Male Institute of Cancer Research (ICR) mice obtained from the animal facilities of Nanjing Medical University (Nanjing, Jiangsu, China) were housed (n=5) in a temperature-controlled room and received water and food ad libitum. Animal welfare and experimental procedures were strictly in accordance with the Guide for the Care and Use of the Laboratory Animals (US National Research Council, 1996) and the associated ethics regulations of Nanjing University of Chinese Medicine (Nanjing, Jiangsu, China).

The tubers of PT were collected from Jiangsu Province (China) with the help of Taizhou Gaogang Pieces Factory Co., Ltd., (Jiangsu, China) on May 25, 2012. The fresh plant was identified by Professor Chungen Wang (Nanjing University of Chinese Medicine).

Mini protean cell and semi-dry transfer cell was supplied by Bio-Rad Laboratories (Hercules, CA, USA). HiPrep™ Phenyl FF, HiTrap™ Q FF, HiTrap™ desalting and AKTA purifier were supplied by GE Healthcare (Uppsala, Sweden). Scanning electron microscope (SEM) was supplied by Nikon (Tokyo, Japan). Fluorescence microplate reader was supplied by BioTek Instruments, Inc. (Winooski, VT, USA). RPMI-1640 was from Gibco (Waltham, MA, USA). FCS was from Sijiqing (Hangzhou, China). Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 ELISA kits were from eBioscience (San Diego, CA, USA). Transwell 24-well sets and 12- and 48-well plates were from Corning Inc. (Corning, NY, USA). Dextran T-500 and Percoll were from Pharmacia (Piscataway, NJ, USA). 2′,7′-Dichlorofluorescein-diacetate (DCFH-DA) was from Nanjing Jiancheng Chemical Industrial Co., Ltd. (Nanjing, China). Anti-GAPDH antibody (ab9485) and anti-NF-κB p65 (ab16502) antibodies were from Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-labeled secondary antibody was from Hangzhou Lianke Biology Technology Ltd. (Hangzhou, China). The ECL chemiluminescence detection kit was from Millipore (Burlington, MA, USA). Distilled water was produced in the EPED Superpure water purifying system (Nanjing EPED Science and Technology Development Co., Nanjing, China). All the reagents were at least analytical grade.

Fresh tubers of PT weighing 50 g were washed thoroughly with tap water and subsequently with distilled water. The combined roots and tubers were separated from the stems and leaves prior to crushing using a juice extractor to extract the juice and centrifugation for 20 min at 4°C. The clear supernatant was dissolved with saturated (NH₄)₂SO₄ (pH 7.0) and was centrifuged for 30 min at 4°C. The precipitate was fully dissolved with 0.6 mol/l (NH₄)₂SO₄ (pH 7.0) and centrifuged at a high speed. The resulting supernatant obtained was applied to a HiPrep™ Phenyl FF (10 ml) column. The mobile phase was a 0.6-0 mol/l (NH₄)₂SO₄ gradient at a flow rate of 1.4 ml/min. The main peak was collected and applied to a column of HiTrap™Q FF. The column was eluted with a gradient of 0–0.4 mol/l NaCl at a flow rate of 2.5 ml/min. Finally, the main peak was desalted with 0.02 mol/l Tris-HCl (pH 8.0) buffer for lectin purification and subsequently freeze-dried.

Purified lectin preparation was checked on an Agilent 1200 system equipped with a DAD detector using an Agilent Zorbax GF-450 column (9.4×250 mm). The mobile phase was 0.1 mol/l phosphate buffer at a flow rate of 1.5 ml/min. PTL was also subjected to SDS-PAGE (pH 8.3), using a 15% (w/v) acrylamide slab gel for subunit molecular mass determination. The sample was heated for 5 min in a boiling water bath. The gel was stained with silver nitrate. Molecular weights of the standard marker proteins were matched with that of the sample protein (4.6–42.0 kDa) to determine the subunit molecular weight of PTL.

The 12-kDa protein band was destained with 400 µl of 30% acetonitrile in 100 mM NH₄HCO₃, reduced with 10 µl of 100 mM dithiothreitol at 56°C for 30 min, dehydrated with 100 µl of acetonitrile, alkylated with 30 µl of 200 mM iodoacetamide at room temperature in the dark for 20 min, dehydrated with 100 µl of acetonitrile, and subsequently dried. In-gel tryptic digestion was carried out at 37°C for 20 h using a 10.0 ng/ml solution of sequencing grade-modified trypsin (Promega, Madison, WI, USA). Tryptic peptides resulting from the digestion were extracted with 100 µl of 60% acetonitrile in 0.1% formic acid for 15 min. The extraction was repeated 3 times. The extracted peptides were dried and reconstituted with 5% acetonitrile and 0.1% formic acid in water.

