The murine macrophage cell lines, RAW264.7 and NR8383 cells (American Type Culture Collection, Manassas, VA, USA), were cultured in Roswell Park Memorial Institute (RPMI)-1640 and F-12K medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA), respectively, and supplied with 10% fetal bovine serum, 2 µM glutamine (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. NR8383 cells were treated with 1 µg/ml LPS (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 4 h at 37°C, with or without pretreatment of 10 mM MAPK p38 inhibitor SB203580 (Selleck Chemicals, Houston, TX, USA) for 1 h at 37°C. Conditioned media and cells were collected for measurement of protein expression levels by enzyme-linked immunosorbent assay (ELISA), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.

A total of 48 10-week-old male wild-type C57BL/6 mice were purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China). All mice were housed at 22°C with 12-h light/dark cycle and free access to food and water. The animal protocol was approved by the Laboratory Animal Care and Use Committee at the Medical College of Fudan University, Zhongshan Hospital. Mice were intratracheally treated with 5 mg/kg LPS with or without 1 h intraperitoneal (i.p.) pretreatment with 20 mg/kg p38 MAPK inhibitor SB203580 (LPS group and SB203580/LPS group) at room temperature. Mice received PBS, and the same doses of SB203580 were used as controls (PBS and SB203580 groups). Following 24 h LPS/PBS treatment, collection of bronchoalveolar lavage fluid (BAL) was performed by intratracheal injection of 0.5 ml of PBS followed by gentle aspiration. The lavage was repeated three times and the recovery rate was >90%. The collected BAL fluid was centrifuged at 400 × g for 5 min at 4°C and cell pellets were suspended in 1 ml of PBS and cell count was manually measured with a hemocytometer by light microscopy. The supernatant of BAL fluid was used for cytokine assay and the lung tissues were collected for further analysis.

SB203580 was purchased from Selleck Chemicals, dissolved with DMSO (Sigma-Aldrich, Merck KGaA) according to the manufacturer's protocol and diluted with PBS as a working solution. A final concentration of 2% DMSO was introduced in the cell culture media for each group, and 10% DMSO in solution was used for administration into the mouse model. There were no signs of cell toxicity and mouse mortality with the concentrations used in vitro and in vivo (data not shown).

The wet and dry weights (W) of the left lung tissues were measured prior to and following drying at 65°C for 48 h. Water content was obtained by calculating the W/D weight ratio.

Lung tissue was fixed in 4% paraformaldehyde and sections of 5 µm thickness were placed onto glass slides and stained with hematoxylin and eosin (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's protocol, for histopathological examination. The lung histology was viewed under a light photomicroscope and evaluated for pathological changes using a double-blind method. The severity of lung injury was evaluated using a semiquantitative histological index, including alveolar edema, hemorrhage, alveolar septal thickening and infiltration of polymorphonuclear leukocytes. Each item was divided into four grades from 0 to 3 (0, normal; 1, mild; 2, moderate; 3, severe) and then calculated for a total acute lung injury score (24).

The collected cells (1×10⁶ cells/ml) were incubated with 100 µl radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) supplied with proteinase inhibitor phenylmethylsulphonyl fluoride (Beyotime Institute of Biotechnology) for 30 min on ice. The proteins of the lung tissues were extracted using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Protein concentration was determined by bicinchoninic assay protein assay kit (Beyotime Institute of Biotechnology). Each 20 µg protein sample was resolved on a 10% SDS-PAGE gel. The protein was transferred onto polyvinylidene fluoride membrane blots (Merck Millipore; Merck KGaA), and the blots were incubated with blocking buffer (Shanghai Beyotime Institute of Biotechnology) for 1 h at room temperature, followed by incubation with the indicated primary antibodies against NLRP3 (1:1,000; NBP2-12446; Novus Biologicals, LLC, Littleton, CO, USA), caspase-1 (1:1,000; Ab1872; Abcam, Cambridge, MA, USA), caspase-3 (1:1,000; 9665s; Cell Signaling Technology, Inc., Danvers, MA, USA) cleaved-caspase-3 (1:1,000; 9664s; Cell Signaling Technology, Inc.), Toll-like receptor (TLR)2 (1:1,000; ab108998; Abcam), TLR4 (1:1,000; sc-293072; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), nuclear factor (NF)-κB p65 (1:1,000; 3033p; Cell Signaling Technology, Inc.), p-p38 (1:1,000; 4511S; Cell Signaling Technology, Inc.) and MAPK p38 (1:1,000; 4511S; Cell Signaling Technology, Inc) at 4°C overnight. The antibody anti-mouse β-actin (1:1,000; BM0627; Boster Biological technology, Pleasanton, CA, USA) was used for internal loading control and blots were incubated with stripping buffer at 37°C for 15 min. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000; anti-rabbit:A0208; anti-mouse A0216; Beyotime Institute of Biotechnology) for 1 h at room temperature. Immune reactivity was visualized using an enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology). Band intensity was quantitatively analyzed by densitometric analysis on ImageJ software version 1.37 (National Institutes of Health, Bethesda, MD, USA).

