Dexmedetomidine attenuates lung apoptosis induced by renal ischemia–reperfusion injury through α₂AR/PI3K/Akt pathway

Experiments were performed on male C57BL/6J mice, 10-weeks old and weighing 25–30 g. The animals were obtained from the laboratory animal center of Third Military Medical. They were kept in a room with lights on from 8:00 to 20:00 at 25 ± 2 °C and a relative humidity of 55 ± 5%, and fed with a standard pellet diet and water ad libitum. The experiments were reviewed and approved by the Animal Care Committee of Third Military Medical University, Chong Qing, China.

All mice were anesthetized with pentobarbital sodium (50 mg/kg i.p.) and placed on a heating pad to maintain the temperature at 36 ± 0.1 °C. Animals were randomly assigned to five groups (n = 6 per group): renal ischemia–reperfusion injury group (IR): bilateral renal pedicles were clamped for 60 min with a microvascular clamps. Then the clamps were gently removed; Sham group (Sham): laparotomy was performed without renal vessels occlusion. Dex group (Dex): Dex (Precedex 100 μg/mL Dex, Orion Pharma, Espoo, Finland) was intraperitoneally injected at 25 μg/kg without surgery. Pre-treatment with Dex group (Dex + IR): 25 μg/kg Dex 15 min prior to ischemia was administered. Combination of the α₂-adrenergic antagonist atipamezole (Atip + Dex + IR): atipamezole (Sigma-Aldrich, St. Louis, MO, USA) 250 μg/kg was administered 10 min prior to Dex pre-treatment. All animals were administered 0.5 mL of sterile saline intraperitoneally, and the incision closed in two layers with a 4–0 silk. At the end of 24 h reperfusion, the mice were euthanized by exsanguination under pentobarbital sodium anesthesia, and tissues were collected for analysis. Blood samples from abdominal aorta were obtained from Sham and IR group animal, and were centrifuged (3000g, 4 °C for 20 min) to obtain serum, which was then stored at − 80 °C.

After paraffin embedding, lung tissues were sectioned into 5-μm thick sections. Then the sections were stained with hematoxylin/eosin and examined under a light microscope. The degree of lung injury was scored on a scale from 0 to 3 using a previously described scoring system: Grade 0, normal pulmonary appearance; Grade 1, mild moderate interstitial congestion and neutrophil leukocyte infiltrations; Grade 2, perivascular edema formation, partial leukocyte infiltration, moderate neutrophil leukocyte infiltration; Grade 3, severe destruction of the lung architecture and massive neutrophil leukocyte infiltration.

TUNEL assay was conducted to evaluate apoptosis using an In Situ Cell Death Detection Kit, POD (Roche, Germany) according to the manufacturer’s instruction. Lung tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Then the tissues were sectioned at 5 μm for TUNEL staining. After deparaffinization and rehydration, the sections were applied with 10 μg/mL protease K for 15 min. Fresh TUNEL reaction mixture was added on the samples which were incubated for 60 min at 37 °C in the dark. After washing, the cell nucleuses were stained with 0.1 μg/mL DAPI (beyotime, China). The samples were analyzed in a drop of PBS under a fluorescence microscope (Olympus, Japan). The number of TUNEL-positive cells per high-power field was calculated and analyzed in a blinded manner by observing eight random visual fields per animal under a magnification of 200×.

Arterial blood samples were obtained for blood gas analysis. A 0.5-mL sample of arterial blood was drawn from the abdominal aorta, and pH, partial pressure of oxygen (PaO₂) and partial pressure of carbon dioxide (PaCO₂) were measured at the end of the reperfusion period with a blood gas analyzer (Beckman Coulter, Inc., USA).

C57BL/6J mice PMVECs were purchased from the Cell Biologics Inc. (Chicago, USA). The cells were cultured in M-1168 medium (Cell Biologics, Chicago, USA) at 37 °C and in a humidified atmosphere of 5% CO₂. Cells between passage 3 and 6 were used in the subsequent experiments.

