*Contributed equally
Thioredoxin 1 (Trx1) serves a central role in redox homeostasis. It is involved in numerous other processes, including oxidative stress and apoptosis. However, to the best of our knowledge, the role of Trx1 in inflammation remains to be explored. The present study investigated the function and mechanism of cell permeable fused Tat-Trx1 protein in macrophages and a mouse model. Transduction levels of Tat-Trx1 were determined via western blotting. Cellular distribution of transduced Tat-Trx1 was determined by fluorescence microscopy. 2',7'-Dichlorofluorescein diacetate and TUNEL staining were performed to determine the production of reactive oxygen species and DNA fragmentation. Protein and gene expression were measured by western blotting and reverse transcription-quantitative PCR (RT-qPCR), respectively. Effects of skin inflammation were determined using hematoxylin and eosin staining, changes in ear weight and ear thickness, and RT-qPCR in ear edema animal models. Transduced Tat-Trx1 inhibited lipopolysaccharide-induced cytotoxicity and activation of NF-κB, MAPK and Akt. Additionally, Tat-Trx1 markedly reduced the production of inducible nitric oxide synthase, cyclooxygenase-2, IL-1β, IL-6 and TNF-α in macrophages. In a 12-O-tetradecanoylphorbol-13-acetate-induced mouse model, Tat-Trx1 reduced inflammatory damage by inhibiting inflammatory mediator and cytokine production. Collectively, these results demonstrated that Tat-Trx1 could exert anti-inflammatory effects by inhibiting the production of pro-inflammatory mediators and cytokines and by modulating MAPK signaling. Therefore, Tat-Trx1 may be a useful therapeutic agent for diseases induced by inflammatory damage.
Inflammation is known to involve a series of physiological responses to various stimuli, including pathogen, chemical, and damaged cells of the host (
Mitogen activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB) signaling pathways are involved in LPS-induced inflammation (
Thioredoxin 1 (Trx1), a redox regulating protein, plays crucial roles in cell growth, cell protection against neurotoxicity, and inhibition of apoptosis (
We obtained LPS and TPA from Sigma-Aldrich. Antibodies were provided by Cell Signaling Technology. All other chemicals were used of high quality reagent grade.
DMEM was used in the culture of Raw 264.7 cells as described previously (
For concentration- and time-dependent transduction of Tat-Trx1, Raw 264.7 cells were treated with different concentrations (0.1-1 µM) of Tat-Trx1 for 1 h or with time periods (15-60 min) of Tat-Trx1 (1 µM). After transduction, the cells were further incubated and intracellular stability of Tat-Trx1 was determined by western blotting using an anti-histidine antibody. Western blotting was performed as described previously (
Equal amounts of sample proteins were separated with 15% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in TBST buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h. The membranes were immunoblotted with the indicated primary and HRP-conjugated secondary antibodies as recommended by the manufacturer. The protein bands were detected using enhanced chemiluminescent reagents (Amersham).
ROS staining was performed as described previously (
TUNEL staining was performed as described previously (
Raw 264.7 cells (105 cells/well) were grown overnight in 12-well plate and total RNA extracted using easy-BLUE according to the manufacturer's instructions (iNtRON). Also, mouse skin tissue (50 mg) was used for total RNA extraction using TRIzol reagent (Life Technologies). The cDNA synthesis was performed using 2 µg of total RNA with cDNA Reverse Transcription Kit (Omniscript® RT Kit) as per the manufacturer's instructions (Qiagen). The mRNA levels were quantified using a SYBR Premix Ex Kit (Bio-Rad) with a CFX Real time PCR Detection Systems (Bio-Rad). The relative gene expression levels were normalized to GAPDH and calculated using the 2-ΔΔCq method (
The primer sequences for PCR were as follows: COX-2 (sense: 5'-CCAGCACTTCACCCATCAGTT-3'; antisense: 5'-AAGGCGCAGTTTATGTTGTCTGT-3'), iNOS (sense: 5'-GGCTGCCAAGCTGAAATTGAAT-3'; antisense: 5'-CGTGATAGCGCTTCTGGCTCTT-3'), IL-1β (sense: 5'-TGTGTTTTCCTCCTTGCCTC-3'; antisense: 5'-TGCTGCCTAATGTCCCCTTG-3'), IL-6 (sense: 5'-AAGGAGTGGCTAAGGACCAAGAC-3'; antisense: 5'-AGTGAGGAATGTCCACAAACTGATA-3'), TNF-α (sense: 5'-CTTGTTGCCTCCTCTTTTGC TTA-3'; antisense: 5'-CTTTATTTCTCTCAATGACCCGTAG-3'), GAPDH (sense: 5'-CTTTGGCATTGTGGAAGGGCTC-3'; antisense: 5'-GCAGGGATGATGTTCTGGGCAG-3').
