2.1 Chemical and reagents
Cell culture media and its supplements were purchased from Lonza Inc. (Walkersville, MD, USA). The major enzymes such as α-amylase and proteinase K were attained from Sigma-Aldrich, St. Louis, MO, USA. The primary antibodies such as anti-phospho-NF-κB p65, anti-phospho-c-Jun N-terminal kinase (JNK), anti-phospho-extracellular signal-regulated kinase (ERK1/2), and anti-phospho p38, and secondary antibody (horseradish peroxidase-conjugated anti-rabbit antibody) were obtained from Abcam, Cambridge, United Kingdom. The antibodies for flow cytometry analysis such as Anti-CD40-APC (1C10) and anti-CD11b (M1/70) were procured from ThermoFisher Scientific, Waltham, MA, USA.
2.2 Isolation of non-starch polysaccharide
A non-starch polysaccharide (CQNP) was isolated from C. quinoa using the α-amylase treatment. The C. quinoa seeds were collected from the local market of XiNing city, QingHai province, China. The seeds thoroughly cleaned with distilled water, dried at 45°C, and were milled into a fine powder. For CQNP extraction, 20 g of powdered sample was extracted with 200 mL of distilled water at 65°C. The collected extract was centrifuged at 4000 × g for 15 min. Afterwards, the extract was mixed with an equal volume of phosphate-buffered saline (PBS, pH 6.0) and incubated with 4 mg of α-amylase (Sigma-Aldrich, St. Louis, MO, USA) for 16 h at 55°C. The reaction was arrested by raising the temperature to 100°C for 10–15 min. Finally, the reaction mixture was filtered through a 110 nm size of the membrane, dialyzed against distilled water and freeze-dried.
2.3 Deproteination of CQNP
About, 1mg of CQNP was dissolved in 0.1 M sodium phosphate buffer and then incubated with 10–20% of proteinase K (w/v) in a water bath at 58°C for 24 h. The reaction was stopped by keep the sample at 100°C for 10 min. Afterwards, the reaction mixture was centrifuged (10000 rpm for 10 min) and the supernatant was dialyzed against distilled water and freeze-dried [21]. The deproteinated CQNP was named as D- CQNP.
2.4 Chemical and Monosaccharide composition analysis
The presence of carbohydrate, protein and uronic acid were examined by phenol-sulfuric acid (Dubois et al., 1956), folin-phenol reagent (Lowry et al., 1951) and m-hydroxydiphenyl reaction [24], respectively. For the monosaccharide composition analysis, three milligrams of the sample was hydrolyzed with 0.5 mL of 4 M trifluoroacetic acid (TFA) at 100°C for 6 h. The hydrolyzed product was reduced by NaBD4 and then acetylated using acetic anhydride. The monosaccharide composition analysis was carried out by a gas chromatography mass- spectrometry analysis (GC-MS, 6890 N/MSD 5973, Agilent Technologies, Santa Clara, CA, USA) equipped with the HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm; Agilent Technologies).
2.5 Molecular weight analysis
The average Mw and Rg values were estimated by following the method of Tabarsa et al (Tabarsa, et al., 2020). Briefly, the sample was solubilized in distilled water (2 mg/mL) and heated for 30 S using a microwave bomb (#4872; Parr Instrument Co., Moline, IL, USA). Then, the samples were filtered through a cellulose acetate membrane (3.0 µm pore size; Whatman International) and injected into a TSK G5000 PW column (7.5 × 600 mm; Toso Biosep, Montgomeryville, PA, USA) coupled with a multi-angle laser light scattering (MALLS) detection (HELEOS; Wyatt Technology Corp, Santa Barbara, CA, USA) and refractive index (RI) detection (#2414; Waters, Milford, MA, USA) system (HPSEC-UV-MALLS-RI). The Mw was calculated using ASTRA 5.3 software (Wyatt Technology Corp.).
2.6 Glycosidic linkage analysis
The glycosidic linkage profile of the highest immunostimulant D-CQNP was investigated using the method of Ciucanu and Kerek [26]. Nearly, 3 mg of D-CQNP was fully dissolved in 0.5 mL dimethyl sulfoxide under a nitrogen stream. Add, 20 mg of NaOH to this solution. The methylation process was carried out by mixing of methyl iodide for 45 min and then hydrolyzed with 4 M TFA at 100°C for 6 h. The hydrolysates were reduced in distilled water with NaBD4 and acetylated using acetic anhydride at 100°C. The partially methylated alditol acetates were subjected to GC-MS analysis (6890 N/MSD 5973, Agilent Technologies) equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm; Agilent Technologies).
