| Objective:Shenling Baizhusan is a commonly used spleen-strengthening prescription for treating spleen-qi deficiency syndrome.It is closely related to intestinal flora and intestinal short-chain fatty acids-producing flora.At present,most of the current researches explore the regulating effect on intestinal flora from the perspective of traditional Chinese medicine,but its pharmacological mechanism is still unclear.Based on the fact that traditional Chinese medicine polysaccharide is an important active ingredient in the compound,and the intestinal flora and intestinal functional flora are important targets of spleen-strengthening formula,this study took the intestinal short-chain fatty acids-producing flora as the object,through the construction of animals with spleen-qi deficiency syndrome model and treatment,combined with in vitro carbon source experiments,aims to further explore the micro-ecological mechanism of Shenling Baizhusan polysaccharide’s spleen-invigorating effect by studying the regulation of intestinal short-chain fatty acids-producing flora by Shenling Baizhusan polysaccharide.Methods:1.Grouping and model building:SD rats were randomly divided into blank group(group B),model group of spleen-qi deficiency syndrome(SQ group),Shenling Baizhusan polysaccharide group(PH group),Shenling Baizhusan decoction group(group D)and sucrose group(SUC group)(n=6).In the modeling stage(Day 1st-14th),all rats except group B were subjected to the"bitter cold diarrhea+overwork+eating disorder"modeling method to establish a model of spleen-qi deficiency syndrome,and group B was given normal saline;(Day 15th-28th)PH group,D group and SUC group were given Shenling Baizhusan polysaccharide,Shenling Baizhusan decoction and sucrose respectively,and group B and SQ group were given normal saline.The body weight,feces,mental state,diet,hair condition and other signs of the rats were recorded.After the treatment,the spleen and thymus of the rats were collected and weighed to calculate the organ index;urine,blood and colon tissue were collected,and the urinary D-xylose excretion rate of the rats in each group was determined by using the D-xylose kit,and the ELISA method was used.The serum CPK activity of the rats in each group was measured,the skeletal muscle damage of the rats with spleen-qi deficiency syndrome was observed,and the changes in the colon tissue of the rats were observed by H.E.staining.2.Structural changes of the total intestinal flora during treatment:Rat feces were collected and DNA was extracted.The Illumina Mi Seq sequencing platform was used to detect the changes of the total intestinal flora of each group of rats in the normal stage,the modeling stage and the treatment stage.group influence.3.Structural changes of intestinal short-chain fatty acid-producing flora during treatment:The fecal DNA was amplified by PCR using the butyryl Co A-Co A transferase gene,and then the TA cloning sequencing platform was used to detect the diversity and structural changes of butyric acid-producing bacteria in the gut of each group of rats at each stage;mmd A,lcd A,pdu P and pdu Q genes were used to amplify fecal DNA by PCR,and then the TA cloning sequencing platform was used to detect the diversity and structural changes of intestinal propionate-producing bacteria in rats at various stages.The effect of microflora and propionic acid-producing bacteria;the content of short-chain fatty acids in the feces of rats in the treatment stage was measured,and the effect of Shenling Baizhusan polysaccharide on the level of intestinal short-chain fatty acids in rats with spleen-qi deficiency syndrome was studied;4.In vitro carbon source experiment:The mixed bacteria Clostridium butyricum and Firmicutes YT2 were cultured in BHI medium and Shenling Baizhusan polysaccharide as the sole carbon source medium,and samples were taken every 2-4 hours to determine the growth quantity of the mixed bacteria in different mediums.and butyric acid-producing concentration,to study the effect of Shenling Baizhusan polysaccharide on the growth of intestinal butyric acid-producing bacteria in vitro.Results:1.Construction results of spleen-qi deficiency syndrome model:Rats with spleen-qi deficiency syndrome were established by the method of"bitter and cold diarrhea+overwork+eating disorder".The rats in the modeling group(SQ group,PH group,D group,and SUC group)showed symptoms such as mental fatigue,diarrhea,and decreased diet.The weight growth rate was slower than that of group B(P<0.05).The spleen index,urinary D-xylose excretion