Male C57BL/6 mice (28 mice in total, 26-30 days old, weighing 11-14 g) were purchased from the Changsha SLAC Experimental Animal Center (Changsha, China). All mice were given water and food ad libitum under a 12/12-h light-dark cycle under specific pathogen-free conditions, and were bred to 10-12 weeks for establishing our model. All in vivo experiments were approved by the local Animal Health Service, the Central South University Animal Care Committee and were performed according to the National Institutes of Health guidelines on animal care and welfare.

The mice (28 mice were randomly divided into 4 groups, n=7 per group, 4 groups in total, 10-12 weeks old) were exposed to air or CS, as previously described (26). Briefly, the whole bodies of the mice were exposed to CS from 8 cigarettes simultaneously (without a filter; Baisha, Changsha, China) in a box (40×60×50 cm), 4 times for 25 min each at 20-min intervals; exposure to CS was carried out 5 days per week for 10 weeks. Baisha Pai cigarettes contain 10 mg of tar, 1.0 mg of nicotine and 13 mg of carbon monoxide (CO). The animals in the control group were exposed to room air (RA). All animals received siRNA-Nur77-lentivirus (siRNA-Nur77 sequence, 5'-CGC CTG GCA TAC CGA TCT AAA -3') or lentiviral vector vehicle (50 µl containing 1×10⁹/mouse/treatment) on the first day of each week.

The mice were anesthetized by an intraperitoneal injection of pentobarbital (40 mg/kg) at 24 h after the final CS exposure. One main bronchus was ligated, and the lung was perfused with 4% paraformaldehyde at a constant pressure of 2.45 kPa for 1 h before being removed from the animal and then was placed into fresh 4% paraformaldehyde for 24 h. The fixed lungs were embedded in paraffin and then were cut into 4-µm-thick lung tissues slices. Some lung slices were dewaxed with xylene, hydrated with ethanol and then stained with 20% hematoxylin for 5 min and 0.5% eosin for 3 min at room temperature (H&E, G1120, Solarbio Science & Technology, Beijing, China) for the analysis of inflammation and emphysema. A total of 10 of random fields were examined using a Leica digital microscope (Leica DM6Bl Leica, Wetzlar, Germany). Histopathology was graded by experienced pathologists using a semi-quantitative histology score system (27). Nur77 expression in lung tissues was detected by IHC according to a previously described protocol (12). Briefly, the lung tissues slices were incubated with an anti-Nur77 antibody (1:400; NB100-56745; Novus Biologicals, Centennial, CO, USA) overnight at 4°C, incubated with anti-rabbit IgG (ZB2301,1:1,000) for 30 min at room temperature, and then stained with 3,3'-diaminobenzidine (DAB, DA1010, Solarbio Science & Technology) as the chromagen. Finially, the slides were counterstained with hematoxylin, dehydrated and mounted. In total, 10 random fields were examined using a Leica digital microscope.

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco/Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco's phosphate-buffered saline (DPBS) was purchased from Invitrogen/Thermo Fisher Scientific. In addition, 3-methyladenine (3-MA) and rapamycin (RAPA) were purchased from Selleck Chemicals (Shanghai, China), and leptomycin B (LMB) was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). The anti-Nur77 antibody was purchased from Novus Biologicals, and anti-LC3 (cat. nos. 12741 and 2775) and anti-GAPDH antibodies (cat. no. 5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Beclin-1 (cat. no. ab210498) and anti-Bcl2 (cat. no. ab692) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-rabbit and mouse IgG (ZB2301, ZB2305, 1:2,000) were purchased from Zhong Shan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).

CSE was prepared using a smoke device as previously described (28). A filtered cigarette (Baisha Pai) was combusted with a vacuum pump (VWR International, West Chester, PA, USA), and the smoke was directed via a tube through 5 ml of PBS. This solution, which was considered to be 100% CSE, was filtered through a 0.22-µm pore filter (Lida Manufacturing Corp., Kenosha, WI, USA) for sterilization. Different preparations of CSE were standardized by measuring the absorbance at a wavelength of 320 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Freshly prepared CSE was diluted with culture medium supplemented with 10% FBS immediately prior to use and was used within 30 min.