The extracted peptides were analyzed using an LC-LTQ system (LTQ VELOS, Thermo Finnigan, San Jose, CA, USA). Briefly, peptides were first enriched on a reverse-phase trap column (Zorbax 300SB-C₁₈ peptide traps; Agilent Technologies, Wilmington, DE, USA) and subsequently eluted to an analytical column (RP-C₁₈, 0.15 × 150 mm; Column Technologies Inc., Lombard, IL, USA). The flow rate of the pump was 0.15 ml/min. The mobile phases used for the reverse phase were (A) 0.1% formic acid in 84% ACN and (B) 0.1% formic acid in water, pH 3.0. The gradient started at 4% solvent A, where it was held for 4 min, went linearly to 50% solvent A in 15 min, and subsequently went linearly to 100% solvent A in 3 min. An electrospray ionization mass spectrometer was used for peptide detection. The positive-ion mode was employed and the spray voltage was set at 3.2 kV. The spray temperature was set at 200°C for peptides. Collision energy was set automatically by the LTQ system. The mass spectrometer was set as one full MS scan followed by 10 MS/MS scans on the most intense ions. Protein identification using MS/MS raw data was performed with the BioWorks Browser 3.3 (University of Washington, Seattle, WA, USA; licensed to Thermo Finnigan) searching program against the UniProt Arecaceae protein database. The protein identification criteria that were used were based on Delta CN (≥0.1) and Xcorr (one charge ≥1.9, two charges ≥2.2, three charges ≥3.75).

Animals were sacrificed and sterilized in 75% alcohol. Each mouse was injected intraperitoneally with 4 ml of cold phosphate-buffered saline (PBS) and massaged softly for 3 min. Harvested peritoneal fluid was centrifuged at 70 × g for 4 min and the precipitate was dissolved and adjusted to 1×10⁶ cells/ml with RPMI-1640 containing 10% fetal bovine serum (FBS). Peritoneal cells were subsequently cultured in a 48-well plate and washed 3 times with cold PBS after incubation at 37°C for 2 h. Macrophage purity was examined with non-specific esterase staining as >98%. The viability of cells was >99%, as determined by the trypan blue exclusion test.

Blood from healthy volunteers (5 ml) was drawn into vacuum blood collection tubes containing EDTA-2Na. The blood was mixed with cold PBS and Dextran T-500 (1.5:1.5:1, v/v/v) at room temperature until sedimentation of the red blood cells. The supernatant was collected 30 min later and centrifuged at 110 × g for 10 min. The precipitate was subsequently centrifuged together with 75% Percoll and 60% Percoll at 250 × g for 20 min. Following centrifugation, neutrophils were purified from the blood cells. This procedure usually produced a cell fraction containing over 99% neutrophils, as determined by quick Giemsa staining. The trypan blue exclusion test showed that the viability of cells was >99% (21).

To confirm that macrophages activated by PTL were involved in the recruitment of neutrophils, an in vitro chemotaxis experiment was conducted as follows: 100 µl of the neutrophil suspension (10⁶ cells/ml) was placed in the upper wells of a 24-well modified Corning Transwell set equipped with a polycarbonate filter (8 µm pore size); in the lower wells, the protocol was set up either with D-Hanks or PTL (50 µg/ml in D-Hanks)-treated fresh medium, or with D-Hanks or PTL (50 µg/ml in D-Hanks)-treated pre-cultured macrophages (10⁶ cells/ml).

Following Transwell plate incubation for 1.5 h at 37°C in 5% CO₂, the cells remaining in the upper wells were removed using cotton swabs and those retained on the lower side of the filter were stained with Giemsa staining. The number of neutrophils reaching the lower side of the filter was counted in 5 random fields using an electron microscope (objective, ×40) for each set of wells.