All experiments utilized ICR mice (male, 6-8 week-old, total used animal = 25) obtained from Experimental Animal Center, Soonchunhyang University. The mice were provided with a commercial diet and water
TPA-induced skin animal models were produced as described elsewhere (
For histological analysis, ear biopsies were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at a thickness of 5 µm, and then stained with hematoxylin and eosin (
Data are expressed as the mean ± SEM of three experiments. The statistical significance analyzed using one-way ANOVA followed by a Bonferroni post hoc test. P-value < 0.05 was considered as significant.
We constructed a fusion protein Tat-Trx1 and purified it. As expected, one single band was detected for the purified protein (
It is known that LPS can ROS production and DNA fragmentation and lead to cell death (
Suppressing MAPK (p38, JNK, and ERK) and NF-κB activation is important for inhibiting inflammation in LPS-activated Raw 264.7 cells (
We also determined levels of Bcl-2, Bax, caspase-3, and cleaved caspase-3 proteins. Bax and cleaved caspase-3 protein expression levels were increased in LPS-treated Raw 264.7 cells, whereas Tat-Trx1 reduced these protein expression levels in LPS-treated cells. In contrast, Tat-Trx1 markedly increased Bcl-2 and caspase-3 protein expression levels in LPS-treated cells, whereas there was no significant difference the expression of Bcl-2, Bax, caspase-3, and cleaved caspase-3 proteins in cells treated with Trx1 or Tat peptide (
We further investigated whether Tat-Trx1 inhibited LPS-induced inflammatory responses in Raw 264.7 cells. As shown in
TPA-induced ear edema animal model is generally used for skin inflammation study. Thus, we examined the anti-inflammatory effect of Tat-Trx1 in TPA-induced ear edema animal model. We designed the experiment to induced mice ear edema using TPA. As shown in
We also determined the effect of Tat-Trx1 in TPA-exposed mouse model (
Inflammatory response plays an important role in the defense against external stimuli including microbial pathogens and chemicals. Also, inflammatory response can lead to various diseases including neurodegenerative diseases (
LPS plays a crucial role in inducing inflammatory responses, leading to various inflammatory diseases (
Many reports have shown that LPS can induce NF-κB activation and its upstream regulator, MAPK. MAPKs pathway can regulate inflammatory responses. Activated p65 can lead to the production of pro-inflammatory mediators and cytokines (
It is well described that TPA, a promotor of skin tumorigenesis, can induce inflammatory symptom and cancer development. In general, TPA-induced mouse model can be used to understand the inflammatory mechanism (
Results of the present study revealed that Tat-Trx1 markedly reduced TPA-induced ear thickness, weight, and inflammatory responses. Taken together, our results indicate that Tat-Trx1 can significantly prevent inflammation in macrophages and in an animal model by inhibiting the production of pro-inflammatory mediators and cytokines as well as NF-κB and MAPK activation. Therefore, Tat-Trx1 can be used as a therapeutic agent for treating diseases induced by inflammatory damage.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
EJY, MJS, WSE and SYC were involved in the conceptualization of the study. HJY, YJC, EJS, LRL and HJC performed the experiments, and acquired and analyzed the data. HJK, YHY, DWK and DSK produced the methodology of the animal model and concurrently conducted animal studies. SHL, SL, JP and KHH were responsible for data analysis and interpretation, and participated in the drafting of the manuscript. EJY and SYC confirmed the authenticity of all the raw data. WSE, MJS and SYC were involved in the writing and editing of the manuscript and provided final approval of the version to be published. All authors read and approved the final manuscript.
The care of animals conformed to the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research and Quarantine Service of Korea and the present study was approved by the Institutional Animal Care and Use Committee of Soonchunhyang University Cheonan, Chungcheongnam, Republic of Korea (approval no. SCH 15-0002-3).
Not applicable.
The authors declare that they have no competing interests.
