The effect of gum Arabic supplementation on cathelicidin expression in monocyte derived macrophages in mice

The study included 30 wild-type male mice aged 4 months, with an average weight of 25 g. Mice were obtained from the National Center For Research (Khartoum, Sudan)and were housed in separate cages. Mice had free access to food and water. After 5 days adaptive period, mice were randomly and equally divided into three groups as follow: The control group; 15% GA group: mice in this group received 30 g/d GA in 200 ml drinking water in a concentration of 15%w/v; and 30% GA group: mice in this group received 60 g/d GA in 200 ml drinking water in a concentration of 30% w/v. GA was provided to the two intervention groups. The lower dose of GA was selected based on previous experiments with GA [42, 44]. The higher dose was used to investigate if the effect of GA was dose related. GA supplementation continued for 28 days in which fresh GA solutions were prepared daily. Mice were weighed weekly. Animals were checked at least daily, even on weekends. At the end of the 28 days period, mice were euthanized via CO₂ inhalation and blood samples were collected under aseptic condition.

Blood samples were collected on day 29.Mice were euthanized and blood collected by cardiac puncture and placed into EDTA tubes. Samples were centrifuged for 10 minutes at 2000 g and the plasma was stored at − 20 C°for further analysis. The buffy coat was transferred into a single tube that can contain at least twice its volume. Then it was diluted and mixed gently with equal amount of PBS.

Peripheral blood mononuclear cells (PBMC) were isolated from the diluted buffy coat using Ficoll–Paque density gradient (3 ml of Ficoll) was added to the bottom of a15 ml conical tube without touching the side of the tube. Gently the Ficoll was overlayed with the diluted buffy coat. The diluted sample was allowed to flow down the side of the tube and pool on top of the density gradient media surface without breaking the surface plane and then the tube was centrifuged at 400 g for 20 min at 18 °C. New sterile conical tubes were labeled, with the same number and same volumes as for the density gradient cell separation, which will contain PBMCs. The plasma layer was removed as much as possible with pipette. The mononuclear cells were collected from the plasma/Ficoll interface with a disposable transfer pipette and transferred into a new sterile conical tube. PBS (3 ml) was added to each tube and the tubes were centrifuged at 400 g for 10 minutes. The supernatant was removed carefully (the pellet is loose because of red blood cells) by tilting the tube and pipetting off the supernatant without touching the pellet. The cell pellet was suspended by gentle pipetting in a 1 mL PBS and the tube was then filled with PBS and centrifuged at 400 g for 10 minutes. The supernatant was discarded and cell concentration and viability were determined by using the Cellometer 2000. PBMCs were washed by using PBS and re-suspended in culture medium containing 10% autologous plasma in RPMI-1640, 1% L-glutamine, 1% sodium pyruvate, 0.5% amphotericin B and 1% penicillin-streptomycin. Cells were plated in two parallel 4-well tissue culture plates (NUNC, Roskilde, Denmark) and incubated for 5 days in 5% CO₂ at 37 °C. Thereafter, the supernatant containing the non-adherent cells was removed. The remaining adherent cells in the culture plate monocyte derived macrophage (MDM) were harvested using a cell scraper and treated with Saponin 0.1% [32].

RNA was extracted from MDM samples as follow: Cells were homogenized in TRI Reagent (easy- BlueTM Total RNA extraction kit) then it was extracted with chloroform in phase lock tubes (5 Prime) following the manufactures protocol and stored at − 80 °C until processed. RNA integrity and concentration were determined on a NanoDrop 2000 (Thermo Scientific). The b-actin primers used to amplify beta actin were, BACTIN-1 (GGT-CGTCGACAACGGCTC) and BACTIN-2 (TGCCATGTTCAATG-GGGTAC). CRAMP-F1 (5′-AGGAGATCTTGGGAACCATGCAGTT-3′) and CRAMP-R1 (5′-GCAGATCTACTGCTCCGGCTGAGGTA-3′) were the primers used to amplify CRAMP [49]. The reverse transcriptase reactions were run with reverse transcription reaction template and oligos using SYBR Green PCR Master Mix (iNtRON Biotechnology). All measurements were performed in triplicate with Rotor- Gene Q Series software 2.3.1 sequence detection system. The thermal profile consisted of 42 °C for 20 min., 95°Cfor 10 min., followed by 45 cycles of 95 °C for 10 s and 60 °C for 60 seconds. Results were analyzed by the comparative cycle threshold (Ct) Method, where Ct is the number of cycles required to reach an arbitrary threshold [49].

Comparison between the three groups regarding the levels of CRAMP constructs revealed significant difference (p = 0.028) (Fig. 1). The group that was supplemented with 15% GA showed significantly higher levels of CRAMP transcripts compared to the control group (p = 0.023). Although the levels of CRAMP transcripts increased in the group that received 30% GA they were not statistically different from the control group (p = 0.055).