Fluorofenidone (AKFPD) was synthesized by Haikou Pharma (Haikou, China). LPS (Escherichia coli O111:B4) was purchased from Sigma Aldrich (#L2630). The antibody against cleaved caspase-3 (#9664), BCL-2 (#2876), Bax (#2772), phospho-ERK (#4370), phospho-JNK (#4668), phospho-P38 (#4631), ERK (#4695), JNK (#9252), P38 (#8690), phospho-IKK (#2697), phospho-IκB (#2859), IKK (#2682), IκB (#9242), P65 (#4764), anti-mouse IgG HRP-linked antibody (#7076), and anti-rabbit IgG HRP-linked antibody (#7074) were purchased from Cell Signaling Technology. The antibody against phospho-P65 (#ab76302) was from Abcam. The antibody against α-tubulin was purchased from proteintech (#66031-1-Ig). The F4/80 antibody was from BD bioscience (#565409), and the MPO antibody was from proteintech (#22225-1-AP). The BCA Protein Assay kit was from Thermo Fisher Scientific (#23225). A terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) kit was purchased from Roche (#11684817910). An FITC annexin V and propidium iodide (PI) staining kit was purchased from BD Biosciences (#556547). An ELISA test kit of IL-1β (#88-7013-88), TNF-α (#88-7324-22), and IL-6 (#88-7064-22) was purchased from Thermo Fisher Scientific. All other chemicals were of analytical grade.

C57/BL mice were purchased from SJA Laboratory Animal Company (Hunan, China) and housed in a specific pathogen-free facility with a light/dark cycle of 12 h/12 h and randomly assigned to four groups: the control group, LPS group, AKFPD preventive group, and AKFPD treatment group (n = 6 per group). ALI models were generated according to previous studies (Ortiz-Munoz et al., 2014; Li T. et al., 2020). After anesthesia with 1% pentobarbital sodium, the mice were injected intratracheally with LPS (5 mg/kg) in 50 μl saline. The control group was treated with saline at the same volume. The AKFPD preventive group was administered orally with AKFPD 2 days (500 mg/kg/day) before LPS injection. The AKFPD treatment group was administered orally with AKFPD (500 mg/kg/day) 1 h after LPS injection for 2 days. LPS group were administered with the same volume of vehicle. Following a duration of 48 h after LPS injection, mice were sacrificed for subsequent assays.

To observe the effect of fluorofenidone on survival, 36 mice were randomly divided into three groups: the control group, LPS group, and AKFPD preventive group (n = 12 per group). Survival was assessed every 6 h. All experimental protocols were performed according to the guidelines of the National Institutes of Health and Central South University, and all procedures were approved by the Institutional Animal Care and Use Committee of Central South University.

Lung tissue samples were fixed in 4% paraformaldehyde for 2 days and then were embedded in paraffin. For the histological examination, the samples were cut into 4 μm sections and stained with hematoxylin and eosin (H&E). The stained slides were analyzed with a light microscope under identical conditions. The sections were examined at 200× magnification for all the groups. Histopathology was scored using a semi-quantitative scale from 0 (normal and no focal inflammatory infiltrates) to 4 (severe infiltration and damage in the lung structure), as described before (Barriga et al., 2021).

Immunohistochemistry for F4/80 and myeloperoxidase (MPO) was performed, as previously described (Liao et al., 2021). After deparaffinization and rehydration, the lung tissue slides were treated with 3% H₂O₂ for 20 min, denatured for 15 min in boiling 10 mM citric acid (pH 6.0), blocked with 5% bovine serum albumin for 30 min at room temperature, and then incubated overnight at 4°C with the F4/80 antibody or MPO antibody. After washing with PBS and incubation with secondary antibodies, the samples were visualized by diaminobenzidine staining. A total of 10 scopes were randomly selected in each sample under 200-fold magnification for quantitative analysis.

BALF was collected by lavaging the lungs with 0.5 ml precooled phosphate buffered saline three times in all groups. The supernatant of BALF was obtained after centrifugation at 3,000 g for 15 min at 4°C and stored at −20°C for further analysis. The total protein concentration in the supernatant was measured using the BCA Protein Assay kit.

Mouse lung epithelial cells (MLE-12) and immortalized mouse bone marrow–derived macrophages (iBMDMs) were used. MLE-12 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM)/F12; iBMDMs were incubated in Roswell Park Memorial Institute 1640 (RPMI 1640) medium. Media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were maintained at 37°C under humidified 5% CO₂.

