Rat ischemia-reperfusion (I/R) injury model
All SD rats were divided into four groups: Sham group, I/R group, DEX + I/R group and DY + I/R group (n = 8 per group, total n = 32 ) using a computer based random order generator. All SD rats were anesthetized with 1% sodium pentobarbital (50 mg/kg, Hengrui Medicine Co., Ltd., China). After a sufficient depth of anesthesia was established, all rats were intravenously injected with saline (10 ml/kg) or DEX (Hengrui Medicine Co., Ltd., China) at a concentration of 50 ug/kg for 1 h prior to the induction of I/R or treatment with yohimbine (Shanghai Yuanye Bio-Technology Co., Ltd., China) at a concentration of 1.0 mg/kg, followed by the infusion of DEX, as described in previous studies.5,16 The tracheal tube was connected to a small animal ventilator for mechanical ventilation (the respiratory rate was 60 breaths/min, the tidal volume was 15 ml/kg, I:E = 1:2.5 and FiO2: 99%). The chest of each rat was opened at the fourth intercostal space, and the subcutaneous tissue was bluntly separated. The pleura was exposed and cut in the expiratory phase. The left thoracic cavity was exposed and the left hilar was carefully separated to expose the trachea. Then, the portal vein was injected with 300 U/kg of unfractionated heparin via the tail vein. After 10 min, the left hilar was clamped with a noninvasive vascular clamp to establish an ischemic model. After 30 min, the noninvasive vascular clamp was released to restore the ventilation and blood supply to the left lung for 2 h. Then the rats were euthanized with an overdose of isoflurane (5%). The left lung was removed immediately and washed with pre-cooled saline to eliminate the blood clot. The left hilar of rats in Sham group were not clamped as a vehicle control. Four different researchers are responsible for anesthesia, administering drugs, surgery and data collection.
At the end of the experiment, the lung tissues were removed. The upper parts of the lung tissues were fixed with 10% formalin, embedded in paraffin and subsequently sectioned. After deparaffinization and rehydration, the tissue sections were stained with hematoxylin and eosin. Finally, the pathological sections were evaluated in a blinded manner.
Measurement of the lung W/D weight ratio.
The wet lung tissues were weighed and then placed in an 80 °C drying oven for 48 h and were subsequently weighed when the tissues were dried. The W/D weight ratio was calculated.
Analysis of the oxygenation index (PaO2/FiO2).
At the end of experiment, arterial blood was acquired from the carotid artery and tested by using a blood gas analyzer (Premier3000, GEM).
In vitro cell viability test
To evaluate the cytotoxicity of DEX and yohimbine against PVECs, DEX was added to the culture medium at concentrations of 2, 4, 6, 8 and 10 nmol/L, and yohimbine was added to the culture medium at concentrations of 0.25, 0.5, 0.75 and 1.0 µmol/L. The viability of DEX/yohimbine-treated PVECs was determined by using the cell counting kit-8 (CCK-8) assay.
Cellular oxygen glucose deprivation (OGD) model.
Rat pulmonary vascular endothelial cells (PVECs) were acquired from ScienCell (San Diego, CA, USA). The PVECs were pretreated with DEX (10 nmol/L) or DEX and yohimbine (1 µmol/L followed by the DEX) for 30 min, and then PVECs were cultured in glucose-free DMEM (GIBCO, New York, USA) without FBS in a hypoxic incubator (95% N2 and 5% CO2) at 37 °C for 10 h. The control group was treated with DMEM. After exposure to OGD, the cells were transferred to absolute medium containing 10% FBS and cultured under normoxic conditions (95% air, 5% CO2, and 21% FiO2) for another 24 h.
Measurement of the mitochondrial membrane potential (MMP) in vitro.
At the end of the experiment, PVECs were stained with the JC-1 probe (2 µmol/L) (Beyotime Institute of Biotechnology) for 30 min, rinsed twice with PBS, and then imaged with a confocal microscope (original magnification, × 400). The MMP was determined by monitoring the dual emissions from the mitochondrial JC-1 monomers (green) and the aggregates (red) under a confocal microscope with excitation using a 488-nm-laser. The MMP was reported as the emission intensity ratio (the ratio of green to red fluorescence), which represents the relative arbitrary MMP level. A higher ratio indicates a lower MMP.
