Male Institute of Cancer Research mice (age, 6–8 weeks; weight, 18–20 g) were provided by the Animal Experimental Center of Wenzhou Medical University (Wenzhou, China). The study was approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University & Laboratory Animal Centre of Wenzhou Medical University (Wenzhou, China). Animals were housed in a room with a 12-h light/dark cycle and allowed free access to tap water and standard laboratory food. The PQ and Res used in the experiment were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-mouse SIRT1 monoclonal antibody (cat. no. 3931) and rabbit anti-mouse Nrf2 polyclonal antibody (cat. no. ab31163) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA) and Abcam (Cambridge, MA, USA), respectively. Polyclonal mouse anti-β actin antibody (cat. no. BS1002) was purchased from Bioworld Technology (St. Louis Park, MN, USA). Formaldehyde, Hematoxylin and Eosin (H&E) Staining kit, agarose gel, SDS-PAGE and Nuclear and Cytoplasmic Protein Extraction kit (no. P0028) were purchased from Beyotime Institute of Biotechnology (Haimen, China)

The dose- and time-dependent effects of Res on SIRT1 expression and lung injury after PQ exposure were examined by two independent experiments. A) Mice were randomly divided into four groups as follows: i) The control group, mice received saline solution by intraperitoneal (i.p.) injection (n=6); ii) The Res group, mice received Res (30 mg/kg i.p.; n=6); iii) The PQ group, mice received PQ (20 mg/kg i.p.; n=6); iv) The PQ + Res group, mice received PQ (20 mg/kg i.p.) and Res (15, 30 or 60 mg/kg i.p.; n=6 for each condition). PQ was dissolved in saline solution and injected intraperitoneally in a single toxic dose of 20 mg/kg of body weight based on preliminary experiments. Mice were anesthetized with 50 mg/kg pentobarbital (Hanlim Pharm Co., Ltd., Seoul, Korea). Pulmonary samples were collected at 24 h subsequent to PQ injection. B) Mice were randomly divided into three groups as follows: i) Control group (n=18); ii) The PQ group, mice received PQ (20 mg/kg i.p.; n=18); iii) The PQ + Res group, mice received PQ (20 mg/kg i.p.) and Res (30 mg/kg i.p.; n=18). Pulmonary samples were collected at 6, 24 and 72 h subsequent to PQ exposure, and tissues from the same mice were used for histopathological, PCR and western blot analyses.

The lower lobes of the right lungs of six mice pre group were removed and then transferred to 4% formaldehyde for 24 h. The lungs were embedded in paraffin (Leica Microsystems, Wetzlar, Germany), and 2–3 butterfly-shaped sections of 4-µm thickness were cut per sample using a microtome (Leica RM2016; Leica Microsystems) and placed on glass microscope slides stained with hematoxylin and eosin (H&E) for histopathological analysis. Lung histopathology images were acquired using a microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan). The severity of lung injury was determined by a histopathologist blinded to the protocol design.

To quantify the magnitude of pulmonary edema, the lung W/D weight ratio was evaluated. The middle lobe of right lung of six animals was excised and the wet weight was determined, following which the lung was placed in a drying oven at 60°C for 72 h to stabilize the dry weight. The ratio of W/D was calculated.

The total RNA of the upper right lung lobe of six mice per group was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA USA). The cDNA was prepared from 2 µg RNA using a PCR master mix (Promega Corp., Madison, WI, USA). The primers used were as follows: SIRT1, F 5′-ACGCTGTGGCAGATTGTTATTA-3′ and R 5′-TTGAAGAATGGTCTTGGGTCTT-3′; NRF2, F 5′-ATTCTTTCAGCAGCATCCTCTC-3′ and R 5′-ACACTTCCAGGGGCACTATCTA-3′, resulting in PCR products of 278 and 403 bp, respectively. The primers for mouse β-actin were F 5′-ATATCGCTGCGCTGGTCGTC-3′ and R 5′-AGGATGGCGTGAGGGAGAGC-3′, resulting in a PCR product of 517 bp. Amplified fragments of expected size were analyzed using a 2% agarose gel and images were captured under ultraviolet (UV) light (302 nm) (Tianneng Co., Shanghai, China). Gels were imaged using the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and quantified by densitometry using Quantity One 4.52 software (Bio-Rad Laboratories, Inc.). Data are presented as fold changes in gene expression normalized to β-actin.

