The human alveolar epithelial cells (A549) were obtained from American Type Culture Collection (ATCC). They were routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (both from Beijing Solarbio Science & Technology Co., Ltd.), 100 U/ml penicillin and 100 pg/ml streptomycin in a humidified incubator with 5% CO₂ at 37˚C.

A H/R-A549 cell model was established. Briefly, A549 cells were exposed to anoxia for 24 h (1% O₂, 5% CO₂ and 94% N₂) at 37˚C followed by 4 h of reoxygenation (5% CO₂ and 95% air) at 37˚C (H/R group) (21,22).

The cell model with the SIRT1/AMPK signaling pathway blocked (H/R-A549 + sirtinol) was established. Sirtinol stock solution (10 mM) (Sigma-Aldrich; Merck KGaA) and MAF (Sigma-Aldrich; Merck KGaA; Fig. 1A) stock solution (50 mM) were prepared in dimethyl sulfoxide and stored at -80˚C until further use. H/R-A549 cells were treated with 15 µM of sirtinol for 72 h at 37˚C (23). Subsequently, the cells were cultured for another 24 h with 20 µM MAF at 37˚C (H/R + MAF + sirtinol group). Additionally, H/R-A549 cells were incubated with 20 µM MAF at 37˚C for 24 h (H/R + MAF group).

Cell viability was measured using MTT assay. A549 and H/R-A549 cells (5x10³/well) were each incubated in 96-well plates with various concentrations of MAF (5, 10, 20 and 50 µΜ). Using the RPMI-1640 medium to dilute the drug stock solution, the cell toxicity of DMSO was negligible due to the use of ≥1,000-fold dilution. After incubation for 24 h, 20 µl MTT (5 mg/ml) was added to each well and cells were incubated for a further 4 h at 37˚C. A buffer solution (10% SDS, 5% isopropanol and 0.1% HCl) was then added to solubilize the MTT formazan crystals overnight. The absorbance in each well was measured at 570 nm using a microplate reader (Thermo Fisher Scientific, Inc.). Untreated cells were used as a control.

A549 and H/R-A549 cells (5x10³/well) were inoculated into 96-well plates and treated with different MAF concentrations (5, 10 and 20 µΜ) for 24 h at 37˚C. Subsequently, the cell culture medium was centrifuged at 2,000 x g for 4 min at 4˚C to remove debris and the supernatant was collected for assay. The levels of IL-6, IL-1β and IL-10 in cells were quantified using their corresponding ELISA kits (cat. no. E-EL-H0102c, E-EL-H0149c and E-EL-H0103c; Elabscience Biotechnology Co., Ltd.) according to the manufacturer's protocols. The absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Inc.).

Apoptotic A549 and H/R-A549 cells were visualized using one-step TUNEL apoptosis detection kit according to the manufacturer's protocol (cat. no. KGA7071; Nanjing KeyGen Biotech Co., Ltd.). Briefly, cells (2x10⁶/ml) that were cultured on cover slips were fixed using 4% neutral-buffered formalin solution at room temperature for 30 min and incubated with 0.3% Triton X-100 at room temperature for 5 min. Subsequently, each sample was supplemented with 50 µl TUNEL detection reagent for 60 min at 37˚C in the dark. The cell nuclei were stained with 5 µg/ml DAPI at 37˚C in the dark for 5 min. An anti-fade solution (Beijing Solarbio Science & Technology Co., Ltd.) was dropped onto the area containing the treated cells and the sections were mounted onto the slides. Finally, all samples were imaged in three random fields per coverslip using a fluorescence microscope (magnification, x100) to view the green fluorescence at 520±20 nm and blue DAPI at 460 nm.

The protein samples from the cells were extracted using the cell lysis buffer (cat. no. P0013; Beyotime Biotechnology Inc.) and the protein content was measured using a BCA Protein Assay Kit (cat. no. P0012S; Beyotime Biotechnology Inc.). The protein samples (40 µg) were subjected to 15% SDS-PAGE for separation and transferred onto nitrocellulose membranes. After blocking in fresh 5% non-fat milk at room temperature for 2 h, the membranes were incubated overnight at 4˚C with primary antibodies, followed by incubation with a HRP-conjugated secondary antibody at room temperature for 2 h. An enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.) was used to visualize the signals. The antibodies (Affinity Biosciences) used included: Anti-SIRT1 (cat. no. DF6033; 1:1,000), anti-phosphorylated (p-)-AMPK (cat. no. AF3423; 1:1,000), anti-AMPK (cat. no. AF6423; 1:1,000), anti-Cox-2 (cat. no. AF7003; 1:1,000), anti-iNOS (cat. no. AF0199; 1:1,000), anti-Bcl-2 (cat. no. AF6139; 1:1,000), anti-Bax (cat. no. AF0120; 1:1,000), anti-cleaved-caspase-3 (cat. no. AF7022; 1:1,000), anti-cleaved-caspase-9 (cat. no. AF5240; 1:1,000), anti-GAPDH (cat. no. AF7021; 1:5,000) and the goat anti-rabbit IgG secondary antibody (cat. no. S0001; 1:5,000). Protein expression levels were semi-quantified using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc.).

