Regulation Of Pseudostellaria Heterophylla On The Intestinal Microecology Of Spleen-deficiency Rats

Objective: In this study,the decoction of different grades of Pseudostellaria heterophylla was administered to rats with spleen-deficiency to explore its effects on immune cytokines and gastrointestinal hormones.The crude polysaccharide of Pseudostellaria heterophylla was purified by enzyme.The effects of different grades of decoction and polysaccharide of Pseudostellaria heterophylla on intestinal microflora and short-chain fatty acids of spleen-deficiency rats were investigated.This study aims to provide theoretical basis for the spleen strengthening and qi supplementing effect of Pseudostellaria heterophylla.Methods: 1.The spleen-deficiency and diarrhea model was prepared by the bitter-cold diarrhea method,and three grades of Pseudostellaria heterophylla decoction(PHD)were administered.The contents of immune cytokines and gastrointestinal hormones in blood samples were detected by ELISA,spleen and thymus indexes were calculated,and the level of inflammation changes of spleen-deficiency rats during modeling and administration were determined by the analysis results of various indexes.2.Crude polysaccharide was extracted by water extraction and alcohol precipitation method,and protein was removed by protease method.The enzymolysis temperature,enzymolysis time and solution p H were selected by Box-Behnken response surface method to optimize the purification method of crude polysaccharide.Response surface was drawn to obtain the optimal conditions for enzymatic purification of polysaccharides,and the prediction conditions were verified.The contents of glucose and galactose in crude polysaccharide were measured by PMP-HPLC.3.The spleen-deficiency rats were administered with different grades of PHD and Pseudostellaria heterophylla polysaccharide(PHP),and cecum contents were collected.The changes of intestinal microflora composition of rats in each group were investigated by high-throughput sequencing,short-chain fatty acid content was detected by GC-MS.Results: 1.Spleen index and thymus index of spleen-deficiency model group decreased significantl;PHD improved spleen index and thymus index to some extent after administration,and grade I decoction had the best effect.The contents of the three pro-inflammatory cytokines increased in the model group,while the contents of the anti-inflammatory factor IL-10 decreased significantly.After administration,the content of pro-inflammatory factors decreased,while the content of anti-inflammatory factors increased significantly(P <0.05),grade I and grade II decoction had the most significant effect on the content of cytokines.2.The optimal conditions predicted by response surface method for protein removal of polysaccharides as follows: enzymatic hydrolysis time 2.0 h,temperature45°C,p H 6.5,and the optimal protein removal rate was 73.83%.The influence of the factors was in the order of temperature > time > solution p H,and the interaction of the three factors was significant.The total contents of glucose and galactose in the crude polysaccharides purified by enzymatic and Sevag methods accounted for more than 58%,and the peaks of other monosaccharides with smaller peak area could be seen in the chromatogram.3.High-throughput sequencing showed that dilution curve and rank abundance curve indicated that the amount of data sequenced was progressive and reasonable,and the species richness and uniformity of the samples were good.Compared with blank group,the α and β-diversity indexes in model group were significantly decreased(p<0.05).After administration,the α andβ-diversity indexes in each group were increased to varying degrees,and the effect of the grade I decoction and polysaccharide group was the best.The ratio of Firmicutes/Bacteroidetes in model group was significantly higher than that in blank group,and the ratio was significantly decreased in all administration groups,especially in the grade I decoction and polysaccharide group.At the classification level of order,family and genus,the model group,blank group and each administration group showed differences in species.The genus level heatmap also directly reflected the composition difference of intestinal flora in each group.The linear discriminant effect size analysis directly reflected the enrichment species in each group,and the principal coordinate analysis and linear discriminant distribution histogram showed the difference between the model group and the blank group,each administration group and the positive control group.The results showed that PHP and PHD can significantly promote the production of acetic acid and butyric acid in caecal contents of spleen-deficiency rats,especially in the grade I decoction group and polysaccharide group.Conclusion: 1.The changes of immune organ index and inflammatory cytokine content in spleen-deficiency rats after receiving water decoction of different grades of Pseudostellaria heterophylla showed that the water decoction of Pseudostellaria heterophylla had certain effects on improving immune function and alleviating inflammation level in rats with spleen-deficiency,and the grade I decoction was the most significant.2.Response surface model predicted the optimal conditions for enzymatic protein removal,and the validation experiment showed that the model had good reliability and predictability.The monosaccharide composition of Pseudostellaria heterophylla was mainly glucose and galactose,and also contained other monosaccharides.3.The community species composition,flora richness,species diversity,and the change of content of short chain fatty acids in spleen-deficiency rats showed that Pseudostellaria heterophylla decoction and polysaccharide improved the intestinal microecological health of spleen-deficiency rats to a certain extent,and the effect of grade I decoction and polysaccharide was the best.