A total of 60 adult male Sprague Dawley rats, 2-4 months old, weighing 200-250 g, were housed at 22˚C, with a 12-h light/dark cycle, and with relative humidity maintained at 40-60% and with free access to food and water in the Henan Provincial Experimental Animal Center, Zhengzhou, China. The experimental protocols complied with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (21).

Animals were anesthetized with 1% pentobarbital sodium (60 mg/kg) by intraperitoneal injection (ip), and their arterial pressure was monitored via the femoral artery. Venous access was established through the femoral vein and the urine volume was monitored by cystostomy. The rats were intubated by tracheotomy mechanically, with an oxygen fraction of 100% (Harvard Apparatus). Next, a 2-mm hole was drilled with a marathon-3 dental grinder (Saeyang Co., Ltd.) through the skull, 4-mm lateral to the sagittal suture. Next, a 3F Fogarty Catheter (Edwards Lifesciences) was placed and inflated with 20 µl fluid every 5 min until BD was achieved (11). During the entire experimental procedure, SpO₂ remained >90% and the SBP remained ≥90 mmHg.

BD was confirmed by induction of a deep coma, spontaneous respiratory arrest, mydriasis, absence of brainstem reflexes and an amplitude of the electroencephalogram <0.02 v.

Animals were randomly assigned into the following groups: i) Sham operation group (sham, n=6); ii) BD groups (the BD subgroups were divided into BD 0.5, 1, 2, 4 and 6 h according to the time of specimen collection; n=6 per group); iii) MG132 groups, in which MG132 (cat. no. m1902; Abmole Bioscience Inc.) was dissolved in DMSO, diluted to 10 mmol/l with PBS and was administered at a dose of 10 mg/kg (ip) ~30 min before BD induction (22) (the MG132 subgroups were divided into BD 2 h + MG132 and BD 6 h + MG132; n=6 per group); and iv) control groups in which an equivalent volume of DMSO to that in the MG132 group 30 min before BD induction (the control subgroups were divided into BD 2 h + control and BD 6 h + control; n=6 per group).

The blood was drawn from the femoral artery to measure partial artery pressure of oxygen (PaO₂). Rats were sacrificed by exsanguination of the abdominal aorta until cardiac arrest was achieved. The right lower lung was fixed with 4% paraformaldehyde at room temperature for 7 days, the right middle lung after removal was quickly placed in liquid nitrogen and then transferred to be frozen in a -80˚C refrigerator for the next experiments within 3 months and the weight of the left lung was measured (wet weight and dry weight after 1 week in a 60˚C oven). Bronchoalveolar lavage fluid (BALF) was obtained by lavage of the left main bronchus with 2 ml normal saline repeated three times after clipping the right main bronchus according to previous research methods (11). After mixing, 10 µl of BALF was collected into the cell counter (Jiangsu Jimbio Technology Co., Ltd.) to measure the cell concentration in BALF, and these data are displayed below. The BALF was centrifuged at 13,800 x g/min for 10 min at 4˚C, the cell sediment was resuspended with 500 µl normal saline to make cell smears to observe the cell morphology by using hematoxylin and eosin (H&E) staining as described below, and the supernatant was obtained to measure the protein concentration using a bicinchoninic acid assay.

The right lower lobe was fixed in 4% paraformaldehyde for 7 days at room temperature, embedded in paraffin, sectioned at 4-µm and stained with H&E staining (11). The paraffin sections were heated at 60˚C oven for 1 h, dewaxed twice in xylene solutions for 10 min each and rehydrated in descending alcohol series. The paraffin slides were stained with hematoxylin for 5 min at room temperature and differentiated with 0.1% hydrochloric acid ethanol for 1 min, followed with eosin for 5 min at room temperature, dehydrated in ascending alcohol series, followed by being dewaxed twice in xylene solutions for 10 min each. The cell smears were fixed with 95% alcohol for 10 min, and the following steps of staining were the same as those for paraffin sections. The slides were captured using a conventional light microscope (Axiolab 5; Zeiss GmbH) at a magnification of x200. A semiquantitative severity-based scoring system was used as previously described in the reference (23): i) Intra- and extra-alveolar hemorrhage; ii) intra-alveolar edema; iii) inflammatory infiltration of the inter-alveolar septa and airspace; iv) over-inflation; and v) erythrocyte accumulation below the pleura. Variables i) -iv) were graded as: 0=Negative, 1=slight, 2=moderate, 3=high and 4=severe. Variable v) was scored as 0=absent or 1=present. Lungs were scored by two blinded investigators, across 10 random, non-coincidental fields per section and then the mean values were analyzed.

