STAT3 signaling pathway by induction of glutathione peroxidase-1

Neurochemistry International 94 (2016) 9e22

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Repeated exposure to far infrared ray attenuates acute restraint stress in mice via inhibition of JAK2/STAT3 signaling pathway by induction of glutathione peroxidase-1 Thai-Ha Nguyen Tran a, 1, Huynh Nhu Mai a, 1, Eun-Joo Shin a, **, Yunsung Nam a, Bao Trong Nguyen a, Yu Jeung Lee b, Ji Hoon Jeong c, Hoang-Yen Phi Tran d, Eun-Hee Cho e, Seung-Yeol Nah f, Xin Gen Lei g, Toshitaka Nabeshima h, i, Nam Hun Kim j, Hyoung-Chun Kim a, * a

Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea Clinical Pharmacy, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea c Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea d Physical Chemistry Department, University of Medicine and Pharmacy, Ho Chi Minh City 760000, Viet Nam e Department of Internal Medicine, Medical School, Kangwon National University, Chunchon 200-701, Republic of Korea f Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, KonKuk University, Seoul 143-701, Republic of Korea g Department of Animal Science, Cornell University, Ithaca, New York 14853, USA h Department of Regional Pharmaceutical Care and Sciences, Graduate School of Pharmaceutical Sciences, Meijo University, Nagoya, Japan i NPO, Japanese Drug Organization of Appropriate Use and Research, Nagoya 468e8503, Japan j College of Forest and Environmental Sciences, Kangwon National University, Chunchon 200-701, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 January 2016 Received in revised form 26 January 2016 Accepted 1 February 2016 Available online 2 February 2016

Exposure to far-infrared ray (FIR) has been shown to exert beneficial effects on cardiovascular and emotional disorders. However, the precise underlying mechanism mediated by FIR remains undetermined. Since restraint stress induces cardiovascular and emotional disorders, the present study investigated whether exposure to FIR affects acute restraint stress (ARS) in mice. c-Fos-immunoreactivity (IR) was significantly increased in the paraventricular hypothalamic nucleus (PVN) and dorsomedial hypothalamic nucleus (DMH) in response to ARS. The increase in c-Fos-IR parallels that in oxidative burdens in the hypothalamus against ARS. Exposure to FIR significantly attenuated increases in the c-Fos-IR, oxidative burdens and corticosterone level. ARS elicited decreases in GSH/GSSG ratio, cytosolic Cu/Znsuperoxide dismutase (SOD-1), glutathione peroxidase (GPx), and glutathione reductase (GR) activities. FIR-mediated attenuation was particularly observed in ARS-induced decrease in GPx, but not in SOD-1 or GR activity. Consistently, ARS-induced decreases in GPx-1-immunoreactivity in PVN and DMH, and decreases in GPx-1 expression in the hypothalamus were significantly attenuated by FIR. ARSinduced significant increases in phosphorylation of JAK2/STAT3, and nuclear translocation and DNAbinding activity of NFkB were observed in the hypothalamus. Exposure to FIR selectively attenuated phosphorylation of JAK2/STAT3, but did not diminish nuclear translocation and DNA-binding activity of NFkB, suggesting that JAK2/STAT3 constitutes a critical target for FIR-mediated pharmacological potential. ARS-induced increase in c-Fos-IR in the PVN and DMH of non-transgenic mice was significantly attenuated by FIR exposure or JAK2/STAT3 inhibitor AG490. GPx-1 overexpressing transgenic mice significantly protected increases in the c-Fos-IR and corticosterone level induced by ARS. However, neither FIR exposure nor AG490 significantly affected attenuations by genetic overexpression of GPx-1. Moreover, AG490 did not exhibit any additional positive effects against the attenuation by genetic overexpression of GPx-1 or FIR exposure. Our results indicate that exposure to FIR significantly protects

Keywords: FIR Acute restraint stress JAK2/STAT3 GPx-1 overexpressing transgenic mice Corticosterone Hypothalamus

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (E.-J. Shin), [email protected] (H.-C. Kim). 1 First two authors contributed equally to this work. http://dx.doi.org/10.1016/j.neuint.2016.02.001 0197-0186/© 2016 Elsevier Ltd. All rights reserved.

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ARS-induced increases in c-Fos-IR and oxidative burdens via inhibition of JAK2/STAT3 signaling by induction of GPx-1. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction

2. Materials and methods

Infrared radiation is subdivided into three categories: 1) nearinfrared radiation (0.8e1.5 mm); 2) middle infrared radiation (1.5e5.6 mm); and 3) far-infrared radiation (FIR; 5.6e1000 mm). FIR therapy uses low energy of light emitted from an artificial radiator. FIR has been utilized to treat vascular (Inoue and Kabaya. 1989; Kihara et al., 2004) and depressive disorders (Tsai et al., 2007). Although the technology of FIR has been widely applied in health promotion (Nagasawa et al., 1999; Udagawa and Nagasawa. 2000), the exact mechanisms of the pharmacological activities of FIR remain poorly understood. The tissue-warming activity of FIR may be regarded as a possible common background. This activity must induce an accelerated blood circulation at peripheral tissues, eventually triggering an improved state of metabolism, and improved transfer of chemical messengers. NMR studies have revealed that water clusters become much smaller in size after an exposure to FIR (Inoue and Kabaya. 1989; Matsushita, 1988). Such a change means that the motility of water molecules is highly activated by the FIR. Hence, it might be speculated that FIR stimulates the penetration of water molecules into various sites inside of the body tissue and also modulates dynamic functions of humoral factors in the body fluid. Stress has been reported to play a role in the development of cardiovascular disease (Rozanski et al., 1999). In addition, the biological response to stress involves activation of both the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic system. Indeed, there is strong evidence that the sympathetic nervous system plays a prominent role in producing stressinduced responses (Crane et al., 2005). Acute restraint is an unavoidable stress model, which is known to elicit several emotional and autonomic responses. The paraventricular nucleus of the hypothalamus (PVN) is in close proximity to the walls of the third ventricle, being divided into parvocellular and magnocellular neurons that regulate autonomic and neuroendocrine functions (Swanson and Kuypers. 1980). There is also research indicating PVN involvement in the modulation of sympathetic responses related to the exposure to stressful conditions (Coote et al., 1998). PVN neurons were also reported to be activated during exposure to stressful stimuli (Day et al., 2005; Girotti et al., 2006). In fact, stress constitutes one of the major contributory factors that stimulate numerous intracellular pathways leading to increased free radical formation. Information exists regarding the contribution of stress to oxidant production in the brain. Earlier reports have demonstrated that restraint stress results in the imbalance of antioxidant status, which ultimately leads to increased oxidative stress and resulting oxidative damage. In the present study, we investigated whether exposure to FIR affects acute restraint stress (ARS)-induced c-Fos-immunoreactivity (c-Fos-IR) and oxidative damage in the hypothalamus of mice. We observed here that exposure to FIR significantly protects ARS-evoked c-Fos-IR and oxidative stress via inhibition of JAK2/ STAT3 signaling by induction of glutathione peroxidase-1 (GPx-1) gene.

