Algal strain and culture
E.gracilis CCAP 1224/5Z was obtained from CCAP (Culture Collection of Algae and Protozoa, UK) and maintained in medium (1.8g/L NH4Cl, 0.6g/L KH2PO4, 0.6g/L MgSO4, 60 mg/L Urea, 0.02g/L CaCl2, 0.48 mg/L Na2EDTA, 2mg/L Fe2 (SO4)3, 10 ml EtOH, 60 μL HCl, 0.01 mg/L Vb1, 0.0005 mg/L Vb12, 20 mg/L CuSO4·5H2O, 0.4 g/L ZnSO4·7H2O, 1.3 g/L Co (NH3)·H2O, 1.6 g/L MnCl2·4H2O). Cells were grown at 22°C photo-incubator under 100 µmol/m2/s with hand shaking twice every day, and cells at the exponential growth period were harvested for further tests.
Lipopolysaccharide, polymyxin B, NG-nitro-L-arginine methyl ester and pyrrolidine dithiocarbamate were purchased from Sigma-Aldrich (St. Louis, MO, USA). The inhibitors: SB 20358, SP 600125 and PD 98059, were purchased from Selleck (Shanghai, China). The Cell Counting Kit (CCK)-8 and Radioimmunoprecipitation Assay buffer were purchased from Beyotime (Jiangsu, China). Antibodies (against iNOS, IκB-α, phosphor-IκB-α, p65, p-p65, p38, p-p38, JNK, p-JNK, ERK, p-ERK) and horseradish peroxidase conjugated secondary antibody were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against alpha-tubulin and GAPDH were obtained from Proteintech (Hubei, China).
Preparation of paramylon
Five-day-old E. gracilis were collected by centrifugation at 5500 g for 5 minutes and washed twice with deionized water. Sonication was then used to break up the cells. Paramylon is insoluble, so to remove lipids and proteins, the sonicate was solubilized in a 1% (w/v) sodium dodecyl sulfate (SDS) solution at 95°C dry bath for 1 hour. The crude paramylon was then precipitated by centrifugation at 5000 g for 5 minutes and treated at 50°C for 30 minutes with a 0.1% SDS solution. Further centrifugation was carried out at 3000 g for 5 minutes, and the pellet was washed separately with water, acetone, diethyl ether and centrifuged . The pellet was then dissolved in 0.5 M NaOH, two volumes of cold 98% ethanol were added to the pellet and the mixture was centrifuged at 12,000 g for 10 minutes at 4°C. The ethanol precipitation was repeated once and the final pellet was suspended in 30 mL of deionized water and the pH value was adjusted to 7.0 with 2 M HCl. Paramylon suspension is ultrasonically treated on the ice for 12 minutes (12 48-second ultrasonic treatment cycles with 12-second cycle intervals). One mL aliquots were made and stored at -20°C.
Cell culture and processing
RAW264.7 macrophages were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic (Cat No. 15240062, Gibco, Grand Island, NY, USA) at 37 °C, 5% CO2. The cells were seeded into 96-well (1×105 cells/well) or 6-well plates (1×106 cells/well) for 4 hours, and then incubated with 10, 50, 100 or 200 μg/mL paramylon or 100 ng/mL lipopolysaccharide (positive controls) for 30 minutes or 24 hours.
Cell viability assay
After treatment with paramylon or lipopolysaccharide for 24 hours, 10 μL of cell counting kit reagents was added to the cells. After 1 hour of incubation, the absorbance of each well was measured at A450 nm using a SpectraMax microplate reader (SpectraMax 190, Molecular Devices, USA).
After treatment with paramylon or lipopolysaccharide for 24 hours, 50μL supernatant was mixed with 50μL sulfonamide reagent (SUL, Sangon Biotech, Shanghai) and then 50 μL naphthalene ethylenediamine reagent (NED, Sangon Biotech, Shanghai) was added into mixture. After a 5 minutes incubation, the absorbance was detected at 545nm, and the NO2- content was calculated according to the standard curve of NaNO2 . In inhibition experiment, RAW264.7 cells were pre-treated with polymyxin B for 2 hours, and then co-cultured with 100 ng/mL LPS or 200 µg/mL paramylon 24 hours.
After treatment with paramylon or lipopolysaccharide, cellular TNF-α and IL-6 levels were measured using an ELISA kit (Neobioscience, Guangdong, China) according to the manufacturer's protocol. In the inhibition experiment, adherent cells were pretreated with the inhibitor for 2 hours and then co-cultured with 200 μg/mL paramylon for 24 hours.
RNA isolation and quantitative Real-time Polymerase Chain Reaction(qRT-PCR)
qRT-PCR was used to quantify gene expression of IL-6, TNF-α and iNOS, and GAPDH was used as reference housekeeping gene. After treating RAW264.7 cells with paramylon or lipopolysaccharide for 12 hours, total RNA was isolated, with an RNeasy Mini Kit (Qiagen, Hilden, Germany), and reversed transcribed, using the iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, California, CA). qRT-PCR was performed with SoFastTM EvaGreen® Supermix (Bio-Rad, Hercules, California, CA) in the StepOne PlusTM Real-Time PCR System (ABI Applied Biosystems, Foster City, CA). Gene primers were designed using Beacon Designer software (Premier Biosoft, Palo Alto, California, USA) (Table 1). Samples were assayed in triplicates and gene expression was calculated using the 2-ΔΔCt relative quantification method . The amount of each transcript was normalized to the GAPDH transcript in the same cDNA sample.
Western blot analysis
After 24 hours of treatment with different concentrations of paramylon or lipopolysaccharide, the total cellular protein (20 μg) in each sample was separated by 8% or 12% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% (w/v) fat-free milk, the membrane was incubated with the primary antibody (1: 1000 to dilute) at 4 °C overnight, and rinsed thoroughly with the secondary antibody (1: 5000 to dilute) for 1 hour at room temperature. The membrane was then detected using an electroluminescent kit (Thermo Fisher Scientific, Waltham, MA, USA).
Immunocytochemistry of NF-kB
RAW264.7 cells were seeded on sterile glass coverslips in 6-well plates (1×106 cells/well) and treated with 200 μg/mL paramylon or 100 ng/mL lipopolysaccharide for 2 hours. The cells on the coverslips were ﬁxed immediately in 4% (v/v) formaldehyde at RT for 30 minutes. After being permeabilized with 0.2% (v/v) Triton X-100 in PBS for 10 minutes, cells were blocked with 10% (w/v) goat serum in PBS, at 37 °C for 1 hour. The cells were then incubated with an NF-κB/p65 primary antibody at 4 °C overnight. After three washes in cold PBS, the cells were incubated with an Alexa Fluor 596-conjugated secondary antibody and DAPI at RT for 2 hours. The nuclear translocation of the NF-κB/p65 subunit was observed by confocal microscopy (Carl Zeiss Jena Gmbh, Jena, Germany) .
All the data were indicated by mean ± standard deviations (mean ± SD) and statistically analyzed by two-tailed Student's t-test in GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). p value less than 0.01 (p < 0.01) was considered extremely significantly different, p < 0.05 indicating statistically different, and p value more than 0.05 (p > 0.05) was not significant.