Isoliquiritigenin, purity > 98%, was obtained from Chengdu Pufei De Biotech Co., Ltd. Compound C (CC, a specific inhibitor of AMPK), brusatol (a specific inhibitor of Nrf2), and tert-butyl hydroperoxide (t-BHP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dexamethasone was obtained from TianJin KingYork Group HuBei TianYao Pharmaceutical Co., Ltd. LPS (Escherichia coli 055:B5) and dimethylsulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Penicillin and streptomycin, fetal bovine serum (FBS), and Dulbecco’s modified Eagle’s medium (DMEM) for cell culture use were obtained from Invitrogen-Gibco (Grand Island, NY, USA). Myeloperoxidase (MPO), malondialdehyde (MDA), GSH, and superoxide dismutase (SOD) determination kit were provided by the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Mouse TNF-α, IL-1β, and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were provided by Biolegend (San Diego, CA, USA). Antibodies against NLRP3, ASC, Caspase-1, IL-1β, p-AMPK, AMPK, p-GSK3β, GSK3β, p-IκBα, IκB, Nrf2, HO-1, NQO1, GCLM, GCLC, β-actin, and Lamin B were obtained from Cell Signaling (Boston, MA, USA) or Abcam (Cambridge, MA, USA). The horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG were obtained from Protein-Tech (Boston, MA, USA). Unless specifically stated, all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

RAW 264.7 cells were obtained from the China Cell Line Bank (Beijing, China). The cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml of penicillin and 100 U/ml of streptomycin in a 5% CO₂-humidified atmosphere at 37°C prior to experiments.

Cell viability were determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RAW 264.7 cells were plated at a density of 2 × 10⁴ cells per well in a 96-well plate and incubated at 37°C for 18 h. The cells were treated with various concentrations of ISL (5, 10, 20 µM) for 18 h with or without brusatol (a specific inhibitor of Nrf2) and then exposed to t-BHP (10 mM) for 3 h. After that, 20 µL of MTT (0.5 mg/mL) was added into each well, and the cells were incubated for another 4 h. The formazan crystals were solubilized in 150 µL DMSO, and the absorbance was measured at 570 nm on a microplate reader. The relative cell viability was calculated as a percentage against the untreated group.

RAW 264.7 cells were plated at a density of 1.5 × 10⁴ cells per well in a 96-well plate and cultured to reach 75% confluence. Using the Viafect transfection reagent (Invitrogen, Carlsbad, CA, USA), pGL4.37 (luc2P/ARE/Hygro vector) and pGL4.74 (hRluc/TK vector) plasmids were transfected into cells. After treatment of ISL at different concentrations (5, 10, 20 µM) for 18 h or at 20 µM for 1, 3, 6, and 18 h, a dual-luciferase reporter assay system (Dual-Glo^® Luciferase Assay System) was put into use to determine the ARE-driven promoter activity.

Wild-type (WT) C57BL/6 mice (18–20 g) were purchased from Liaoning Changsheng Technology Industrial Co., Ltd. (Certificate SCXK2010-0001; Liaoning, China), and Nrf2^−/− (knockout) C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The animals were housed in certified, standard laboratory cages and fed with food and water ad libitum before using for experiments. All of the experiments were approved by Animal Use Committee of Jilin University (license number: SYXK (Ji) 2014-0006), in accordance with International Guiding Principles for Biomedical Research Involving Animals.

To establish ALI model, an intranasal instillation of LPS (0.5 mg/kg) was administered to the mice. In the treated groups, mice received a single dose of ISL or dexamethasone (Dex) via intraperitoneal (i.p.) route 1 h before the administration of LPS. Briefly, WT were divided into five groups randomly: (1) control, (2) ISL (30 mg/kg), (3) LPS (0.5 mg/kg), (4) LPS + ISL (30 mg/kg), and (5) LPS + dexamethasone (5 mg/kg). For further comparison between WT and Nrf2^−/− (knockout) mice, they were, respectively, divided into four groups: (1) control, (2) ISL (30 mg/kg), (3) LPS (0.5 mg/kg), and (4) LPS + ISL (30 mg/kg). At 12 h after LPS treatment, the mice were sacrificed by diethyl ether.

