In total, 20 male C57BL/6J mice (8-week-old) were obtained from the Animal Center of Xinjiang Medical University (Urumqi, China). Animals were housed (one per cage) under standard conditions of 21–25°C and 50±5% humidity with a 12/12 h light/dark cycle (lights on at 8:30 a.m.) in a specific-pathogen-free facility in the Research Institute of Uygur Pharmaceutics (Urumqi, China). All mice were provided tap water and standard chow diet ad libitum (18% fat, 24% protein, 58% carbohydrates). The study protocol was approved by the Animal Care and Use Committee of the People's Hospital of Xinjiang Uygur Autonomous Region (protocol no. KY201803703; Urumqi, China). The study was completed based on the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health.

The mice were randomly divided into the control (n=10) and chronic restraint stress groups (CRS, n=10). Each of the control mice were left undisturbed and remained in a single cage, while stressed mice were isolated in individual cages and subjected to immobilization stress for 2 h per day (6 days per week, between 10:00 a.m. and 12:00 p.m.) over a period of 14 consecutive days, as described in detail previously (21,22). Body weight and food intake were monitored every 2 days during the stress period.

In the morning following the last restraint stress, all mice underwent a 16 to 18-h fasting period, anesthetized by intraperitoneal injection of sodium pentobarbital (150 mg/kg) and then euthanized. Subsequent to euthanasia, blood samples were collected from the inferior vena cava, and stored at −80°C. Esophageal samples were also collected under aseptic conditions and stored in RNase-free tubes at −80°C for extraction of total RNA, analysis of biological marker expression levels and histopathological examination.

The esophageal tissues of all mice were excised, weighed, fixed with 10% formalin, dehydrated at room temperature by ethanol series and then embedded in paraffin. Next, tissues were cut into 5-µm sections and stained with hematoxylin and eosin (H&E) or Masson's trichrome (MT) for the evaluation of esophageal inflammation and fibrosis, respectively. Images of H&E and MT staining were obtained from 10 different, randomly selected microscopic fields per section under a light microscope at a magnification of ×200 using a digital camera (Eclipse E200; Nikon Corporation, Tokyo, Japan). The extent of stress-induced inflammatory damage was evaluated by histologic scoring performed by an investigator who was blinded to the group. For statistical analysis, three independent measurements for each specimen were performed. The histologic findings were scored in terms of the epithelial damage, submucosal edema and submucosal inflammatory cells observed (23). The epithelial damage scores were assigned as follows: 0, normal morphology; 1, mild surface lifting; 2, intraepithelial separation and surface lifting; and 3, epithelial cell loss to basal cell layer or deeper. Submucosal edema was scored as follows: 0, normal tissue; 1, mild focal edema; 2, moderate diffuse edema; and 3, severe edema. Finally, the score for submucosal inflammatory cells was as follows: 0, 0–5 cells/high-power field (HPF); 1, 5–10 cells/HPF; 2, 10–15 cells/HPF; and 3, ≥15 cells/HPF. MT-positive areas, including the mucosal and epithelial layers, were analyzed by Adobe Photoshop (Adobe Inc., Mountain View, CA, USA) and quantified in 10 random fields-of-view per section using NIH ImageJ version 1.62 software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry was performed according to the streptavidin-biotin complex method, as described previously (22,24). Briefly, esophageal sections were deparaffinized with xylene and dehydrated with ethanol. All sections were incubated overnight at 4°C with primary antibodies, including anti-Nox-4 (1:100; cat. no. ab133303; Abcam, Cambridge, UK), anti-TRPV-1 (1:100; cat. no. NBP1-71774; Novus Biologicals, Ltd., Cambridge, UK); and anti-PAR-2 (1:100; cat. no. ab180953; Abcam). The localization of Nox-4, TRPV-1 and PAR-2 was visualized using 3,3-diaminobenzidine tetrahydrochloride (DAB tablet; Merck KGaA, Darmstadt, Germany) at a concentration of 30 mg/ml containing 0.03% H₂O₂. Then, sections were counterstained in 2% methylene green for 12 min at room temperature. Sections were dehydrated in descending ethanol series, washed with xylene for 5 min at room temperature three times, and mounted in mounting media (Mount-quick; Daido Sangyo Co., Ltd., Kawasaki, Japan). Images of all sections were captured under a light microscope (magnification, ×200) with a digital camera (Eclipse E200; Nikon Corporation).