cDNA was synthesized from 1 µg total RNA with a ReverTra Ace qPCR RT Master Mix kit (Toyobo Life Science, Osaka, Japan). RT-qPCR was performed using SYBR-Green PCR Master Mix-Plus (Toyobo Life Science, Osaka, Japan). All the primers were synthesized by Shanghai BioSune Biotechnology Co. Ltd. Primer sequences are listed in Table I. qPCR reaction was performed on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the condition of 95°C 1 min followed by 40 cycles of 95°C 15 sec, 60°C 60 sec, 72°C 45 sec. The expression level was quantified using the 2^−ΔΔCq method (25), relative to the internal control β-actin.

IL-1β, tumor necrosis factor (TNF)-α and IL-6 expression levels in the BAL fluid or the supernatant of treated cells were measured by ELISA assays (IL-1β: Mouse: MLB00C, rat: RLB00; TNF-α: Mouse: MTA00B; IL-6: Mouse: M6000B, rat: R6000B; R&D Systems Inc, Minneapolis, MN, USA) according to the manufacturer's protocols.

RAW264.7 (1×10⁴ cells/ml) were stimulated with 1 µg/ml LPS for 4, 8, 12 and 24 h. Following treatment, the cells were incubated with fixing buffer and were blocked with blocking buffer (Shanghai Beyotime Institute of Biotechnology) for 30 min at room temperature. Subsequently, the cells were incubated with primary antibodies against rabbit anti-NLRP3 (1:1,000; NBP2-12446; Novus Biologicals, LLC) and mouse anti-caspase-1 (1:1,000; sc-514; Santa Cruz Biotechnology, Inc.) for 2 h at 4°C. The cells were incubated with secondary antibodies Cy3-conjugated anti-rabbit IgG (1:1,000; A0516; Shanghai Beyotime Institute of Biotechnology) and FITC-conjugated anti-mouse IgG (1:1,000; A0568; Shanghai Beyotime Institute of Biotechnology) for 1 h at room temperature following incubation with the primary antibody and washing. DAPI was used for staining nuclei for 10 min at room temperature. Finally, the stained cells were visualized under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Pyroptotic cells were characterized by greater expression of cleaved caspase-1 and positive propidium iodide (PI) staining (26) and early apoptotic cells were Annexin V+PI- (27). Detection of apoptotic and pyroptotic cells by flow cytometry was performed with Annexin V: FITC Apoptosis Detection kit I (556547; BD Pharmingen; BD Biosciences, San Jose, CA, USA) and FLICA 660-YVAD-FMK far-red caspase-1 reagent (9122; ImmunoChemistry Technologies, LLC, Bloomington, MN, USA) according to the manufacturer's protocols in vitro (BD Pharmingen; BD Biosciences). Briefly, the cells (1×10⁵ cells/ml) were resuspended in 100 µl 1X binding buffer and incubated with 5 µl FITC-Annexin V (FL1 channel) and 5 µl PI in the dark for 20 min at room temperature. The stained cells were washed with PBS twice and fixed in 4% paraformaldehyde. To analyze pyroptotic cells, a 150 µl single cell suspension was incubated with 5 µl FITC-conjugated Annexin V, 5 µl PI (FL2 channel) and 5 µl FLICA 660-YVAD-FMK far-red caspase-1 reagent (FL4 channel) in the dark at room temperature for 20 min, then washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Analysis was performed on a FACScan cytometer (Becton Dickinson; BD Biosciences), and data were analyzed with FlowJo software version 7.6 (FlowJo LLC, Ashland, OR, USA).

All experimental values were presented as the mean ± standard error of the mean. Statistical comparison between two groups was performed by Student's t-test, and one-way analysis of variance followed by Bonferroni post hoc analyses was performed among multiple groups for parametric data. P<0.05 was considered to indicate a statistically significant difference.