Human bronchial epithelial (HBE) cells and A549 cells were kindly provided by the Central South University Advanced Research Center (Changsha, China) and maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. CSE and other reagents (rapamycin, 3-MA, LMB, siRNA) were added to the HBE or A549 cells at the indicated concentrations and for the indicated periods of time (see corresponding figure legends for details).

siRNA-Nur77 (sequence, 5′-TCGAGGACTTCCAGGTGTA-3′) and a negative control sequence (cat. no. siN05815) were obtained from RiboBio (riboFect™CP; Guangzhou, China). The cells in the exponential growth phase were seeded in 6-well plates at 4×10⁵ cells per well, grown to 40-50% density for 24 h, and transfected with siRNA-Nur77 or negative control for a further 36 h according to the manufacturer's recommended protocol. siRNA-Nur77, pre-mixed with riboFECT™CP buffer and riboFECT™CP reagent, was added to the medium at a final concentration of 100 nM. Western blot analysis was performed to evaluate the transfection efficiency.

The inhibition ratio of CSE was measured with a Cell Counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay, as specified. A total of 1×10⁴ HBE cells or 5×10³ A549 cells were seeded into 96-well plates, grown to nearly 40% confluence and transfected with siRNA or negative control for 36 h. Subsequently, the cells were treated with or without rapamycin (1 mM) or 3-MA (2.5 mM) or left untreated for 2 h and then exposed to 5% CSE for a further 24 h. After all the treatments were performed, 10 µl of CCK-8 were added to each well for 1 h at 37°C, and the absorbance was detected at 450 nm with a microplate reader (BioTek Instruments, Winooski, VT, USA).

A total of 1×10⁵ HBE cells or 5×10⁴ A549 cells were seeded in 6-well glass-bottom dishes and subjected to various treatments for the indicated periods of time. The treated cells were washed and fixed in 4% paraformaldehyde for 20 min, treated with TritonX-100 for 15 min, and blocked with 5% bovine serum albumin (BSA) for 30 min. The cells were then incubated with primary antibodies (anti-Nur77, 1:200; anti-Bcl2, 1:200; anti-LC3, 1:200) overnight at 4°C, washed with PBS, and then incubated with fluorescent secondary antibodies (anti-rabbit IgG: 711-025-152, anti-mouse IgG: 715-545-150; 1:200 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h at 37°C; after washing with PBS, and the cells were stained with DAPI (C1005, Beyotime Institute of Biotechnology, Haimen, China) for 5 min at room temperature. Following washing with PBS, the coverslips were sealed by anti-fade mounting Medium (P0126-5 ml, Beyotime Institute of Biotechnology). Images were captured under a fluorescence microscope (Leica, Buffalo Grove, IL, USA).

Total proteins were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology) and quantified with the bicinchoninic acid (BCA) method to ensure equal concentration. Loading buffer and total proteins were mixed and heated at 95°C for 10 min to denature albumin. Equal quantities of the samples (30-50 µg/well) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10-15%) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membranes were incubated with corresponding primary antibodies (anti-Nur77, 1:1,000; anti-LC3, 1:1,000; anti-Beclin-1, 1:1,000; anti-GAPDH, 1:1,000) at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies (anti-rabbit IgG: 1:2,000) for a further 1 h at 37°C, and the proteins were detected with an enhanced chemiluminescence (ECL) reagent (Millipore) and an ECL system (Syngene, Cambridge, England, UK). ImageJ 1.48v software (National Institutes of Health) was used to measure the gray value of each band.

The HBE and A549 cells were collected and fixed with 2.5% glutaradehyde overnight at at 4°C; they were then washed, and fixed with 1% osmium tetroxide for 1 h. Subsequently, the cells were dehydrated with a graded ethanol series, and embedded in epoxy resin (CAS 25068-38-6; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Ultrathin sections were detected using a transmission electron microscope (Hitachi Ltd., Tokyo, Japan).

The HBE cells were treated with LMB (10 ng/ml) for 2 h or left untreated, and were then exposed to 5% CSE for a further 24 h. The cells were collected, lysed in immunoprecipitation (IP)-RIPA lysis buffer, and then centrifuged at 10,000 × g for 10 min at 4°C. The supernatants were collected and quantified with the BCA method to ensure equal concentration; 10% and another 3% of total proteins were used as input experiments. The remaining were incubated with anti-Nur77 (1:50), anti-Bcl2 (1:20), or anti-Beclin-1 (1:30) antibodies or with the same species IgG (anti-GAPDH rabbit antibodies, cat. no. 5174; and anti-GAPDH mouse antibodies, cat. no. 51332; 1:20 dilution; Cell Signaling Technology) as a negative control overnight at 4°C. Protein G agarose beads were pre-washed 3 times in IP-RIPA lysis buffer and then incubated with the protein/antibody mixtures at 4°C for 4 h on shaking tables. The supernatant was discarded and the precipitate was retained and washed 4 times with IP-RIPA lysis buffer. Loading buffer was added to the precipitate; and the mixture was then heated at 95°C for 5 min. After the beads were discarded, the supernatant was analyzed by western blot analysis.