For NF-κB/MAPK pathway evaluation, iBMDMs were stimulated with LPS (1 μg/ml) for 0.5–3 h, and AKFPD (400 μg/ml) were added 24 h before LPS treatment. For apoptosis level detection, MLE-12 were stimulated with LPS (1 μg/ml) for 24 h, and AKFPD (400 μg/ml) were added 24 h before LPS treatment. Each experiment was replicated at least three times.

Concentrations of IL-6, IL-1β, and TNF-α of BALF and the cell supernatant were detected by using the ELISA kit according to manufacturer’s instructions.

TUNEL assay was performed in 4 μm paraffined sections with the In Situ Cell Death Detection Kit, fluorescein, according to the manufacturer’s instructions. After deparaffinization and rehydration, lung slides were denatured in 10 mM citric acid buffer, following by blocking in 3% goat serum. Reagent 1 (TDT) and reagent 2 (dUTP) were mixed at 1:10 and added to the slices at 4°C overnight. After washing with PBS, slides were stained with DAPI for 10 min. A total of five scopes were randomly selected in each sample under 200-fold magnification for quantitative analysis.

After deparaffinization and rehydration, lung slides were denatured for 15 min in boiling 10 mM citric acid (pH 6.0) and blocked with 5% bovine serum albumin for 30 min at room temperature and then incubated overnight at 4°C with the P65 antibody. After washing with PBS, slides were incubated with secondary antibodies for 1 h and then were stained with DAPI for 10 min.

MLE-12 cells were seeded on 6-well culture plates in a complete medium containing 10% FBS for 24 h. Wells were randomly separated into three groups: control, LPS, and LPS + AKFPD. The LPS + AKFPD group was pretreated with AKFPD (400 μg/ml) for 24 h. After AKFPD pretreatment, the LPS group and LPS + AKFPD group were incubated with LPS (1 μg/ml) for 24 h to induce cell apoptosis. After that, cells were harvested, stained with the FITC Annexin V and PI staining kit according to the manufacturer’s instructions, and analyzed by flow cytometry to identify cell apoptosis. Annexin V⁺ and PI⁻ cells were considered as early apoptotic cells, and annexin V⁺ and PI⁺ cells were considered as late apoptotic cells. The experiment was replicated at least three times.

Protein concentrations were determined using the BCA Protein Assay Kit. For Western blot analysis, 20–40 μg of protein was separated on sodium dodecyl sulfate–polyacrylamide (SDS-polyacrylamide) gel followed by transfer to polyvinylidene difluoride membranes. The membranes then were blocked in 5% skim milk for 1 h at room temperature and were incubated overnight at 4°C with the primary antibodies against p-IKK/IKK (1:1000), p-IκB/IκB (1:1000), p-P65/P65 (1:1000), p-ERK/ERK (1:1000), p-P38/P38 (1:1000), p-JNK/JNK (1:1000), caspase-3 (1:1000), Bax (1:1000), Bcl-2 (1:1000), and α-tubulin (1:5000). Then, the membranes were subsequently incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The bands were visualized by the ECL substrate and analyzed using ImageJ software.

Results were presented as the mean value ±standard deviation. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests with Prism software (version 6.01; GraphPad, United States) was used for comparison among all different groups. The log-rank test was used to compare the survival rate. p value <0.05 was considered as statistically significant.

To determine whether fluorofenidone has a protective effect on ALI, we evaluated the histopathologic feature of the lungs in the LPS-induced ALI mouse model. As showed in H&E staining (Figure 1A), LPS administration induced extensive and serious inflammation with massive inflammatory cell infiltration, alveolar architecture disorder, and partial consolidation, while the control group showed a normal pulmonary architecture. Compared with the LPS group, fluorofenidone treatment remarkably relieved those pathological symptoms and decreased lung injury scores (Figure 1B). In order to confirm the late protective effect of fluorofenidone in ALI, we further observed the survival rate in ALI mice, and the results showed that treatment with fluorofenidone remarkably reduced LPS-induced mortality in ALI mice (Figure 1C).