2.1. Animals All mice were treated in strict accordance with the NIH Guide for the Human Care and Use of Laboratory Animals (NIH Guide for the Care and Use of Laboratory Animals). Eight-week-old male C57BL/6 (wild-type) mice (Bio Genomics, Inc., Charles River Technology, Gapyung-Gun, Gyeonggi-Do, Republic of Korea), male glutathione peroxidase-1 overexpressing transgenic (GPx-1 TG) and nontransgenic (non-TG) mice, weighing about 25 ± 3 g, were maintained on a 12 h/12 h light/dark cycle and fed ad libitum. The GPx-1 TG mice, which carry three copies of the transgene, were derived from B6C3 (C57BL/6
C3H) hybrid mice (Cheng et al., 1997, 1998; Pepper et al., 2011; Xiong et al., 2004; Yan et al., 2012). Breeding pairs of GPx-1 TG mice were kindly provided by professor Xin Gen Lei (Dept. of Animal Science, Cornell University, Ithaca, New York, U.S.A.). Polymerase chain reaction (PCR) analysis was performed for genotyping of GPx-1 transgene using genomic DNA extracted from mouse tail. PCR primers for genotyping were as follows; 50 -CTC AAA CAA TGT AAA ATT GGG CTC GAA CCC GC- 30 , and 50 e GAA AGC GAT GCC ACG TGA TCT CAG CAC CAT CC-30 (Bioneer Corporation, Daejeon, Republic of Korea). The band intensity of PCR products was determined by PhotoCapt MW (version 10.01 for Windows; Vilber e, France), and then compared with the Lourmat, Marne la Valle intensity of PCR products of non-TG mice. 2.2. Far-infrared (FIR) irradiation FIR irradiation was performed as described previously (Nagasawa et al., 1999; Udagawa and Nagasawa. 2000). The FIR panel (S-warmer, i-ONE FILM, Anyang, Gyeonggi-Do, Republic of Korea) was positioned at a height of 40 cm above the mice. This panel emits FIR ranging from 5 to 20 mm as measured and verified by the Korea Far Infrared Association (Seoul, Republic of Korea) and the Korea Institute of Ceramic Engineering and Technology (Jinju, Republic of Korea). The panel surface temperature was controlled at 40  C. The temperature was approximately 25.5  C in the control cages and 27.0  C in the FIR cages. 2.3. Exposure to FIR, acute restraint stress (ARS), and drug treatment Because a single exposure to FIR did show significant pharmacological effects in our pilot study, mice were exposed to FIR for 20 min, twice a day by the 3 h-time interval (i.e., 10:00e10:20 and 13:00e13:20) consecutively for 5 d (days 1e5). On day 6, the mice were restrained horizontally in a 50-ml conical tube containing many holes (0.4-cm-diameter) for breathing (Anglen et al., 2003; Yang et al., 2014). Mice were not able to move forward or backward in this device, but they were not pressed and could breathe freely (Hare et al., 2014; Kim and Han. 2006; Yang et al., 2014). Mice were stressed for 4 h (i.e., 13:00e17:00). Thirty minutes before being subjected to ARS, the mice received AG490 (10 mg/kg, i.p., Tocris Bioscience, Ellisville, MO, U.S.A.), an inhibitor of JAK2/STAT3. AG490 was dissolved in 50% dimethyl sulfoxide in sterile saline

