Adult male Sprague-Dawley (SD) rats weighing 200–220 g were provided by the Animal center of the Fourth Military Medical University (FMMU, Xi'an, China). Rats were captured in air-filtered, temperature-controlled units with free access to food and water. All experimental protocols were approved by the Animal Care and Use Committee of the FMMU according to the Declaration of the National Institutes of health guide for Care and use of Laboratory Animals (publication no. 85–23, revised 1985).

Seawater was prepared according to the formula provided by Chinese Ocean Bureau: osmolality 1,300 mOsm/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. Artificial seawater was sterilized before experiments. Resveratrol (3,5,4′-trihydroxystilbene, Res, structure is shown in Fig. 1A), was purchased from Xi'an grass Plant Technology Corp. (Xi'an, China) with purity >98%. AC-Res, structure (Fig. 1B), was synthesized by the Pharmacy Department of Medicinal Chemistry of FMMU with HPLC purity >99%. Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β were purchased from the R&D Systems Inc. (Minneapolis, MN, USA). MDA and T-SOD activity analyzing kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against Nrf2, Trx-1, Txnip and β-actin were purchased from the Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

SD rats were randomly assigned into 4 groups (n=8): control (Ctl) group; seawater (SW) inhalation group; SW + AC-Res group; AC-Res group. Rats from SW + AC-Res group and AC-Res group were pretreated with AC-Res (50 mg/kg/day) for 7 days, and rats from Ctl and SW group were treated with equal amount of normal saline. Seawater was instilled into the trachea of rats from SW and SW + AC-Res group 90 min after the last administration of normal saline or AC-Res.

The seawater inhalation rat models were built according to a previous study in our laboratory. Briefly, the trachea was exposed after the rat was anesthetized with 3% pentobarbital sodium (100 mg/kg body weight, i.p.). A 1 ml syringe was gently stabbed into the trachea and 4 ml/kg body weight of artificial seawater was instilled into both lungs within 4 min. Rats were held in the supine position with the head elevated 30° degree and maintained anesthesia with 25 mg/kg/h pentobarbital sodium. Rats from all groups were sacrificed 4 h after modeling.

The alveolar macrophage cell line NR8383 and alveolar epithelial cell line A549 were cultured to evaluate the protecting effects of Res, intermediate of AC-Res. NR8383 cells were maintained in Ham's F12 and A549 cells in RPMI-1640 medium containing 10% fetal calf serum at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. Cells within the logarithmic growth phase were divided into four groups: control (Ctl); 25% seawater treated only (SW); 25% seawater + 40 µg/ml resveratrol (SW + Res); 40 µg/ml Res. Cells from SW and SW + Res group were stimulated with 25% seawater with the presence of Res or not. All cells were collected after being treated for 4 h.

In order to quantify the magnitude of pulmonary edema, W/D weight ratio of lung samples were calculated. Briefly, lung tissues of the same lob from each rat in every group were obtained and weighed immediately. After that, lung samples were kept in an oven at 70°C for 72 h. Finally, samples were weighed again and W/D was calculated by dividing the wet weight with the dry.

Contents of TNF-α and IL-1β in lung tissues and cells stimulated by seawater was measured to assess the degree of lung injury. Lung tissues were homogenized in cold phosphate-buffered saline (PBS) (lung tissue to PBS 1:5) and cells were collected and homogenized with repeated freeze-thaw method. Supernatants from tissues and cells were collected by centrifuging at 12,000 rpm for 5 min at 4°C. Contents of TNF-α and IL-1β in supernatant were measured according to the manufacturer's instructions.

T-SOD activity and MDA content were measured to evaluate the status of oxidative stress in lung tissues stimulated by seawater. The lung samples were homogenized in cold PBS (lung tissue to PBS 1:10) and homogenate supernatant was collected by centrifuging at 12,000 rpm for 5 min at 4°C. T-SOD activity and content of MDA were measured at 550 and 523 nm, respectively.

Immunohistochemistry was carried out to evaluate the expression of Trx-1 in lungs stimulated by seawater or not, as well as the effects of AC-Res on its expression. Briefly, slices of rat lung tissues were deparaffinized, rehydrated in graded alcohol and soaked in 0.3% H₂O₂ at room temperature for 30 min. Slices were blocked with goat serum albumin at 37°C for 30 min, and then incubated with the primary antibody against Trx-1 (1:100) at 4°C overnight. After that, sections were washed with PBS and incubated in biotin-labeled secondary antibody derived from goat at 37°C for 30 min. Then, slices were washed with PBS, incubated in horseradish enzyme-labeled streptavidin working solution and detained with diaminobenzidine (DAB). All slices were checked under a light microscope (DMI6000; Leica, Wetzlar, germany).

Activity of Trx-1 was detected by immunofluorescence in A549 cells challenged with or without seawater. Briefly, confluent cells grown on coverslips were fixed with methanol at room temperature for 20 min. After being blocked with 0.5% BSA, cells were incubated with the primary antibody against Trx-1 (1:100) at 37°C for 1 h. Then, cells were incubated with CY3 conjugated secondary antibody. Then, nucleus was detained with DAPI at room temperature for 5 min. Finally, cells were examined by using an inverted fluorescence microscope (DMI6000B; Leica).

