Alkali treated antioxidative crude polysaccharide from Russula alatoreticula potentiates murine macrophages by tunning TLR/NF-κB pathway

Abstract

In our previous research, Russula alatoreticula was demonstrated as a novel species, ethnic myco-food and reservoir of hot water extractable polysaccharides. However, residue after the hydrothermal process still offer plenty of medicinal carbohydrates that could easily be extracted by using alkali solvent. Thus, the present work was attempted to prepare crude polysaccharide using remainder of the conventional method and subsequently a β-glucan enriched fraction, RualaCap, was isolated. The bio-polymers displayed pronounced therapeutic efficacy as evident by radical scavenging, chelating ability, reducing power and total antioxidant capacity. In addition, strong immune-enhancing potential was also observed indicated by augmentation in macrophage viability, phagocytic uptake, nitric oxide (NO) production and reactive oxygen species (ROS) synthesis. Alongside, the polysaccharides effectively triggered transcriptional activation of Toll like receptor (TLR)-2, TLR-4, nuclear factor kappa B (NF-κB), cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, Iκ-Bα, interferon (IFN)-γ and interleukin (IL)-10 genes explaining mode of action. Taken together, our results signify possibility of RualaCap as a potent nutraceutical agent and enhance importance of R. alatoreticula especially in the field of innate immune stimulation.

Figure 1

Structural and molecular characterization of cold alkaline extracted crude polysaccharide, RualaCap, isolated from Russula alatoreticula. (a) Identification of monosaccharides in hydrolysed polysaccharides by HPTLC, Lanes: 1: L-arabinose, 2: D-fructose, 3: D-fucose, 4: D-galactose, 5: RualaCap, 6: D-glucose, 7: D-mannose, 8: D-rhamnose, 9: D-xylose (b) GC-MS chromatogram of derivatized polysaccharide (Retention time of D-mannose: 16.6 min, D-glucose: 16.7 min, D-galactose: 16.8 min) (c) FT-IR spectrum (d) Changes in absorption maximum of Congo red-polysaccharide complex at various concentrations of sodium hydroxide solution.

Figure 2

Antioxidant activity of cold alkaline extracted crude polysaccharide, RualaCap, prepared from Russula alatoreticula. (a) Hydroxyl radical scavenging activity (b) DPPH radical scavenging activity (c) ABTS radical scavenging activity (d) Chelating ability of ferrous ion (e) Reducing power.

Figure 3

Effect of cold alkaline extracted crude polysaccharide, RualaCap, isolated from Russula alatoreticula on activity of macrophages. (a) Proliferation was monitored using water soluble tetrazolium (WST) in treatment of polysaccharide at different concentrations and time and expressed in relation (%) to negative control. (b) Phagocytosis in relation (%) to control was determined by neutral red method. (c) Release of NO in cell supernatant was quantified using Griess reagent. (d) Intracellular ROS generation was determined by flow cytometry (See also Supplementary Fig. S2). In all assays. LPS at the concentration of 5 µg/ml was used as a positive control. Results represent the mean ± standard deviation of at least three independent experiments. ANOVA p < 0.05; as regards Tukey’s post-hoc test the sign ‘*’ indicates significant differences compared to untreated control group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Effect of cold alkaline extracted crude polysaccharide, RualaCap, isolated from Russula alatoreticula on morphology of macrophages. Cells were incubated for 24 h with different concentrations of extract where LPS at the level of 5 µg/ml was used as a positive control. Afterwards cells were fixed, subjected to fluorescence microscopy and images were captured. (a) Negative control (b) LPS (c) 50 µg/ml (d) 100 µg/ml (e) 200 µg/ml.

Figure 5

Effect of cold alkaline extracted crude polysaccharide, RualaCap, prepared from Russula alatoreticula on mRNA expression. Total RNA was isolated from macrophage cells after 24 h incubation either with LPS (5 µg/ml concentration) or RualaCap (50, 100 and 200 µg/ml concentration) along with untreated cells. cDNA was prepared from respective RNA samples and semi-quantitative reverse transcriptase PCR was performed to analyse the expression of seven different genes such as TLR-2, TLR-4, NF-κB, COX-2, iNOS, Iκ-Bα and IFN-γ where β-Actin was considered as a house keeping gene. Full-length gel pictures are presented in Supplementary Fig. S4.

Figure 6

Analysis of mechanism of action by cold alkaline extracted crude polysaccharide, RualaCap, prepared from Russula alatoreticula. Real time PCR was implemented to detect expression level of five genes quantitatively (a) TLR-2 (b) iNOS (c) Iκ-Bα (d) TNF-α (e) IL-10. Values were represented as mean ± standard deviation of at least three independent experiments. ANOVA p < 0.05; as regards Tukey’s post-hoc test the sign “*” indicates significant differences compared to untreated control group. **p < 0.01, ***p < 0.001. (f) The schematic diagram represents immune stimulatory activity of RualaCap mediated through TLR/NF-κB pathway. The polysaccharide fraction being enriched in β-glucan acts as ligand for TLR2 and TLR4 and consequently activates downstream signalling pathway. As a result the transcription factor, NF-κB, becomes stimulated that subsequently binds to promoter region of a number of genes resulting synthesis of important mediators. These mediators play a significant role for activation of adaptive immune response and thus enhance overall host defence mechanism.