A total of 32 female C57BL/6 mice (age, 6–8 weeks; weight, 20–22 g) were purchased from the Experimental Animal Center Medical College of Xi'an Jiaotong University (Xi'an, China). All animals were maintained in steel cages in a room with controlled temperature at 23–25°C, under a 12-h light/dark cycle in a specific pathogen-free facility, with access to food and water ad libitum. All experimental protocols were approved by the medical ethics committee of The Second Affiliated Hospital of Xi'an Jiaotong University. Mice were maintained in an animal facility under standard laboratory conditions for 1 week prior to experiments, and were randomly assigned into four groups (n=8/group): i) Control, ii) OVA, iii) EX-527 and iv) rapamycin groups. The OVA group was sensitized by subcutaneous injection of OVA (50 µg/kg) adsorbed to 2 mg aluminum hydroxide in 200 µl normal saline at 0, 7 and 14 days, then challenged by exposure to an aerosol containing 1% OVA (w/v) in PBS for 30 min using an ultrasonic nebulizer (Soniclizer305, ATOM) on days 21, 23, 25 and 27 after the initial sensitization. In the EX-527 group, mice received EX-527 administration (10 mg/kg) by intraperitoneal injection 1 h prior to OVA inhalation. The control group mice were sensitized to PBS and challenged with PBS aerosols.

To further define the role of mTOR activation in the effect of EX-527 on allergic airway inflammation, asthmatic mice were intraperitoneally treated with the mTOR inhibitor rapamycin (4 mg/kg) 30 min prior to EX-527 administration. A total of 24 h after the last OVA challenge, the mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg; Sigma-Aldrich; Merck KGaA) followed by cervical dislocation. Mice were considered dead when respiratory arrest and cardiac arrest were observed, their nerve reflexes disappeared, and muscles relaxed. Mice health and behavior were monitored every day for 28 days, and none of the rats became severely ill or moribund. Fig. 1 shows the treatment regimen through the course of the experiment. BALF was collected for determining cytokine levels and cell counts. The left lungs were preserved and fixed with 10% formalin for 24 h at room temperature and then stained with hematoxylin for 5 min and eosin for 3 min at room temperature for histopathological analysis under a light microscope (Olympus Corporation). The right lungs were collected and frozen at −80°C for further experiments.

OVA and rapamycin were purchased from Sigma-Aldrich (Merck KGaA). ELISA kits for detecting IL-4 [cat. no. KGEHC006(H)-1], IL-13 (cat. no. KGEMC124-1) and interferon (IFN)-γ (cat. no. KGEBC101g-1) were purchased from Yufeng Technology Co., Ltd. Rabbit anti-mouse SIRT1 (1:1,000; cat. no. ab110304), microtubule-associated protein 1 light chain 3β (LC3B; 1:1,000; cat. no. ab243506) and Beclin-1(1:1,000; cat. no. ab114071) antibodies, and EX-527 (cat. no. ab141506) were purchased from Abcam. Antibodies against total (t)-mTOR (1:1,000; cat. no. 2972) and p-mTOR (1:1,000; cat. no. 5536) were purchased from Cell Signaling Technology, Inc. Anti-β-actin antibodies (1:5,000; cat. no. AM33096PU-S) were from OriGene Technologies, Inc.

The left lobe of the lung was fixed in formalin and paraffin embedded. The sections were stained with hematoxylin and eosin for histopathological evaluation under a light microscope (Olympus Corporation).

A tracheal tube was inserted for a 1-ml ice-cold PBS lavage, and the obtained BALF was centrifuged at 500 × g at 4°C for 5 min. Next, the supernatant was collected and stored at −80°C for subsequent cytokine measurements. The levels of IL-4, IL-13 and IFN-γ were determined by ELISA kits, according to the manufacturer's instructions. The cell pellets were obtained and resuspended in PBS for total cell and eosinophil counts. The slides were fixed and stained with Diff-Quik Stain (cat. no. G1541; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol, and differential cell counts were obtained using light microscopic evaluation of 300 cells/slide. Total BALF cells were counted on a haemocytometer.

Lung tissue samples were lysed in RIPA Lysis Buffer (Guangzhou Fansi Biotechnology Co., Ltd.). Lysates were centrifuged at 6500 × g at 4°C for 15 min, and supernatant was collected and quantified with a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). 40 µg of proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (cat. no. IPVH00005; EMD Millipore) and blocked with 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature. The membranes were probed with polyclonal antibodies against SIRT1, LC3B, Beclin-1, t-mTOR, p-mTOR, and a monoclonal antibody against β-actin in Tris-buffered saline containing Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.) for 2 h at room temperature. then incubated with goat anti-rabbit secondary antibody in TBST for 1 h at room temperature. Reactions were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.), followed by exposure to autoradiographic films. Signals were semi-quantified from scanned films using Quantity One software (v4.6.6, Bio-Rad Laboratories, Inc.).