At the end of the experiment, lung and cell samples from different groups were collected and total proteins were extracted according to the manufacturer's instructions (Beyotime Institute of Biotechnology, Jiangsu, China). Equal amount of proteins from each group were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to PVDF membranes by wet transfer method. Then, membranes were blocked with 5% non-fat dry milk melted in Tris-buffered saline with 0.1% Tween-20, and incubated with primary antibodies overnight at 4°C against Txnip (1:200), Nrf2 (1:200) and β-actin (1:2,000). After that, membranes were incubated with secondary anti-body for 2 h at room temperature followed by 3 times washing. Results of western blot analysis were examined by the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Arlington Heights, IL, USA).

Statistical analysis was performed with SPSS 17.0 for Windows. Numeric variables were expressed as means ± SD. Differences between groups were performed by one-way analysis of variance (ANOVA) followed by Dunnett's test. Statistical significance was accepted as P<0.05.

Water content in lungs from each group was manifested by W/D ratios. Seawater inhalation dramatically increased the W/D ratios of lung tissues (P<0.05), while AC-Res pretreatment significantly inhibited the increasing of water content in lungs manifested by decreased W/D ratios compared with that of seawater inhalation group (P<0.05) (Fig. 2). On the other hand, administration of AC-Res alone did not affect the content of water in lungs.

Content of inflammatory cytokines was taken as the symbol of lung injury (Fig. 3) the content of TNF-α (Fig. 3A) and IL-1β (Fig. 3B) significantly increased in lungs stimulated by seawater (P<0.05). While pretreatment of AC-Res inhibited the formation and secretion of TNF-α and IL-1β in lungs stimulated by seawater (P<0.05). Also, treatment of AC-Res alone did not affect the secretion of cytokines in lung.

In order to evaluate the oxidative stress in lungs challenged by seawater and the protecting effects of AC-Res, The content of MDA and activity of T-SOD were measured by TBA method and hydroxylamine method, respectively. The results (Fig. 4) showed that seawater inhalation led to increased content of MDA (Fig. 4A) together with the decreased activity of T-SOD (Fig. 4B), while pretreatment of AC-Res increased the activity of T-SOD (P<0.05) and inhibited the formation of MDA (P<0.05) compared with those in lungs stimulated by seawater. In addition, administration of AC-Res alone did not affect the content of MDA and activity of T-SOD in lungs.

In order to explore the mechanisms of oxidative stress in lungs stimulated by seawater and to illustrate how AC-Res inhibited the oxidative stress, we further examined the expression of Trx-1 axis in lungs. As shown in Fig. 5A, immunohistochemistry staining revealed that Trx-1 expressed abundant alveoli epithelium from normal rat lungs, while the expression of Trx-1 dramatically decreased (Fig. 5B) when lungs were challenged by seawater. However, pre-administration of AC-Res strikingly maintained the relative high level expression of Trx-1 in lungs when exposed to seawater (Fig. 5C). Administration of AC-Res alone did not affect the expression of Trx-1 in lungs.

Besides, the expression of key regulators for Trx-1, Nrf2 and Txnip, has also been measured by western blot analysis. As shown in Fig. 6, seawater inhalation upregulated the expression of Txnip (Fig. 6A) and downregulated the expression of Nrf2 (Fig. 6A) (P<0.05). While pretreatment of AC-Res reversed this trend by inhibiting the express of Txnip increasing Nrf2 expression when lungs were stimulated by seawater (P<0.05). AC-Res administration alone did not markedly influenced the expression of the two regulators.

Based on the findings that seawater inhalation resulted in pulmonary edema and lung inflammation, and pre-administration of AC-Res could alleviate lung injuries by interfering with the Trx-1 axis in lungs. We further investigated the protecting effects of Res, intermediate of AC-Res, by in vitro experiments. As shown in Fig. 7, formation of TNF-α (Fig. 7A) and IL-1β (Fig. 7B) in NR8383 cells increased when stimulated by 25% seawater (P<0.05), while co-incubation of Res decreased the generation of TNF-α and IL-1β (P<0.05) in seawater stimulated cells. In addition, Res treatment alone did not affect the secretion of cytokines in cells.

Activity of T-SOD and content of MDA were measured to manifest the oxidative stress in NR8383 cells stimulated by seawater. Results (Fig. 8) showed that seawater stimulation increased the content of MDA (Fig. 8A) and inhibited the activity of T-SOD (Fig. 8B) in NR8383 cells (P<0.05), while Res co-incubation decreased the content of MDA and increased the activity of T-SOD in cells (P<0.05), which was also similar to the effects of AC-Res on seawater stimulated lungs.

The expression of Trx-1 in seawater-stimulated A549 cells were measured by immunofluorescence detection, and results (Fig. 9B) showed that the expression of Trx-1 decreased in A549 cells 4 h after seawater exposure while treatment of Res dramatically inhibited the decrease of Trx-1 expression manifested by the relatively higher fluorescence intensity (Fig. 9C).

Also, we detected the expression of Nrf2 and Txnip in seawater-stimulated NR8383 cells. The results showed that seawater stimulation increased the expression of Txnip (Fig. 10A) and decreased Nrf2 expression (Fig. 10B) compared with that of control, while Res co-incubation upregulated the expression of Nrf2 (P<0.05) and inhibited the expression of Txnip (P<0.05) in NR8383 cells when stimulated by seawater.