Mouse macrophages were harvested with RPMI-1640, cultured in plastic dishes (48-wells of 500 µl capacity), washed and replaced with 450 µl/well fresh RPMI-1640 containing 10% FBS prior to the experiment. The protocol for the assay of TNF-α and IL-1β was conducted using 6 groups (3 wells/group) of macrophages treated as follows: Group 1, 50 µl/well PBS alone; and groups 2–6, 50 µl/well PTL (25, 50, 100, 200 and 400 µg/ml). The protocol for the assay of IL-6 was conducted using 8 groups (3 wells/group) of macrophages treated as follows: Group 1, 50 µl/well PBS alone; and groups 2–6, 50 µl/well PTL (6.25, 12.5, 25, 50, 100, 200 and 400 µg/ml). After 3 h of incubation, all the supernatants were harvested and stored at −80°C. The levels of TNF-α, IL-1β, IL-6 and nitric oxide (NO) released by macrophages were measured using ELISA kits according to the manufacturer's instructions.

Mouse macrophages were harvested with RPMI-1640, cultured in plastic dishes (48-wells of 500 µl capacity), washed and replaced with 450 µl/well fresh RPMI-1640 containing 10% FBS prior to the experiment. The protocol was conducted using 18 groups (3 wells/group) of macrophages treated half with 50 µl of 100 µg/ml PTL and the others with 50 µl of PBS as control groups. The incubating times were as follows: 0.5, 1, 1.5, 2, 2.5, 3, 6, 9 and 12 h. After the incubation, all the supernatants were harvested and stored at −80°C. The levels of TNF-α, IL-1β, IL-6 and NO released by macrophages were measured using the ELISA kits according to the manufacturer's instructions.

Intracellular ROS production was measured using DCFH-DA (22). DCFH-DA penetrates into cells and is hydrolyzed by intracellular esterase to the non-fluorescent DCFH, which can be rapidly oxidized to the highly fluorescent 2,7-dichlorofluorescein (DCF) in the presence of ROS. Peritoneal macrophages were seeded at 1×10⁶ cells/well in 12-well plates. PTL was dissolved and diluted in D-Hanks. Mouse macrophages were harvested with RPMI-1640, cultured in plastic dishes (48-wells of 500 µl capacity), washed and replaced with 450 µl/well fresh RPMI-1640 containing 10% FBS prior to the experiment. Peritoneal macrophages were seeded at 1×10⁶ cells/well in 12-well plates. The protocol was conducted using 4 groups (3 wells per group) of macrophages treated as follows: Group 1, 50 µl/well D-Hanks alone; and groups 2–4, 50 µl/well PTL (25, 50 and 100 µg/ml). Following stimulation and incubation with or without different treatments for 1 h, the cells were treated with 4 µM DCFH-DA at 37°C for 20 min and examined using a fluorescence microscope (Nikon, Tokyo, Japan) with an emission wavelength of 598 nm. The fluorescence intensity was measured with a fluorescence microplate reader with an excitation wavelength of 420 nm and an emission wavelength of 560 nm.

To confirm that pro-inflammation induced by PTL was associated with the NF-κB signaling pathway, the content of p65 in the cytoplasm and nucleus was analyzed by western blot analysis, which was conducted using 4 groups (3 wells/group) of macrophages treated as follows: Group 1, 50 µl/well PBS alone; and groups 2–4, 50 µl/well PTL (12.5, 25 and 50 µg/ml). After 0.5 h of incubation, the culture supernatant was discarded, and the macrophages were washed with PBS buffer. Subsequently, the cytoplasmic and nuclear proteins of the macrophages were extracted (23). The cytoplasmic and nuclear protein solutions were denatured by boiling at 100°C for 5 min, mixed evenly with 5X bromophenol blue loading buffer, and were loaded (10 µl for each lane) onto 10% SDS polyacrylamide gel followed by electroblotting onto nitrocellulose membrane. Following blocking of the non-specific binding with 5% non-fat milk (prepared in TBS containing 0.1% Tween-20) for 3 h, the membrane was applied with antibodies against p65 and GAPDH, followed by incubation with HRP-conjugated anti-rabbit antibody. Subsequently, the protein was detected using an ECL chemiluminescence detection kit and cultured for 5 min in the dark. Densitometry was performed using the software Quantity One (Biorad).

Mouse macrophages were harvested as indicated. For SEM, the cells were cultured on the sterile slides placed in 6-well plastic plates. The assay was performed with 5 groups (3 wells/group) of macrophages treated with 500 µl of 6.25, 12.5, 50 and 100 µg/ml PTL. All the supernatants were harvested following incubation for 0.5, 1 and 3 h. Each slide was washed with cold PBS 3 times and was fixed with 2.5% glutaraldehyde overnight. Sample slides were prepared through sequential dehydration in ethanol (30, 50, 70, 80, 90 and 100%). The sample slides were subsequently coated with gold-palladium prior to SEM observation. The morphological changes of macrophages were evaluated by capturing images focusing on a single macrophage at (magnification, ×3,000–5,000).

