| Ganoderma lucidum is regarded as the treasure of "supporting the energy and strengthening the body" by medical experts in past dynasties,the immunomodulation of which is an important embodiment of "supporting the energy".Ganoderma lucidum polysaccharide is one of the most important active components of ganoderma lucidum.The immune regulation of ganoderma lucidum is mainly caused by polysaccharide.Due to the large molecular weight of ganoderma lucidum polysaccharide,the human body lacks relevant digestive enzymes.Thus,it is difficult to be directly digested and absorbed,so the gastrointestinal tract may be the main place where ganoderma lucidum polysaccharide plays its role.Although the in vivo and in vitro experiments of ganoderma lucidum polysaccharides in existing studies can explain the effects of ganoderma lucidum polysaccharides on tumor cells,immune cells,cytokine secretion and immune system,etc.,the polysaccharides of ganoderma lucidum are difficult to be absorbed and directly act on related cells,so the detailed mechanism of its action is still difficult to explain.The gastrointestinal tract is not only an important digestive organ of the body,but also an important immune organ of the body as well as the body's first line to defend against the outside pathogenic antigens.In addition,intestinal colonization of a large number of symbiotic microbial communities can also affect the function of intestinal mucosal immunity through a variety of different pathways.In this study,ganoderma lucidum polysaccharide was obtained by water-extraction and alcohol-precipitation,which was then hydrolyzed by hydrochloric acid degradation.The polysaccharide and its hydrolysates were characterized by molecular weight,reducing sugar content,infrared absorption spectrum and monosaccharide composition.The effects of GLP and DGLP on intestinal mucosal and systemic immunity of immunosuppressive mice induced by cyclophosphamide were compared.Effects of ganoderma lucidum polysaccharide on intestinal mucosal immunity and secretion of SIgA and IL-4 were investigated by isolated intestinal model.This study lays a foundation for elucidation of the mechanism of polysaccharides from ganoderma lucidum exerting its pharmacological effects through gastrointestinal tract.(l)Preparation and characterization of ganoderma lucidum polysaccharide and its hydrolysatesThe impure ganoderma lucidum polysaccharide was obtained by water-extraction and alcohol-precipitation.After that,GLP(45.95%)was made through deproteinization,dialysis and other steps.DGLP was made by HCI degradation(80 ?,5 h).The reducing sugar content detected by DNS method was 8.02%and 13.07%respectively.The molecular weight of DGLP measured by GPC method is one order of magnitude lower than that of GLP.The average molecular weight range of GLP is 84 Da-23269 Da,the distribution range of weight average molecular weight is 114 Da-76347 Da,and the range of Z average molecular weight is 113 Da-222865 Da.The average molecular weight range of DGLP was 87 Da-2409 Da,the distribution range of heavy average molecular weight was 126 Da-5292 Da,and the range of Z average molecular weight was 150 Da-23956 Da.The results of IR spectra showed that the absorption peaks of GLP and DGLP were different.DGLP contains a-pyran glucan.The results of composition and relative content of monosaccharides showed that GLP and DGLP had no difference in the composition of monosaccharides,but there were significant differences in the relative content of various monosaccharides.(2)Effects of ganoderma lucidum polysaccharide and its hydrolysates on intestinal mucosal immune function of immunosuppressed miceThe curative effects of GLP and DGLP were observed in immunosuppressive mice induced by cyclophosphanide with levanisole hydrochloride as the positive drug.?Effects of ganoderma lucidum polysaccharide and its hydrolysates on intestinalmucosal immunity:Intestinal mucosal immune effects of ganoderma lucidum polysaccharide and its hydrolysates were observed by detecting intestinal mucosal mechanical barrier and immune-related factors.The experimental results show that both GLP and DGLP can improve immunosuppressive mice 's small intestine tissue morphology,small intestinal villus length(V)?crypt depth(C)?V/C,increase the intestinal mucous the content of IL-2,IL-4,IFN-?,SIgA,mucoprotein and lysozyme and SCFAs content in feces.Among them,the effect of GLP was better than that of DGLP in SCFAs content results(P<0.01).The results showed that ganoderma lucidum polysaccharide could enhance the intestinal mucosal barrier function in immunosuppressed mice.?Effects of ganoderma lucidum polysaccharides and their hydrolysates on the immune system:The immune system related indicators were detected.GLP and DGLP can improve the visceral index,PPs number,cecum weight and cecum index of immunosuppressed mice,increase the content of IL-2,IL-4,IFN-? and IgA in serum,and reduce the content of LPS.The results showed that ganoderma lucidum polysaccharide could enhance the immune function of immunosuppressive mice,but there was no significant difference between GLP and DGLP.(3)Establishment of isolated intestinal model for mucosal immune function detectionIsolated intestinal model was used to investigate intestinal mucosal immunity.LPS,levamisole hydrochloride and Gln were used to treat isolated intestine to explore the optimal conditions for isolated intestinal culture and its effect on intestinal mucosal immunity.?The isolated intestine was treated with LPS for different duration.With the increase in time,the medium color turned yellow,intestinal activity gradually disappeared,IL-4 content gradually decreased.Finally,0.5 h was determined as the most appropriate time,?In vitro intestines were treated with different concentrations of LPS,levamisole hydrochloride and Gln.The results showed that LPS,levamisole hydrochloride and Gln had regulatory effects on intestinal mucosal immunity.However,the effects of LPS and levamisole hydrochloride were significant.It is suggested that isolated intestinal model can be used for the detection of intestinal mucosal immune function.(4)Effects of ganoderma lucidum polysaccharides and its hydrolysates on immune function of isolated intestinal mucosa?After 0.5 h isolated intestinal culture with GLP and DGLP,SIgA content was detected.The results showed that both GLP and DGLP could promote the secretion of SIgA.?The SIgA content was detected after the intestinal contents collected in the GLP-H group was treated for 0.5 h in vitro.The results showed that intestinal contents also promoted the increase of SIgA secretion.Conclusion:both GLP and DGLP can restore intestinal mucosal immune function and systemic immune function of immunosuppressed mice to varying degrees,but there is no significant difference in the effect of the two.Isolated intestinal experiment results showed that ganoderma lucidum polysaccharides can directly stimulate the secretion of SIgA by enter-related cells,and also indirectly promote the secretion of SIgA by influencing the content of intestinal flora metabolite SCFAs,thereby regulating intestinal mucosal immunity.In this study,the isolated intestinal model was used for the first time in the study of intestinal mucosal immunity.Compared with the in vivo experiment,the effect of intestinal flora on intestinal mucosal immunity could be excluded.The regulation mode of ganoderma Iucidum polysaccharide on intestinal mucosal immunity was tested,and the direct regulation effect of ganoderma lucidum polysaccharide on intestinal mucosal immunity was verified.Secondly,isolated intestine can be treated with intestinal contents.As intestinal contents contains a large number of metabolites of intestinal flora,the regulation mode of intestinal flora on intestinal mucosal immune function can be detected to verify the indirect regulation effect of ganoderma lucidum polysaccharide on intestinal mucosal immunity.Based on the above experimental results,it can be inferred that ganoderma lucidum polysaccharide may have a clear regulatory effect on the regulation mode of intestinal mucosal immune function,which can not only directly affect the intestinal epithelial cells to play a role.This study provides new experimental evidence for elucidation of the mechanism of“supporting the energy”of ganoderma lucidum polysaccharides. |
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