To construct the rat CPB model, 45 adult male Sprague-Dawley rats weighing 350–450 g and aged 6–8 weeks were obtained from the Animal Center of Shandong University, China. All subsequent animal experiments were in compliance with the National Institute of Health guidelines and approved by the Animal Care and Use Committee of Linyi Hospital, China. The rats were housed in an air-conditioned room and fed a standard diet. Prior to the surgical procedures, the rats were randomly divided into three equal groups (15 in each group), including a sham (control) group, a CPB group and an RIPC treatment group.

Both the CPB group and the treatment group were individually annulated and underwent CPB surgery. Briefly, the rats were intraperitoneally injected with 2% butaylone at a dose of 50 mg/kg body weight and maintained under anesthesia with additional butaylone. A 24-gauge inflow catheter and a 22-gauge outflow catheter were inserted into the right carotid artery and right femoral vein, respectively. Additionally, a 22-gauge catheter was inserted into the right femoral artery for blood monitoring and sampling. The CPB circuit consisted of a 20-mL sterile venous reservoir, a roller pump and a membrane oxygenator with around 4-mL pre-charge and a gas exchange area of 0.05 m² (Xijing Medical Co. Ltd, Xi’an, Shaanxi, China), all of which were connected to each other through sterile silicone tubes. The circuit was primed with a 10-mL mixed solution comprising 1 mL of 250 IU/kg heparin, 0.5 mL of 5% sodium bicarbonate, 3.5 mL of Lactated Ringer’s Solution and 5 mL of 6% hydroxyethyl starch in 0.9% sodium chloride. The flow rate was then gradually increased to 100 mL/kg body weight/min and maintained at this level for 60 min, where the mean arterial pressure stayed relatively stable in the range of 60 to 80 mmHg. The body (rectal) temperature of each rat was monitored and maintained between 36.5–38.5°C by placing a warm blanket on its back and a heat lamp nearby. After the CBP procedure was completed, the outflow cannula was removed and the right femoral vein was ligatured. The remaining priming solution was slowly infused into the carotid artery until stable circulation was achieved. The inflow catheter was subsequently removed and the carotid artery was ligatured, followed by intramuscular administration of 2000 U/kg body weight of penicillin.

The anesthetized rats in the treatment group then were subjected to three cycles of 5-min ischemia followed by 5-min reperfusion in their right hind limbs via a tourniquet. The animals in the sham group were annulated but did not receive surgery or treatment. As a result, the sham group served as a negative control that should represent normal baseline pulmonary function, whereas the CPB group was a positive control indicative of unmitigated bypass-induced lung injury.

Evaluation of pulmonary function in rats was performed 24 h after the CPB using an invasive pulmonary function device (Buxco Research Systems, Wilmington, NC, USA) based on a previously described procedure with minor modifications [10]. Briefly, the rats were anesthetized, subjected to tracheal intubation and then placed on the device. An average respiration rate of 50 breaths/min was applied. The following parameters were recorded by the built-in software, including the total lung capacity (TLC), airway resistance (R_aw) and dynamic lung compliance (C_dyn).

At the end of pulmonary function tests, rats were sacrificed by exsanguination under anesthesia. The lung was dissected from the thorax and bronchoalveolar lavage (BAL) was performed. Differential leucocyte counts were performed on the obtained BAL fluid samples.

The fluid samples were centrifuged and total protein was determined by using the BCA Protein Assay Kit (Beyotime, Jiangsu, China). The levels of granulocyte-macrophage colony stimulating factor (GM-CSF), IL-1α, IL-1β, IL-2, IL-4, IL-10, IL-12, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in BAL fluid were quantified using Cytokine Rat 10-Plex Panel for the Luminex platform (Thermo Fisher Scientific, Waltham, MA, USA).

The right lung of each rat was removed following the euthanasia. After the removal of the trachea and esophagus by blunt dissection, the wet weight of the lung was determined. Subsequently, the lung was incubated at 60°C for 3 to 4 days to remove all moisture, after which its dry weight was measured and the wet-to-dry weight ratio was calculated.

P-V curves were obtained based on a previously reported protocol [11]. In brief, 20 mL normal saline containing 5 mM EDTA was injected into the vena cava to flush the lung vasculature. A 16-gauge angiocatheter (BD, Franklin Lakes, NJ, USA) was inserted into the trachea through the neck incision, immobilized with a suture, and then connected to a syringe pump (World Precision Instruments, Sarasota, FL, USA) with an in-line pressure sensor (Flowplus, Intertronics, Oxfordshire, England). The lung P-V curves were monitored and recorded on a ViscoTec Flowscreen Evaluation Unit (Intertronics, Kidlington, Oxfordshire, UK). The flow rate of the injected air was around 25 mL/min and the direction of the pump was reversed when the pressure exceeded 40 cm H₂O or started to increase dramatically [12].

The left lung was dissected and fixed. Lung damage was assessed by quantitative stereological analysis. In brief, pulmonary circulation was rinsed with phosphate buffer and perfused with 4% paraformaldehyde. Then, ligation was performed between the pulmonary artery and trachea, followed by the inflation of the lung with 4% paraformaldehyde. After the fixation, the lung was embedded in paraffin and cut into 7-μm-thick slices, which were then mounted on slides and stained with hematoxylin and eosin (H&E). Images were acquired randomly at 20× and 40× magnification. Six high-power fields were randomly selected in each photograph and lung injury scores were calculated based on the algorithm proposed by Faller et al [13].