Plasma samples of all mice were obtained and properly processed according to the protocols, as described previously (21,22). Plasma levels of Nox-4, interleukin (IL)-6, IL-8, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were determined with a competitive ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the protocol provided by the manufacturer.

Normal human esophageal epithelial cells (HEECs) were purchased from The American Type Culture Collection (cat. no. ATCC CRL-2629; Manassas, VA, USA), and maintained in keratinocyte serum-free medium, purchased from Gibco (Thermo Fisher Scientific, Inc.), containing 10% FBS supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin under standard cell culture conditions of 37°C and a humidified atmosphere with 5% CO₂. The medium was replaced every three days until confluence was reached. Cells between passages 2 to 5 were used in all cell culture experiments.

HEECs were seeded at a density of 5×10³ cells/well in 96-well plate in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) for 48 h. HEECs were serum-starved for 24 h, and were subsequently were stimulated with trypsin (0.05 and 0.1 nM), which served as an endogenous PAR-2 agonist, for 0, 2, 4 and 8 h. Next, cells were lysed in 200 µl cell lysis buffer, and the lysate was harvested using a cell scraper and centrifugation for 15 min at 12,000 × g and at 4°C. Finally, the supernatant was collected, and used to determine the mRNA expression levels of Nox-4, PAR-2 and TRPV-1 by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

The role of PAR-2 on Nox-4, TRPV-1, IL-6, IL-8, IFN-γ and TNF-α production by HEECs was investigated using a blocking antibody against the amino-terminal cleavage region of PAR-2 (SAM11; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Briefly, HEECs were grown to confluence at a density of 5×10⁵ cells/well on 96-well culture plate. HEECs were serum-starved for 24 h, and were subsequently treated with or without anti-PAR-2 blocking antibody (1:20,000) for 2 h, followed by incubation with trypsin (0.1 nM) for 4 h. Subsequently, culture supernatants were centrifuged for 15 min at 12,000 × g and at 4°C. Supernatants were treated with 0.05% trypsin and maintained at 37°C with 5% CO₂ for 5 min. The mRNA expression levels of the genes of interest were measured by RT-qPCR.

siRNA specific to TRPV-1 was purchased from Santa Cruz Biotechnology, Inc., and Lipofectamine RNA iMAX (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to transfect the siRNA into HEECs according to the manufacturer's protocol. Briefly, cells were seeded at a density of 5×10⁵ cells/well on a 6-well culture plate in keratinocyte serum-free medium containing 10% FBS, and grown to confluence. HEECs were incubated with control siRNA or TRPV-1 siRNA (20 nmol/l). The siRNAs were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Cells were incubated for 48 h prior to trypsin treatment (0.1 nM) for 4 h. Subsequently, culture supernatants were harvested, and the mRNA expression levels of the aforementioned proteins were measured by RT-qPCR.

HEECs were lysed in lysis buffer [containing 65 mmol/l Tris-HCl (pH 6.8), 3.3% sodium dodecyl sulfate (SDS), 10% glycerol and 2.2% bromophenol blue], and the supernatant was collected and stored at −80°C. The protein concentration was determined using the bicinchoninic acid protein assay kit. Next, proteins were fractionated by SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Immobilon-P; EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline Tween-20 (TBS-T) at room temperature for 1 h. Subsequent to washing with TBS-T, the membranes were treated with mouse polyclonal anti-TRPV-1 antibody (1:1,000; cat. no. NBP1-71774; Novus Biologicals, Ltd., Cambridge, UK) and β-actin (1:1,000; cat. no. 3700; Cell Signaling Technology, Inc., Danvers, MA, USA). The membranes were further incubated with HRP-linked secondary antibody (1:10,000; cat. no. 7076; Cell Signaling Technology, Inc.) at room temperature for 1 h. Following washing with TBS-T three times, the protein expression levels were visualized using the Chemi-Lumi One enhanced chemiluminescence system (Nacalai Tesque, Inc., Kyoto, Japan).