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immediately before use (Park et al., 2013). The final FIR irradiation was performed for 20 min after the ARS (i.e., 17:00e17:20), and then the mice were sacrificed immediately after the final exposure to FIR (Suppl. Fig. 1). 2.4. Immunocytochemistry Immunocytochemistry was performed as described previously (Shin et al., in press, 2014). Mice were perfused transcardially with 50 mL of ice-cold PBS (10 mL/10 g body weight) followed by 4% paraformaldehyde (20 mL/10 g body weight). Brains were removed and stored in 4% paraformaldehyde overnight. The brain was cut into 35-mm-thick coronal sections. Sections were blocked with PBS containing 0.3% hydrogen peroxide for 30 min and then incubated in PBS containing 0.4% Triton X-100 and 1% normal serum for 20 min. After a 48-h incubation with primary antibody against cFos (1:5000; Merck Millipore, Billerica, MA, U.S.A.) (Inagaki et al., 2014; Kobayashi et al., 2015) or GPx-1 (1:500; Abcam, Cambridge, UK), sections were incubated with the biotinylated secondary antibody (1:1000; Vector Laboratories, Burlingame, CA, U.S.A.) for 1 h. The sections were then immersed in a solution containing avidinebiotin peroxidase complex (Vector Laboratories) for 1 h, and 3,30 -diaminobenzidine was used as the chromogen. Digital images were acquired under an upright microscope (BX51; Olympus) using an attached digital microscope camera (DP72; Olympus) and an IBM-compatible PC (Shin et al., in press). ImageJ version 1.47 software (National Institutes of Health, Bethesda, MD, U.S.A.) was utilized to count c-Fos-immunoreactive cells and to examine GPx-1-immunoreactivity as described previously (Maximova et al., 2006; Pellegrino et al., 2007; Wang et al., 2012). Briefly, the entire paraventricular hypothalamic nucleus (PVN) and dorsomedial hypothalamic nucleus (DHM) (Figs. 1, 5 and 7) from each section were selected as the region of interest. c-Fospositive cells were counted blindly by two investigators, and results were obtained from the average. For examining GPx-1immunoreactivity, threshold values for hue, saturation, and brightness were set in the “Adjust Color Threshold” dialog box, and then the mean density was measured (Shin et al., in press). 2.5. Determination of reactive oxygen species (ROS) The extent of reactive oxygen species (ROS) formation in the hypothalamus was assessed by the method described by Lebel and Bondy (1990). Ten percent (w/v) homogenates, in phosphate buffered saline (PBS), of hypothalamic tissues were incubated with 5 mM 20 ,70 -dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR, U.S.A.) for 3 h at 37  C. The excess unbound probe and the flocculent precipitate were removed by centrifugation at 12,500
g for 10 min. The fluorescent intensity was measured at an excitation wavelength of 488 nm and emission wavelength of 528 nm. 20 ,70 -Dichlorofluorescein (DCF) was used as a standard. Protein level in each homogenate was measured using the BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, U.S.A.). 2.6. Determination of malondialdehyde (MDA) The amount of MDA was determined by the methods of Jareno et al. (1998) with some modification (Halliwell, 1992; Kim et al., 1999). In brief, each hypothalamic tissue was homogenized in PBS, and 0.1 ml of this homogenate (or standard solutions prepared daily from 1,1,3,3-tetra-methoxypropane) and 0.75 ml of the working solution (thiobarbituric acid 0.37% and perchloric acid 6.4%, 2:1, v:v) were mixed and heated to 95  C for 1 h. After cooling (10 min in ice water bath), the flocculent precipitate was removed

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by centrifugation at 3200
g for 10 min. The supernatant was neutralized and filtered prior to injection on an octadecylsilane 5 mm column. The mobile phase consisted of 50 mM PBS (pH 6.0): methanol (58:42, v/v). Isocratic separation with 1.0 ml/min flow rate and detection at 532 nm using a UVeVIS high-performance liquid chromatography detector (model LC-20AT and SPD-20A, Shimadzu, Kyoto, Japan) were performed. Protein level in each homogenate was determined using the BCA protein assay kit. 2.7. Determination of protein carbonyl The extent of protein oxidation was assessed by measuring the content of protein carbonyl groups, which was determined spectrophotometrically with the 2,4-dinitrophenylhydrazine (DNPH)labeling procedure (Kim et al., 2000; Shin et al., 2005) as described by Oliver et al. (1987). The results are expressed as nanomole of DNPH incorporated/mg protein based on the extinction coefficient for aliphatic hydrazones of 21 mM1 cm1 (Kim et al., 2000; Tran et al., 2012). 2.8. Determination of glutathione peroxidase (GPx) activity Hypothalamic tissues were homogenized in 50 mM potassium phosphate buffer (pH 7.0) and centrifuged at 13,000
g for 20 min. The resulting supernatant was used to measure GPx activity. GPx activity was analyzed by a spectrophotometric assay described by Lawrence and Burk (1976), using 2.0 mM reduced glutathione and 0.25 mM cumene hydroperoxide as substrates (Shin et al., 2008, 2014). The reaction rate at 340 nm was determined using the NADPH extinction coefficient (6.22 mM1 cm1) at 25  C. One unit of GPx activity was defined as the amount required to oxidize 1 mmol NADPH/min. Protein level in each sample was quantified using the BCA protein assay kit. 2.9. Determination of superoxide dismutase (SOD) Hypothalamic tissues were homogenized in 50 mM potassium phosphate buffer (pH 7.8) and centrifuged at 13,000
g for 20 min. The resulting supernatant was used to measure SOD activity. SOD activity was determined on the basis of inhibition of superoxidedependent reactions as described previously (Shin et al., 2008, 2014). The reaction mixture contained 70 mM potassium phosphate buffer (pH 7.8), 30 mM cytochrome-c, 150 mM xanthine, and tissue extract in phosphate buffer diluted 10 times with PBS in a final volume of 3 mL. The reaction was initiated by adding 10 mL of 50 units of xanthine oxidase, and the change in absorbance at 550 nm was recorded. One unit of SOD is defined as the quantity required inhibiting the rate of cytochrome c reduction by 50%. For estimating total SOD, 10 mM potassium cyanide (KCN) was added to the reaction mixture to inhibit cytochrome oxidase activity (McCord and Fridovich. 1969). For estimating Mn-SOD (SOD-2) activity, 1 mM KCN was added to the incubation mixture to inhibit Cu/Zn-SOD (SOD-1) activity (Kondo et al., 1997). The SOD-1 activity was calculated by the subtraction of the SOD-2 activity from the total SOD activity. Protein level in each sample was measured using the BCA protein assay kit. 2.10. Determination of glutathione and glutathione disulfide levels GSH and GSSG were immediately examined from dissected hypothalamic tissues as described previously (Reed et al., 1980; Tran et al., 2012). Briefly, a sample of the acidified supernatant was added to the internal standard (1 mM cysteic acid) and 0.88 M iodoacetic acid. Excess potassium hydrogen bicarbonate was added to the reaction to precipitate potassium perchlorate. Subsequently

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Fig. 1. Effect of FIR on the increase in c-Fos-positive cells induced by acute restraint stress (ARS) in the paraventricular hypothalamic nucleus (PVN; A) and dorsomedial hypothalamic nucleus (DHM; B) of wild-type mice (for details, refer to Materials and methods). Each value is the mean ± S.E.M. of six animals. *P < 0.01 vs. respective control or FIR without ARS. &P < 0.01 vs. Control with ARS (two-way ANOVA followed by Fisher's LSD pairwise comparisons). Scale bar ¼ 400 mm.