The data are expressed as the means ± standard error of the mean (SEM). The data were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison tests with GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). Differences with P-values <0.05 were regarded as statistically significant.

Nur77, which plays a critical role in inflammation and in cell proliferation, survival and death, is overexpressed in the majority of lung cancer and PASMC proliferation models (11). However, limited information of the mechanisms through which CS affects the expression of Nur77 is available. Thus, in this study, in order to shed light into this matter, C57BL/6 mice were bred to 10-12 weeks and were randomly divided into 4 groups, and the weight growth of each group did not differ significantly (compared to weight at purchase, data not shown). The C57BL/6 mice were exposed to RA or CS for 10 weeks, and lung tissues were collected the day after the final CS exposure. We found that exposure to CS significantly decreased the weight gain of the mice; however, lentivirus infection did not (Fig. 1B, compared to initial weight: Weight of 26-30-day-old mice, 11-14 g). Nur77 expression in the lung tissues was examined by immunohistochemical staining and western blot analysis. The number of Nur77-positive pulmonary epithelial cells in the CS-exposed group was higher than that in the RA group (Fig. 1A and C). Western blot analysis confirmed that CS increased the Nur77 levels in the lung tissues (Fig. 1D and E). We also examined the effects of various concentrations of CSE on Nur77 expression at the cellular level. The Nur77 levels significantly increased with the increasing CSE concentrations, and the group of HBE cells treated with 5% CSE, expressed higher Nur77-levels than the other groups (Fig. 1F and G). To further confirm the upregulation of Nur77 by CSE, we also examined the Nur77 levels in A549 cells exposed to various concentrations of CSE. Consistent with our findings with the HBE cells, the Nur77 levels were elevated with the increasing CSE concentrations (Fig. 1H and I). These results demonstrated that CS and CSE increased the Nur77 levels in pulmonary epithelial cells.

Exposure to CS results in pulmonary epithelial cell dysfunction and eventual cell death. Lung tissues and pulmonary epithelial cells treated with CS and CSE overexpressed Nur77; however, whether the high Nur77 levels participate in the pathophysiological processes of pulmonary epithelial cell dysfunction and cell death remained unclear. The results of this study revealed that CS-exposed mice exhibited more severe destruction of the pulmonary alveoli and bronchial tubes than RA-exposed mice; however, intratracheal inoculation with siRNA-Nur77 lentivirus mitigated the observed CS-induced lung injury (Fig. 2A and B). We further found that CSE reduced the viability of the HBE and A549 cells, an effect that was attenuated by the knockdown of Nur77 (Fig. 2C and D).

The autophagy of lung epithelial cells is involved in the pathological process of CS-induced COPD. In this study, combined treatment with CSE and RAPA (an autophagy activator) accelerated cell death, while 3-MA (an autophagy inhibitor) effectively attenuated CSE-induced cell death (Fig. 3A). It has been demonstrated that Nur77 mediates autophagic cell death in mammalian cells (29). Hence, we hypothesized that Nur77 may be involved in CSE-induced autophagy. To investigate the role of Nur77 in CSE-induced autophagy, the HBE cells were treated with 5% CSE for 24 h, and the expression levels of LC3 (an autophagy marker; an increasing LC3II/LC3I ratio indicates autophagy induction) and Beclin-1 (responsible for autophagy initiation) were then examined. We found that the LC3I to LC3II conversion and Beclin-1 expression increased synchronously with Nur77 upregulation (Fig. 3B-D). siRNA-Nur77 inhibited the expression of Nur77 (Fig. 3B), and the knockdown of Nur77 with siRNA-Nur77 attenuated the ability of CSE to upregulate LC3II/LC3I and Beclin-1 expression (Fig. 3B-D). These data indicate that the CSE-induced upregulation of Nur77 is linked to CSE-induced autophagy. A similar result was observed in the A549 cells co-treated with siRNA-Nur77 and CSE (Fig. 3E-G). Moreover, we found that punctate LC3 staining was greater in the CSE-treated HBE and A549 cells than in the control cells, as indicated by immunofluorescence staining, while transfection with siRNA-Nur77 diminished punctate LC3 staining, and the conversion of LC3-I to LC3-II (Fig. 3H and I).