The paraffin sections were heated at 60˚C oven for 1 h, dewaxed twice in xylene solutions for 15 min each and rehydrated in descending alcohol series. Antigen retrieval was performed with sodium citrate buffer (cat. no. C1031; Beijing Solarbio Science & Technology Co., Ltd. China) at 100˚C for 10 min. The slides were immersed in 3% H₂O₂ at room temperature for 30 min to inhibit endogenous peroxidase activity. The slides were then blocked with 10% normal goat serum (cat. no. WGAR1009-5; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 1 h. The slides were incubated with the primary antibody at 4˚C overnight followed with secondary antibody at room temperature for 1 h. The primary antibody used was a 20S proteasome β1 antibody (1:100 diluted in 1% BSA; cat. no. sc-374405; Santa Cruz Biotechnology, Inc.). The secondary antibody used was Biotin-conjugated Affinipure goat anti-mouse IgG (1:100; cat. no. SA00004-1; ProteinTech Group, Inc.). DAB was added for color development for 10 min and then counterstained with hematoxylin for 5 min at room temperature. Single sections from five rats per a group were evaluated. The slides were captured using a Nikon ECLIPSE Ni-E400 fluorescence microscope (Nikon Corporation). Semi-quantitative image analysis was performed by using the open-source software Image J 1.53e (National Institutes of Health) plugin the IHC profiler (24). The staining score was scored as 4 (high positive), 3 (positive), 2 (low positive) and 1 (negative); the staining positive number score was defined in at least five areas (x400 magnification per section) in a blinded manner and scored as 1 (<10%), 2 (10-49%), 3 (50-74%) and 4 (75-100%). The final score was defined as staining number score multiplied by staining color score as described before (25).

Immunofluorescence staining was performed as described previously (26). The paraffin sections were heated at 60˚C oven for 1 h, washed twice in xylene solutions for 15 min each, rehydrated in descending alcohol series, blocked with sodium citrate buffer (cat. no. C1031; Beijing Solarbio Science & Technology Co., Ltd. China) at 100˚C for 10 min, incubated with primary antibodies for 6 h at 4˚C, followed by secondary antibodies for 6 h at 4˚C in an opaque wet box and stained with DAPI for 10 min at room temperature in the dark. The primary antibodies used were 20S proteasome β1 (1:100 diluted with 1% BSA; cat. no. sc-374405, Santa Cruz Biotechnology, Inc.) and inducible nitric oxide synthase (iNOS; 1:100; cat. no. GB11119; Wuhan Servicebio Technology Co., Ltd.), myeloperoxidase (MPO; 1:100; cat. no. GB11224; Wuhan Servicebio Technology Co., Ltd.) and CD31 (1:100; cat. no. GB113151; Wuhan Servicebio Technology Co., Ltd.). The secondary antibodies used were Cy3-conjugated Affinipure goat anti-rabbit IgG (1:100; cat. no. SA00009-2; ProteinTech Group, Inc.) or Coralite488-conjugated goat anti-mouse IgG (1:100; cat. no. SA00013-1; ProteinTech Group, Inc.). An Olympus fluorescence microscope (Olympus Corporation) was used to obtain images at excitation/emission wavelengths of 547/570 nm (Cy3, red), 494/520 nm (Coralite488, green), and 360/460 nm (DAPI, blue) (original magnification x400).

The mRNA levels of Psmb1 were measured using RT-qPCR. Total RNA was extracted from lung tissues using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), and reverse transcription was performed as described previously (27). Synthesis of cDNA and sample preparation were performed according to the manufacturer's instructions for the PrimeScript RT reagent kit (Takara Bio, Inc.) and SYBR Premix Ex Taq kit (Takara Bio, Inc.), respectively. qPCR was performed using a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Inc.). The thermocycling conditions were 95˚C for 30 sec, 95˚C for 5 sec and 60˚C for 35 sec (40 cycles). The method of quantification (2^-ΔΔCq method) was performed as described previously (28). The primer sequences (Invitrogen; Thermo Fisher Scientific, Inc.) used in this experiment are provided in Table SI.