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Fig. 2. Effect of FIR on the increases in the level of reactive oxygen species (ROS; A), malondialdehyde (MDA; B), protein carbonyl (C) and GSSG (D), and the decreases in GSH level (E) and GSH/GSSG ratio (F) induced by acute restraint stress (ARS) in the hypothalamus of wild-type mice. Each value is the mean ± S.E.M. of six animals. *P < 0.05, **P < 0.01 vs. respective Control or FIR without ARS. &P < 0.05, &&P < 0.01 vs. Control with ARS (two-way ANOVA followed by Fisher's LSD pairwise comparisons).

0.5 ml of an alcoholic solution of 1.5% (v/v) 2,4dinitrofluorobenzene was added to the samples and incubated for 4 h. Diethyl ether (1.0 ml) was added and the samples were shaken and centrifuged at 2000
g for 20 min at room temperature. The residual aqueous phase containing derived glutathione was separated and analyzed by HPLC-UV/VIS detection system (model LC20AT and SPD-20A, Shimadzu). Separation of 5-carboxymethyl glutathione was carried out at room temperature with a flow rate of 1.2 mL/min. Chromatographic separation of derivatives was performed by injecting samples (10 ml) of the aqueous phase onto a Spherisorb NH2 5 mm analytical column (Waters, Milford, MA, U.S.A.). Glutathione was subsequently detected using a UV detector at 365 nm. Glutathione derivatives (GSH and GSSG) were quantified in relation to the internal standard (cysteic acid). Protein level in

each sample was determined using the BCA protein assay kit. 2.11. Western blot analysis Western blotting analysis was performed as described previously (Shin et al., in press; Tran et al., 2012). Hypothalamic tissues were homogenized in lysis buffer, containing 200 mM Tris HCl (pH 6.8), 1% SDS, 5 mM ethylene glycol-bis(2-aminoethyl ether)N,N,N0 ,N0 -tetraacetic acid (EGTA), 5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1
phosphatase inhibitor cocktail I (SigmaeAldrich, St. Louis, MO, U.S.A.), and 1
protease inhibitor cocktail (SigmaeAldrich). Lysate was centrifuged at 12,000
g for 30 min, and supernatant fraction was used for Western blot analysis. Proteins (20e50 mg/lane) were separated by 8% or 10% sodium

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Fig. 4. Effect of FIR on acute restraint stress (ARS)-induced corticosterone level in the plasma of wild-type mice. Each value is the mean ± S.E.M. of six animals. *P < 0.01 vs. respective Control or FIR without ARS. &P < 0.01 vs. Control with ARS (two-way ANOVA followed by Fisher's LSD pairwise comparisons).

dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto the PVDF membranes. Following transfer, the membranes were preincubated with 5% non-fat milk for 30 min, and incubated overnight at 4  C with primary antibody against JAK2 (1:500, Cell Signaling Technology, Inc., Danvers, MA, U.S.A.), phospho-JAK2 (1:500, Cell Signaling Technology, Inc.), STAT3 (1:500, Cell Signaling Technology, Inc.), phospho-STAT3 (1:1000, Cell Signaling Technology, Inc.), GPx-1 (1:500, R&D Systems, Minneapolis, MN, U.S.A.) or b-actin (1:50000, SigmaeAldrich). Then, membranes were incubated with HRP-conjugated secondary antirabbit IgG (1:1000, GE Healthcare, Piscataway, NJ, U.S.A.), antimouse IgG (1:1000, SigmaeAldrich), or anti-goat IgG (1:1000, SigmaeAldrich) for 2 h. Subsequent visualization was performed using an enhanced chemiluminescence system (ECL Plus®, GE Healthcare, Arlington Heights, IL, U.S.A.). Relative intensities of the bands were quantified by PhotoCapt MW (version 10.01 for Wine, France), and then normaldows; Vilber Lourmat, Marne la Valle ized to the intensity of b-actin (Shin et al., 2014). 2.12. Analysis of nuclear translocation of nulear factor kB (NFkB) Nuclear and cytosolic fractions were extracted using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Rockford, IL, U.S.A.) according to the manufacturer's instructions (Tran et al., 2012). The cytosolic and nuclear fractions were subjected to 8% SDS-PAGE (20e50 mg protein/lane), and the separated proteins were transferred onto a PVDF membrane. The membranes were immunoblotted with the primary antibody against NFkB p65 subunit (1:1000; AbD Serotec, Raleigh, NC, U.S.A.). An anti-histone H4 antibody (1:1000; Cell Signaling Technology, Inc.) was used as an internal loading control for the nuclear fraction, and an anti-b-actin antibody (1:5000; SigmaeAldrich) was used as an internal loading control for the cytosolic fraction. 2.13. NFkB p65 DNA-binding activity

Fig. 3. Effect of FIR on the changes in Cu/Zn-superoxide dismutase (SOD-1; A), Mnsuperoxide dismutase (Mn-SOD; SOD-2), and glutathione peroxidase (GPx; C) activities induced by acute restraint stress (ARS) in the hypothalamus of wild-type mice. Each value is the mean ± S.E.M. of six animals. *P < 0.05, **P < 0.01 vs. respective Control or FIR without ARS. &P < 0.01 vs. Control with ARS (two-way ANOVA followed by Fisher's LSD pairwise comparisons).