Mice received starch broth of 6% via intraperitoneal (i.p.) route 2 or 3 days before isolation. Serum-free DMEM was injected into the abdominal cavity of the mice, while the abdomen was kneaded softly for 2 min. The culture medium was drawn out and collected, and then centrifuged for 5 min at 1,500 rpm. Sediment cells were resuspended in DMEM, which regulated the concentration of the cells at 2 × 10⁶ cells/ml. The cells were cultivated in 96-well plates at 37°C for 2 h until they had adhered, then the cultivation holes were washed with PBS. The adherent cells were peritoneal macrophages.

For the histological analysis of lung tissue, mice were euthanized and the lower lobes from left lungs were fixed in 4% formalin, followed by dehydrated with ethanol. Subsequently, 5 µm sections were cut after paraffin embedding, and stained with hematoxylin and eosin (H&E) according to previous description (30). The H&E-stained sections were observed under a light microscope to evaluate pathological changes. At the same time, we applied a standard assessment method to judge the lung injury (31). In detail, lung injury was scored based on edema, neutrophil infiltration, hemorrhage, bronchiole epithelial desquamation, and hyaline membrane formation, and five visual fields were observed for each slice. The works were performed in a blinded manner. A score scaled from 0 to 4 represents the severity: 0 for no damage, 1 for mild damage, 2 for moderate damage, 3 for severe damage, and 4 for very severe damage.

Right lungs of the mice were separated by blunt dissection, and the wet weights were measured. And then the lungs were dried in an oven continuously at 60°C for 3 days to determine the dry weights. Ultimately, we calculated the ratios of wet/dry weight to evaluate the degree of pulmonary edema.

Trachea was exposed after the mice were euthanized. Gently we injected 0.5 mL PBS into the trachea and aspirate the liquid for three times to obtain BALF (32). BALF were centrifuged to pellet cells, the supernatants were used to measure total protein concentration by BCA (Bicinchoninic acid) method. The cells were then lysed by ACK Lysis Buffer for 5 min, washed twice with ice-cold PBS, and then centrifuged again at 3,000 rpm for 10 min at 4°C. After that, sediment cells were resuspended in PBS to count total cell number with a hemocytometer or conduct Wright–Giemsa staining using a cytospin and count differential inflammatory cell numbers, as well as measure the ROS level. Meanwhile, the supernatants were conserved at −80°C to measure cytokine levels by ELISA.

Cells from BALF were spun onto glass slides by a cytospin. After the slide was mixed in methyl alcohol, Wright–Giemsa A was added onto it for 1 min, and then Wright–Giemsa B was added on top of Wright–Giemsa A for 5 min. The dye liquor was washed off gently by running water, followed by drying and observation under a microscope. To count differential inflammatory cell number, 300 cells in total were counted, and 100 of the cells in each microscopic field were scored. The mean number of cells per field was reported.

Applying a commercially available mouse ELISA kits, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in BALF were detected. The absorbance was read at 450 nm with a microplate reader.

Right lungs of the mice were excised and homogenized in saline. The levels of MPO, MDA, GSH, and SOD were determined by commercially available test kits according to the manufacturer’s kit protocols.

Reactive oxygen species scavenging activity of ISL was assayed with the oxidant-sensitive fluorescent probe, DCFH-DA (20, 70-Dichlorofluorescein diacetate). ISL-treated cells with or without brusatol were stained with DCFH-DA (5 µM) for 40 min prior to t-BHP (10 mM) for 5 min, or stimulated with LPS (1 µg/mL) for 24 h prior to DCFH-DA (5 µM) for 40 min, the fluorescence intensity was read by a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. As well, resuspended cells in BALF were stained with the same fluorescent dye, but the ROS levels were determined by both the microplate reader and flow cytometry.

Cytoplasmic and nuclear extracts were prepared using an NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce Biotechnology, Rockford, IL, USA), following the manufacturer’s instructions. All steps were performed on ice.

For western blot analysis, homogenized lung tissues and treated cells were lysed in RIPA buffer with protease and phosphatase inhibitors for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4°C. The supernatants were collected, and BCA method was used to determine protein concentration. Protein samples were separated by 10–12.5% SDS-PAGE, and were transferred to a PVDF membrane. The membrane was blocked in 5% skim milk at room temperature for 1 h, blotted with each primary antibody (1:1,000) overnight at 4°C and the corresponding secondary antibody (1:5,000) at room temperature for 1 h. Finally the blots were visualized with an enhanced chemiluminescence western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and band intensities were quantified using Image J gel analysis software.

