AC-Res with an HPLC purity of >99%, was obtained from Department of Medicinal Chemistry, School of Pharmacy, Fourth Military Medical University (Xi'an, Shaanxi, China). Seawater (osmolality 1,300 mmol/l, pH 8.2, SW 1.05, NaCl₂ 6.518 g/l, MgSO₄ 3.305 g/l, MgCl₂ 2.447 g/l, CaCl₂ 1.141 g/l, KCl 0.725 g/l, NaHCO₃ 0.202 g/l, NaBr 0.083 g/l) was prepared as per the composition of the East China Sea provided by the Chinese Ocean Bureau. Additionally, seawater was filtered prior to inspiration by the animals. Enzyme-linked immune adsorbent assay (ELISA) kits of TNF-α and interleukin-1 β (IL-1β) were purchased from R&D Systems Inc. (Minneapolis, MN, USA). The NO assay kit was obtained from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Anti-pNF-κB p65, anti-i-NOS and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Other chemicals were of high purity and were used without purification.

Male adult Sprague-Dawley (SD) rats, weighing 180–220 g each, were obtained from the Animal Center of the Fourth Military Medical University. The rats were acclimatized to their environment for 1 week and housed in air-filtered, temperature-controlled units with free access to standard rodent chow and water. The experimental protocols were approved by the Animal Care and Use Committee of the Fourth Military Medical University and all experiments complied with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals (publication no. 85–23, revised 1985).

Thirty-two SD rats were randomly divided into four groups. Rats in the control (Ct) group were treated without any intervention. In the seawater inhalation (SW) group, seawater inhalation-induced ALI models were established in rats. Briefly, the rats were anesthetized with pentobarbital sodium [100 mg/kg of body weight, administered intraperitoneally (i.p.)] and maintained in the supine position during the experiment with the head elevated at 30°. A syringe (1 ml) was inserted into the trachea and seawater (4 ml/kg) was instilled at a steady speed within 4 min into the lungs.

Rats from the seawater inhalation plus 3,5,4′-tri-O-acetyl-resveratrol pretreatment (SW + AC-Res) group were pretreated with AC-Res (50 mg/kg body weight) for 7 days prior to modeling. SW-ALI models were established 90 min after the previous administration of AC-Res and the modeling process was the same as that of the SW group.

In the fourth group, the rats were treated with 50 mg/kg AC-Res for 7 days. These rats were euthanized 90 min after the final administration of AC-Res.

The rats that suffered from seawater stimulation were anesthetized and exsanguinated through aortic transection 4 h after modeling. The lungs were removed rapidly from the thoraxes and processed as described below.

The rats were sacrificed 4 h after seawater exposure (n=8 in each group). The same right lower lung lobes from all the animals were preserved in 4% formalin for 24 h and then embedded in paraffin. Lung tissues were cut into 5 µm sections with a microtome and stained with hematoxylin and eosin after deparaffinisation and dehydration. Microscopic evaluation was performed to characterize the lung injury.

In order to quantify the magnitude of pulmonary edema, we evaluated the wet to dry weight ratios of the lung samples from each group. Samples were obtained 4 h after seawater instillation and weighed immediately after removal prior to being subjected to desiccation in an oven at 70°C until a stable dry weight was achieved after 72 h. Wet to dry weight ratios were calculated to evaluate the level of pulmonary edema.

Lungs were removed intact and lavaged with 7 ml ice-cold phosphate-buffered saline (PBS) 5 times 4 h after seawater inhalation. The recover ratio of PBS was >90%. BALF from each group was centrifuged at 500 × g for 10 min at 4°C.

The protein content in the supernatant was determined by measuring the absorbance at 630 nm following the addition of bromocresol green. The values were calculated according to a standard curve and data were presented as µg protein/ml (µg/ml).

The cell pellet was resuspended in 1 ml of red blood cell-lysis buffer to eliminate red cells. White cells were then re-pelleted by centrifugation at 520 × g for 20 min at 4°C. The cell pellet was again resuspended in PBS and cells were counted using a hemocytometer (XFA6100; Perlong Medical Equipment Co., Ltd., Nanjing, China).

Vascular permeability was evaluated by extravasation of the Evans blue dye into lung tissues. Briefly, Evans blue (20 mg/kg) was administered intravenously (1 ml/kg) via a tail vein prior to modeling. Aliquots of lung tissues from each animal were dried in an oven for 24 h at 37°C. Evans blue was then extracted in 1 ml formamide for 24 h at room temperature. Evans blue contents were quantified by measuring absorbance at 620 nm and the concentration of Evans blue was calculated according to a standard curve (1–100 mg/ml). The results were presented as the amount of Evans blue µg/g tissue.

NO content, assessed by determining NO2⁻ concentration in the plams of the lung tissues, reflected the degree of lung damage and expression of i-NOS. Briefly, the lung tissue samples were homogenized in cool normal saline (lung tissue to normal saline, 1:10). The homogenate was assessed according to the manufacturer's instructions. NO content was measured by absorbance at 550 nm and expressed as mmol/mg protein.