Total RNA was extracted from the esophageal tissues of mice and HEECs using TRIzol reagent (Thermo Fisher Scientific, Inc.) and subjected to FastLane Cell cDNA Kit (Qiagen GmbH, Hilden, Germany). Total RNA (1 µg) was reverse transcribed to cDNA with oligo (dT) primers according to the manufacturer's protocol (Qiagen GmbH). The obtained cDNA was subjected to qPCR analysis. The thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min, followed by 40 cycles of 12 sec at 95°C and 60 sec at 60°C, using the Bio-Rad CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and Power SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). Serial dilutions of a control sample of cDNA were used as the standard curve for each reaction. All experiments were performed in triplicate. Changes in gene expression were calculated by the 2^−ΔΔCq method (25), and the values were normalized to the levels of β-actin and GAPDH. The primer sequences used in the present study are listed in Tables I and II. The amount of each mRNA was normalized to β-actin for the RNA extracted from mice tissues and GAPDH for the RNA extracted from HEECs, respectively.

All data are expressed in terms of the mean ± standard deviation. The quantitative analysis of histological damage score, MT staining and body weight gain was conducted by Student's t-test. The mRNA and plasma levels of Nox-4, antioxidant enzymes, inflammatory cytokines, as well as TRPV-1 and PAR-2, were also analyzed by Student's t-test. The mRNA levels of Nox-4, PAR-2 and TRPV-1 in cell culture experiments were analyzed by one-way analysis of variance with Fisher's protected least significant difference test. Differences between groups were considered as statistically significant at P<0.05.

In the current study, 8-week-old male C57BL/6J mice were subjected to restraint stress for 2 weeks to observe the stress-induced inflammatory response in the esophagus. The histopathological analysis by H&E staining revealed that pathological changes in the esophagus were not observed in the control group. In the esophagus of the CRS group, however, mild infiltration of inflammatory cells in the submucosa, basal cell hyperproliferation, papillary hypertrophy, epithelial hyperkeratinization, squamous cell expansion and an increase in the number of fibrin cells were observed (Fig. 1A). The mucosal damage scores in the esophageal tissue of mice are displayed in Fig. 1B. Compared with the control group, the histological damage score in the CRS group was markedly increased (0.38±0.09 in the control vs. 1.23±0.05 in the CRS group; P<0.01). These results were consistent with the typical histological findings associated with low-grade reflux esophagitis (26). In addition, the MT staining results revealed that stress increased esophageal interstitial fibrosis as compared with the control mice (Fig. 1C and D).

The body weight of mice was monitored and measured during the 2-week stress period. Body weight gain was significantly reduced in CRS mice as compared with that in the non-stressed control mice (Table III). Each group of mice consumed similar amounts of food (~130 mg/g/day; P>0.05).

It has previously been reported that chronic stress evokes ROS production in adipose tissue and the colon (22,24). To determine whether stress induces ROS generation in the esophagus, the current study analyzed the expression of Nox-4 in mice by immunohistochemical analysis, RT-qPCR and ELISA. The data revealed that 2 weeks of restraint stress resulted in an increase in Nox-4 expression in the mucosal and epithelial layers of the esophagus (Fig. 2A), significantly elevated plasma levels of Nox-4 (Fig. 2B) and marked upregulation of Nox-4 mRNA expression (Fig. 2C), when compared with the levels in control mice.