Purification and transduction of Tat-Trx1 protein. (A) Purified Tat-Trx1 and Trx1 proteins were identified by 15% SDS-PAGE and western blotting. (B) Cell culture media were treated with Tat-Trx1 (1 µM) for different time periods (15-60 min). (C) Intracellular stability of transduced Tat-Trx1. The cells were incubated for 12 h after transduction of Tat-Trx1 for 1 h. Transduction of Tat-Trx1 was measured by western blotting and the intensity of the bands was measured by densitometry. (D) Cells were treated with Tat-Trx1 (1 µM) for 1 h and the cellular distribution of transduced Tat-Trx1 was confirmed by fluorescence microscopy. Scale bar, 50 µm. Trx1, thioredoxin 1.
Effects of Tat-Trx1 on LPS-induced ROS production and DNA damage. Treatment with Tat-Trx1 (1 µM) was followed by 1 h treatment with LPS (1 µg/ml). (A) Intracellular ROS levels were measured by DCF-DA staining and (B) DNA fragmentation was detected by TUNEL staining. The fluorescence intensity was measured using an ELISA plate reader. Scale bar, 50 µm. *P<0.05 and **P<0.01 vs. LPS-treated cells. DCF-DA, 2',7'-dichlorofluorescein diacetate; LPS, lipopolysaccharide; ROS, reactive oxygen species; Trx1, thioredoxin 1.
Effects of Tat-Trx1 on LPS-induced NF-κB (p65), MAPK (p38, JNK and ERK) and Akt phosphorylation in Raw 264.7 cells. The cells were pretreated with Tat-Trx1 (1 µM) for 1 h and then treated with LPS (1 µg/ml). Phosphorylation levels of (A) p65, (B) MAPK (p38, JNK and ERK) and (C) Akt were determined by western blotting. The band intensity was measured by densitometry. *P<0.05 and **P<0.01 vs. LPS-treated cells. LPS, lipopolysaccharide; p-, phosphorylated; Trx1, thioredoxin 1.
Effect of Tat-Trx1 on LPS-induced Bcl-2, Bax, caspase-3 and cleaved caspase-3 production in Raw 264.7 cells. The cells were pretreated with Tat-Trx1 (1 µM) for 1 h and then treated with LPS (1 µg/ml). The production levels of Bcl-2, Bax, caspase-3 and cleaved caspase-3 were determined by western blotting. The band intensity was measured by densitometry. *P<0.05 and **P<0.01 vs. LPS-treated cells. LPS, lipopolysaccharide; Trx1, thioredoxin 1.
Effects of Tat-Trx1 on LPS-induced inflammatory responses in Raw 264.7 cells. The cells were treated with Tat-Trx1 (1 µM) for 1 h before being exposed to LPS (1 µg/ml). (A) Protein expression levels of COX-2 and iNOS were analyzed by western blotting. The band intensity was measured by densitometry. (B) mRNA levels of cytokines were determined using reverse transcription-quantitative PCR. The mRNA levels were normalized to GAPDH and subsequently represented as the fold change relative to the control group. *P<0.05 and **P<0.01 vs. LPS-treated cells. COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; Trx1, thioredoxin 1.
Effects of Tat-Trx1 on TPA-induced mice ear edema. Ears of mice were treated with TPA (1 µg/ear) once a day for 3 days. Tat-Trx1 protein (10 µg) was topically applied to mice ears 1 h prior to TPA exposure over 3 days. (A) Schematic representation of the experimental procedure. Protective effects of Tat-Trx1 were demonstrated by (B) hematoxylin and eosin staining, as well as changes in (C) ear weight and ear thickness in a TPA-induced mice ear edema model. Scale bar, 50 µm. *P<0.05 and **P<0.01 vs. TPA-treated mice. TPA, 12-O-tetradecanoylphorbol-13-acetate; Trx1, thioredoxin 1.
Effects of Tat-Trx1 on TPA-induced pro-inflammatory mediator (iNOS and COX-2) and cytokine (IL-1β, IL-6 and TNF-α) expression in mice ears. Mice were stimulated with TPA (1 µg/ear) after which Tat-Trx1 (10 µg) was topically applied to mice ear for 3 days. The mRNA levels of (A) pro-inflammatory mediators and (B) cytokines were determined using reverse transcription-quantitative PCR. The mRNA levels were normalized to GAPDH and subsequently represents as the fold change relative to the control group. *P<0.05 and **P<0.01 vs. TPA-treated mice. COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; TPA, 12-O-tetradecanoylphorbol-13-acetate; Trx1, thioredoxin 1.