TNF-α and IL-1β content were evaluated in the lung tissues collected at 4 h after seawater instillation. Briefly, portions of lung tissues were homogenized in cool phosphate-buffered saline (lung tissue to normal saline, 1:5). The assay was carried out using a commercially available ELISA kit, according to the manufacturer's instructions.

The NR8383 alveolar macrophage cell line, obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), was maintained in Ham's F12 medium supplemented with 10% fetal calf serum, 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. The cells were stimulated with seawater (25%) for 4 h with or without resveratrol (40 µg/ml) pretreatment. The total protein was extracted from the cells to evaluate the expression of p-65NF-κB and i-NOS on a protein level. Additionally, cytokines and NO content in medium were tested by ELISA and nitrate reductive enzymatic method, and the results were expressed as pg/ml and µmol/l, respectively.

Lungs were perfused to remove the blood cells from pulmonary circulation. Tissue samples from each group were collected and the total proteins were extracted. Proteins from the lung samples and cells were quantified using a BCA protein assay kit. Subsequently, 100 µg protein from each group was boiled in loading buffer. The proteins from each group were loaded and separated on a 12% SDS-PAGE gel, transferred to nitrocellulose membranes, blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20 and probed overnight at 4°C with antibodies against p65NF-κB (1:1,000), i-NOS (1:500) and β-actin (1:5,000). The secondary antibody (anti-rabbit IgG peroxidase conjugated, 1:10,000) was incubated and the relative content of the target proteins was detected by the enhanced chemiluminescent detection system according to the manufacturer's protocol.

Data were presented as means ± SD. Statistical analysis was performed with analysis of variance. Statistically significant differences between groups were identified using one-way ANOVA followed by the Dunnett's test. A statistical difference was accepted as significant if P<0.05.

No histological alteration was observed in the lung tissues in the Ct group (Fig. 2A). Alveolar damage, pulmonary edema and infiltration of inflammatory cells in the lung tissues and alveoli were evident 4 h after seawater instillation (Fig. 2B). AC-Res significantly reduced pulmonary edema and alveolar damage and inhibited inflammatory cells and blood cell infiltration (Fig. 2C).

To observe lung edema resulting from seawater inhalation and the protective effects of AC-Res, we measured the lung wet to dry weight ratios of the tissues from each group (Fig. 3). The ratios were significantly increased in the seawater exposure group compared with the control group (n=8, P<0.05). Compared with SW, the wet to dry ratio of the AC-Res group was significantly decreased (P<0.05).

Vascular permeability was evaluated to determine the reason for lung edema and to assess the effects of AC-Res (Fig. 4). The results showed that seawater stimulation increased the protein content in BALF (Fig. 4A), enhanced Evans blue dye and cell infiltration from blood vessels to lung tissues (Fig. 4B and C). By contrast, AC-Res pretreatment reduced the protein content in BALF and inhibited Evans blue and cell infiltration (n=8, P<0.05).

To determine the reason for the increase in vascular permeability, inflammation in lung samples was examined. The contents of TNF-α and IL-1β in lung tissues were tested using ELISA (Fig. 5). The results showed that seawater inspiration increased inflammatory TNF-α and IL-1β in lung tissues (n=8, P<0.05). By contrast, AC-Res treatment reduced the seawater stimulation, resulting in cell infiltration and TNF-α and IL-1β in lungs stimulated by seawater.

The results from the NR8383 cells showed that seawater stimulation increased TNF-α and IL-1β (Fig. 6) secretion. AC-Res treatment inhibited the release of those cytokines (P<0.05).

Since NO is associated with inflammation and plays an important role in the body, we investigated the effects of seawater on NO content and whether AC-Res attenuated that effect (Fig. 7A). The results showed that seawater inhalation increased the NO production (n=8, P<0.05), while AC-Res pre-treatment decreased NO content compared with that of SW (n=8, P<0.05).

On the other hand, NO content in medium significantly increased when NR8383 cells were challenged with seawater (Fig. 7B). In addition, AC-Res pre-treatment reduced NO production (P<0.05).

Western blot analysis was performed to examine the status of p-65 NF-κB and i-NOS expression as well as the effects of AC-Res. The results (Figs. 8 and 9) showed that seawater instillation significantly upregulated the expression of p-65 NF-κB and i-NOS (P<0.05), while the pretreatment of AC-Res reversed the trend and decreased p-65 NF-κB and i-NOS at the protein level (P<0.05).

Similarly, the p-65 NF-κB and i-NOS expression levels were enhanced in the NR8383 cells following stimulation with seawater (Figs. 10 and 11). AC-Res inhibited the expression of p-65 NF-κB and i-NOS in cells challenged with seawater (P<0.05).