The nuclear fraction was extracted using a nuclear extraction kit (#40410; Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, fresh hypothamamus was homogenized in the hypotonic buffer provided in the kit, and the homogenate was incubated on ice for 15 min. After centrifugation for 10 min at 850
g, the pellet was resuspended in the complete lysis buffer provided in the kit. This suspension was incubated for 30 min on ice, and then centrifuged for 10 min at 14,000
g at 4  C.

Fig. 5. Effect of FIR on the decreases in glutathione peroxidase-1-immunoreactivity (GPx-1-IR) in the paraventricular hypothalamic nucleus (PVN; A) and dorsomedial hypothalamic nucleus (DHM; B) and GPx-1 expression in the hypothalamus (C) induced by acute restraint stress (ARS) in wild-type mice. Each value is the mean ± S.E.M. of six animals. *P < 0.05, **P < 0.01 vs. respective Control or FIR without ARS. &P < 0.05, &&P < 0.01 vs. Control with ARS (two-way ANOVA followed by Fisher's LSD pairwise comparisons). Scale bar ¼ 400 mm.

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Fig. 6. Effect of FIR on the level of p-STAT3 (A), p-JAK2 (B), cytosolic NFkB p65 (C), nuclear NFkB p65 (D), and NFkB p65 DNA-binding (E) induced by acute restraint stress (ARS) in the hypothalamus of wild-type mice. Each value is the mean ± S.E.M. of six animals. *P < 0.05, **P < 0.01 vs. respective Control or FIR without ARS. &P < 0.05, &&P < 0.01 vs. Control with ARS (two-way ANOVA followed by Fisher's LSD pairwise comparisons).

The supernatant (nuclear fraction) was stored at 80  C until use. The protein concentration was measured using the Pierce 660 nm Protein Assay™ reagent (Thermo Scientific). NFkB DNA-binding activity was measured using the TransAM® NFkB transcription factor ELISA kit (Active motif) (Kim et al., 2013) according to the manufacturer's instructions. Briefly, 10 mg of each nuclear protein extract were added to the well coated with oligonucleotides containing an NFkB consensus binding site. The plate was incubated for 1 h at room temperature, and then washed with the 1
wash buffer provided in the kit. After incubation with the primary antibody against the NFkB p65 subunit for 1 h at room temperature, the plate was incubated with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit IgG for 1 h. Colorimetry was performed using the developing solution provided in the kit. The absorbance at 450 nm was measured. 2.14. Determination of plasma corticosterone Mice were anesthetized with pentobarbital sodium (40 mg/kg,

i.p.), and blood samples were collected by cardiocentesis. After collection, blood samples were fractionated by centrifugation at 1500
g for 15 min at 6  C to separate plasma from other blood components. Plasma corticosterone content was assessed using a corticosterone ELISA (Enzo Life Sciences, New York, NY, U.S.A.). Each plasma sample was initially diluted in the assay buffer (1:50) supplied in the kit, and denatured at 75  C for 1 h. Thereafter, the assay procedure precisely followed that outlined in the manufacturer's handbook (Longden et al., 2014; Suberbielle et al., 2013; Sun et al., 2013). 2.15. Statistics Data were analyzed using IBM SPSS ver. 21.0 (IBM, Chicago, IL, U.S.A.). Two-way analysis of variance (ANOVA) (acute restraint stress
FIR) or four-way ANOVA (GPx-1 overexpressing transgene
acute restraint stress
FIR
AG490) was employed for the statistical analyses. Post-hoc Fisher's least significant difference pairwise comparisons tests were then conducted. P-values

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< 0.05 were considered to be significant.

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3.4. Exposure to FIR attenuated the increase in corticosterone level induced by acute restraint stress (ARS) in the plasma of wild-type mice

3. Results 3.1. Exposure to FIR attenuates increase in c-Fos-immunoreactivity (c-Fos-IR) induced by acute restraint stress (ARS) in the paraventricular hypothalamic nucleus (PVN) and dorsomedial hypothalamic nucleus (DMH) of wild-type mice It has been reported that PVN neurons are activated during exposure to stressful stimuli (Day et al., 2005; Girotti et al., 2006), and the role played by the PVN in the modulation of stress-evoked response is well established (Ziegler and Herman. 2000). Experimental design for understanding the effects of FIR against ARS was shown in Suppl. Fig. 1. A little c-Fos-IR in the PVN was observed in the absence of stress. FIR alone did not affect c-Fos induction. ARS significantly increased (P < 0.01 vs. control without ARS) c-Fos-IR in the PVN. FIR exposure significantly attenuated (P < 0.01 vs. control with ARS) c-Fos-IR induced by ARS in the PVN of mice (Fig. 1A). Consistently, FIR-mediated effects in the PVN are comparable to those in DMH area (Fig. 1B).

3.2. Exposure to FIR attenuates acute restraint stress (ARS)-induced oxidative damage in the hypothalamus of wild-type mice As it is well-recognized that restraint-induced oxidative stress mediates vascular (Busnardo et al., 2014) and emotional disorders (Tsai et al., 2007), we examined whether exposure to FIR affects oxidative damage induced by ARS. We investigated reactive oxygen species (ROS), lipid peroxidation (malondialdehyde; MDA), protein oxidation (protein carbonyl), GSSG, GSH, and GSH/GSSG ratio in the hypothalamus of mice. FIR did not significantly alter ROS, MDA, protein carbonyl, GSSG, GSH, and GSH/GSSG ratio in the hypothalamus. ARS significantly increased ROS (P < 0.05), MDA (P < 0.01), protein carbonyl (P < 0.05) and GSSG (P < 0.05), but significantly decreased GSH (P < 0.05), and GSH/GSSG ratio (P < 0.01). However, exposure to FIR significantly attenuated increases in ROS (P < 0.05), MDA (P < 0.01), protein carbonyl (P < 0.05) and GSSG (P < 0.05), and decreases in the GSH (P < 0.05) and GSH/GSSG ratio (P < 0.01) induced by ARS, respectively (Fig. 2).