The data are presented as mean ± standard error of the mean and compared using t-test by SPSS (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Hydrophobic interaction, ion exchange and desalting chromatographic steps were applied to purify PTL. The crude protein extract was eluted by hydrophobic interaction. The main peak was eluted with a linear gradient of NaCl (0–0.4 mol/l). A single peak on HPLC and a single band of ~13 kDa on SDS-PAGE (Fig. 1) were observed, suggesting that the isolated PTL was quite pure. Gel electrophoresis was combined with LC-MS/MS for the proteomic analysis of PTL. The 13-kDa band was subjected to in-gel trypsin digestion. The peptides extracted from each band were loaded to LC-MS/MS for protein identification. Through a protein database search, the 13-kDa band protein was identified as PTL, with the molecule matching those previously reported (24).

In order to investigate the involvement of macrophages in neutrophil infiltration induced by PTL treatment, neutrophil migration assay was used with a 24-well Transwell set. Compared to the control (D-Hanks) group, significantly more neutrophils were retained on the lower surface of the filter following treatment with PTL (PTL+M_Φ+D-Hanks). Macrophages untreated with PTL (D-Hanks+M_Φ) and the group in which PTL did not coexist with macrophages (D-Hanks+PTL) did not show any neutrophil chemotaxis (Fig. 2), therefore, PTL-stimulated neutrophil chemotaxis was mediated by macrophages.

Peritoneal macrophages were treated with indicated doses of PTL and the levels of released cytokines, such as TNF-α, IL-1β and IL-6, were measured. PTL activated macrophages to release TNF-α, IL-1β and IL-6 in a dose-dependent manner. Compared to the control group (PBS), the levels of TNF-α, IL-1β and IL-6 significantly increased following exposure of the macrophages to PTL (50, 100, 200 and 400 µg/ml) for 3 h (Fig. 3). Notably, IL-6 was more prone to induction compared with TNF-α and IL-1β.

To confirm whether PTL could cause acute inflammation in animal models, macrophages were treated by PTL for different periods of time and the levels of cytokines, TNF-α, IL-1β and IL-6, were measured. As shown in Fig. 4, the levels of TNF-α and IL-1β significantly increased 1.5 h after stimulation compared to the control. The levels of TNF-α and IL-1β reached maxima after 3 h of treatment with PTL, and subsequently gradually decreased (Fig. 4A and B). The time curve of IL-6 changed in a similar manner. In addition, PTL significantly increased the level of IL-6 starting from 1 h (Fig. 4C). Therefore, PTL may cause inflammation in macrophages at an early stage.

To evaluate the effects of PTL on ROS production, macrophages were stimulated with different doses of PTL (25, 50 and 100 µg/ml) for 1 h and observed under an inverted fluorescence microscope following the addition of a fluorescent probe, DCFH-DA. The fluorescence intensity was measured with a fluorescence microplate reader. Compared with the D-Hanks group, elevating the PTL dose significantly increased the intracellular ROS contents (Fig. 5). Thus, PTL stimulated macrophages to undergo intense oxidative stress, releasing considerable ROS eventually.

Following stimulation with PTL (5, 12.5 and 25 µg/ml) for 0.5 h, the content of p65 in the macrophage cytoplasm reduced compared with that of the blank group (Fig. 6B), however, this content in the macrophage nucleus markedly increased (Fig. 6A), all showing evident dose-effect associations. Therefore, PTL stimulation caused transfer of p65 from the macrophage cytoplasm to the nucleus, which suggested that the inflammation induced by PTL-stimulated macrophage was associated with the NF-κB signaling pathway.

The morphological changes of macrophages following treatment with PTL were observed using SEM. As shown in Fig. 4, the mouse peritoneal macrophages were normal at 0 h of PTL treatment. From 0.5 h of treatment, 6.25, 12.5 and 50 µg/ml PTL caused apparent wrinkles on the macrophage surface and more pseudopodia, accompanied by clear morphological changes. At 3 h, 50 and 100 µg/ml PTL evidently damaged or even destructed the macrophage surface (Fig. 7). Therefore, PTL at appropriate doses could induce the activation of macrophages, and at a high dose induced cell death.