The upper parts of the left lung from six animals per group were harvested and homogenized immediately. Total proteins were extracted from the lungs using the Nuclear and Cytoplasmic Protein Extraction kit according to the manufacturer's protocol. Equal amounts of total proteins (40 µg) per lane were separated by 8% SDS-PAGE (80 V, 30 min; 120 V, 90 min) and then transferred to polyvinylidene fluoride membranes (Solarbio, Beijing, China). Membranes were incubated with rabbit anti-mouse SIRT1 monoclonal antibody (1:1,000 dilution) or rabbit anti-mouse Nrf2 polyclonal antibody (1:500 dilution) at 4°C for 24 h and washed with Tris-buffered saline containing Tween 20 (Solarbio). Subsequent to washing, samples were incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (cat. no. sc-2030; 1:2,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature and visualized using Western Blotting Luminol reagent (cat. no. sc-2048; Santa Cruz Biotechnology, Inc.). Images of the blots were captured using the ChemiDoc™ MP Imaging System and quantified by densitometry using Quantity One 4.52 software.

The heme oxygenase-1 (HO-1) activity in samples of the lower part of the left lung of six mice was measured by commercially available ELISA kits (Westang Biotechnology Co., Shanghai, China). ELISA was performed following the protocols provided by the manufacturer.

The MDA and GSH contents in samples of the lower part of the left lung of six mice were detected according to the instructions of the MDA and GSH assay kits purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The SOD and CAT activities were detected according to the instructions of the SOD and CAT assay kits (Nanjing Keygen Biotech Co., Ltd., Nanjing, China).

All data are described as the mean ± standard deviation and analyzed using SPSS 19.0 software (International Business Machines, Armonk, NY, USA). The differences between groups were analyzed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To assess the potential role of SIRT1 in PQ-induced lung injury, the SIRT1 levels in lung tissue prior and following Res injection were determined. As demonstrated in Fig. 1A and B, the expression of SIRT1 mRNA and protein levels were elevated at 6 and 24 h after PQ exposure compared with the saline group (P<0.05). At 72 h, the SIRT1 mRNA and protein levels were significantly decreased compared with the saline group (P<0.05). Additionally, administration of 30 mg/kg Res significantly increased the mRNA and protein levels of SIRT1 at all time points compared with the PQ group (P<0.05). Administration of 15, 30 or 60 mg/kg Res upregulated SIRT1 expression levels in the lung at 24 h compared with the PQ group (P<0.05; Fig. 1C and D).

Histopathological and W/D weight ratio analyses were performed to determine the effect of Res on PQ-induced lung injury. Compared with the control group, PQ administration caused marked lung hemorrhage, edema, alveolar septal thickening, influx of inflammatory cells and fibrin deposition (Fig. 2A). In the PQ+Res group, similar changes were identified, however to a lesser degree. Additionally, the W/D weight ratios of the lung tissues were significantly increased following PQ administration compared with the ratios in the NS group (P<0.05; Fig. 2B). In the PQ+Res group, the lung W/D weight ratio was significantly lower than that in the PQ group at 24 and 72 h subsequent to PQ exposure (P<0.05; Fig. 2B).

To investigate whether the protective role of Res was mediated through NRF2, the NRF2 expression levels in the mouse lung tissues were determined. NRF2 protein and mRNA expression levels were significantly elevated at 6 and 24 h after PQ administration but were decreased at 72 h, compared with the NS group (P<0.05; Fig. 3). However, Res administration significantly increased NRF2 protein expression levels compared with the PQ group at all time points (P<0.05; Fig. 3).

Compared with the NS group, HO-1 activity in the lung tissue was upregulated markedly at 6 and 24 h after PQ administration, but was decreased at 72 h (P<0.05; Fig. 4). In the PQ+Res group, HO-1 activity was upregulated compared with that in the PQ group (P<0.05; Fig. 4).

SOD and CAT activity levels in the mouse lung tissue were measured. Compared with the NS group, the PQ group exhibited a significant decrease in SOD and CAT activity levels at all time points (P<0.05; Fig. 5). Following Res administration, SOD and CAT activity levels significantly increased in the lung tissue, compared with the NS group (P<0.05; Fig. 5).

Following PQ administration, GSH protein levels were decreased in the lung tissue compared with the NS group (P<0.05; Fig. 6A). The GSH protein levels were significantly increased in the PQ+Res group compared with those in the PQ group (P<0.05; Fig. 6A). By contrast, PQ administration led to an increase in MDA activity levels compared with the NS group (P<0.05; Fig. 6B). Res administration decreased MDA activity levels compared with those in the PQ group (P<0.05; Fig. 6B).