The Isolation And Purification Of Lycium Barbarum Polysaccharides And The Study Of The Immune Mechanism Based On The Regulation Of Intestinal Flora

ObjectiveThe water-solubility polysaccharides of Lycium barbarum L.is the main bioactive component of L.barbarum,which has a good immune enhancing effect.However,the mechanism of immunoregulation of the polysaccharides of L.barbarum(LBP)is still in the research.In recent years,with the development of gut microbiota research,it have been found that gut microbiota is closely related to immunity.Polysaccharides,as a low bioavailability product,could regulate the gut microbiota by staying in the intestinal tract.How does LBP affect gut microbiota and whether it is related to immune function?It is very important to study the immune mechanism of LBP.In this study,RAW 264.7 cells and cyclophosphamide(CTX)induced immunosuppressive mice model were used to investigate the immunoregulation function of LBP.Moreover,for the first time,the transmembrane transport ability of LBP were studied to reveal the immunomodulatory pathway of LBP.The structure of polysaccharides is closely related to its activity.In order to ensure the effectiveness,stability,uniformity and safety of polysaccharides products,it is necessary to establish a quality control method of polysaccharides.However,due to the complexity of polysaccharides structure,the research progress of its quality control method is slow.In this study,the primary structure of LBP was detected by using a variety of analytical techniques.The key structural characteristics of LBP which infect immune function has been obtained by the analysis of structure-activity relationship to establish the quality control method,which can play a guiding role in the preparation of LBP.In addition,LBP from different producing areas was compared with the in vitro immune and antioxidant activities,which provide reference for the quality evaluation of L.barbarum resources.Methods1.In this study,LBP was extracted by hot water and precipitated by ethanol.The phenol sulfuric acid method was used to determine the total sugar content;carbazole sulfuric acid method was used to determine the acid sugar content;PMP derivatization combined HPLC-PDA method was established to determine the monosaccharide composition and molar ratios in LBP;GPC-RI-MALLS method was used to determine the relative molecular weight(Mw),dispersion coefficient(PDI)and spatial conformation(?)of LBP;methylation GC/MS method was established to determine the glycoside bond of LBP.2.Ethanol precipitation,membrane separation and chromatography methods were investigated to obtain homogeneous LBP with high purity3.In order to investigate the stability of LBP in the digestive fluid,the artificial gastric juice and artificial intestinal juice were used to simulate the digestive state in vitro.The LBP was labeled by using FITC,then Caco-2 cell model was used to study the transmembrane absorption of FITC-LBP,and the apparent permeability coefficient Papp was obtained.4.The effects of LBP on cell proliferation,phagocytosis and expression of related cytokines were investigated in the model of RAW264.7 mouse monocyte macrophage,and the related cell pathway proteins were studied by Western-Blot methods.5.The effects of LBP on immune organs,phagocytic index of macrophages,lymphocyte,T cell subsets in spleen and IgG,IgM levels in serum were studied in the CTX induced immunosuppressive mice model.6.16S rDNA V3+V4 hypervariable region technique was used to analyze the diversity and community structure of gut microbiota in different experimental groups.Pearson statistical method was used to analyze the correlation between gut microbiota and immune function.In addition,HE staining was used to observe the morphology of colonic mucosa in mice.7.LBP was extracted and purified from L.barbarum of different producing areas(Ningxia,Xinjiang,Qinghai),and its yield was calculated.The primary structure of LBP was determined,and the monosaccharide composition and glycoside linkage fingerprint of LBP were evaluated.At the same time,the immune activity and antioxidant activity in vitro of LBP from different habitats were measured.Results1.LBP is an acid heteropolysaccharides with total sugar content of 66.9%.It is composed of nine kinds of monosaccharides,such as mannose,ribose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,xylose and arabinose.The results of Mw by GPC-RI shows two chromatographic peaks,the Mw of peak 1 is 1.37×106 Da(PDI=1.58)and Mw of peak 2 is 8.83×104 Da(PDI=1.48).?(slope value of Rg-M curve)is 0.237,which indicated that LBP presents a compact spherical shape in 0.1%NaCl solution.14 glycosidic bond have been found in LBP,(1-4)GalA type is the main glycosidic bond.The quantitative determination method used in this experiment has high sensitivity,good accuracy and meets the requirements of methodology.2.Separation and purification of LBP have been finished by DEAE-52 cellulose column and HW-65 column.Two high purity homogeneous components were obtained,LBP1(Mw=1207400 Da)and LBP2(Mw=125000 Da).3.LBP1 and LBP2 were not degraded in artificial gastric juice and artificial intestinal juice by the determination of Mw,which indicated that the LBP1 and LBP2 were stable in digestive fluid.The transmembrane transport ability of LBP in Caco-2 cells was verified by FITC-LBP