Cell viability was monitored using a CCK-8 assay (Dojindo, Japan) according to the manufacturer’s protocol. PMVECs were plated at a density of 5 × 10³ cells/well in 96-well plates. After overnight culture, the culture medium was removed, after which the cells were incubated with different doses of Dex (0.1, 1, 10 μM) or different concentrations of IR serum (10, 15, 20, 25%) for 24 h, or pre-incubated with different concentrations of Dex for 1 h before exposure to 15% IR serum for 24 h. Meanwhile, cells incubated with 15% sham serum for 24 h was used as the control group. After chemical stimulation, the supernatant was removed, CCK-8 reagent (10 μL) was added to each well of a 96-well plate containing 100 μL culture medium and the plate was incubated for 3 h at 37 °C. Cell viability was evaluated by absorbance measurements at 450 nm.

PMVECs were cultured in 6-well plates at a density of 4 × 10⁴ cells per well overnight, after which cells were exposed to various concentrations of Dex for 1 h, followed by exposure to 15% IR serum for 24 h. After these treatments, PMVECs were fixed in paraformaldehyde (4%) for 10 min, washed with PBS buffer, and then stained with the Hoechst 33258 (Beyotime Biotechnology Company, China) for 5 min. Photomicrographs were captured under an inverted fluorescence microscope Nikon EclipseTE300 inverted microscope (Nikon, Tokyo, Japan) at an excitation wavelength of 350 nm excitation and an emission wavelength of 460 nm Annexin V-FITC/PI double staining study using flow cytometry.

Cells apoptosis was examined by double staining with Annexin V-FITC Apoptosis Detection Kit (Bestbio, China). PMVECs were seeded in 6-well plates at a density of 1 × 10⁵ cells/mL, and incubated overnight. Then cells were treated with different concentrations of Dex for 1 h prior to exposure to 15% IR serum for 24 h. After being washed twice with cold PBS, cells were suspended in 400 μL binding buffer at a concentration of 1 × 10⁶ cells/mL, after which cells were incubated in 5 μL Annexin V-FITC for 10 min and 10 μL PI for 15 min at 4 °C in the dark. Cells were subsequently analyzed using flow cytometry (ACEA, Biosciences Inc., China).

A mitochondrial membrane potential assay kit with JC-1 (Beyotime, China) was used to detect the mitochondrial membrane potential. According to the manufacturer’s protocol, PMVECs were seeded in 6-well plates at a density of 1 × 10⁵ cells/mL, and incubated overnight. Cells incubated with 15% Sham serum or IR serum for 24 h were used as the control group or IR group, respectively. The Dex + IR group was pretreated with 0.1 μM Dex 1 h before the IR group. In the Atip + Dex + IR group, 1 μM atipamezole was administered 10 min prior to dexmedetomidine pre-treatment. The LY294002 + Dex + IR group was treated with 20 μM LY294002 for 30 min before Dex treatment. After these treatment, cells were incubated with JC-1 working solution for 20 min at 37 °C in the dark after treatment, and then washed twice with ice-cold JC-1 buffer solution before analysis with flow cytometry.

The lung tissues and cells were harvested at the end of the experiment. Protein was extracted from tissue or cells in a lysis buffer. After estimate protein concentrations with a bicinchoninic acid protein assay kit, equal amounts of protein (40 μg) from each sample were separated and electrotransferred onto a PVDF membranes. The membranes were then blocked in 5% BSA and incubated overnight at 4 °C with primary antibodies. After washing three times in TBST, the membranes were then incubated with HRP-coupled secondary antibodies. The antibodies are as follows: rabbit monoclonal anti-cleaved caspase-3 antibody (1:1000, Cell Signaling Technology, USA), rabbit monoclonal anti-Bcl-2 antibody (1:1000, Cell Signaling Technology, USA), rabbit monoclonal anti-Bax antibody (1:1000, Cell Signaling Technology, USA), rabbit monoclonal anti-phospho-Akt antibody (1:1000, Ser473, Cell Signaling Technology, USA), anti-rabbit IgG, HRP- linked antibody (1:2500, Cell Signaling Technology, USA), rabbit polyclonal anti-β-actin antibody (1:2000, Solarbio, China). Bands were detected using standard ECL (Millipore, USA) and quantified by densitometric analysis using Image J software (Version 1.44p, National Institutes of Health, Bethesda, MD, USA).

Data were expressed as mean ± SD. All statistical analyses were performed using GraphPad Prism 5.01. One-way analysis of variance followed by post hoc Newman–Keuls test were used for comparison, otherwise, unpaired two tailed Student test was used wherever appropriate. A p value less than 0.05 was considered to be of statistical significance.