduced ischemia-induced ROS and lipid peroxidation levels. We also determined endogenous Trx1 protein expression levels. Compared with the vehicle group, endogenous Trx1 protein levels were similar in other treatment groups (Fig. 4E). These results indicate that Tat-Trx1 protein plays a role in reducing ischemia-induced cell damage by inhibiting oxidative stress in an ischemic injury animal model.
Thioredoxin 1 (Trx1) is a multifunctional protein with MW of 12 kDa. It is expressed in all living cells including prokaryotic and eukaryotic cells. Trx1 has two redox-active cysteine residues within a conserved active site having a sequence of Cys-Gly-Pro-Cys. It plays key roles in cellular growth, regulation of gene expression, and apoptosis (Susanti
We showed that Tat-Trx1 protein transduced into HT-22 cells and markedly inhibited HT-22 cell death, ROS generation, and DNA fragmentation caused by oxidative stress. Recent studies have shown that knockdown of Trx1 can decrease astrocyte cell viability in an oxygen glucose deprivation/reperfusion (OGD/R)-induced cell model, suggesting that Trx1 can protect astrocyte cells from oxidative stress by exerting anti-oxidant effects (Wang
Several studies have demonstrated that excessive ROS play a key role in ischemic injury and that ROS are associated with the induction of MAPKs, NF-κB, and Akt activation in neuronal cells (Kwon
Next, we examined effects of Tat-Trx1 protein against H2O2-induced apoptotic cell death. Tat-Trx1 protein markedly inhibited Bax and cleaved Caspase-3 expression, whereas Tat-Trx1 protein increased Bcl-2 and Caspase-3 expression in H2O2 exposed HT-22 cells. In Trx1 knockdown EMT6 cells, cleaved Caspase-3 expression is markedly increased, meaning that knockdown of Trx1 can increase apoptosis and cell death (Yoo
We further investigated the effect of Tat-Trx1 protein on ischemic insults using an animal ischemia model. Tat-Trx1 protein markedly inhibited neuronal cell death and reduced astrocytes and microglia activation in the ischemic animal model. In a previous study, we have shown that various PTDs fused with proteins can transduce into animal ischemia model brain and inhibit neuronal cell death (Shin
In summary, we demonstrated that Tat-Trx1 protein transduced into HT-22 cells and significantly inhibited oxidative stress-induced cell death. In addition, Tat-Trx1 protein prevented hippocampal neuronal cell death in an animal ischemia model. Our resutls suggest that Tat-Trx1 protein may represent a potential therapeutic strategy against brain ischemic injury.
This research was supported by Basic Science Research Program (2018R1D1A3B07049265 & 2019R1A6A1A11036849) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education.
The authors declare no conflict of interest.