Dexmedetomidine protects gastric mucosal epithelial cells against ischemia/reperfusion-induced apoptosis by inhibiting HMGB1-mediated inflammation and oxidative stress

Purpose: To investigate the role of dexmedetomidine in gastric ischemia/reperfusion injury using gastric mucosal epithelial cell (GES-1) model. Methods: GES-1 were subjected to oxygen-glucose deprivation conditions, followed by increasing dexmedetomidine concentrations (0.5, 1.0, or 1.5 μ M) for 4 h of reoxygenation. Cell viability and apoptosis were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide and flow cytometry, respectively. Oxidative stress and inflammation were analyzed by enzyme-linked immunosorbent assay (ELISA). Results: Oxygen-glucose deprivation conditions induced cytotoxicity in GES-1 by decreasing cell viability and increasing apoptosis. Dexmedetomidine treatment significantly increased the cell viability of hypoxia/reoxygenation-induced GES-1 (p < 0.01) but reduced apoptosis. Dexmedetomidine also attenuated the hypoxia/reoxygenation-induced increase in malondialdehyde and myeloperoxidase, but the decrease in superoxide dismutase and glutathione in GES-1. Moreover, upregulated tumor necrosis factor- α , interleukin (IL)-1 β , and IL-18 in hypoxia/reoxygenation-induced GES-1 was downregulated by dexmedetomidine treatment. Dexmedetomidine also enhanced IL-10 levels and inhibited pro-inflammatory factor production (p < 0.01). High-mobility group box 1 (HMGB1) protein in GES-1 was upregulated by hypoxia/reoxygenation but decreased by dexmedetomidine. HMGB1 over-expression attenuated the dexmedetomidine-induced increase in cell viability and the decrease in apoptosis, oxidative stress, and inflammation in hypoxia/reoxygenation-induced GES-1 (p < 0.01). Conclusion: Dexmedetomidine protects GES-1 against ischemia/reperfusion-induced apoptosis, inflammation, and oxidative stress by inhibiting HMGB1, thus providing a potential strategy for treating gastric ischemia/reperfusion injury.


INTRODUCTION
Gastric ischemia-reperfusion injury is a common clinical problem caused by distinct hemorrhagic diseases, such as vascular rupture, peptic ulcer bleeding, and hemorrhagic shock [1]. Gastric ischemia-reperfusion injury with significant morbidity and mortality lacks satisfactory treatment [2]. Pathophysiological manifestations of gastric ischemia-reperfusion injury include dysregulation of intracellular calcium homeostasis, increased mitochondrial permeability, and increased cytoskeletal and structural vulnerability [3]. Oxidative stress and the resultant inflammatory response that leads to cellular death and mucosal injury are implicated in the pathogenesis of gastric ischemia-reperfusion injury [4]. Therefore, strategies to suppress oxidative stress show promising effects against gastric ischemia-reperfusion injury [4].
Dexmedetomidine is a selective α-2 adrenergic receptor agonist that exerts sedative, antianxiety, analgesic, and antihypertensive properties [5]. Pretreatment with dexmedetomidine has been reported to also attenuate ischemia-reperfusion-induced intestinal injury [6]. However, the role of dexmedetomidine in gastric ischemia-reperfusion injury remains unknown. High mobility group box 1 (HMGB1) participates in the immune process by binding to Toll-like receptors and activating NF-κB signaling [7]. HMGB1 is implicated in the pathogenesis of noninfectious inflammation-associated diseases, including trauma, cancer, and ischemia reperfusion injury [7]. For example, HMGB1 contributes to ischemia-reperfusion-induced injury in the heart [8], and inhibiting HMGB1 reduces ischemia-reperfusion-induced inflammation and apoptosis in the lung [9].

