Study On Immune Response Of Carrageenan Oligosaccharides On Mouse Monocytes /Macrophages

Objective:Carrageenan is extracted from red alga polysaccharide sulfate, widely used in food, chemical and pharmaceutical industries. It is rich in resources, and has a variety of biological activities. The study found that after the degradation of carrageenan, its activity is improved, due to its polysaccharide chain is broken and active group is exposed. At the same time, it has some new characteristics, like strong solubility, easy absorption. So the study of carrageenan oligosaccharides is an important direction for future development of carrageenan. But the study of carrageenan oligosaccharides is concentrated in oligosaccharide synthesis and biological activity analysis of oligosaccharides mixture. The biological activity of carrageenan oligosaccharides are closely related to their types, molecular weight and sulfate content, therefore, using carrageenan oligosaccharides with specific type and structure, uniform molecular weight to study the relationship between the activity of carrageenan oligosaccharides and its structure, which is the necessary condition to realize the active development.Methods:MTT colorimetric method was used to screen the types with strongest promoting proliferation of mouse monocytes/macrophages RAW264.7 from three kinds of K-carrageenan oligosaccharides (carrageenan three sugar, eleven sugar and nineteen sugar) with clear structure. Then through the detection of the MTT method, the total and release amount of LDH, determination of ATP levels and cell growth curve, observe the effect of carrageenan eleven glucose on cell growth, and determine the optimal condition. Use general morphological observation, neutral red and ink phagocytosis experiment, Griess method and fluorescence method, enzyme linked immunosorbent assay (ELISA), to study the role of carrageenan eleven sugar on immune response of RAW264.7 cells. Then through the TLR4 blocking experiment and the fluorescence method, explore its mechanism.Results:The type with strongest promoting proliferation of mouse monocytes/ macrophages RAW264.7 in three kinds of ?- carrageenan oligosaccharides is carrageenan eleven sugar. It was found that, the cell proliferation rate, membrane integrity, activity and proliferation ability were all promoted,100 g/mL,24h was the best condition. In this condition, We explore the RAW264.7 cell immune response to carrageenan eleven sugar. Results show that the morphology of the treated group was round, and the condition was good, but the volume change was not obvious. The ink and the neutral red phagocytosis increased. NO content and nitric oxide synthase (NOS) activity increased. The secretion of tumor necrosis factor-a(TNF-a) was enhaned. Also we found that, the use of TLR4 specific inhibitors can significantly reduce the TNF-a secretion increasing caused by carrageenan eleven sugar. And after treated with carrageenan eleven sugar, RAW264.7 cell TLR4 expression was increased, and the main subunit of NF-?B P65 was transferred.Conclusion:We screend out the best proliferation ability from three kinds of carrageenan oligosaccharides in RAW264.7 cells. And we found that the proliferation activity of carrageenan oligosaccharides on RAW264.7 is influenced by molecular weight and sulfuric acid group, and the molecular weight was stronger. The experiment found that carrageenan eleven sugar not only can promote cell proliferation, but also can improve the cell viability and proliferation. At the same time, it can activate RAW264.7, improve its ability of phagocytosis, NO production and TNF-a secretion. And the immune response of RAW264.7 cells to carrageenan eleven sugar is through the TLR4-NF-?B pathway.