Measurement of ROS production in vitro.
After treatment, the PVECs were measured using MitoSOX™ Red mitochondrial superoxide indicator (Molecular Probes, Inc., Eugene, OR, USA) at 37 °C for 20 min, washed gently 3 times with warm buffer, and imaged in × 200 magnification using Leica microscope. Mitochondrial ROS generation was expressed as the mean fluorescence intensity of the red color, according to previously described.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). After treatment, total RNA was extracted from lung tissues and PVECs with Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Total RNA (1 µg) was reverse transcribed into cDNA using the PrimeScript™ RT reagent kit (Takara Bio, Dalian, China). And then, the cDNA templates were amplified with SYBR Premix Ex Taq™ (Takara Bio) by using the following cycling conditions: 30 sec at 95 °C for 1 cycle, following 40 cycles of 5 sec at 95 °C and 30 sec at 60 °C. The following primer sequences were used: peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) sense, 5'-CGG AGC AAT CTG AGT TAT ACG-3' and antisense, 5'-CAG TCA CAG GAG GCA TCT-3'; mitochondrial transcription factor A (Tfam) sense, 5'-ATC TCA TCC GTC GCA GTG-3' and antisense, 5'-GCA CAG TCT TGA TTC CAG TTC-3'; PINK1 sense, 5'-CAA TGC CGC TGT GTA TGA A-3' and antisense, 5'-CCG TCT CTG GAT CTT CTG TAA-3'; Parkin sense, 5'-CTC AGA CAA GGA CAC ATC AGT A-3' and antisense, 5'-GCG GTG GTT ACA TTG GAA G-3'; dynamin-related protein 1 (Drp1) sense, 5'-ACT GGC CCC CGT CCA GCT TA-3' and antisense, 5'-TGG ACC AGC TCC ACA CAG CG-3'; and GAPDH sense, 5'-GTG AAG GTC GGT GTG AAC-3' and antisense, 5'-GGT GAA GAC GCC AGT AGA-3'. The threshold cycle (Ct) values were set in the exponential phase of the PCR. Relative gene expressions were calculated by comparing the cycle times for each target PCR. The Ct values of the target PCR were normalized by subtracting the Ct value of GAPDH; and then, the relative mRNA expression of each sample was calculated by the 2−(∆Ct sample − ∆Ct control) method.
Western blot analysis. Total cellular protein was extracted from lung tissues and PVECs using standard protocols, and protein concentration from the supernatant was determined by the BCA protein assay kit (Thermo Fisher Scientific). Equal amounts (30 µg) of purified protein were electrophoresed on 8% SDS-PAGE gels, then transferred and blotted onto polyvinylidene difluoride membranes. After blocking (5% nonfat milk, 2 h), membranes were incubated with the following primary antibodies at 4 °C overnight : anti-PGC-1α antibody (1:1,000; ab54481; Abcam), anti-Tfam antibody (1:2,000; ab131607; Abcam), anti-dynamin 1-like protein 1 (Drp1) antibody (1: 1,000; ab56788, Abcam), anti-PINK1 antibody (1:1,000; Abcam, ab23707), anti-Parkin antibody (1:1,000; Abcam, ab179812) and anti-GAPDH antibody (1:1,000; 5174P; Cell Signaling Technology, Inc, Danvers, MA, USA). After four washes, membranes were then incubated with HRP-conjugated secondary antibodies, containing the anti-rabbit antibody (1:5,000; ab6721, Abcam) and anti-mouse antibody (1:2,000; ab97023, Abcam); all steps were performed at room temperature. Finally, the signals were detected by the chemiluminescence substrate (Super Signal West Pico, Thermo Fisher Scientific, USA), the intensities of bands were calculated by Gel-Pro Analyzer software(Media Cybernetics, Rockville, MD, USA). In addition, all intensity values were normalized to GAPDH and then reported as relative expression.
Statistical analysis. All data are presented as the means values ± SD, with the number of independent experiments indicated in the figure legends. Statistical analyses were performed with Student’s t-test or one-way ANOVA, followed by Dunnett’s post hoc tests. Differences are considered statistically significantat p < 0.05.