3.3. Acute restraint stress (ARS) significantly decreased Cu/Znsuperoxide dismutase (SOD-1) and glutathione peroxidase (GPx) activities in the hypothalamus, and exposure to FIR selectively attenuated decrease in GPx activity induced by ARS in wild-type mice Knowing that restraint stress constitutes a typical psychophysiological burden, in which this stress response is initiated in the brain as a result of being unable to move freely, this study aimed to determine whether FIR alters oxidative changes and anti-oxidant activity in the brain of confined mice. We examined SOD and GPx activities in the hypothalamus of mice to elucidate whether FIR alters the enzymatic antioxidant system induced by ARS. ARS induced significant decreases in the SOD-1 (P < 0.05), and GPx (P < 0.01) activities. Although exposure to FIR did not significantly affect ARS-induced decrease in SOD-1 activity (Fig. 3A), it selectively attenuated (P < 0.01) ARS-induced decrease in GPx activity (Fig. 3C). SOD-2 activity appeared to be decreased by ARS; however, exposure to FIR did not significantly alter SOD-2 activity (Fig. 3B).

Exposure to FIR did not alter significantly. ARS-induced significant increase (P < 0.01) in plasma corticosterone level was observed. Exposure to FIR significantly attenuated (P < 0.01) increase in corticosterone level induced by ARS (Fig. 4). 3.5. Exposure to FIR significantly attenuated acute restraint stress (ARS)-induced decrease in glutathione peroxidase-1immunoreactivity (GPx-1-IR) in the PVN and DMH and decrease in GPx-1 expression in the hypothalamus of wild-type mice Exposure to FIR did not significantly affect GPx-1-IR in the PVN. ARS significantly decreased GPx-1-IR (P < 0.01) in the PVN. Exposure to FIR significantly attenuated (P < 0.05) decrease in GPx-1-IR induced by ARS in the PVN (Fig. 5A); This phenomenon was also observed in DMH (Fig. 5B). Consistently, exposure to FIR significantly attenuated (P < 0.01) ARS-induced decrease in GPx-1 expression (P < 0.05) in the hypothalamus of mice (Fig. 5C). 3.6. Exposure to FIR attenuates phosphorylation of JAK2/STAT3 induced by ARS in the hypothalamus of wild-type mice; it does not significantly alter nuclear translocation and DNA-binding activity of NFkB p65 ARS significantly increased phosphorylation of JAK2/STAT3 and nuclear translocation of NFkB p65. Exposure to FIR significantly prevented phosphorylation of JAK2 (P < 0.01) and STAT3 (P < 0.05) (Fig. 6A and B). However, exposure to FIR did not significantly change cytosolic expression of NFkB p65 and nuclear translocation of NFkB p65 (Fig. 6C and D). Further, ARS-induced significant increase in NFkB p65 DNA-binding activity was not significantly altered by exposure to FIR (Fig. 6E). 3.7. GPx-1 overexpressing transgenic (TG) mice protect increase in c-Fos-immunoreactivity (c-Fos-IR) induced by ARS in the PVN and DMH; neither FIR exposure nor JAK2/STAT3 inhibitor AG490 alters protective effects mediated by genetic overexpression of GPx-1 Exposure to FIR did not affect the basal level of c-Fos-IR in nonTG and GPx-1 TG mice. However, ARS significantly increased c-FosIR (P < 0.01) in the PVN of non-TG mice. This increase was significantly attenuated by FIR exposure (P < 0.01) or AG490 (P < 0.01). AG490 treatment did not significantly change attenuation mediated by FIR in non-TG mice. Importantly, GPx-1 TG mice showed significantly reduced c-FosIR compared to non-TG mice (P < 0.01) induced by ARS. However, neither FIR nor AG490 showed significant additional effects on the protection by genetic overexpression of GPx-1. AG490 did not exhibit any additional positive effect against FIR-mediated protective activity against c-Fos-IR in GPx-1 TG mice receiving ARS (Fig. 7A). Consistently, these phenomena in the PVN paralleled those in the dorsomedial hypothalamic nucleus (DMH) (Fig. 7B). 3.8. GPx-1 overexpressing transgenic (TG) mice protect increase in plasma corticosterone level induced by ARS; neither FIR exposure nor JAK2/STAT3 inhibitor AG 490 affects the protective effect mediated by genetic overexpression of GPx-1 Exposure to FIR did not significantly alter the basal level of corticosterone in non-TG and GPx-1 TG mice. ARS significantly increased corticosterone level (P < 0.01) in the plasma of non-TG mice. This increase was significantly inhibited by FIR or AG490.

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Fig. 8. Effect of JAK2/STAT3 inhibitor AG490 on FIR-mediated pharmacological activity in response to corticosterone level induced by acute restraint stress (ARS) in the plasma of glutathione peroxidase-1 gene overexpressing transgenic (GPx-1 TG)- and non-TG mice. Veh ¼ Vehicle (50% DMSO in saline, the solvent for AG490). AG490 ¼ AG490 (10 mg/kg, i.p.). Each value is the mean ± S.E.M. of six animals. *P < 0.05, **P < 0.01 vs. respective Control or FIR without ARS. #P < 0.01 vs. non-TG mice treated with Vehicle/Control with ARS (four-way ANOVA followed by Fisher's LSD pairwise comparisons).

