Immune response mechanism of mouse monocytes/macrophages treated with κ-carrageenan polysaccharide

Highlights

• Cell proliferation rate was highest treated with polysaccharide (100 μg/mL)at 24 h

• The mechanisms of immune function induced by polysaccharide were discussed.

• The immune response of RAW264.7 cells is via the TLR4–NF-κB pathway.

Abstract

This study investigated the function of κ-carrageenan polysaccharide in immune regulation. The immune response of RAW 264.7 cells treated with κ-carrageenan polysaccharide was explored by MTT assay, general morphological observation, neutral red phagocytosis assay, Griess method, fluorescence method, and enzyme-linked immunosorbent assay. In addition, TLR4 blocking experiment and double-fluorescence immunostaining were performed on cells to demonstrate their immune response mechanism. Results show that κ-carrageenan polysaccharide not only promotes cell proliferation but also activates RAW 264.7 cells, thereby improving the cells’ phagocytic capability, NO production, and tumor necrosis factor-α (TNF-α) secretion. In addition, the use of TLR4-specific inhibitors can significantly mediate the increased TNF-α secretion induced by κ-carrageenan polysaccharide. The RAW 264.7 cells treated with κ-carrageenan polysaccharide show upregulated TLR4 expression, and the main subunit of NF-κB (p65) is translocated. These results support the immunomodulatory function of κ-carrageenan polysaccharide in RAW 264.7 cells.

Introduction

Carrageenan, as a polysaccharide of sulfuric acid (Velde, 2008), exhibits antitumor, immunoregulatory, antiangiogenetic, antioxidant, antiviral, antibacterial, anticoagulant, blood fat, and other biological activities (Thomson and Fowler, 1981, Carlucci et al., 1997). Carrageenan can be dissolved in water at above 60 °C. Carrageenan at concentrations less than 1% can be stable in solution state, and its solubility is only affected by solution viscosity. Carrageenan exhibits good chemical stability in dry powder state under neutral and alkaline conditions, but this stability of carrageenan decreases and its degradation is promoted when the powder is dissolved and the pH is less than 4. The most remarkable stability of carrageenan is observed at pH 9, when heating does not easily result in hydrolysis (Arfors and Ley, 1993, Yuan et al., 2006b). However, the molecular weight, viscosity, solubility, poor absorption, and complex structure of carrageenan limit its applications in medicine. The degradation of carrageenan oligosaccharides due to polysaccharide chain break results in active group exposure, increased activity, low toxicity, strong solubility, and easy absorption, which broaden the applications of carrageenan. Thus, research on carrageenan oligosaccharides is becoming considerably important (Yuan et al., 2006a). A previous study found that the degradation of carrageenan oligosaccharides in mice generates a tumor-suppressing effect stronger than that produced by carrageenan; this degradation leads to thymic weight increase, which prompts the antitumor activity of carrageenan oligosaccharides via immune regulation (Hu et al., 2008).

Existing studies on carrageenan and oligosaccharides focused on the preparation and degradation of oligosaccharides and mixed oligosaccharides, as well as on the analyses of their biological activities. The biological activities of carrageenan oligosaccharides, their types, molecular weights, and sulfate group contents are closely related. Therefore, using carrageenan oligosaccharides of a specific type, structure, and molecular weight uniformity is necessary to realize active development. Although the immune response induced by carrageenan polysaccharides has been demonstrated in rat models (Mou et al., 2003, Hu et al., 2008), the mechanism of this response in vitro remains unknown.

Monocyte/macrophage cells (RAW 264.7) are distributed throughout the body of a mouse, allowing the identification of exogenous substances for the first time. Given their immune defense, regulation, and surveillance functions, RAW 264.7 cells play important roles in the immune system. These cells not only serve a non-specific immune function but also connect the innate immune defense and adaptive immune responses to an important signaling hub. Recent studies have shown that polysaccharides interact with cell-surface receptors to trigger downstream signaling molecules that affect information transfer and cell gene expression, thereby regulating immune function. In the present study, mouse monocyte/macrophage RAW 264.7 cells were treated with polysaccharide, with carrageenan as the carrier, to observe the effects of carrageenan polysaccharide on cellular immune response and determine the mechanism underlying this response.