Rat alveolar macrophages (NR8383 cell line; The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) have been demonstrated to closely mimic the important biological characteristics of normal alveolar macrophages previously, and have been used instead of primary alveolar macrophages (29,30). The cells were cultured in a semi-suspension with Ham's F-12 Nutrient Mixture (cat. no. GNM21700; Genom) supplemented with 20% FBS (cat. no. WGG8001-100; Wuhan Servicebio Technology Co., Ltd.) at 37˚C, in a humidified incubator supplied with 20% O₂ and 5% CO₂. NR8383 cells were serum starved for 6 h to ensure synchronization of the cell cycle and were pretreated with 10 µM MG132 (MG132 group) or an equivalent volume of DMSO vehicle (control group) for 1 h. The cells were then cultured in an incubator supplied with 1% oxygen for 2 h or 6 h to mimic hypoxia (30). Cells were then treated as follows: Half of the medium was absorbed, and the same amount of 40% FBS and medium along with the same drug concentration was added and reoxygenated in the normoxic incubator for 2 h.

A total of 1x10⁶ cells/ml NR8383 cells were prepared according to the instructions of the Annexin V-FITC/PI Apoptosis Detection kit (cat. no. ca1020; Beijing Solarbio Science & Technology Co., Ltd.). Briefly, NR8383 cells were harvested and resuspended with 100 µl binding buffer. Cells were incubated with 5 µl Annexin V-FITC at room temperature for 5 min in the dark, then 5 µl PI and 400 µl of PBS were added. Apoptosis was detected using a flow cytometry (BD FACSCantoII; BD Biosciences) and analyzed using BD FACSDiva Software v8.0.1 (BD Biosciences).

The proteins were extracted from lung tissue or cells and lysed by using RIPA buffer (high; cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) on ice for >30 min. Protein levels were quantified using BCA reagent. Samples were loaded 10 µl per lane and electrophoresed by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% non-fat milk for 2 h at room temperature. The membranes were incubated with primary antibodies at 4˚C overnight. The primary antibodies used were: 20S proteasome β1 (1:500; cat. no. sc-374405; Santa Cruz Biotechnology, Inc.), iNOS (1:1,000; cat. no. AF0199; Affinity Biosciences), cleaved caspase 3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), p-JNK (1:1,000; cat. no. ET1609-42; HUABIO, Inc.), total JNK Antibody (1:500; cat. no. ET1601-28; HUABIO, Inc.) and GAPDH (1:5,000; cat. no. 60004-1-lg; ProteinTech Group, Inc.). The secondary antibodies used were horseradish peroxide-conjugated goat anti-mouse IgG (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.) and goat anti-rabbit IgG (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.). The bands were visualized using the Chemiluminescent Substrate kit (cat. no. PE0010; Beijing Solarbio Science & Technology Co., Ltd.) combined with a Bio-Rad exposure system (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ 1.53e.

All experiments were repeated three times. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.). The ordinal data (hispathological injury scores, IHC scores) are presented as median and range, and differences between sham group and BD groups were analyzed using the Kruskal-Wallis test followed by Dunn post hoc tests. Other data are presented as the mean ± SD, and differences in characters [PaO₂/fractional concentration of inspired oxygen (FiO₂), Wet/Dry weight ratio of left lung, BALF cells count, BALF protein content, relative protein level, relative expression of Psmb1) between sham group and BD groups were analyzed using a one-way ANOVA followed by Bonferroni's test in the post-hoc comparison. Differences in other characters between control groups and MG132 groups were analyzed using a one-way ANOVA followed by unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the lung injury after BD in rats, the oxygenation index (PaO₂/FiO₂), the wet/dry weight ratio, H&E staining, and histological lung injury scores were used to evaluate the standard of lung injury in lung tissue at different time points after BD. The PaO₂/FiO₂ of the BD groups were significantly lower compared with that of the sham group (Fig. 1A). The wet/dry weight ratios of the left lung in the BD groups were significantly higher compared with that of the sham group (Fig. 1B). Compared with the sham group, pulmonary alveolar wall thickening, pulmonary hemorrhage, and neutrophil infiltration were observed in the BD rats (Fig. 1C); the histological lung injury scores increased over time and were significantly higher compared with that in the sham group (Fig. 1D). Based on the microscopic images, a notable increase in the proportion of cells in the BALF was observed (Fig. 1E). The cell count and the protein content in the BALF was higher in the experimental group compared with that in the sham group (Fig. 1F and G). These results indicated that lung injury gradually increased in rats after BD.