To establish an acute lung injury mouse model, 8- to 10-week-old male mice were injected with 5 mg/kg LPS intratracheally (i.t.). The mice injected with PBS were used as controls. Massive inflammatory infiltrates, perivascular edema and severe alveolar space destruction were observed compared to the PBS-treated group 24 h following LPS injection (Fig. 1A). However, pretreatment with the p38 MAPK inhibitor SB203580 significantly reversed the severity of acute lung injury compared with the mice that received LPS treatment alone (Fig. 1A and B). Additionally, LPS treatment significantly induced more total BAL protein content (Fig. 1C) and cell counts (Fig. 1D). The weight loss (Fig. 1E) and lung wet/dry ratio (Fig. 1F) were also significantly elevated after i.t. LPS injection. The beneficial effects were consistent with the reduced total BAL protein content (Fig. 1C), cell counts (Fig. 1D), weight loss (Fig. 1E) and lung wet/dry ratio (Fig. 1F) in the group of mice that received both SB203580 and LPS.

To determine whether the reduced acute lung injury and inflammation in the SB203580-treated mice was supported by the decreased expression of proinflammatory cytokines, IL-1β, tumor necrosis factor (TNF)-α and IL-6 expression levels in BAL and lung tissues were measured by ELISA and RT-qPCR analysis. Supporting the observation above, it was demonstrated that the expression levels of IL-1β (Fig. 2A and B), TNF-α (Fig. 2C and D) and IL-6 (Fig. 2E and F) were greatly elevated at the mRNA and protein levels in the mice treated with LPS. However, blockage of p38 MAPK signaling pathway by SB203580 pretreatment significantly attenuated their expression (Fig. 2).

IL-1β is a downstream proinflammatory cytokine of the NLRP3 inflammasome and caspase-1 activation (28). To further investigate the effects of p38 MAPK signaling pathway blockage on the NLRP3 inflammasome, the relative protein expression levels of NLRP3, pro-caspase-1, cleaved-caspase-1 (Fig. 3A) and TLR2 (Fig. 3B) was analyzed by western blot analysis and the mRNA of NLRP3 (Fig. 3C) and TLR2 (Fig. 3D) by RT-qPCR. SB203580 pretreatment significantly reversed the LPS-induced upregulation of NLRP3, cleaved caspase-1 and TLR2, indicating a beneficial role of blockage of the p38 MAPK signaling pathway in reducing the formation of the NLRP3 inflammasome and pyroptosis.

To further confirm the effects of LPS in inducing macrophage pyroptosis, the mouse macrophage cell line, RAW264.7 cells were treated, with 1 µg/ml LPS for 4, 8, 12, and 24 h. The NLRP3 expression was analyzed by immunofluorescence staining. As a result, the NLRP3-expressing cells were increased 4 h following LPS treatment that was gradually increased from 10.1±1.1% untreated cells to a maximal 20.2±1.7% cells treated for 24 h (P<0.01; Fig. 4A and B). In addition, the increasing number of NLRP3+ and cleaved caspase-1+ cells was observed by immunofluorescence staining following LPS treatment, indicating a role of LPS in NLRP3 inflammasome formation and activation (Fig. 4C).

Pyroptosis and early apoptosis of NR8383 after treatment were determined by flow cytometry. Further analysis indicated that LPS treatment resulted in 1.7-fold increases in caspase-1+PI+ pyroptotic cells as well as Annexin V+PI- early apoptotic cells (Fig. 5A and B). The present analysis further indicated that pretreatment with SB203580 significantly reversed LPS-induced caspase-1+PI+ pyroptotic cells (Fig. 5A and C) but enhanced LPS-induced Annexin V+PI- apoptotic cells (Fig. 5B and D). Thus, SB203580 treatment reduced cell pyroptosis but increased cell apoptosis by blockage of the p38 MAPK signaling pathway. LPS induced uncontrolled lung inflammation, possibly by inducing more cell pyroptosis and less cell apoptosis through p38 MAPK signaling, but this needs to be investigated further.

Additional analysis by western blot revealed that the blockage of p38 MAPK signaling pathway suppressed LPS-induced p38 MAPK activation (Fig. 6A) and inhibited the LPS-induced upregulation of NLRP3 and TLR2 proteins (Fig. 6B), consistent with the results of pyroptosis analyzed by flow cytometry (Fig. 5). However, higher expression levels of cleaved caspase-3 were detected in the cells treated with both SB203580 and LPS (Fig. 6C), which was in line with the results analyzed by flow cytometry, demonstrating a larger Annexin V+PI- apoptotic cell population following SB203580 pretreatment (Fig. 5B and D).

Further analysis of cytokines in the supernatant and the cell lysate of the treated cells indicated that LPS treatment induced higher mRNA expression levels of IL-1β and IL-6 (Fig. 7A and B, respectively) and higher protein expression levels (Fig. 7C and D). However, the elevated expression of IL-1β and IL-6 were significantly suppressed by SB203580 pretreatment (Fig. 7; P<0.05), further supporting the effects of p38 MAPK blockage in reducing macrophage pyroptosis in vitro, but this needs to be validated further.