The data were analyzed using SPSS19.0 (IBM) and expressed as mean ± SEM. The statistical analysis of comparison among groups was performed with the one-way analysis of variance, whereas multiple comparisons were made using the LSD method. Statistical significance was accepted when P < 0.05 or P < 0.01.

First, the cytoprotective effects of ISL were confirmed in t-BHP-treated RAW 264.7 cells. Pretreatment with ISL (18 h) attenuated the cell death induced by t-BHP in an MTT assay (Figure 1B). Meanwhile, incubation with t-BHP significantly raised the intracellular ROS levels, which were inhibited by pretreatment with ISL (Figures 1C,D). These results suggest that ISL may alleviate cytotoxicity through its intracellular ROS scavenging activity. However, brusatol pretreatment abolished the protective effects of ISL on cell viability and ROS (Figures S1A,B in Supplementary Material), which indicated that the cytoprotective and antioxidant effects of ISL in t-BHP-treated RAW cells were dependent on Nrf2.

Under conditions of oxidative stress, the expressions of antioxidant enzymes regulated by Nrf2, such as HO-1, GCLM, GCLC, and NQO1, are clearly increased. As expected, we discovered that ISL significantly increased the ARE-mediated transcriptional activities of antioxidant enzyme genes (Figures 2A,B). In addition, the results of western blot showed that ISL increased the protein expression of total Nrf2 and HO-1, GCLM, GCLC, and NQO1 in a time-dependent manner, as well as resulted in an increase in the nuclear expression and a decrease in cytoplasmic expression of Nrf2 (Figures 2C,D).

Furthermore, previous studies have shown that the AMPK pathway may work upstream of Nrf2. In the current study, we also found that ISL increased the phosphorylation of AMPK slightly prior to the induction of Nrf2 (Figure 2E), while pretreatment with an AMPK inhibitor, CC decreased the ISL-induced Nrf2 activation (Figure 2F). The results suggest that in ISL-mediated Nrf2 activation, AMPK signaling works as an upstream regulator.

Recently, a series of studies have shown that the potent anti-inflammatory effect of ISL is partly derived from the inhibition of the caspase-1/IL-1β and NF-κB pathways. However, due to the crosstalk between Nrf2 and anti-inflammatory pathways, we deemed it worthwhile to investigate whether the Nrf2 activator ISL exerts anti-inflammatory effects through Nrf2 signaling. We confirmed that ISL also inhibited the induction of two important pro-inflammatory enzymes inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 cells (Figure 3A). The results showed that AMPK/Nrf2 signaling was activated in the ISL-treated group after stimulation with LPS, preliminarily suggesting that ISL does exert its anti-inflammatory effects through Nrf2 signaling (Figure 3B). Subsequently, we observed that the protein expression levels of NLRP3, p-IκBα and nuclear NF-κB (p65) were significantly increased by the combination of LPS and ATP or by just LPS stimulation, while this increase was blocked by pretreatment with ISL. Furthermore, we used a specific inhibitor of Nrf2, brusatol, to inhibit ISL-induced Nrf2 activation (Figure 3C), and the inhibitory effects of brusatol on the antioxidant enzymes regulated by Nrf2 were also investigated (Figure S2 in Supplementary Material). As might be expected, ISL failed to inhibit the increase in NLRP3 expression after brusatol pretreatment (Figure 3D). Nevertheless, we were surprised to discover that the inhibitory effects on the NF-κB pathway were not affected (Figure 3E). These results suggest that ISL suppresses the LPS-induced inflammation in RAW 264.7 cells through the inhibition of NLRP3 in a Nrf2-dependent way and the suppression of the NF-κB pathway in a Nrf2-independent way.

Previous studies have shown that the pathological characteristics of LPS-induced ALI in a mouse model are fairly similar to those in human ALI (33). Therefore, LPS instillation was applied to induce ALI in our study and to investigate the protective effects of ISL. To observe the tissue damage, the lower lobes of the left lungs were taken for histology 12 h after the LPS challenge. The administration of LPS led to diffuse pathological changes in the lung, which were characterized by alveolar congestion and inflammatory cell infiltration. However, pretreatment with ISL significantly reduced these changes, just as the positive control drug dexamethasone did. The evaluation of lung injury severity was also performed by calculating a lung injury score (Figure 4A). In accordance with the pathological analysis, the lung wet/dry weight ratio and total protein concentration in BALF showed similar results (Figures 4B,C). These results indicate that ISL effectively alleviates LPS-induced pathological changes in ALI.

