A total of 40 specific-pathogen-free-grade male Sprague-Dawley rats (age, 6–8 weeks; weight, 180–200 g) were provided by the Model Animal Research Center of Nanjing University (Nanjing, China). All animals were housed in individually ventilated cages (n=2 in each cage) under a controlled temperature (20–25°C), humidity (20–30%) and 12-h light/dark cycle conditions with free access to food and water. Rats were allowed to adapt to the environment for one week before the experiments. Animal experimental procedures were performed according to the Guide for Care and Use of Laboratory Animals (15,16) and protocols were approved by the Animal Experiment Ethics Committee of The First People's Hospital (Wuhan, China).

The rat sepsis model was established using the cecal ligation and puncture (CLP) method, as previously described (17). Briefly, all rats were deprived of from food 12 h before surgery, but had free access to water during this time. Rats were anesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital. Following anesthesia, a 2-cm incision was made along the middle of the abdomen to expose the cecum. Subsequently, the cecum was ligated with the No. 4 suture below the ileocecal valve and double-punctured using a sterile 18-gauge needle, which released a small amount of feces. The intestinal contents within the cecum were squeezed through the puncture wound and the cecum was restored, followed by suturing of the abdominal incision, layer by layer. Rats in the sham groups underwent identical laparotomy and resuscitation procedures; however, ligation and perforation were not performed. All rats were allowed free access to food and water following CLP. The behavior of the rats was observed every 2 h. There were no abnormal deaths during the experiment. All animals were euthanized with an intraperitoneal injection of 200 mg/kg sodium pentobarbital 24 h after CLP surgery, which was in accordance with previous studies (18,19). Death was determined when the animals' hearts stopped completely and pupils dilated. Lung tissues were collected for further experimentation. If the rats went into shock, or showed decreased activity, lethargy or dyspnea, the animals were euthanized prior to the experimental endpoint.

All rats were randomly allocated into four groups (10 rats in each group) as follows: i) Sham group; ii) sham + sufentanil group; iii) CLP group; and iv) CLP + sufentanil group. Sufentanil was purchased from Yichang Humanwell Pharmaceutical Co., Ltd. Rats in the sham + sufentanil and CLP + sufentanil groups were intravenously injected with sufentanil (3 µg/kg; 1 ml) 30 min prior to surgery, whereas rats in the sham only and CLP only groups were intravenously injected with normal saline at the same time.

In order to assess the magnitude of lung tissue edema, the upper right lobe of lung tissues was immediately excised and the surface fluid and blood was absorbed using filter paper. After weighing, the tissue samples were placed in an incubator at 80°C for 48 h to acquire the dry weight, and the W/D ratio was subsequently calculated using the following calculation: Lung W/D weight ratios=wet weight/dry weight.

Lung tissues were fixed in 10% buffered formalin for 24 h at 4°C, conventionally dehydrated, cleared and waxed. The tissue samples were subsequently embedded in paraffin and cut into 5-µm-thick sections. The tissue sections were deparaffinized in xylene and rehydrated in a descending ethanol series. The sections were stained with hematoxylin (5 min, room temperature) and eosin (3 min, room temperature), prior to dehydration in a graded ethanol series and xylene. The stained slides were observed under a light microscope (Olympus Corporation; magnification, ×400).

Alveolar epithelial type II cells (AEC II) A549 cells (cat. no. SCSP-503) were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂.

In order to mimic the process of ALI, AEC II were treated with 50 µg/ml lipopolysaccharide (LPS; Sigma-Aldrich; Merck KGaA) for 24 h at 37°C. Cells in the control group were not manipulated, while cells in the treatment groups were stimulated with sufentanil (5, 10, 20 and 40 µM) for 2 h prior to treatment with LPS at 37°C in 5% CO_2.

Cells were seeded into 6-well plates (1×10⁶ cells/well) and cultivated in a humidified chamber until they reached 70% confluence. AEC II were transfected with KNG1 overexpression pcDNA3.1 plasmid (Oe-KNG1-1 or Oe-KNG1-2; 5 µl; 50 ng) or empty vector plasmid (Oe-NC; both from Shanghai GenePharma Co., Ltd.) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cells were harvested and analyzed 48 h post-transfection. Transfection efficiency was determined via reverse transcription-quantitative (RT-q) PCR analysis.

