2b). At 8 μg mL−1 apigenin, the α-hemolysin could not be detected in the culture supernatant. Alpha-hemolysin is encoded by the hla gene, which is regulated by the Agr two-component system. Consequently, a real-time RT-PCR p38 MAPK activity assay was performed to examine whether apigenin can affect the transcription

of the hla and agrA genes. As shown in Fig. 2c and d, the transcription of hla and agrA was remarkably inhibited when increasing concentrations of apigenin were added. When cells were co-cultured with 8 μg mL−1 apigenin, the transcriptional levels of the hla and agrA genes were reduced 14.03- and 9.13-fold, respectively. Human A549 alveolar epithelial cells are widely used in pulmonary disease models (Nizet et al., 1996; Hirst et al., 2002). Previous studies have demonstrated that α-hemolysin can cause A549 cell injury in a dose-dependent manner (Liang et al., 2009). Therefore, apigenin was assayed for its ability to protect A549 cells from α-hemolysin-mediated cell injury. In this study, A549 cells Doxorubicin solubility dmso were co-cultured with S. aureus and different concentrations of apigenin. Cells were strained with a live/dead (green/red) reagent. As shown in Fig. 3a, uninfected cells retained a green fluorophore, while dead cells were red (Fig. 3b). As

shown in Fig. 3c, apigenin conferred significant protection from cell injury at the concentration of 8 μg mL−1. Furthermore, a LDH release assay was performed to quantify cell injury, and as shown in Fig 3e, apigenin provided a dose-dependent protection to co-cultures of A549 cells with concentrations from 1 to 8 μg mL−1. Alpha-hemolysin has been established as the main virulence factor in mouse models of S. aureus pneumonia (McElroy et al., 2002; Gomez et al., 2004). Alpha-hemolysin has also been shown to damage the air–blood dipyridamole barrier in a rat model of S. aureus lung infection (McElroy et al., 1999). On the foundation of in vitro research that apigenin can reduce the expression of α-hemolysin at very low concentrations, a S. aureus-mediated mouse pneumonia model was used to investigate

the in vivo protective effects of apigenin. Mice were infected intranasally with a 30-μL S. aureus 8325-4 suspension as described in the ‘Materials and methods’. Next, mice were subcutaneously administered either PBS or 50 mg kg−1 apigenin. The hla− strain DU1090 was used as a negative control. The bacteria burden was quantified to evaluate the influence of apigenin on the survival in the lungs. As shown in Fig. 4a, the CFUs of lungs from infected mice treated with 50 mg kg−1 were remarkably lower than those treated with PBS. The lung tissues of S. aureus 8325-4-infected mice that had been treated with apigenin were pink and spongy. However, the lung tissues of mice that were treated with PBS were kermesinus and had a firm texture (Fig. 4b).