All data were analyzed with SPSS 19.0 software (IBM Corp.). Values are presented as the mean ± SD (n=8 mice/group). Data were assessed by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

BALF samples were collected 24 h after the final OVA challenge. In order to determine the effects of EX-527 on airway inflammation, total cell and eosinophil counts in BALF samples from OVA-induced asthmatic mice were examined. As shown in Fig. 2A, OVA administration significantly increased total cell counts in the BALF compared with the control group (P<0.05). After EX-527 administration, total cell counts were significantly reduced compared with the OVA group (P<0.05; Fig. 2A). BALF eosinophil levels were consistent with total cell counts. Specifically, OVA treatment resulted in markedly increased eosinophil levels in BALF samples in comparison with the control group (P<0.05; Fig. 2A). Meanwhile, the EX-527 group exhibited decreased eosinophil levels compared with the OVA treatment group (P<0.05; Fig. 2A). In order to evaluate the role of mTOR in the EX-527-mediated protective effect on asthma, OVA-induced allergic mice were treated with EX-527 in combination with rapamycin. As shown in Fig. 2A, rapamycin restored the decreased amounts of total inflammatory cells and eosinophils induced by EX-527 (P<0.05).

A Th1/Th2 imbalance is believed to play a vital role in asthma pathogenesis, including changes in the levels of Th1 cytokines (IFN-γ) and Th2 cytokines (IL-4 and IL-13) (2). To determine the effects of EX-527 on cytokine release in OVA-induced asthmatic mice, BALF IL-4, IL-13 and IFN-γ levels normalized to total protein amounts in BALF were evaluated by ELISA 24 h after the final OVA challenge. Asthmatic mice exhibited marked increases in IL-4 (0.77±0.03 vs. 0.12±0.03) and IL-13 (0.86±0.06 vs. 0.23±0.02) levels, and a significant decrease in IFN-γ (0.11±0.01 vs. 0.27±0.03) levels (P<0.01; Fig. 2B-D). Treatment with EX-527 resulted in markedly reduced IL-4 (0.28±0.03) and IL-13 (0.44±0.01) levels, and significantly increased IFN-γ levels (0.20±0.02) in BALF samples in comparison with the OVA group (P<0.05; Fig. 2B-D). Consistent with the aforementioned results, rapamycin treatment partially reversed the reductions in IL-4 (P<0.05; Fig. 2B) and IL-13 (P<0.05; Fig. 2C) levels, and the increase in IFN-γ levels (P<0.05; Fig. 2D).

Histopathological examination of mouse lungs was performed in order to confirm the inhibitory effect of EX-527 on airway inflammation. The extracted lung tissues exhibited marked infiltration of inflammatory cells in perivascular and peribronchiolar connective tissue samples from the OVA group compared with control mice, and most leukocytes were eosinophils (Fig. 3A and B). Meanwhile, mice treated with EX-527 prior to OVA challenge exhibited markedly decreased inflammatory cell infiltration around the airways and blood vessels (Fig. 3C). As expected, co-administration of rapamycin partly abolished the anti-inflammatory effects of EX-527 (Fig. 3D). These findings indicated that the beneficial effects of EX-527 on OVA-induced allergic mice may be inhibited by rapamycin.

Because EX-527 is considered a selective SIRT1 inhibitor, the study next assessed whether its beneficial effects are associated with SIRT1 inhibition. Western blot analysis indicated that OVA exposure resulted in increased SIRT1 expression, which was significantly reversed by treatment with EX-527 (Fig. 4A). These data suggested that EX-527 suppressed airway inflammation in asthmatic mice by inhibiting SIRT1, since EX-527 is a well-known specific inhibitor of SIRT1-1 (22). LC3B, a mammalian homolog of yeast Atg8, is comprised of two forms: LC3-I (18 kDa) and LC3-II (16 kDa). During autophagy the cytoplasmic form LC3-I is processed and recruited to autophagosomes, where LC3-II is generated by site-specific proteolysis near the C-terminus (24). The ratio of LC3-II to LC3-I is commonly used to analyze autophagic activity, of which Beclin-1 is also considered a hallmark (17). Furthermore, it has been reported that autophagy suppression may inhibit airway inflammation in allergic mice (16). Therefore, the present study assessed whether EX-527 exerted anti-inflammatory effects by suppressing autophagy. In this study, the LC3-II/LC3-I ratio was significantly increased in mice in the OVA group compared with the control group (P<0.01), and this upregulation was alleviated by EX-527 administration (P<0.05; Fig. 4B). Similarly, Beclin-1 expression was increased by OVA and decreased by EX-527 (Fig. 4C). Thus, the beneficial effects of EX-527 might be attributed to LC3-II and Beclin-1 suppression. These findings indicated that autophagy was upregulated in OVA-induced allergic asthmatic mice but alleviated by SIRT1 inhibition.

mTOR activation was induced after treatment with EX-527 in OVA-stimulated mice. To investigate whether mTOR-mediated autophagy was involved in the protective effect of EX-527 on OVA-induced allergic airway inflammation, the OVA group was administered rapamycin 30 min prior to treatment with EX-527. Subsequently, SIRT1, LC3-II/LC3-I, Beclin-1 and p-mTOR expression levels were determined by western blotting. Notably, rapamycin significantly reversed the inhibited autophagic flux induced by EX-527, as reflected by increased SIRT1 (Fig. 4A; P<0.05), LC3-II/LC3-I (Fig. 4B; P<0.05) and Beclin-1 expression (Fig. 4C; P<0.05), and decreased levels of p-mTOR (Fig. 4D; P<0.05). These results suggested that the inhibitory effect EX-527 on autophagy was significantly suppressed by rapamycin, which indicated that EX-527 inhibited autophagy through mTOR signaling suppression.