Cell viability was detected using Cell Count Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions. After transfection, cells were seeded in 96-well plates (1×10⁴ cells/well). Cells were incubated with sufentanil for 2 h followed by treatment with 50 µg/ml LPS for 24 h at room temperature. A total of 10 µl CCK-8 solution was added to each well. Following incubation for 1 h at room temperature, the absorbance was detected at 450 nm.

The rats were euthanized 24 h after CLP and the left lungs were washed three times with 0.5 ml saline. Subsequently, BALF was collected and centrifuged at 850 × g for 10 min at 4°C. The supernatant was collected and preserved at −80°C. The concentrations of the inflammatory factors TNF-α (cat. no. F16960), IL-1β (cat. no. F15810), IL-6 (cat. no. F15870) and MCP-1 (cat. no. F16120) in BALF were assessed using ELISA kits (Shanghai Xitang Biotechnology Co., Ltd.), according to the manufacturer's protocol.

The partial lung tissue specimens were collected and homogenized (10%, w/v) in cold saline. Malondialdehyde (MDA; cat. no. A003-1-2) content and the activity levels of superoxide dismutase (SOD; cat. no. A001-1-2), catalase (CAT; cat. no. A007-1-1) and glutathione peroxidase (GSH-Px; cat. no. A005-1-2) were determined in the tissue homogenates or AEC II using colorimetric commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using the PrimeScript RT kit (Takara Bio, Inc.) at 37°C for 15 min and 85°C for 5 sec. The cDNA solution was stored at −80°C. Subsequently, qPCR was performed using the iTaq™ Universal SYBR-Green Supermix (Bio-Rad Laboratories, Inc.) on an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 95°C for 3 min, 95°C for 30 sec and 58°C for 30 sec. Relative expression levels were calculated using the 2^−ΔΔCq method and normalized to the internal reference gene GAPDH (20). The sequences of all primers were as follows: GAPDH: Forward, 5′-CGCCTGGAGAAAGCTGCTA-3′ and reverse, 5′-ACGACCTGGTCCTCGGTGTA-3′; and KNG1: Forward, 5′-TAATACGACTCACTATAGGG-3′ and reverse, 5′-TAGAAGGCACAGTCGAGG−3′.

Total proteins were extracted from lung tissue samples or AEC II using RIPA lysis buffer (Beyotime Institute of Biotechnology) and protein concentrations were measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). An equal amount of protein sample (40 µg per lane) was separated via SDS-PAGE on a 10% gel and subsequently transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat milk at room temperature for 2 h, prior to incubation with primary antibodies (overnight at 4°C) for the target proteins. Following incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h (1:10,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.), the protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) and detected using ImageJ software (version 1.8.0; National Institutes of Health). Anti-KNG1 antibody (1:1,000; cat. no. ab175386) was obtained from Abcam. Anti-nuclear factor erythroid 2-related factor 2 (Nrf2; 1:1,000; cat. no. 12721T), anti-phospho-nuclear factor-κB (p-NF-κB p65; 1:1,000; cat. no. 3033T), anti-NF-κB p65 (1:1,000; cat. no. 8242T); anti-heme oxygenase-1 (HO-1; 1:1,000; cat. no. 43966S), anti-Lamin B1 (1:1,000; cat. no. 13435S) and anti-GAPDH (1:1,000; cat. no. 5174T) antibodies were purchased from Cell Signaling Technology, Inc. Protein expression was normalized to the internal reference gene GAPDH or Lamin B1.

All results were obtained from at least three independent experiments. Data are presented as the mean ± standard deviation. All statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, Inc.). A Student's t-test was used to compare differences between two groups, while one-way ANOVA, followed by Tukey's post hoc test was used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

H&E staining of lung tissue sections was performed to determine the pathological changes in each group in order to investigate the effects of sufentanil in sepsis-induced ALI. Normal pulmonary alveoli structure was observed in sham-operated rats, whereas no significant difference in lung tissue pathology was observed between rats in the sham only and sham + sufentanil groups (Fig. 1A). However, animals presented with destructive alveolar structure, thickened alveolar septal walls, visible vascular congestion and inflammatory cell infiltration following CLP. Notably, the morphological changes observed in rat lung tissues following CLP were attenuated after treatment with sufentanil. Furthermore, the degree of lung tissue edema was calculated using the W/D ratio. The results demonstrated that the W/D ratio significantly increased following CLP, whereas treatment with sufentanil notably attenuated this (Fig. 1B). Taken together, these results suggested that sufentanil may effectively alleviate lung injury and edema in sepsis-induced ALI rats.

