The function role of ubiquitin proteasome pathway in the ER stress-induced AECII apoptosis during hyperoxia exposure

Sprague–Dawley rats (SD, 90–100 days old, 250–300 g) were provided by the Animal Center of the Jiangsu University (Zhenjiang, China). The BPD animal model was constructed as previously described [18]. Newborn SD rats were randomly divided into two groups, and were exposed to hyperoxia (80–85% O₂; hyperoxia group) and room air (21% O₂; normoxia group) at the beginning of the day of their birth. Three newborn rats from different litters per group were dissected and their lungs were removed at the postnatal day 7 and day 14 (P7 and P14). The left lungs were fixed with 4% paraformaldehyde and the right lungs were stored at − 80 ˚C.

Tissues were fixed with 4% paraformaldehyde for 24 h at 4˚C and washed with PBS. Subsequently, samples were dehydrated using an alcohol gradient (75% alcohol, 1.5 h; 95% alcohol, 1.5 h; 100% alcohol, 1.5 h; 100% alcohol, 1 h; two xylene washes, 0.5 h each) and embedded in paraffin. Sections were sliced at 3 µm, followed by conventional dewaxing in water. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) and boiled for 20 min. The tissue sections were stained with hematoxylin and eosin (Solarbio Science & Technology Co., Ltd., China) 2–3 min each for histological analysis. All steps were performed at a room temperature. The sections were acquired by confocal light microscopy (Olympus Corporation, Japan) at magnification of × 400. Radial alveolar counts (RACs), representing alveolar septation and alveologenesis, were determined by standard morphometric techniques [19]. From the center of the respiratory bronchiole, a perpendicular was drawn to the edge of the acinus, defined by a connective tissue septum or the pleura, and the number of septa intersected by this line were counted.

TUNEL assay was applied to detect the apoptotic cells in lung tissue samples. Lung tissues were fixed with 4% paraformaldehyde, embedded in paraffin wax, and sectioned into 4 μm thick slices. The tissue section was then dewaxed and rehydrated. A Dead End Fluorometric TUNEL System (Vazyme Biotech Co., Ltd., China) was used to perform the TUNEL assay on the isolated lung tissue, following the manufacturer’s instructions. Finally, 5 random fields were selected in each section and the TUNEL-positive cells were calculated by an inverted fluorescent microscope at magnification of × 400. The apoptosis index was determined as the percentage of the total cells positive for TUNEL.

At the gestational age of 19–20 days, the lung of fetal rat was in the small tubular phase. In this period, AECII produce a large number of lamellar bodies and secrete large amounts of alveolar surfactant. The structure and functional characteristics of human fetal lung at 34–35 weeks are similar with that of prenatal day 19–20. So we selected 19 fetal rat for this study. Isolation and culture of fetal AECIIs was performed as previously described [20, 21]. In brief, lungs of 19-day gestation fetal rats (Term 22 days) were removed, dissected free from connective and nonparenchymal pulmonary tissues. Cells were dispersed using a solution of trypsin, Dnase and collagenase. AECIIs were extracted from a cell suspension utilizing a property of fibroblasts and other lung cells to adhere to plastic. Freshly isolated AECIIs were plated at 5 × 10⁵ cells/mL in 50 ml culture flasks in 2 ml of MEM containing 10% fetal bovine serum. The cells were incubated for 18–20 h at 37 °C in 5% CO₂ atmosphere. AECIIs were characterized by their morphologic appearance and the presence of lamellar bodies. All cultures contained 94 ± 2% (mean ± SE) AECIIs as determined under phase contrast microscope.

AECIIs were divided into two groups according to the conditions of their maintenance, “normoxia group” and “hyperoxia group”. The normoxia group was cultured in 5% CO₂ incubator at 37 oC.The hyperoxia group was exposed to the stream of 95% O₂ and 5% CO₂ at a speed of 3 l/min for 10 min, then sealed and cultured in a parallel with normoxia group in 5% CO₂ incubator for 24, 48 and 72 h at 37° C. To explore ubiquitin proteasome pathway during hyperoxia exposure, AECII cells were divided into Control group (hyperoxia group), DMSO group (hyperoxia + DMSO) and MG132 group (hyperoxia + MG132). MG132 was purchased from MCE (MedChemExpress, USA) and dissolved in dimethyl sulfoxide (DMSO). The method of hyperoxia exposure is the same as before. The oxygen concentration in the cells of hyperoxia group was detected by CYS-1 digital oxygen monitor when the cells were harvested. The samples with oxygen concentration less than 90% were discarded and the remaining cells were harvested for the next experiment.