AG490 treatment did not show additive effects against FIRmediated protective potentials in non-TG mice. GPx-1 TG mice showed significantly reduced corticosterone level compared to non-TG mice (P < 0.01) induced by ARS. Neither FIR nor AG490 significantly altered the protective activity by genetic overexpression of GPx-1. Importantly, AG490 did not exhibit any additive effects in response to FIR-mediated protective potentials against corticosterone level induced by ARS in the plasma of GPx-1 TG mice (Fig. 8).

4. Discussion We have observed for the first time that exposure to FIR significantly protects stimulatory responses induced by ARS. ARSinduced increase in c-Fos-IR parallels that in oxidative stress in the hypothalamus and in corticosterone level in the plasma of wildtype mice. ARS promoted decreases in GSH/GSSG ratio, SOD-1, and GPx activities. Exposure to FIR significantly attenuated ARSinduced increases in the c-Fos-IR, oxidative burdens, and corticosterone level. FIR attenuated decreases in GPx activity, GPx-1immunoreactivity, and -expression after ARS. In addition, we observed that JAK2/STAT3 signaling, but not NFkB signaling, is essential for FIR-mediated protective potentials against ARS. ARSinduced increase in c-Fos-IR in the PVN and DMH of non-TG mice was significantly attenuated by FIR exposure or JAK2/STAT3 inhibitor AG490. Genetic overexpression of GPx-1 (using GPx-1 TG mice) significantly protected c-Fos-IR and plasma corticosterone level induced by ARS. AG490 did not exhibit any additive effects on the amelioration by genetic overexpression of GPx-1 or FIR exposure, suggesting that interactive signaling between JAK2/STAT3 and GPx-1 is critical for the protective activity afforded by FIR. Therefore, we propose here that exposure to FIR significantly protects ARS-induced psychostimulatory responses (i.e., c-Fos-IR, oxidative burdens, and corticosterone level) via inhibition of the JAK2/STAT3 pathway by induction of GPx-1 (Fig. 9).

The neurons in the PVN play an integral role as the final common pathway in the CNS mediating the activation of the adrenal cortex that is considered a hallmark of the stress response (Whitnall, 1993). Most of the corticotropin releasing hormone (CRH)-containing neurons that release their contents into the hypophyseal portal system to cause secretion of ACTH from the anterior pituitary are localized in the PVN. A host of diverse stressors have been shown to increase the expression of Fos (Ceccatelli et al., 1989) and mRNA for CRH (Makino et al., 1995) in neurons in the parvocellular PVN, suggesting that these neurons are, indeed, activated by stress. In addition, several lines of evidence point to the possibility that neurons in the DMH play a crucial role in activation of the neurons in the PVN that are responsible for recruitment of the HPA axis in some forms of stress, indicating that the DMH constitutes a major source of afferent input to the PVN that is activated in stress. In this regard, therefore, we have selected PVN and DMH of the hypothalamic area. ARS-induced c-Fos induction in PVN was comparable to that in DMH. In addition, we have suggested that increase in c-Fos-IR leads to the production of oxidative products, although the exact mechanism of this phenomenon remains to be further characterized. Current results confirmed the pro-oxidant activity of restraint stress and established that decreased anti-oxidant enzyme activities in restraint stress-treated mice enhance oxidative burdens of the brain. Since GPx appears to be of major importance in the detoxification of peroxides in the brain (Pillai et al., 2014; Shin et al., 2014), the depletion of hypothalamic GSH or GSH/GSSG might result in a significant decrease in GPx activity. Consistently, a significant decrease in GPx activity in the brain has also been reported in response to restraint in rats (Derin et al., 2006; Sahin and Gumuslu. 2007). ROS, lipid peroxidation, and protein oxidation may increase due to the depletion of intracellular GSH content, which is considered as a first line of defense as an endogenous non-enzymatic antioxidant. It has been reported that restraint stress significantly increased

Fig. 7. Effect of JAK2/STAT3 inhibitor AG490 on FIR-mediated pharmacological activity in response to c-Fos induction by acute restraint stress (ARS) in the hypothalamic paraventricular nucleus (PVN; A) and dorsomedial hypothalamic nucleus (DHM; B) of glutathione peroxidase-1 gene overexpressing transgenic (GPx-1 TG)- and non-TG mice. Veh ¼ Vehicle (50% DMSO in saline, the solvent for AG490). AG490 ¼ AG490 (10 mg/kg, i.p.). Each value is the mean ± S.E.M. of six animals. *P < 0.01 vs. respective Control or FIR without ARS. #P < 0.01 vs. non-TG mice treated with Vehicle/Control with ARS (four-way ANOVA followed by Fisher's LSD pairwise comparisons). Scale bar ¼ 400 mm.

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Fig. 9. A schematic depiction of protective potential of FIR in response to acute restraint stress (ARS) in mice. ARS stimulates c-Fos induction in the hypothalamic area followed by oxidative damage, as shown by increases in reactive oxygen species, lipid peroxidation and protein oxidation, and decrease in the ratio of GSH/GSSG. Mice are subjected to ARS exhibited decreases in SOD-1 (Fig. 3) and GPx (Fig. 3) activities. In particular, ARS-induced decreases in the GPx activity and GPx-1 (a major type of GPx) expression might accumulate peroxide (including H2O2). Meanwhile, this accumulation of peroxide might facilitate oxidative damage. Consequently, ARS-induced failure in the induction of GPx activity and GPx-1 expression might potentiate plasma corticosterone. Importantly, exposure to FIR selectively induced GPx activity, and it inhibited phosphorylation of JAK2/STAT3 (but not NFkB-signaling) (Fig. 6). Furthermore, genetic overexpression of GPx-1 (using GPx-1 overexpressing transgenic mice) or JAK2/STAT3 inhibitor AG490 attenuated the stimulatory responses (surges in the c-Fos-IR and plasma corticosterone) induced by ARS. AG490 did not significantly alter FIR-mediated protective activity against stimulatory ARS insult in non-TG mice. “AG490” or “FIR” and treatment of AG490 in the presence of FIR did not exhibit any additional positive effects against the protective potential mediated by genetic overexpressing GPx-1 against ARS insult. Therefore, interactive modulation between GPx-1 gene and JAK2/STAT3 signaling pathway is critical for FIR-mediated protective potentials against ARS insult.