EXPERIMENTAL Cell culture and treatments
GES-1 was acquired from ScienCell (San Diego, CA, USA) and cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (Gibco). Cells were incubated in a 37 °C incubator with 5 % CO2. Hypoxic conditions were induced by culturing cells in glucose-free medium and incubating them in 1% O2, 5% CO2, and 94 % N2 for 2 h. Cells in the control group were cultured in RPMI 1640 under normal conditions with 5 % CO2 and 95 % atmosphere. Cells in the treatment groups were treated with increasing concentrations (0.5, 1.0, or 1.5 mM) of dexmedetomidine (Sigma-Aldrich, St. Louis, MO, USA) for 4 h of reoxygenation.

Cell viability and apoptosis assays
GES-1 were seeded into 96 well plates and then was incubated with 5 mg/ml of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (10 ml; Beyotime, Beijing, China) for 4 h. Absorbance at 490 nm was measured using a microplate reader (Thermo Fisher Scientific). For flow cytometry, GES-1 were resuspended in the binding buffer of the Annexin V FITC and PI Staining Kit (Thermo Fisher Scientific) and then stained with 5 µL PI and 5 μL FITC-labeled annexin V. The apoptosis ratio was evaluated using a FACS flow cytometer (Life Technologies, Gaithersburg, MD, USA).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Cells were lysed in TRIzol kit (Invitrogen) to isolate RNAs. The RNAs were then synthesized into cDNAs, and the PreTaq II kit (Takara, Dalian, Liaoning, China) was used to determine the mRNA expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-18 and IL-10. The relative expression was calculated using the 2 -∆∆Cq method with normalization to GAPDH. The primers used are shown in Table 1.

Statistical analysis
All the data were expressed as means ± standard error of the mean (n = 3) and analyzed by Student's t-test or one-way analysis of variance (ANOVA) using SPSS 11.5. A p value of < 0.05 was considered statistically significant.

DISCUSSION
α-2 Adrenergic receptors are widely distributed throughout the whole body and exert pathologic effects in various diseases [11]. The agonists of α-2 adrenergic receptors are widely used to treat pain and panic disorders, hypertension, and alcohol withdrawal [11]. Dexmedetomidine, an α-2 adrenergic receptor, protects against ischemiareperfusion-induced intestinal injury [6]. The present study revealed that dexmedetomidine also attenuates gastric ischemia/reperfusion injury by inhibiting HMGB1-mediated inflammation and oxidative stress.
Hypoxia/reoxygenation induces cytotoxicity in gastric epithelial cells by decreasing cell viability and increasing inflammation and oxidative stress [12]. Therefore, hypoxia/reoxygenation-induced GES-1 was used as a cell model of gastric ischemia/reperfusion injury [12]. Here, GES-1 was also subjected to hypoxia/reoxygenation conditions. The cell viability of GES-1 was decreased, while cell apoptosis was increased, by hypoxia/reoxygenation. Moreover, hypoxia/reoxygenation reduced the levels of SOD and GSH and enhanced the levels of MDA and MPO to promote oxidative stress in GES-1. The levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-18, were upregulated in hypoxia/reoxygenation-induced GES-1.
Suppression of hypoxia/reoxygenation-induced inflammation and oxidative stress in GES-1 provided potential value in preventing acute gastric mucosal lesions [12].
Here, dexmedetomidine increased the cell viability of hypoxia/reoxygenation-induced GES-1 and decreased cell apoptosis to protect against cytotoxicity. Moreover, a previous study has shown that dexmedetomidine attenuated ischemia/reperfusion-induced hepatic inflammation and oxidative stress [13]. Dexmedetomidine in this study enhanced the levels of MDA and MPO while reducing SOD and GSH in hypoxia/reoxygenation-induced GES-1. Dexmedetomidine also increased IL-10 to downregulate TNF-α, IL-1β, and IL-18, suggesting antioxidant and anti-inflammatory effects against gastric ischemia/reperfusion injury.

CONCLUSION
Dexmedetomidine exerts anti-apoptotic, antiinflammatory, and antioxidant effects against hypoxia/reoxygenation-induced GES-1 by downregulating HMGB1, providing a potential strategy to manage gastric ischemia/reperfusion injury. However, the role of dexmedetomidine in an animal model of gastric ischemia/reperfusion injury should be investigated in further studies.