AECII cells were treated by MG132 with varying concentration (0, 5, 10, 15 and 50 μmol/L) and cell viability was detected by MTT assay. The MTT assay involves the reduction of the soluble yellow dye (MTT) to an insoluble purple formazan salt. Cells were cultured in a sterile 96 wells plate in 100 μl media and incubated overnight for attachment. AECII cells were treated by MG132 for 24 h, and at the end of reaction 50 μl of MTT dye (5 mg/ml) was added to each well and incubated further for 4 h at 37 °C in a CO₂ incubator. The formazan products formed in cells were dissolved in DMSO (100 µl) and absorbance was measured at 540 nm using multimode plate reader (Perkin Elmer, USA).

The cells were treated with 0.25% trypsin non-supplemented with EDTA (Invitrogen, USA), washed and resuspended in PBS. Apoptotic cells were identified by double supravital staining with recombinant FITC-conjugated Annexin V and PI, using the Annexin V/PI-FITC Apoptosis Detection kit (Becton, Dickinson and Company, USA) according to the manufacturer’s recommendations. After 15 min incubation in the dark, the samples were subjected to flow cytometry analysis using BD FACS CantoII flow cytometer (Becton, Dickinson and Company, USA), the data obtained were analyzed by Flow Jo software.

Activity assays were carried out in a 200 μL reaction volume. Different concentrations of test compounds were added to a black flat/clear bottom 96-well plate containing 1 nM of constitutive 20S proteasome in 50 mM Tris–HCl at pH 7.5 and allowed to sit for 10 min at RT. Fluorogenic substrates were then added and the enzymatic activity measured at 37 °C on a Spectra Max M5e spectrometer by measuring increase in fluorescence unit per minute for 1 h at 380/460 nm. The fluorescence units for the vehicle control were set to 100%, and the ratio of MG132-treated sample set to that of vehicle control was used to calculate the fold change in enzymatic activity.

Lung histology of the rats exposed to hyperoxia was characterized by decreased septation, distal air space enlargement and a reduction in complexity, which was similar to the histology observed in human infants with BPD [22]. Three newborn rats from different litters were dissected in each group. The survival rate of pups in normoxia and hyperoxia group were both 100%. To quantity the alveolar septation and alveologenesis, Radial alveolar count (RAC) were performed. Compared with the rats exposed to room air, RAC were lower in the rats exposed to hyperoxia (Fig. 1A). These results demonstrated the animal model of BPD was successful. Apoptosis was analyzed using a TUNEL assay following exposure to hyperoxia or room air for P 7 and P 14. As shown by the TUNEL results in Fig. 1B, apoptosis was induced in the lungs of the rats exposed to hyperoxia conditions. A few TUNEL-positive cells were observed in the control group, however, numerous positive cells were observed in the hyperoxia group (Fig. 1B). Under hyperoxia conditions, the levels of total ubiquitinated proteins were significantly increased relative to normal oxygen conditions at both P7 and P14 (Fig. 1C, which is consistent with our results on the apoptosis index in the lung tissue. Further reagent kit with specific fluorescent substrate was utilized to measure the activity of 20 s proteasome and revealed that the level of activity was significantly decreased during hyperoxia conditions (Fig. 1D). These results indicated that ubiquitin or ubiquitin proteasome pathway plays a vital role in apoptosis during the development of BPD.

To determine whether ubiquitin or ubiquitin proteasome pathway affects BPD development by participating in the apoptosis of AECII, AECII cells were cultured in vitro in the current study and exposed to hyperoxia. As shown by flowcytometry analysis (Annexin V/PI double staining), the apoptosis rate of AECII was low in normoxia group. However, time prolonged exposure to hyperoxia increased number of apoptotic cells and the differences between hyperoxia and normoxia group at 24, 48 and 72 h were statistically significant (P < 0.05), pointing out that hyperoxia may induce AECII apoptosis in a time-dependent manner (Fig. 2A). In contrast to the normoxia group, the hyperoxia group showed increased expression of total ubiquitinated proteins after 24, 48 and 72 h exposure to 95% oxygen (Fig. 2B). Compared with it in normoxia group, the level of 20 s proteasome activity was significantly decreased in hyperoxia group (Fig. 2C). Taken together, these results suggested that ubiquitin or ubiquitin proteasome pathway participate in apoptosis of AECII during hyperoxia condition.