It is well known that inflammation plays a critical role in the development of ALI. Numerous pro-inflammatory cytokines and chemokines would be released to damage pulmonary epithelial cells and bronchial epithelial cells and further recruit inflammatory cells such as macrophages, to induce the inflammatory cascade response (Ma et al., 2010; Wang et al., 2017). To further evaluate the effects of fluorofenidone on ALI, we investigated the inflammatory level in BALF of ALI mice. We found that fluorofenidone treatment significantly diminished the elevated levels of IL-1β, TNF-α, and MCP-1 in BALF of LPS-induced ALI mice (Figures 2A–C). Moreover, the percentage of inflammatory cells in the lungs was determined by immunohistochemistry. As showed in Figures 2E–H, fluorofenidone administration obviously decreased the macrophage marker F4/80 and neutrophil marker MPO positive cell counts in the lungs of mice. These results suggest that fluorofenidone can interrupt the LPS-induced inflammatory cascade and reduce the inflammatory response in ALI.

Previous studies have revealed that excessive AEC apoptosis is observed in LPS-induced ALI (Yanagihara et al., 2001; Luo et al., 2019). AEC apoptosis could be mediated by various pathways, such as the mitochondrial dysregulation–mediated pathway or DNA damage-mediated pathway (Galani et al., 2010). Inhibition of pulmonary epithelial apoptosis would improve the pulmonary function and survival rate of ALI mice (Gong et al., 2017). Thus, our study further investigated whether fluorofenidone could reduce cell apoptosis in ALI mice. The results showed that fluorofenidone markedly reduced the number of TUNEL positive cells in the lungs of ALI mice induced by LPS (Figures 3A,B). Furthermore, we found that fluorofenidone could significantly increase the expression levels of the mitochondrial anti-apoptosis factor B-cell lymphoma 2 (Bcl-2), decrease the pro-apoptosis factor Bcl-2 X-associated protein (Bax), the ratio of Bax to Bcl-2, and cleaved caspase-3 in the lung tissue of ALI mice (Figures 3C–G), which suggests that fluorofenidone could ameliorate the mitochondrial-mediated apoptosis in LPS-induced ALI mice.

It has been reported that LPS can induce the production and secretion of various inflammatory mediators by stimulating the NF-κB pathway and MAPK pathways (Guo et al., 2018). Hence, we further explored whether fluorofenidone exerts its inhibitory effect on NF-κB and MAPK signaling pathway in LPS-induced ALI mice. In our study, the expressions of phosphorylated ERK, JNK, and P38 in the LPS group were remarkably increased compared with those of the control group, while fluorofenidone administration significantly reduced those changes (Figures 4A–D). Moreover, we also found that fluorofenidone remarkably suppressed phosphorylation of IκB and NF-κB induced by LPS in lung tissues, but the expression of phosphorylated IKK was not affected (Figures 4E–H). Meanwhile, immunofluorescence also showed that fluorofenidone obviously prevented nuclear translocation of NF-κB in the lungs of ALI mice (Figure 4I).

To further validate the effects of fluorofenidone on apoptosis, we mimicked LPS-induced lung injury in mouse intrinsic alveolar epithelial cell lines (MLE-12) by treating cells with LPS. The results of Western blot showed that LPS treatment stimulated the expression of Bax and cleaved caspase-3, while decreased that of Bcl-2 and increased the ratio of Bax to Bcl-2. However, pretreatment with fluorofenidone markedly diminished those changes (Figures 5A–E). Meanwhile, we measured the proportion of apoptotic cells by flow cytometry assay. As shown in Figures 5F,G, more apoptotic cells were observed in LPS-induced MLE-12 cells, while fluorofenidone pretreatment significantly reduced the proportion of apoptotic alveolar epithelial cells.

It is widely accepted that the inflammatory cascade response in ALI majorly occurs and initiates in macrophages, including the inflammatory signaling pathway activation and the proinflammatory factor release (Zhang et al., 2016). Therefore, we determined the anti-inflammatory effects of fluorofenidone in BMDMs. As shown in Figure 6, preincubation with fluorofenidone markedly reduced the expression levels of IL-1β, TNF-α, and IL-6 in the supernatant of BMDMs elevated by LPS (Figures 6A–C). Moreover, the effect of fluorofenidone on NF-κB/MAPK pathway activation on LPS-induced BMDMs was detected. The results of Western blot showed that compared with the control group, the phosphorylation of IκB, NF-κB, ERK, JNK, and P38 was significantly increased upon LPS stimulation, and pretreatment with fluorofenidone markedly reversed those effects (Figures 6D–J). Those results suggest that fluorofenidone could alleviate the LPS-induced inflammation response via suppressing NF-κB/MAPK pathway activation in macrophages.