To further confirm whether ISL combats LPS-induced ALI through its antioxidative effect, we examined the levels of ROS in different groups. After 12 h, stimulation with LPS significantly increased the ROS levels of sediment cells in BALF, and ISL significantly reduced this increase (Figures 5A,B). Additionally, we measured indexes of oxidative stress, including MPO, a marker of neutrophil accumulation that can cause tissue injury by itself or through derived oxidants; MDA, a product of lipid peroxidation considered as a marker of oxidative stress; and glutathione (GSH) and SOD, two essential antioxidant factors (34–37). The results showed that pretreatment with ISL markedly decreased LPS-induced MPO production and MDA formation and reduced the depletion of GSH and SOD (Figures 5C–F). These results reveal that ISL attenuates lung injury through the inhibition of oxidative stress.

The number of total and differential inflammatory cells as well as the levels of pro-inflammatory cytokines in BALF are involved in the pathogenesis of LPS-induced ALI and indicate the severity of lung inflammation. As evidenced by Wright–Giemsa staining and cell counting, LPS induced a significant increase in the number of total cells, neutrophils, and macrophages in BALF, while pretreatment with ISL caused a notable reduction in cell number compared with that after LPS treatment alone (Figure 6A). Moreover, the results showed that ISL inhibited the production of COX-2 and iNOS in the lung tissue, and TNF-α, IL-1β, and IL-6 in BALF (Figures 6B,C). These results suggest that ISL alleviates lung injury by inhibiting the recruitment of inflammatory cells and the production of pro-inflammatory mediators in LPS-induced ALI.

Various cellular and mouse models have been used to investigate the protective effects of Nrf2 against ALI/ARDS (38, 39). As expected, ISL treatment also evoked a notable increase in the expression of nuclear Nrf2 and its target enzymes (e.g., HO-1, GCLM, GCLC, and NQO1) in lung homogenates (Figure 7A). Moreover, the results showed that ISL markedly increased the phosphorylation of AMPK, GSK3β, and Akt (Figure 7B), which have been confirmed to be upstream regulators of Nrf2 (11). Such findings suggest that ISL may protect against LPS-induced ALI through the upregulation of the AMPK/Nrf2 signaling.

Accumulated evidence has revealed that the NLRP3 inflammasome and NF-κB pathways are essential for the full development of ALI. Hence, we analyzed the expression levels of proteins in the NLRP3 and NF-κB pathways. The results showed that in the lung tissue, the administration of LPS substantially increased the protein expression levels of NLRP3, ASC, and caspase-1, which have been proven to be three important components of the inflammasome. ISL successfully suppressed the activation of the NLRP3 inflammasome induced by LPS. In agreement with this, the increase in both pro-IL-1β (31 kDa) and mature IL-1β (17.5 kDa) after the LPS challenge was blocked by ISL pretreatment (Figure 8A). Moreover, ISL reduced the LPS-stimulated increase in p-IκBα and the nuclear expression of NF-κB, both of which are important proteins in the NF-κB pathway (Figure 8B).

To further investigate whether the antioxidative and anti-inflammatory effects of ISL on LPS-induced ALI are due to Nrf2 activation, we used WT and Nrf2^-/- C57BL/6 mice, and the western blot results confirmed the knockout of Nrf2 (Figure 9A). Histological analysis provided a clear impression of the protective effect: ISL notably alleviated the histopathological changes in WT mice, but the inhibitory effect was fairly weaker in the Nrf2^−/− mice (Figure 9B). Furthermore, ISL significantly suppressed the LPS-induced ROS production in peritoneal macrophages of WT mice but not in the peritoneal macrophages of the Nrf2^−/− mice (Figure S3 in Supplementary Material), showing that the antioxidative capacity of ISL was dependent on Nrf2 activation. In addition, in accordance with the in vitro experiments, the inhibitory effect of ISL on the NLRP3 pathway was substantially blocked in Nrf2^−/− mice compared with WT mice, while the inhibitory effect on the NF-κB pathway still existed (Figure 10). Meanwhile, in the Nrf2^−/− mice, we were surprised to find that the ISL + LPS group had increased expression levels of the NLRP3 inflammasome components compared with their levels in the LPS group, but the reason remains unclear. Such findings further support the results that ISL alleviates LPS-induced ALI through the inhibition of NLRP3 in a Nrf2-dependent way and the suppression of the NF-κB pathway in a Nrf2-independent way.