GRP-78 is a sentinel marker of ER stress. To determine the effect of hyperoxia on the expression of the ER chaperone GRP-78, the protein levels were analyzed by Western blotting. The results indicated that the protein expression of GRP-78, was elevated after 24, 48 and 72 h exposure to 95% oxygen in comparison with it in normoxia group (P < 0.05) (Fig. 3A, B). Thus, 24 h exposure to hyperoxia was sufficient to activate ER stress in the AECIIs. The protein expression of CHOP was increasing gradually over time in the hyperoxia group compared with it in normoxia group (P < 0.05) (Fig. 3A, F), indicating that the exposure of AECIIs to 95% oxygen increased the expression of CHOP, a major pro-apoptotic transcription factor induced by ER stress. PERK and ATF6 were ERS sensors, ATF4 were their downstream-activated proteins that activated CHOP expression according to the previously reported mechanism [23‐25]. As shown in Fig. 3A, the PERK, ATF4 and ATF6 proteins were also increased after 24, 48 and 72 h exposure to hyperoxia versus the normoxia group (P < 0.05) (Fig. 3A, C, D, E). Combined with the above results in Fig. 2, we found that ERS is activated and 20S proteasome activity of UPP is damaged in AECII after hyperoxia exposure, which blocks the degradation of ubiquitinated proteins and causes accumulation in cells, resulting in increased AECII apoptosis. These results suggested that UPP may participate in the ERS mediated AECII apoptosis under hyperoxia condition.

MG132 is a natural peptide aldehyde proteasome inhibitor, which inhibits its activity by covalent binding with the β subunit of 20 s proteasome. AECII cells were treated by MG132 with varying concentration (0, 5, 10, 15 and 50 μmol/L) and cell viability was detected by MTT assay. After MG132 treatment for 72 h cell viability was decreased significantly and continued to show a significant decline with the increasing concentration of MG132 (P < 0.05). Compared with no 5 μmol/L MG132 treatment, cell viability were decreased after the 50 μmol/L treatment(P < 0.05), while no significant difference varying concentration of 5, 10 and 15 μmol/L(P > 0.05) (Fig. 4A). Furthermore, the activity of 20 s proteasome showed a decline with the increasing concentration of MG132 and were significantly decreased after 10 μmol/L MG132 treatment (P < 0.05), while slightly decreased after 15 and 50 μmol/L treatment compared with that after 10 μmol/L treatment (Fig. 4B). In view of these results, we finally chose 10 μmol/L as the experimental concentration of MG132 treatment. Compared with hyperoxia group (Control group) and hyperoxia group treated with DMSO (DMSO group), hyperoxia group treated with MG132 (MG132 group) showed a decreased activity of 20 s proteasome and an increased expression of total ubiquitinated proteins (Fig. 4C, D).

To examine whether MG132 treatment affects AECII apoptosis, flow cytometry analysis and an Annexin V-FITC/PI apoptosis detection kit was used. As shown in Fig. 5A, MG132 treatment promoted apoptosis compared with Control group and DMSO group (P < 0.05) (Fig. 5A). In contrast, the level of Caspase-3 protein expression and the ratio of Bax/Bcl-2 were increased after MG132 treatment (P < 0.05) (Fig. 5B). These results suggest that the inhibition of 20S proteasome activity leads to AECIIs apoptosis induced by hyperoxia. At the same time, Western blot were performed to analyze protein level of CHOP, ER chaperone GRP-78, as well as ERS sensors PERK, ATF4 and ATF6. As shown in Fig. 6, GRP78, PERK, ATF4, ATF6 and CHOP expression were all increased in varying degree after MG132 treatment(P < 0.05) (Fig. 6). Overall, it is valid to consider that MG132 can aggravate the ERS and AECII apoptosis under hyperoxia exposure, and this process is related to UPP.