Study On The Immunomodulatory Effects Of Wheat Bran Polysaccharide On Macrophages

Wheat,which is monocotyledonousplant,is one of the earliest cultivated crops in the world.A large number of literatures have reported that wheat bran contains dietary fiberthat can lower the blood sugar and serum cholesterol,prevent constipation,regulate blood glucose levels and strengthen immunity.However,little attention has been paid to the immunoregulatory activity of wheat bran polysaccharide on cells and its mechanism.In this paper,wheat bran(Huaimai 33)is used as experimental material.First,four different extraction methods(alkali extraction,acid'extraction,enzymatic extraction,and hot water extraction)were used to extract polysaccharides from wheat bran under the same extraction conditions.The extraction yield,uronic acid content,sulfate content,protein content,monosaccharide composition,and the immunoregulatory activities of four samples were investigated.Secondly,the study was designed to examine the effect of wheat bran polysaccharide(WBP)on RAW 264.7 cells and the underlying signalling pathways.In addition,we also investigated the immuno-enhancement effects of WBP on cyclophosphamide(CTX)-induced immunosuppression in mice.Finally,the preliminary purified wheat bran polysaccharide to further purify by DEAE-52 cellulose column chromatography and Sephadex G-100 gel column chromatography.At the same time,the molecular weight of the wheat bran polysaccharide and the ratio of the composition of the monosaccharide and the amount of the substance were determined by high performance liquid chromatography(HPLC)and gas chromatography(GC).The structural characteristics of wheat bran polysaccharides were analyzed by infrared spectroscopy,to provide a basis for further study on the relationship between polysaccharide structure and pharmacological activity of wheat bran.The main findings are as follows:(1)Comparing the different extraction methods,polysaccharide by alkali extraction revealed the highest extraction yield,which reached up to 31.73%;total polysaccharides content was 57.79%.The contents of uronic acid and sulfate of four different samples were ranked as followed order:alkali extraction,enzymatic extraction,acid extraction,and hot water extraction.Monosaccharide composition of wheat bran polysaccharides were analyzed by GC-MS under different extraction,which is composited of rhamnose,arabinose,and glucose.Four different samples didn't show any cytotoxicity on RAW 264.7 cells and the polysaccharide prepared by water-extraction could significantly promoted RAW264.7 cells to produce NO production.Furthermore,it could promote the expression of iNOS and COX-2 protein.(2)In vitro experimental results show that wheat bran polysaccharide has no toxic effect on macrophage RAW264.7 cells,and it can promote the immune function of macrophages by regulating NF-?B,AP-1 and p38MAPK signaling pathways.In addition,in vivo experiments showed that wheat bran polysaccharide could enhance the thymus and spleen organ index of cyclophosphamide-induced immunocompromised mice and up-regulate the secretion of cytokines.(3)The fractions of CWBP-1,CWBP-2 and CWBP-3 were obtained after separation and purification of the crude wheat bran polysaccharide.The average molecular weight of the three components of wheat bran polysaccharide was 182.73 kDa,90.104 kDa,40.721 kDa,respectively.The results of gas chromatographic analysis showed that the three components of wheat bran polysaccharide were composed of arabinose and xylose,and the molar mass ratio was 1:0.68,1:1.45,1:1.14.And the results of infrared spectroscopy showed that the absorption peak at 1045 cm-1 was the characteristic absorption peak of arabinoxylan.(4)The three components of wheat bran purified polysaccharide can improve the immunological activity of RAW264.7 cells,the immunoregulatory function of CWBP-2 is particularly significant.