Female C57/BL6 mice, 6–8 weeks of age, were purchased from Shanghai Laboratory Animal Centre at the Chinese Academy of Sciences, Shanghai, China. The mice were maintained in a temperature-controlled room (22±2°C) with a 12-h light/dark cycle and a relative humidity of 40–60%. The mice were given free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Diseases (no. SKL20160021) and the animal protocol was designed to minimize the pain and discomfort of the animals.

Silybin and LPS (Escherichia coli: Serotype O55:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Anti-actin (4970), anti-phosphorylated (p-)p65 (3033) and anti-p65 (8242) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NLRP3 (ab4270) and anti-caspase-1 (ab179515) antibodies were purchased from Abcam (Burlingame, CA, USA). Anti-ASC (sc-514414) antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-mouse CD3-FITC (11-0032), CD11b-PE (12-0112) and Gr1-APC (53-5931) antibodies were obtained from eBioscience (San Diego, CA, USA). Alexa Fluor 488 anti-mouse IgG (A-11001) was from Invitrogen, Thermo Fisher Scientific (Waltham, MA, USA). The DAB staining kit was from KeyGen (Nanjing, China). All other chemicals were obtained from Sigma-Aldrich.

The mice were randomly divided into 4 groups (n=8/group): the control group receiving saline, the model group receiving LPS only, and the 2 experimental groups receiving silybin followed by LPS. The animals inhaled 0.9% NaCl, or 1,000 µg/ml LPS for 1 h at 3 h intervals over a period of 8 h. The mice received silybin (50, 100 mg/kg), dissolved in the vehicle (0.5% carboxy methyl cellulose) via intragastric (i.g.) administration once per day for 3 consecutive days prior to LPS sensitization. We selected the dose of silybin according to the findings of previous studies in which the dose of silybin for OVA-sensitized mice ranged from 25–200 mg/kg (19,23,24). The mice were sacrificed at 6 h post-LPS administration under anaesthesia by an intraperitoneal injection of 30 mg/kg pentobarbital to collect bronchoalveolar lavage fluid (BALF), blood plasma and tissue samples. Serum was isolated from whole blood to assess cytokine levels. After the trachea was cannulated and the chest cavity was opened via a midline incision, the lung was lavaged 3 times with 1 ml of ice-cold sterile saline. The total BALF cell number was counted and the BALF composition was evaluated by FACS analysis as follows: BALF cells were resuspended with 100 µl PBS containing 1 µl CD3-FITC, 1 µl CD11b-PE and 1 µl Gr1-APC antibodies for 30 min on ice. The cells were then washed twice and subjected to FACS analysis. The remaining BALF was centrifuged at 1,000 × g for 5 min at 4°C, and the cell-free supernatant was stored at −80°C for ELISA.

Lungs from 4 animals in each experimental group were fixed by 10% buffered formalin, embedded in paraffin, and then sectioned to reveal the maximum longitudinal view of the main intrapulmonary bronchus of the left lung lobe. Histopathological analysis was carried out using hematoxylin and eosin (H&E)-stained lung sections.

RAW264.7 mouse macrophages and THP-1 human monocytes were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences and cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (both from Gibco, Paisley, UK), 100 U/ml penicillin G, and 100 µg/ml streptomycin at 37°C with 5% CO₂. The RAW264.7 cells were treated with silybin in the absence or presence of 500 µg/ml LPS for 24 h and RNA and cell culture supernatant was collected for use in reverse transcription-quantitative PCR (RT-qPCR) and ELISA for the analysis of cytokine levels. For the determination of the phosphorylation levels of NF-κB and its nuclear translocation by western blot analysis and immnuofluorescence staining, the RAW264.7 cells were treated with silybin (50 and 100 µM) for 3 h and stimulated with 500 µg/ml LPS for 30 min. For the analysis of NLPR3 inflammasome activation, the THP-1 cells were first induced to differentiate by 500 nM PMA for 3 h and then stimulated with 100 ng/ml LPS for 3 h, followed by treatment with various concentrations of silybin for 1 h, and subsequent incubation with 5 mM ATP for a further 1 h. The cells and cell culture supernatant were then collected for use in western blot analysis, immunoprecipitation and FACS analysis.