BASCs were isolated from the lungs following a previously described protocol [14]. Dulbecco’s Modified Eagle Medium: Nutrient Mixture F12 (DMEM-F12), 10% FCS-Joklik’s MEM, PE-conjugated anti-Sca-1, FITC-conjugated anti-CD34, PE-CY5-conjugated anti-CD45 and PE-CY7-conjugated anti-CD31 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Dispace was purchased from Roche (Basel, Switzerland) and 0.1% protease type-XIV was procured from Sigma-Aldrich (St. Louis, MO, USA). Cell sorting was performed on a FACSAria I flow cytometer (BD, Franklin Lakes, NJ, USA).

The BASCs isolated from different animal groups were monitored by the xCELLigence RTCA DP station (ACEA Biosciences, San Diego, CA, USA). Roughly 5*10³ cells were added to each well of a 96-well sample plate containing 100 μL MEM with 10% fetal bovine serum. The presence of the cells could modify the local ionic environment around the electrodes located at the bottom of the well, causing the electrode impedance to increase. The amount of increase was correlated to the total number of cells attached to the electrodes and the total overlapping areas between the cells and the electrodes.

Continuous variables are expressed as mean ± SD. Statistical analysis was performed with the aid of SPSS (version 20.0; SPSS Inc, Chicago, IL, USA) using either ANOVA or Student-Newmann-Keuls multiple comparison test. P<0.05 was considered statistically significant.

We began our study by comparing the extents of lung injury in the three experiment groups. As expected, the total BAL protein concentration in rats increased significantly as a result of CPB surgery (Fig 1a), which indicated increased permeability of the alveolar-capillary membrane, and concomitantly, pulmonary injury. This was further confirmed by the H&E staining results that showed intra-alveolar infiltration of neutrophils and thickening of the alveolar walls in animals of the CPB group compared to the control subjects (Fig 1b and 1d). However, it was also apparent that the treatment with limb RIPC could significantly mitigate the CPB-induced lung injuries as reflected by the reduction in the total BAL protein level and the alleviation of the abovementioned histological abnormalities (Fig 1a, 1b and 1d). Similarly, we measured the lung wet/dry weight ratios in the three groups as an indicator of pulmonary edema. As illustrated in Fig 1c, rats in the CPB group exhibited a significant elevation in the lung wet-to-dry weight ratio compared to the control, suggesting an abnormal accumulation of fluids in their lungs. In comparison, the same ratio in the treatment group showed a significant decline in comparison to that of the CPB group (Fig 1c). These data combined demonstrated that limb RIPC could attenuate CPB-induced lung injury.

We then investigated whether the RIPC-treated rats would also show improvement in their lung function. Indeed, the average TLC dropped significantly from 1.3 mL in the control group to 1.0 mL in the CPB group (Fig 2a). Meanwhile, the rats subjected to limb RIPC showed a partial restoration of TLC to 1.2 mL (Fig 2a). On the other hand, CPB was also found to significantly increase R_aw and lower C_dyn of the animal subjects, both of which served as further evidence of impaired lung function (Fig 2b and 2c). In these cases, the average R_aw and C_dyn of the treatment group displayed intermediate values between those of the control and of the CPB group (Fig 2b and 2c). Measurement and comparison of the lung P-V curves also corroborated the above findings as a downward shift was observed for the CPB group in comparison to the control, the trend of which was partially reversed in the treatment group (Fig 2d). Taken together, we concluded that the mitigation of pulmonary injury by limb RIPC in rats that underwent CPB surgery could also significantly ameliorate their lung function.

BASCs were directly isolated from the lungs that were dissected from euthanized rats and monitored on a real-time cell analyzer. The results showed that BASCs obtained from the CPB group proliferated at a much slower rate compared to those isolated from the control group during the 20-h observation period (Fig 3). In contrast, treatment with limb RIPC substantially improved the proliferative capacity of the cells (Fig 3). These results, taken together, suggested that the application of limb RIPC might improve the viability and proliferative capacity of BASCs, leading to enhanced tissue repair in the CPB-injured lungs.

To probe the possible mechanism through which limb RIPC helped reduce pulmonary injury and the loss of lung function as a result of CPB surgery, we performed blood gas analysis and also measured the levels of various inflammatory cytokines. As summarized in Table 1, the rats that underwent surgical procedure with CPB exhibited significantly lower arterial pH, O₂ tension and overall arterial pressure than the control group. The treatment with limb RIPC was found to restore these matrices to levels similar to those observed in the control subjects. On the other hand, we also found a decline in the mean arterial CO₂ tension and the level of serum bicarbonate, as well as a rise in the concentration of serum lactate among the rats in the CPB group compared to those in the control group, although the differences were not statistically significant. The results of the serum cytokine assay revealed that two anti-inflammatory cytokines, IL-4 and IL-10, were significantly down-regulated in the CPB group compared to the control group (Fig 4). As expected, limb RIPC was shown to improve the levels of both cytokines (Fig 4). The results suggested that the pulmonoprotective benefits of limb RIPC were likely associated with its regulation of lung inflammation, particularly its up-regulation of anti-inflammatory cytokines.