labeling.The compactness and integrity of Caco-2 cell were good by.determination,which can be used as the intestinal epithelial cell.The successfully labeled FITC-LBP 1 and FITC-LBP2 were used in this experiment,the cumulative transport rates of FITC-LBP1 and FITC-LBP2 were 0.99%and 1.15%,respectively.Papp of FITC-LBP1 and FITC-LBP2 both less than 1.0×10-6 cm.s-1,indicated that two LBP components were not easily absorbed in the small intestine.4.Study on the immunoregulation and mechanism of LBP on macrophages showed that LBP1 and LBP2 both can significantly promote the proliferation of RAW 264.7 cells and enhance their phagocytic capacity.LBP1 and LBP2 also can increase the mRNA expression of IL-6,TNF-?,IL-1? and iNOS in cells in a dose-dependent manner.In addition,LBP1 and LBP2 can induce macrophages to activate p-JNK MAPKs,and the protein expression gradually increases with the increase of concentration.The immunoregulation effect of LBP1 on RAW264.7 cells was better than LBP2(P<0.05).5.Compared with CTX model group,LBP1 and LBP2 significantly increased the thymus and spleen index,promoted the proliferation of T and B lymphocytes in spleen,increased the content of IgG and IgM in serum,and regulated the number of CD4+,CD8+T lymphocytes and the ratio of CD4+/CD8+in dose-dependent manner.The immunoregulation effect of LBP1 on CTX induced immunosuppressive mice was better than LBP2(P<0.05).6.The effect of LBP on gut microbiota and intestinal mucosa in immunosuppressed mice showed that high dose LBP1 and LBP2 administration group could reverse the gut microbiota disorder which caused by CTX,and restore the diversity of OTU to the level of control group.The results also revealed that LBP significantly increased the relative abundance of Bifidobacteria,Lactobacilli,Bacteroides,Prevoridae and Rikenellaceae,and decreased the relative abundance of some groups,such as Escherichia,Paraberoides and Ruminococcus.The correlation analysis shows that Lactobacillus,Bacteroides,Prevotella and Bifidobacterium have a significant positive correlation with immune regulation,while Escherichia,Parabacteroides and Ruminococcus have a significant negative correlation with immune regulation.In addition,it was found that the low dose LBP group had no significant regulatory effect on gut microbiota.And LBP can repair the intestinal mucosa damage caused by CTX.7.LBP structure and immune and antioxidant activity in vitro from different areas have high similarity,and the similarity of monosaccharide composition and glycoside fingerprint was greater than 0.90.However,the LBP yield among 1.6%?4.4%of L.barbarum from different areas was significantly different,the average LBP yield from Qinghai L.barbarum was significantly lower than that from Xinjiang and Ningxia(P<0.05).Conclusions1.Based on the results of structure determination of LBP from different producing areas and structure-activity relationship analysis,a quality control method was preliminarily formed.The content limits of GalA,Ara,Gal and Glc monosaccharides in LBP could be made,the range of Mw and PDI of LBP should be detected,and the similarity of LBP monosaccharide composition fingerprint and LBP reference fingerprint should be not less than 0.90.This quality control method with high specificity can reflect the key structural characteristics of LBP and effectively distinguish LBP and related products from other traditional Chinese medicine polysaccharides.2.The structure and immune activity in vitro of LBP in L.barbarum from different habitats are highly consistent,but the yield of LBP is quite different.It is indicated that species have a key influence on the structure and biological activity of polysaccharides,while regions have a greater influence on the content of polysaccharides.Ningxia has always been regarded as the Daodi area of L.barbarum.This study also partly explain the advantage of L barbarum in Ningxia based on polysaccharides research.3.The in vitro and in vivo experiments showed that LBP had strong immunoregulatory function.According to RAW264.7 cell experiment,LBP can affect the immune regulation of macrophages by activating p-JNK MAPKs cell pathway.The immunosuppressive mice model experiment shows that LBP can improve the atrophy of immune organs in mice caused by CTX,and enhance the immune function though body fluid immunity and cell immunity.Meantime,it was found that the effect of LBP1 was better than that of LBP2,indicating that MW was one of the key factors affecting the immune activity of LBP.4.High dose LBP can improve the diversity and relative abundance of flora in immunosuppressed mice,which can directly activate the related immune pathway and promote the differentiation of T lymphocytes,or it could be related to the production of SCFA,which can enhance the intestinal mucus barrier function.Moreover,LBP can reduce some potential pathogenic bacteria,which are also related to colitis,colon cancer and other diseases.5.According to the Caco-2 transmembrane transport experiment,the results showed that the absorption rate of LBP in the intestine is very low.It indicated that the regulation of LBP on the gut microbiota is one of the important ways to play its immune function.However,the low-dose LBP has no obvious regulation effect on the gut microbiota,revealing that the immune pathway of LBP is closely related to its dose.