The 20S proteasome β1 is a subunit of the 20S proteasome with caspase-like activity that is inhibited by MG132(31). Immunohistochemical semi-quantitative analysis, western blotting and RT-qPCR were used to detect the expression of the 20S proteasome β1. As presented in Fig. 2, induction of BD significantly increased the expression of the 20S proteasome β1 in the lung tissues compared with the sham group (Fig. 2A, B, F and G).

Notably, the 20S proteasome β1 positive cells exhibited an alveolar macrophage-like morphology. To confirm the upregulation of the 20S proteasome β1 in alveolar cells from the lung tissues following BD, immunofluorescence staining was used to detect the co-localization of 20S proteasome β1 and iNOS/MPO/CD31 in lung tissue sections of rats after BD (MPO is considered as one of markers of neutrophil activation, CD31 is one of the markers of endothelial cell and iNOS is considered one of the markers of macrophages). The results indicated the presence and the differences in the co-localization of the 20S proteasome β1 and iNOS/MPO/CD31 in the lung tissues, and 20S proteasome β1 mostly colocalized with iNOS, but not with MPO and CD31 (Fig. 2C-E) to rule out the increased high expression of 20S proteasome β1 on neutrophils/endothelial cells. Compared with the sham group, induction of BD significantly increased the protein expression of the 20S proteasome β1 and iNOS (Fig. 2F and G) and also increased the relative Psmb1 levels (Fig. 2H).

To explore the effect of proteasomal inhibition on lung injury following BD in rats, a proteasome inhibitor, MG132, was administered before induction of BD and the specimens were collected for analysis at different time points following BD. Compared with the control group, MG132 treatment was revealed to significantly increase the PaO₂/FiO₂ after BD (Fig. 3A) and significantly reduce the left lung wet/dry weight ratio 6 h after BD (Fig. 3B). However, MG132 treatment also alleviated the thickening of the alveolar wall, pulmonary hemorrhage, and neutrophil infiltration (Fig. 3C), whilst also significantly decreasing the histological lung injury scores compared with the control group (Fig. 3D). As presented in Fig. 3E and F, MG132 treatment significantly reduced the protein expression levels of the 20S proteasome β1 and iNOS in lung tissues following BD at 2 and 6 h. These results suggested that inhibition of the proteasome by MG132 decreased lung injury after BD.

Previous studies suggest that in the acute stage of infection or BD, alveolar macrophages are rapidly recruited and they secrete a large quantity of harmful substances, which in turn attack the lung tissues and induce lung tissue injury (32,33). As the upregulation of 20S proteasome β1 in alveolar macrophages was confirmed, whether MG132 treatment protected lung tissues via induction of apoptosis of alveolar macrophages was next assessed. To test this hypothesis, NR83883 cells (rat alveolar macrophage cells) were used as an in vitro model. Flow cytometry and western blotting were used to detect the apoptosis of NR8383 cells after hypoxia treatment for 2 or 6 h, followed by re-oxygenation for 2 h. Compared with the control group, MG132 treatment significantly increased the apoptotic rate of NR8383 cells (Fig. 4A and B) after 2 h of hypoxia and 2 h of re-oxygenation (H/R-2/2), and after 6 h of hypoxia and 2 h of re-oxygenation (H/R-6/2). In addition, MG132 administration significantly increased the protein expression levels of p-JNK and cleaved-caspase 3 in NR8383 cells after H/R-2/2 and H/R-6/2 compared with the control group (Fig. 4C and D). These results highlighted that inhibition of proteasomal activity using MG132 increased cell apoptosis after H/R in cultured rat alveolar macrophages.