| Dendrobium officinale Kimura et Migo belongs to the family of Orchidaceae genus,and it is renowed as the top of "the nine immortality herbs" in folk.D.officinale polysaccharide(DOP)was subjected for the study in this research,and its effects on gut-associated lymphoid tissue,gut micrbiota immunity and peripheral immune system,as well as the immunomudulatory menchaism were studied.The main contents and conclusions of our study can be summarized as follows:1.The polysaccharide content detecting method was optimized.Phenol-sulfuric acid method and anthrone-sulfuric acid method were compared from the aspects of stability,precision,and recovery.The results showed that phenol-sulfuric acid method was better in polysaccharide content determination.2.The extraction conditions of D.officinale polysaccharide were optimized.Response Surface Method was used to optimize the extraction technique of D.officinale polysaccharide.The results indicated that the optimum extraction condition could be described as follows:extraction temperature 100℃,extraction time 2 h,water material ratio 31:1.Under this condition,the extraction rate could reach to 30.55%with one extraction process.3.The purification method of crude polysaccharide was established,and related physical and chemical properties of purified polysaccharide were analyzed.Starch was removed with heat resistant a-amylase to achieve purified polysaccharide.The related physical and chemical properties of this polysaccharide were determined.And the results proved that this purified polysaccharide is a homogeneous sugar composed of mannose and glucose in a molar ratio of 6.9:1,and it contained 88.21%neutral saccharide,3.18%protein,1.32%water and 0.48%ash.The results of inorganic element analysis showed that the content of Ca was higher in the polysaccharide.4.The immunomodulatory activity and antioxidant activity of fractions from D.officinale in healthy mice were studied.D.officinale powder,crude polysaccharide,and purified polysaccharide were administrated to normal mice to evaluate their safety.The results found that these three materials could stimulate the proliferation and differentiation of spleen lymphocytes,increase the serum immunoglobulin content,stimulate the phagocytosis of peritoneal macrophages,and promote the antioxidant activity of thymus and liver.These indicated that D.ofcinale powder,crude polysaccharide,and purified polysaccharide were nontoxic to health body and could even promote the immunity and antioxidant activity of healthy body.5.The immunomodulatory activity and antioxidant activity of fractions from D.officinale in immunosuppressed mice were studied.Immunosuppressed mice model was constructed by intraperitoneal injection of cyclophosphamide.D.officinale powder,crude polysaccharide,and purified polysaccharide were administrated to these immunosuppressed mice to evaluate their immunomodulatory activity.The results found that all these three materials could promot the proliferation and differentiation of spleen lymphocytes,balance the serum cytokine levels,increase the serum immunoglobulin and haemolysin,stimulate the phagocytotic function of peritoneal macrophages,and increase the antioxidant activity of serum,thymus and liver.These results proved that D.ofcinale powder,crude polysaccharide,and purified polysaccharide had significant immunomodulatory activity and antioxidant activity.6.The transportation of DOP in imitate the intestinal epithelium in vitro was studied.Caco-2 cells were used to imitate the intestinal epithelium in vitro.And this constructed intestinal epithelium was used to analyze transport ability of DOP.The results indicated that DOP can not realize the direct transportation and absorption in intestine.7.The effects of DOP on intestinal mucosal immune system were studied.AOM/DSS was used to construct colitis-associated colorectal cancer model.D.officinale polysaccharide was administrated to colitis-associated colorectal cancer mice in different pattern to study the effects of polysaccharide on gut-associated lymphoid tissue.The results demonstrated that DOP could modulate the abnormal lipid metabolism of colon cancer mice,increase the colon length,decrease the tumor formation probability in gut,promote the secretion of intestinal slgA,balance the cytokine levels in gut,suppresse the excessive active of TLR4/MyD88/NF-κB pathway,and adjust the tumor cell apoptosis.These proved that DOP could modulate the immune response in gut-associated lymphoid tissues.8.The effects of DOP on the properties of intestinal metabolite in colorectal cancer mice were studied.DOP was administrated to colorectal cancer mice in different pattern.Mice fecal was collected termly during the experiment process.The fecal pH,moisture and short chain fatty acid content were tested to evaluate the effects of DOP on cancer mice intestinal metabolite.The results suggested that DOP could modulate the abnormal change of pH and moisture of mice fecal,and increase the content of short chain fatty acid.9.The effects of DOP on gut microbiota community of colorectal cancer mice were studied.DOP was administrated to colorectal cancer mice,stools from Normal group,Model group and Whole process treated group were collected.DNA sequencing analysis method was used to test the gut microbiota community in mice feces.The results found that DOP could improve the abundance and diversity of microbes in fecal,up-regulate the ratio of Proteobacteria,Lachnospiraceae and Prevotellaceae,and down-regulate the ratio of Bacilli and Verrucomicrobiales.10.The effects of DOP on peripheral immune system of colorectal cancer mice were studied.DOP was administrated to colorectal cancer mice in different ways,then the immune-related parameters were tested.The results showed that DOP could balance the ratio of spleen T lymphocyte subsets,adjust the secretion of cytokines in immune cells,down-regulate the serum IgG and IgM levels.In addition,it was also found by metabonomics study that DOP had the effects on amino acid metabolism,lipid metabolism,and glycometabolism of colorectal cancer mice.11.The immunomodulatry mechanism of DOP was discussed based on gut immunity.DOP could affect the body immune system and modulate the tumor development by modulating gut microbiota community and the immune response in gut-associated lymphoid tissues. |
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