To determine whether Nur77 plays a key role in CS-induced autophagy in vivo, mice were exposed to CS and treated with siRNA-Nur77 lentivirus for 10 weeks, and LC3, and Beclin-1 protein levels were detected in the lung tissues by western blot analysis. The lung tissues from the group of mice co-treated with siRNA-Nur77 lentivirus and CS demonstrated lower levels of LC3I to LC3II conversion and a lower Beclin-1 protein expression than those from the group exposed to CS only, indicating that autophagy was inhibited (Fig. 4).

To confirm that autophagy is indeed induced by CSE, the HBE and A549 cells treated with 5% CSE were analyzed by TEM for the evidence of autophagy (Fig. 5). A greater abundance of double-membrane structures containing organelles and more mature autophagosomes containing electron-dense regions were evident in the CSE-exposed HBE cells than in the control cells (Fig. 5A). Similar to the HBE cells, the CSE-exposed A549 cells exhibited much more autophagic features compared to those of the untreated group (Fig. 5B). We then examined whether Nur77 is involved in morphological changes consistent with autophagy. The presence of autophagic structures was markedly reduced in the CSE-exposed HBE and A549 cells following transfection with siRNA-Nur77 (Fig. 5). This finding supports our hypothesis that CSE-induced Nur77 upregulation is responsible for CSE-induced autophagy.

Bcl-2 family proteins are key regulators of programmed cell death. It has been previously clarified that Nur77 interacts with Bcl2 and can convert the function of Bcl2 from a protective function to a pro-death function; in addition, some stimuli can regulate the interaction of Nur77 and Bcl2 to induce cell apoptosis (30). The Nur77-Bcl2 complex also induces autophagy (30). In this study, we observed the Nur77 transfer to the cytoplasm in CSE-exposed HBE cells, while LMB prevented the CSE-induced Nur77 nuclear export (Fig. 6A). To clarify whether CSE promotes the interaction between Nur77 and Bcl2, double immunofluorescence was first used to examine whether Nur77 co-localizes with Bcl2 in the cytoplasm of HBE cells that were exposed or not to CSE. When the HBE cells were exposed to CSE, Nur77 and Bcl2 co-localized in the cytoplasm, while in the control cells, Nur77 existed mainly in the nucleus, and Bcl2 was distributed in the nucleus and cytoplasm (Fig. 6B). Subsequently, we investigated whether the interaction between Nur77 and Bcl2 was enhanced in CSE-exposed cells with a co-immunoprecipitation assay. In the absence CSE, only endogenous Nur77 and a small amount of Bcl2 were pulled down by an anti-Nur77 antibody. Upon CSE exposure, a greater amount of Nur77 co-immunoprecipitated with Bcl2 (Fig. 7A). The same outcome was detected when the cell lysates were immunoprecipitated with an anti-Bcl2 antibody (Fig. 7B). These results indicate that CSE promotes the interaction between Nur77 and Bcl2.

The autophagic function of Beclin-1 is inhibited by Bcl-2 and is induced when Bcl-2 dissociates from Beclin-1 (24,30). It has been verified that the interaction of Nur77 and Bcl2 family proteins augments autophagy (30). The results of this study indicated that CSE induced autophagy by the elevating Nur77 levels and increasing the interaction between Nur77 and Bcl2. To determine whether the CSE-induced increase in Nur77-Bcl-2 complexes increases dissociation of Beclin-1 from Bcl2, we then triggered Beclin-1-dependent autophagy. We first performed co-immunoprecipitation experiments with the HBE cells with or without CSE exposure to detect the endogenous levels of both Bcl2 and Beclin-1. We observed that an anti-Bcl2 antibody and an anti-Beclin-1 antibody pulled down both Bcl2 and Beclin-1 in the absence of CSE; in addition, smaller amounts of Bcl2 and Beclin-1 were pulled down following exposure to CSE (Fig. 8). We then sought to determine whether the binding of Nur77 to Bcl2 prompts the dissociation of the Bcl2-Beclin-1 complex in HBE cells exposed to CSE. LMB inhibits Nur77 nuclear export and then prevents Nur77 from interacting with Bcl2. HBE cells were thus treated with CSE in the presence or absence of LMB. Through co-immunoprecipitation assays, we discovered that the amounts of Bcl-2 and Beclin-1-pulled down by the anti-Bcl2 antibody were lower in the CSE-exposed group than in the control and LMB groups (Fig. 8). These observations confirm that CSE induces the interaction between Nur77 and Bcl-2, which leads to the dissociation of Bcl-2 from Beclin-1.