| Astragalus polysaccharides(APS)is an important pharmacologically active component of Radix Astragali,and it has a variety of different physiological functions:such as regulating immunity,reducing blood glucose,anti-inflammatory,anti-oxidant,and anti-viral.Previous studies have found that,compared with Astragalus polysaccharides,the hydrolyzed Astragalus polysaccharides(DAPS)exerts better regulatory effect on intestinal mucosal immunity in immunosuppressed mice induced by cyclophosphamide(CY).In order to solve the problem of the APS hydrolysis process,this study intends to test the specific parameters and hydrolysis effects of the APS acid hydrolysis process,which is conducive to the subsequent development of related products.In order to further explore the mechanism of APS regulating intestinal mucosal immune function,this study used ex-vivo intestinal models and animal models to detect the changes in intestinal mucosal immunity-related indexes in each model after APS treatment,and further explained the medicinal mechanism of APS.DAPS was prepared by hydrolyzing APS with hydrochloric acid(HCl),trifluoroacetic acid(TFA),and acetic acid(HAc).The molecular weight distribution of APS and DAPS were detected by high-performance gel permeation chromatography(HPGPC)method to determine the appropriate acid hydrolysis conditions.To investigate the effects of APS,DAPS,and intestinal contents on intestinal mucosal immune-related cytokines(SIgA,?-DF,MUC,LYZ,and IFN-y)secretion in ex-vivo intestinal models.After APS treatment,the levels of cytokines such as IL-4,IL-6,IL-2,IFN-y,and LYZ in normal mice and CY induced immunosuppressed mice were measured.The main research methods and results are as follows:(1)Astragalus polysaccharide acid hydrolysis process:?The results of HPGPC method showed that APS was mainly composed of three different molecular weight segments of high,medium and low,and their relative molecular weight ranges were 1.2×105?1.7×105Da,2.0×103?4.2×104Da,and less than 224 Da,the ratios were 82.6%,11.9%,and 5.5%.?The optimum hydrolyzation procedure of APS was configured with 0.16 mol/L HCl hydrolyzing 3 hours at 80?.The results of HPGPC method showed that DAPS was mainly composed of three different molecular weight segments of high,medium and low,and their relative molecular weight ranges were 1.1×105?1.6×105 Da,103?3.3×104 Da,and less than 200 Da,the ratios were 15%,65%,and 20%,indicating that most of the APS is hydrolyzed,and the products were mainly 103?3.3×104 Da medium molecular weight APS.? The optimum hydrolyzation procedure of APS was configured with 0.10 mol/L TFA hydrolyzing 3 hours at 80 ?.The results of HPGPC method showed that DAPS was mainly composed of three different molecular weight segments of high,medium and low;and their relative molecular weight ranges were 1.2×105?1.7×105 Da,103?3.5×104 Da,less than 200 Da,the ratios were 15%,75%,and 10%,indicating that most APS is hydrolyzed,and the products were mainly 103?3.5×104 Da medium molecular weight APS.?The optimum hydrolyzation procedure of APS was configured with 0.16 mol/L HAc hydrolyzing 3 hours at 80?.The results of HPGPC method showed that DAPS was mainly composed of three different molecular weight segments of high,medium and low,and their relative molecular weight ranges were 1.1×105?1.6×105 Da,2.0×103?3.6×104 Da,less than 210 Da,the ratios were 20%,70%,and 10%,indicating that most APS is hydrolyzed,and the products were mainly 2.0×103?3.6×104 Da molecular weight APS.(2)The ex-vivo intestine was treated with APS and DAPS for 0.5 hours,and the content of immune factors in the intestinal mucosa was detected by ELISA.The results showed that compared with APS,DAPS could promote the secretion of SIgA,IFN-?,MUC,and LYZ increased,and the difference was significant(p<0.05).It was suggested that DAPS has a more significant regulatory effect on the intestinal mucosal immune function.(3)The intestinal contents of the animal were isolated after APS treatment.Then treat ex-vivo intestine with the intestinal contents and intestinal contents+APS mixture for 0.5 hours h.The secretion of immune factors in the intestinal mucosa was detected by ELISA.The results showed that compared with intestinal contents,intestinal contents+APS mixture could promote the secretion of ?-DF,LYZ and MUC increased in the ex-vivo intestine,the difference is significant(p<0.05)or extremely significant(p<0.01).It is suggested that APS affects intestinal mucosal immunity is mainly through direct action,while the effect mediated by intestinal bacteria is possible small.(4)100 mg/kg and 400 mg/kg APS were given to immunosuppressed mice induced by CY,400 mg/kg APS were given to normal mice,and the level changes of transcription of immune factors in the small intestine of each group of mice was detected by q-PCR two weeks later.The specific results are as follows:APS can upregulate the mRNA expression levels of IL-4,IL-6,and IL-2 in the intestinal mucosa of immunosuppressed mice,and can downregulate the mRNA expression levels of IFN-y and LYZ in the intestinal mucosa of immunosuppressed mice.At the same time,APS can downregulate the mRNA expression levels of IL-4,IL-6,IL-2,IFN-y,LYZ,and Cryp4 in the intestinal mucosa of normal mice,and can upregulate the mRNA expression levels of MUC2 in the intestinal mucosa of normal mice.It is suggested that APS can regulate the expression of intestinal mucosal immune-related cytokines.In this study,the process of hydrolyzing APS by hydrochloric acid,trifluoroacetic acid,and acetic acid was optimized,and the large molecular weight APS was hydrolyzed to a medium molecular weight segment of 103?3.6×104 Da,and the ratio of APS in the total molecular weight hydrolysate was more than 50%.In the in vitro experiments,APS and DAPS mainly regulate the intestinal mucosal immune function directly,and the intestinal flora is weakly mediated,and the DAPS regulation effect is better than APS;APS can regulate the expression of intestinal mucosal immune-related cytokines in immunosuppressed mice and normal mice,among which the effects on IL-2,FN-yand LYZ are more prominent.This study laid the foundation for the development and utilization of APS products and provided experimental evidence for further understanding of the molecular mechanism of APS in regulating immune function.Related detailed mechanisms need to be further explored. |
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