The effect of sufentanil on inflammation was assessed by detecting the concentrations of inflammatory cytokines, TNF-α, IL-6, IL-1β and MCP-1, in the BALF. The results demonstrated that the levels of inflammatory cytokines significantly increased in the CLP group compared with the sham group, whereas treatment with sufentanil significantly decreased the levels of cytokines in the CLP group (Fig. 2A-D). Furthermore, commercial kits were used to assess the levels of oxidative stress-associated markers. Treatment with sufentanil significantly decreased MDA content, whereas the activity levels of the antioxidant enzymes SOD, CAT and GSH-Px increased compared with the CLP group (Fig. 2E-H). Collectively, these results suggested that sufentanil may relieve inflammation and oxidative stress in CLP-induced ALI rats.

In order to investigate the potential molecular mechanisms of sufentanil function during ALI, KNG1 expression was determined in lung tissues via western blotting. No significant difference in KNG1 expression was observed between the sham and sham + sufentanil groups (Fig. 3A). However, KNG1 expression was significantly upregulated in the CLP group, which was reversed following treatment with sufentanil.

AEC II were subsequently treated with different concentrations of sufentanil (5, 10, 20 and 40 µM) and cell viability was assessed via CCK-8 assays. The results indicated that treatment with a series of concentrations of sufentanil had little effect on the cell viability of AEC II (Fig. 3B). It was also found that cell viability significantly decreased following treatment of AEC II with LPS, compared with the control group, while sufentanil dose-dependently enhanced cell viability (Fig. 3C).

Subsequently, the expression of KNG1 was examined by western blot analysis. As exhibited in Fig. 3D, KNG1 was significantly upregulated in AEC II treated with LPS, whereas sufentanil downregulated KNG1 expression in a dose-dependent manner. Next, KNG1 was successfully overexpressed by transfection with an overexpression plasmid (Fig. 3E and F). Cells transfected with Oe-KNG1-1 were used for all subsequent experiments. Additionally, cell viability was assessed using a CCK-8 assay after KNG1 overexpression with and without LPS. As shown in Fig. 3G, overexpression of KNG1 and/or LPS treatment significantly reduced the viability of AEC II, and the lowest viability was observed in LPS + Oe-KNG1-1 group. Taken together, these results indicated that sufentanil downregulated KNG1 expression in CLP-induced ALI rats and AEC II.

In order to determine whether the inhibitory effects of sufentanil on inflammation and oxidative stress in ALI were achieved by regulating KNG1 expression, the levels of inflammatory- and oxidative stress-associated markers were assessed in AEC II treated with LPS. As demonstrated in Fig. 4A-D, KNG1 overexpression or LPS treatment significantly increased the levels of TNF-α, IL-6, IL-1β and MCP-1, and the highest levels of the aforementioned factors were found in the LPS + Oe-KNG1-1 group. Sufentanil significantly decreased the levels of TNF-α, IL-6, IL-1β and MCP-1 in LPS-treated cells, which was consistent with the results of the CLP-induced ALI rat model. Overexpression of KNG1 significantly alleviated the inhibitory effects of sufentanil on the levels of inflammatory factors. Furthermore, overexpression of KNG1 in LPS-induced AEC II significantly elevated MDA content, which was accompanied by a decline in the activity levels of SOD, CAT and GSH-Px, compared with the control group (Fig. 4E-H). Taken together, these results indicated that overexpression of KNG1 significantly attenuated the inhibitory effects of sufentanil on inflammation and oxidative stress.

In order to further investigate the molecular mechanisms underlying inflammation and oxidative stress regulated by sufentanil in ALI, the expression levels of NF-κB and Nrf2/HO-1 signaling pathway proteins were measured via western blot analysis, following overexpression of KNG1 in LPS-induced AEC II. The expression levels of p-NF-κB p65 significantly increased in the LPS group, which was decreased following treatment with sufentanil. The effects of sufentanil treatment were reversed following overexpression of KNG1 (Fig. 5A). In addition, expression levels of Nrf2 and HO-1 were significantly elevated following treatment with LPS + sufentanil compared with the LPS group (Fig. 5B). Overexpression of KNG1 significantly reversed the regulatory effects of sufentanil on the Nrf2/HO-1 signaling pathway. Taken together, these results suggested that sufentanil relieved inflammation and oxidative stress in ALI by downregulating KNG1 expression.