Total RNA from the lung tissue of each animal and the cells using TRIzol reagent (Takara, Dalian, China). RNA samples were reverse transcribed into cDNA and analyzed by qPCR, and the relative expression of specific genes was determined using the real-time PCR master mix (Roche, Bromma, Sweden) on an ABI 7500 Fast Real-Time PCR system. The program for amplification was 1 cycle of 95°C for 2 min followed by 40 cycles of 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control to normalize for differences in the amount of total RNA present in the samples. The primer sequences used in this study were as follows: IL-1β, 5′-CTTCAGGCAGGCAGTATCACTC-3′ (forward) and 5′-TGCAGTTGTCTAATGGGAACGT-3′ (reverse); IL-6, 5′-ACAACCACGGCCTTCCCTAC-3′ (forward) and 5′-TCTCATTTCCACGATTTCCCAG-3′ (reverse); TNF-α, 5′-CGAGTGACAAGCCTGTAGCCC-3′ (forward) and 5′-GTCTTTGAGATCCATGCCGTTG-3′ (reverse); IL-17, 5′-TCGAGAAGATGCTGGTGGGT-3′ (forward) and 5′-CTCTGTTTAGGCTGCCTGGC-3′ (reverse); GAPDH, 5′-AACGACCCCTTCATTGAC-3′ (forward) and 5′-CACGACTCATACAGCACCT-3′ (reverse).

Freshly isolated lung tissues were homogenized in the presence of protease inhibitors and protein content of the supernatant was determined by BCA protein assay kit (Pierce, Rockford, IL, USA). The protein sample (20 µg) from the lung homogenates was loaded per lane on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Electrophoresis was then performed. The proteins were then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature. The blocked membranes were then incubated with the indicated primary antibodies overnight at 4°C, followed by incubation with HRP-coupled secondary antibody (7074, Cell Signaling Technology). The binding of all the antibodies was detected using an ECL detection system. For the extraction of nucleoprotein, the cells were collected and lysed in lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 2% NP-40 and 1 mM PMSF) for 30 min. The homogenate was centrifuged for 10 min at 3,000 rpm at 4°C. The supernatant was collected as cytoplasmic protein. The pellet was the nuclear fraction. The pellet was washed twice before being resuspended in 50 µl lysis buffer containing Triton X-100 for 30 min on ice with vortexing at 10 min intervals. This was followed by centrifugation for 30 min at 14,000 × g at 4°C. The supernatant was collected as the nuclear fraction. Actin was used as the internal control for cytoplasmic protein and histone for the nuclear fraction (Santa Cruz Biotechnology, Inc.).

Immunohistochemical analysis was performed on paraffin-embedded colonic tissue sections (5-µm-thick). Briefly, the sections were deparaffinised, rehydrated and washed in 1% phosphate-buffered saline (PBS)-Tween-20, and they were thyen treated with 2% hydrogen peroxide, blocked with 3% goat serum and incubated overnight at 4°C with monoclonal rabbit anti-p-NF-κB antibody (1:200). The slides were then processed using the DAB staining kit from KeyGen according to the manufacturer's instructions. Images were obtained using an Olympus IX51 light microscope (Olympus, Tokyo, Japan). The settings for image acquisition were identical for the control and experimental tissues.

The cells grown on cover glasses were fixed with 4% paraformaldehyde (20 min, room temperature), stained with the anti-NF-κB antibody (1:100), and detected with secondary antibody (Alexa Fluor 488 anti-mouse IgG; Invitrogen). The coverslips were counterstained with DAPI and imaged using a confocal laser scanning microscope (Olympus).

For co-immunoprecipitation, the cells were lysed in lysis buffer containing Triton X-100 and cell lysates were immunoprecipitated with antibody to ASC or control IgG with protein A/G-Sepharose (sc-2003; Santa Cruz Biotechnology, Inc.). The beads were washed, separated by SDS-PAGE and analyzed by western blot analysis with antibodies to caspase-1 and NLRP3. Protein bands were visualized using the western blotting detection system (ChemiDoc™ MP Imaging System 17001402; Bio-Rad, Hercules, CA, USA).

LPS-primed THP-1 cells were treated with various concentrations of silybin for 1 h, and then incubated with 5 mM ATP for 1 h. The cells were then harvested and incubated with 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Life Technologies, Carlsbad, CA, USA) at 37°C for 20 min and washed twice with cold PBS. DCF fluorescence level was detected by FACS.

The data are presented as the means ± SD. The significance of differences between experimental groups was assessed using the two-tailed Student's t-test. The value of statistical significance was set at p<0.05.

