Binding screening of Phillygenin and PXR
Target of phillygenin was predicted using the BATMAN-TCM online tool. It was showed that phillygenin could bind to PXR (Fig 2A). To further determine relationship between Phillygenin and PXR, binding screening assay was performed as previously described[19]. In brief, the hollow fiber-based ligand fishing (HFLF) strategy was used to determine the binding between phillygenin and PXR. Hollow fiber membranes with an inner diameter of 500 μm and a pore size of 0.2 μm were purchased from GE Healthcare. PXR protein was injected into the lumen of hollow fibers treated with acetone, methanol, and ddH2O. Hollow fiber membranes filled with proteins were sealed by heating and placed into phillygenin extract (10 mg/ml), incubated in an ultrasonic bath at 37oC for 30 min. After washing with phosphate-buffered saline (PBS), bound compounds formed in the lumen were dissociated with 30 μl of methanol. The fluid was centrifuged at 10000×g for 10 min. Twenty microliters of supernatant was used for liquid chromatography–mass spectrometry (LC-MS) analysis. The flow chart is shown in Fig 2B.
Transient transfection and luciferase assays
A549 cells were used for luciferase assays. Recombinant pGL-3 plasmids containing CYP3A4 promoter and pcDNA3.1 plasmid containing PXR cDNA were transiently co-transfected into A549 cells. After 48 h, different concentrations of PHI were used to treat the transfected A549 cells for 16 h. Each treatment was repeated three times. The cells were lysed, and luciferase activity was quantified using the Dual-Luciferase Reporter Assay System (Promega, WI, USA) according to the manufacturer's instructions. Relative luciferase activities were shown by normalizing the firefly luciferase activity to Renilla luciferase activity. To evaluate whether PHI could inhibit NF-κB by PXR, NF-κB p65 activity was detected using NF-κB (p65) transcription factor assay kit (Cayman Chemical, MI, USA). A549 cells were cultured in 24 well plate and then transfected with siRNA against PXR. 24 hours later, the cells were treated with or without PHI and LPS for 24 hours. According to the manufacture’s instruction, 10 μl of nuclear extracts were used for detection.
LPS-induced acute pneumonia rat model
Thirty-six male Sprague Dawley (SD) rats (Rattus norvegicus) aged 8-10 weeks with an average weight of 220-250 g were purchased from Vital River Laboratory (Vital River Laboratory Animal Technology, Beijing, China). The animals were housed at the Center for New Drug Safety Evaluation, Lunan Pharmaceutical (Lunan Pharmaceutical Group Co., Ltd., Linyi, China). The experimental protocols were approved by the Animal Ethics Committee of Lunan Pharmaceutical Group Co. Ltd. The animals were randomly divided into the following 3 groups (n=12): control (vehicle treatment), LPS (LPS treatment), and PHI (LPS+PHI treatment) group. The LPS and PHI groups were intranasally administered with LPS (20 mg/kg bodyweight). Next, the animals were intragastrically administered with vehicle or PHI (1.3 mg/kg bodyweight; three times a day) continuously for 7 days. According to the drug instruction and the conversion of human body surface area into rat body surface area, the dosage of rat is 1.3 mg/kg. At the end of experiment, the bronchoalveolar fluid (BALF), blood samples and lung tissue were collected from all animals.
Staining
The pathological changes in the lung tissue were evaluated by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining as described previously[20]. Briefly, the lung tissues of rats were fixed in 4% paraformaldehyde buffer overnight and embedded in paraffin. The paraffin-embedded tissue was cut into 2 μm sections. The sections were subjected to H&E and TUNEL staining.
The expression levels of target proteins predicted by bioinformatic and network pharmacology analyses were validated by immunohistochemical (IHC) staining. The expression levels of tumor necrosis factor (TNF)-α signaling pathway-related proteins were evaluated (Supplementary Materials and Methods).
Immunofluorescence and western blotting
To investigate the expression levels of TNF-α signaling pathway-related protein, such as phosphorylated JNK, P38, ERK, P65, Bax, Bcl-2, caspase-3, and caspase-9, different treatment groups of A549 cells were subjected to immunofluorescence and western blotting analyses (Supplementary Materials and Methods).
Flow cytometry and ELISA
The BALF and serum samples were obtained from all experimental animals. The levels of inflammatory factors in the BALF and serum samples were detected by flow cytometry and ELISA.
To determine the levels of cytokines, the rat BALF sample was filtered to obtain single-cell suspensions and washed with 10 mL of Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS). After incubated with red blood cell lysis buffer (0.01 M KHCO3, 0.15 M NH4Cl, and 0.1 M EDTA-Na2, pH 7.4), the cells were resuspended in RPMI-1640 medium and counted. Next, the cells (5×106 cells/well) were cultured in 6-well plates and incubated with phorbol 12-myristate 13-acetate (PMA; 50 ng/mL), ionomycin (100 ng/mL), and Brefeldin A (BFA; 10 μg/mL) for 6 h at 37°C. The cells were harvested and detected using flow cytometry (Supplementary Materials and Methods)..
The respective ELISA kits were used to detect serum levels of TNF-α, IFN-γ, IL-1β, IL-6, and IL-18 in rats following the manufacturer’s instructions (Supplementary Materials and Methods).
Cell culture and cell inhibition, viability assays
The human lung adenocarcinoma epithelial-like cell line A549 (#TCHu150) was obtained from China Center For Type Culture Collection (CCTCC, Wuhan University, Wuhan, Hubei province, China) and cultured as previously described[21].
Cell proliferation was analyzed by using the CellTiter 96®Aqueous Non-radioactive Cell proliferation Kits (#TB169, Promega Corporation, Madison, WI, USA.), following the manufacturer’s instructions. The half-maximal effective concentration (EC50) of PHI was calculated in the Graphpad Prism 5.0 software.
The effect of PHI on LPS-induced apoptosis of A549 cells was determined by flow cytometry using the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, CA).
Statistical analysis
The data were expressed as mean ± standard deviation (Mean ± S.D.). Statistical analyses of all data were performed with SPSS 18.0 (IBM Corporation, USA). If the data passed the tests for normality and homogeneity of variance, the one-way ANOVA was selected for statistical analysis, otherwise, a nonparametric test was applied. The difference was considered statistically significant when the P-value was less than 0.05.