Antioxidant enzymes serve an important role against oxidative stress in various types of cells and tissues (22,24). Herein, the expression levels of antioxidant enzymes were examined using RT-qPCR. After a 2-week period of stress, the mRNA expression levels of antioxidant enzymes, including Mn-superoxide dismutase (MnSOD), Cu/Zn-SOD, glutathione peroxidase (GPx) and catalase, were significantly reduced in stressed mice compared with those in non-stressed control mice (Fig. 2D-G).

Earlier studies have demonstrated that TRPV-1 and PAR-2 receptors regulate VH (12). In the present study, the esophageal expression levels of TRPV-1 and PAR-2 were determined by immunohistochemistry and RT-qPCR. The results revealed that TRPV-1 and PAR-2 were localized in the luminal layers of the esophageal squamous epithelia of stressed mice (Fig. 3A and B). Furthermore, stress significantly increased the esophageal mRNA expression levels of TRPV-1 and PAR-2 as compared with those in non-stressed control mice (Fig. 3C and D).

After 2 weeks of restraint stress, significant increases were observed in the mRNA expression levels of IL-6, IL-8, IFN-γ and TNF-α in the esophageal tissues of the CRS group, as compared with those in the non-stressed control group (Fig. 4A, B, C and D). Furthermore, the plasma concentration levels of IL-6, IL-8, IFN-γ and TNF-α were significantly elevated in stressed mice, in parallel with the changes in mRNA expression in the esophagus (Fig. 4E, F, G and H).

Reflux of proteases, including trypsin, has been previously reported to cause damage to the esophageal mucosa (16,18); however, the mechanism underlying this effect remains unclear. In the present study, the effect of trypsin on the expression levels of Nox-4, PAR-2 and TRPV-1 were investigated. HEECs were incubated with trypsin at the indicated concentrations and time periods. Nox-4, PAR-2 and TRPV-1 expression levels were examined at 4 h after incubating with different concentrations of trypsin (0, 0.05 and 0.1 nM), or at the different time points (0, 2, 4 and 8) following incubation with trypsin (0.1 nM). It was observed that trypsin increased the mRNA expression levels of Nox-4, PAR-2 and TRPV-1 in HEECs in a time- and dose-dependent manner (Fig. 5A-F). Trypsin at a concentration of 0.1 nM was used in subsequent in vitro experiments.

As shown in Fig. 6A-F, the mRNA expression levels of Nox-4, TRPV-1, IL-6, IL-8, IFN-γ and TNF-α were significantly increased when the cells were stimulated with trypsin, serving as a natural PAR-2 agonist, for 4 h. Notably, pretreatment with a blocking antibody against PAR-2 resulted in significant inhibition of the mRNA expression of these inflammatory cytokines in HEECs stimulated with trypsin.

The effects of TRPV-1 knockdown on the mRNA levels of PAR-2, Nox-4 and inflammatory cytokines were examined in cultured HEECs. HEECs were incubated with or without TRPV-1 siRNA (20 nmol/l) for 48 h, followed by the stimulation with trypsin (0.1 nM) for 4 h. In cells without trypsin treatment, transfection with siRNA specific to TRPV-1 suppressed the gene and protein expression levels of TRPV-1 as compared with that of control-transfected HEECs (Fig. 7A). The trypsin-treated cells exhibited significantly increased mRNA expression levels of Nox-4, PAR-2 and inflammatory cytokines, including IL-6, IL-8, IFN-γ, and TNF-α, compared with the control group. Importantly, knockdown of TRPV-1 significantly suppressed the mRNA expression levels of Nox-4, PAR-2 and inflammatory cytokines (IL-6, IL-8, IFN-γ, and TNF-α) compared with the trypsin-treated cells. (Fig. 7B-G).

Collectively, the present findings suggested that 2-week stressed mice showed esophageal inflammation and fibrosis via ROS accumulation and antioxidants suppression. Furthermore, stress-activated PAR-2 and TRPV-1 were involved in ROS production and inflammatory cytokine expression.