In the present study, we used a mouse model of LPS-induced ALI to evaluate the therapeutic effects of silybin. Mice were challenged with LPS and lung tissue samples were collected, and the sections stained with H&E as shown in Fig. 1A. The lung tissues from the LPS group exhibited significant pathological alterations, including notable inflammatory cell infiltration, interstitial and intra-alveolar edema, and patchy hemorrhage, inter-alveolar septal thickening, hyaline membrane formation and some collapsed alveoli. By contrast, a marked attenuation of these responses was observed in the lungs of mice treated with either 50 or 100 mg/kg silybin (Fig. 1A). Next, the infiltration of inflammatory cells in BALF was examined. As shown in Fig. 1B, the number of total cells, macrophages (CD11b⁺), T cells (CD3⁺) and neutrophils (Gr1⁺) in BALF was significantly increased following LPS stimulation, while treatment with silybin dose-dependently reduced the infiltration of inflammatory cells.

Next, the anti-inflammatory properties of silybin were evaluated by determining the mRNA and protein levels of inflammatory cytokines, including TNF-α and IL-1β. LPS stimulation significantly increased the levels of TNF-α and IL-1β in BALF, as well as the serum levels, whereas treatment with silybin significantly decreased these cytokine levels (Fig. 2A and B). In addition, LPS stimulation significantly elevated the mRNA levels of TNF-α IL-1β, IL-17 and IL-6 in lung tissue, whereas treatment with silybin dose-dependently decreased the mRNA levels of these cytokines (Fig. 2C).

NF-κB is one of the major components mediating the inflammatory process. Thus, we examined NF-κB activation in lung samples from mice with LPS-induced lung injury. Immunohistochemical analysis revealed an increased level of p-NF-κB (p-p65) following LPS stimulation; however, treatment with silybin markedly inhibited the phosphorylation of NF-κB (Fig. 3).

To further investigate the protective effects of silybin against LPS-induced lung injury and the underlying mechanisms, we used RAW264.7 cells. LPS stimulation significantly elevated the mRNA and protein levels of TNF-α and IL-1β in the RAW264.7 cells (Fig. 4). Co-incubation with silybin and LPS dose-dependently decreased the mRNA expression levels of TNF-α and IL-1β (Fig. 4A), as well as the protein levels in the cell culture medium (Fig. 4B).

As observed in our mouse model, treatment with silybin inhibited the phosphorylation of NF-κB. Thus, we wished to determine whether it can also inhibit NF-κB signaling in vitro. Upon exposure to LPS, the levels of phosphorylated NF-κB (p65) were significantly increased. However, pre-treatment with silybin for 3 h dose-dependently suppressed the phosphorylation of NF-κB (Fig. 5A). Moreover, silybin decreased the nuclear trans-location of NF-κB (p65) (Fig. 5B). The results of western blot analysis of the cytoplasmic and nuclear content revealed that the level of NF-κB (p65) in the nucleus was reduced by silybin treatment (Fig. 5B). Using immunofluorescence staining, strong NF-κB (p65) staining in the nucleus was observed following stimulation with LPS, whereas in the silybin-treated cells, NF-κB (p65) was mainly located in the cytosol (Fig. 5C). These results suggest that the anti-inflammatory effects of silybin may result from the inhibition of NF-κB.

It is worth noting that there is evidence to indicate that IL-1β is processed as an inactive cytoplasmic precursor (pro-IL-1β and pro-IL-18) that requires cleavage by caspase-1 to produce the mature active forms (25,26). As caspase-1 needs to be activated by the NLRP3 inflammasome, we then evaluated the effects of silybin on caspase-1 activation in THP-1 cells. Treatment with silybin inhibited caspase-1 activation in a concentration-dependent manner (Fig. 6A). Furthermore, immunoprecipitation analysis revealed that the process of NLRP3 inflammasome formation was also interrupted by silybin (Fig. 6B). It has been reported that in the presence of signal I (NF-κB signaling), the NLRP3 inflammasome is activated by ROS signaling which leads to the production of caspase-1 (27). Treatment with LPS and ATP led to ROS generation, whereas treatment with silybin significantly scavenged ROS (Fig. 6C). These results indicate that silybin may reduce ROS generation and interrupt the activation of the inflammasome in macrophages, thereby inhibiting the cleavage of caspase-1 and the production of cytokines.