All data were expressed as the mean ± standard deviation. statistical analysis was performed with GraphPad Prism 8.0 software (GraphPad Software, Inc.). Differences between the means of the groups were compared using a one-way ANOVA followed by Tukey's test. Each experiment was repeated ≥ three times. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1B, MAF at concentrations of <50 µM, could not exert toxicity on A549 cells. However. 50 µM MAF was found to be significantly toxic to cells. According to Fig. 1C, cell viability after H/R induction was significantly decreased compared with that in the control group. By contrast, the viability of H/R-A549 cells was increased significantly after treatment with 20 µM MAF. However, after treatment with 50 µM MAF, cell viability was markedly decreased compared with that in the 20 µM MAF treatment group. Therefore, in the subsequent experiments, the maximum concentration of MAF used was 20 µM.

The levels of the inflammatory markers in H/R-A549 cells treated with different concentrations of MAF were measured. As shown in Fig. 2A-C, ELISA analysis showed that compared with those in the control group, H/R significantly upregulated the expression levels of IL-6 and IL-1β, whilst significantly downregulating the expression level of IL-10. By contrast, the H/R-induced upregulation of IL-6 and IL-1β and downregulation of IL-10 were significantly reversed in the MAF-treated groups in a dose-dependent manner. In addition, the protein expression levels of Cox-2 and iNOS were also significantly increased in the H/R induced group compared with those in the control group, whilst those in the MAF-treated groups were significantly reversed compared with those in the H/R group. Taken together, these data suggest that MAF treatment can inhibit the-H/R induced inflammatory response.

Nuclear DNA fragmentation is an important biochemical event during apoptosis in many cell types and was therefore measured by TUNEL assay in the present study. The TUNEL results showed that A549 cell apoptosis was markedly increased after H/R induction (Fig. 3A). By contrast, after the addition of MAF, the H/R-induced cell apoptosis was reduced (Fig. 3A). In addition, compared with those in the control group, the expression levels of Bax, cleaved-caspase-3 and cleaved-caspase 9 protein were significantly increased in the H/R group, whilst those of Bcl-2 were reduced (Fig. 3B). Notably, the expression level of Bcl-2 was significantly increased by 10 and 20 µM MAF treatment compared with that in the H/R group (Fig. 3B). In addition, the expression levels of Bax, cleaved-caspase-3 and cleaved-caspase-9 were significantly decreased in 20 µM MAF treatment group compared with those in the H/R group (Fig. 3B). These results suggest that MAF can inhibit H/R-induced apoptosis in A549 cells.

To verify the hypothesis that MAF may serve a role in the SIRT1/AMPK signaling pathway, the protein levels of SIRT1 and ratio of p-AMPK/AMPK were evaluated in H/R cells by western blotting assay. As shown in Fig. 4, compared with those in the control group, the protein expression of SIRT1 and ratio of p-AMPK/AMPK were significantly deceased in the H/R-induced group. By contrast, compared with the H/R-induced group, the protein levels of SIRT1 and ratio p-AMPK/AMPK were significantly increased in the 10 and 20 µM MAF treatment groups (Fig. 4). Since a concentration of 20 µM MAF resulted in a highly significant increase in SIRT1 and p-AMPK/AMPK levels, 20 µM MAF was used subsequently to explore the effects of MAF on the SIRT1/AMPK signaling pathway.

To understand whether the SIRT1/AMPK signaling pathway affected H/R-induced inflammation and apoptosis, the SIRT1 inhibitor sirtinol was used to intercept the SIRT1/AMPK signaling pathway following MAF treatment. As shown in Fig. 5A-D, after blocking the SIRT1/AMPK signaling pathway, the protective effects of 20 µM MAF against inflammation were reversed. Compared with those in the MAF treatment group (H/R + MAF), the pathway inhibition group (H/R + MAF + sirtinol) significantly upregulated the expression levels of IL-6 and IL-1β, whilst significantly downregulating the expression level of IL-10 (Fig. 5A-C). In addition, the protein expression levels of Cox-2 and iNOS were also significantly increased in the H/R + MAF + sirtinol group compared with those in the H/R + MAF group (Fig. 5D). After inhibiting the SIRT1/AMPK signaling pathway, the rate of apoptosis in MAF treatment was markedly increased compared with that in the H/R + MAF group (Fig. 5E). Compared with those in the MAF treatment group, the protein expression levels of Bax, cleaved-caspase-3 and cleaved-caspase-9 were significantly increased after the addition of sirtinol, whereas Bcl-2 expression was significantly reduced (Fig. 5F). Altogether, these results suggest that MAF can activate the SIRT1/AMPK signaling pathway to inhibit H/R-induced A549 cell injury.