2.7 Cell proliferation and NO production
In this study, the immunostimulatory activities of CQNP and D-CQNP were tested on RAW 264.7 macrophage cells (ATCC; Manassas, VA, USA and ATCC, Rockville, MD, USA). RAW 264.7 cells were grown in RPMI-1640 medium in the presence of 10% fetal bovine serum (FBS) under 5% CO2 atmosphere condition. The cell proliferation activity was tested using WST-1 assay. RAW 264.7 cells were cultured in a 96-well microplate (1×106 cells/mL) with different concentration of CQNP or D-CQNP (12, 25 and 50 µg/mL) for 24 h at 37°C. Add 100 µL of 10% WST-1 solution into the wells and extend the incubation for 1 h. The absorbance was read at 450 nm and the proliferation activity (%) was calculated using the following equation. Similarly, the cells were treated with CQNP or D-CQNP and the NO production was measured using Griess reaction (Green et al., 1982).
Cell proliferation (%) = Absorbance of sample/Absorbance of control x 100
2.8 Real-time PCR analysis
RAW 264.7 cells (1×106 cells/mL) were incubated with polysaccharide or LPs (1 µg/mL) at the concentration of 50 µg/mL in a 96-well microplate under 5% CO2 atmosphere for 18 h at 37°C. The total RNA content was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was constructed using oligo-(dT) 20 primer and Superscript III RT (Invitrogen) by following the steps provided in the kit. PCR amplification was performed by a Real-Time PCR system using Fast Start DNA Master TB Green II kit (Takara Bio Inc., Shiga, Japan) using the specific primers (Table. 1)
Table 1
Sequences of the primers used in real-time PCR analysis
Gene
|
Sequences of the primers
|
iNOS
|
5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′ (forward)
5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′ (reverse)
|
IL-1
|
5′-ATGGCAACTATTCCTGAACTCAACT-3′ (forward)
5′-CAGGACAGGTATAGATTCTTTCCTTT-3′ (reverse)
|
IL-6
|
5′-TTCC TCTCTGCAAGAGACT-3′ (forward)
5′-TGTATCTCTCTGAAGGACT-3′ (reverse)
|
IL-10
|
5′-TACCTGGTAGAAGTGATGCC-3′ (forward)
5′-CATCATGTATGCTTCTATGC-3′ (reverse)
|
TNF-α
|
5′-ATGAGCACAGAAAGCATGATC-3′ (forward)
5′-TACAGGCTTGTCACTCGAATT-3′ (reverse)
|
β-actin
|
5′-TGGAATCCTGTGGCATCCATGAAAC-3′ (forward)
5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (reverse)
|
2.9 Western blot analysis
RAW 264.7 cells (1×106 cells/mL) were treated with CQNP or D-CQNP (50 µg/mL) for 18 h at 37°C. Then, the cells were lysed in RIPA buffer for 30 min and centrifuged at 14000 g (4°C) for 15 min. The protein content was estimated by Micro BCA™ Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s guidelines. Simultaneously, the cell lysates containing nearly 30 µg of protein was loaded on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the protein band was transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with particular primary antibodies such as anti-phospho-NF-κB p65, anti-phospho-c-Jun N-terminal kinase (JNK), anti-phospho-extracellular signal-regulated kinase (ERK1/2), and anti-phospho p38 (Abcam, Cambridge, United Kingdom) followed by secondary horseradish peroxidase conjugated anti-rabbit antibody. The proteins were identified by Pierce ECL Plus Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA, USA) and the expression was visualized by Bio-Rad image analysis system (Bio-Rad Laboratories).
2.10 Flow cytometry analysis
The polysaccharides treated RAW 264.7 cells (50 µg/mL) were washed with flow cytometry staining buffer and then incubated with specific antibodies such as Anti-CD40-APC (1C10) and anti-CD11b (M1/70) for 30 min at 4°C under the dark condition. The flow cytometry analysis was carried out by CytoFLEX flow cytometer (Beckman Coulter, High Wycombe, United Kingdom), and the data were analyzed using the CytExpert 2.3.0.84 software (Beckman Coulter).
2.11 Statistical analysis
All experiments were performed in triplicate (n = 3), and the value was expressed as the average and standard deviation (SD). The statistical analysis was carried out using SPSS software (Version 16; SPSS Inc., Chicago, IL, USA). The differences between groups were analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple-range test, and a p-value of < 0.05 was considered statistically significant.