oxidative burdens and decreased GSH content in the brain. As we observed that GPx-1 gene is a molecular target of FIR-mediated protective potentials, it may be possible that a significant induction of GPx activity by FIR may lead to an inhibition of H2O2 accumulation, which could block Fenton reactions, leading to the inhibition of the stimulation of lipid peroxidation/protein oxidation. In this study, ARS induced significant decreases in SOD-1 and GPx activity in the hypothalamus of wild-type mice, but the decrease in GPx activity was more pronounced than that in SOD-1 activity. As the brain has low-level catalase activity and only moderate amounts of SOD and GPx (Coyle and Puttfarcken. 1993; Halliwell, 1992), we have mainly focused on SOD and GPx. Our observation of increased ROS/lipid peroxidation/protein oxidation products implies that GPx activity, rather than decreased cytosolic SOD-1, modulates these endpoints. Furthermore, a significant reduction of cytosolic SOD-1 activity induced by ARS was observed in wild-type mice, suggesting that this inactivation of SOD-1 might facilitate impairments in homeostasis between oxidant and antioxidant systems. However, ARS did not significantly alter mitochondrial SOD-2 activity in the hypothalamus of wild-type mice,

although the relationship between oxidative stress and neuronal stimulation has been recognized by the impaired antioxidant activity within mitochondria. Therefore, this issue requires further study. Activation of the transcription factor NFkB is known to be regulated by oxidative stress. Oxidative stress can modulate NFkB activation both positively and negatively. Under basal conditions, NFkB is localized in the cytoplasm in an inactive form in response to stimuli, NFkB is allowed to translocate into the nucleus, where it activates target genes (Kabe et al., 2005). In addition to NFkB, numerous studies have confirmed that the JAK2/STAT3 signal pathway is hyper-activated in cellular and animal models of oxidative stress, suggesting an important role of this signaling pathway in regulating oxidative stress responses (Carballo et al., 1999; Tawfik et al., 2005). Accordingly, FIR-mediated modulation of the JAK2/STAT3 signaling pathway may provide an effective therapeutic strategy in the attenuation of ARS. Since we showed that exposure to FIR exerts GPx (1)-dependent antiperoxidative effects against ARS, we have evaluated the role of NFkB and JAK2/STAT3 in this study. Exposure to FIR selectively and significantly inhibited ARS-induced increase in JAK2/

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STAT3 signaling. Furthermore, JAK2/STAT3 signaling is also abolished by genetic overexpression of GPx-1. Therefore, we propose here that FIR provides protective effects against ARS via GPx-1dependent inhibition of JAK2/STAT3 signaling. Earlier studies demonstrated that application of FIR improves endothelial function (Imamura et al., 2001; Kihara et al., 2002) and increases mRNA expression and protein levels of endothelial nitric oxide synthase (eNOS) of Syrian golden hamsters (Ikeda et al., 2001). Thus, the improvement in endothelial function and increased eNOS expression by FIR may lead to the normalization of autonomic nervous activity (Kuwahata et al., 2011). Interestingly, Oelze et al. (2014) demonstrated that GPx-1 deficiency results in a phenotype of endothelial and vascular dysfunction by using oxidative stresseprone GPx-1 knockout (/)-mice (a model representing decreased breakdown of cellular hydrogen peroxide). They provided stronger mechanistic links between oxidative stress, eNOS dysfunction, and vascular dysfunction. In their study (Oelze et al., 2014), the striking endothelial dysfunction observed in aged GPx-1 (/)-mice could be partly explained by the adverse phosphorylation of eNOS. Moreover, increasing GPx-1 activity might prevent, at least in part, eNOS dysfunction in the aging vasculature (Ullrich and Kissner. 2006). Inoue and Kabaya (1989) demonstrated that FIR can penetrate through skin and transfer energy into deep tissue gradually through a resonance-absorption mechanism of organic and water molecules. Thereafter, the technology of FIR irradiation has been widely applied in many fields. For example, FIR applications can be found in FIR sauna therapy (Imamura et al., 2001). In addition, it was recognized that FIR therapy promotes microvascular blood flow and angiogenesis in various animal models (Akasaki et al., 2006; Yu et al., 2006). Although clinical studies have mainly indicated that FIR radiation can exert beneficial effects in the cardiovascular system (Kuwahata et al., 2011; Miyata and Tei. 2010) and carcinogenic condition (Nagasawa et al., 1999; Udagawa et al., 1999), it remains to be further explored whether FIR therapy positively modulates restraint stress-associated disorders. Combined, our results suggest that GPx-1 gene is a critical mediator for protective activity mediated by FIR, and that GPx-1dependent JAK2/STAT3 modulation is important for the protection of ARS. Further study may be required to achieve a thorough understanding of the complex molecular pharmacological mechanism mediated by FIR against restraint stress-related emotional disorders.

Conflict of interest disclosure There is no conflict of interest.

Acknowledgments Thai-Ha Nguyen Tran, Huynh Nhu Mai, Yunsung Nam, and Bao Trong Nguyen were supported by the BK21 PLUS program, National Research Foundation of Korea, Republic of Korea. Equipment at the Institute of New Drug Development Research (Kangwon National University) was used for this study. The English in this document has been checked by at least two professional editors, both native speakers of English.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuint.2016.02.001.

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