rate and CPK activity of rats in SQ group were significantly lower than those in group B(P<0.05).The difference(P<0.05);in the SQ group,obvious inflammatory cell infiltration was seen in the muscle layer,mucosa layer and submucosa layer;according to the general signs of the rats,the results of related biochemical indicators and the staining results of colon tissue sections,it was shown that the model of spleen-qi deficiency syndrome was constructed.success.2.Changes in the total intestinal flora of rats treated with spleen-qi deficiency syndrome:The Illumina Mi Seq(PE300)sequencing platform was used for sequencing,and the total number of taxa was obtained from 5 groups of 90 samples:Kingdom:1;Phylum:11;Class:18;Order:49;Family:90;Genus:201;Species:376;OTU:1179.(1)F/B ratio change:There was no significant change in F/B value in group B at each stage;after modeling,the F/B value of SQ group(20.83%)was significantly different from that of PH group(20.29%),D group(37.50%)and SUC group(30.14%)compared with the normal stage in this group(P<0.05);after treatment,the F/B value of the PH group(155.88%)was closer to the normal stage than the D group(89.13%)and the SUC group(62.07%).Group normal stage(P<0.05),suggesting that Shenling Baizhusan polysaccharide can restore the ratio of main intestinal flora in rats with spleen-qi deficiency syndrome,and the recovery degree is better than that of Shenling Baizhusan decoction and sucrose.(2)Variation ofαdiversity:There was no significant difference in species abundance and diversity in the three-stage comparison of group B(P>0.05);group SQ:compared with SQ1,SQ2 Ace index(315.77±54.25),Chao index(309.25±45.55),number of OTUs(234),Shannon index(3.84±0.23)decreased,Simpson index(0.07±0.02)increased,the species abundance and diversity in the SQ group decreased significantly during the modeling stage(P<0.05),SQ3 Compared with SQ2,SQ3 Ace index(386.56±81.17),Chao index(372.27±61.59),OTU number(308),Shannon index(3.35±0.47)all increased,Simpson index(0.10±0.07)decreased,it can be seen that SQ The species abundance in the treatment group increased(P<0.05),and the diversity had no significant change(P>0.05);The number of OTUs(228)and the Shannon index(2.72±0.47)decreased,while the Simpson index(0.17±0.09)increased.The species abundance and diversity of the PH group decreased significantly during the modeling stage(P<0.05).Compared with PH2,PH3,PH3 Ace index(495.08±32.08),Chao index(501.49±32.21),number of OTUs(380),Shannon index(3.71±0.38)all increased,Simpson index(0.08±0.03)decreased,it can be seen that the species in the treatment stage of the PH group Abundance and diversity increased(P<0.05);group D:compared with D1,D2 Ace index(336.2±91.33),Chao index(315.66±68.18),OTU number(244),Shannon index(2.74±0.46)decreased,the Simpson index(0.08±0.15)increased,the species abundance and diversity of group D decreased significantly(P<0.05)in the modeling stage.Compared with D2,D3 Ace index(421.10±70.54),Chao index(420.87±74.8),the number of OTUs(335),and the Shannon index(3.16±0.51)all increased,and the Simpson index(0.15±0.08)decreased.It can be seen that the species abundance in the treatment stage of group D increased(P>0.05),and the diversity increased(P<0.05);SUC group:Compared with SUC1,SUC2 Ace index(301.86±63.25),Chao index(286.09±62.75),OTU number(222),Shannon index(2.51±0.17)decreased,Simpson index(0.19±0.06)increased,the species abundance and diversity in the SUC group decreased significantly during the modeling stage(P<0.05).Compared with SUC2,SUC3 Ace index(397.76±21.35),Chao index(406.94±33.61),OTU number(299),Shannon index(3.05±0.37)all increased,Simpson index(0.13±0.04)decreased,it can be seen that the species abundance and diversity in the SUC group increased during the treatment period(P<0.05).(3)βdiversity:PCA analysis showed that the main species composition of SQ group was significantly different from the other four groups;compared with D group and SUC group,PH group and B group had the closest distance,suggesting that PH group and B group had the most similar species composition.It can be seen that Shenling Baizhusan polysaccharide can restore the composition of total intestinal flora in rats with spleen-qi deficiency syndrome,and the recovery degree is better than that of Shenling Baizhusan decoction and sucrose.(4)Main species at the phylum level:Bacteroidetes and Firmicutes were the main species in the three stages of group B,SQ,PH and SUC;Bacteroidetes,Firmicutes and Verrucomicrobiota were the main species in group D in the normal stage,and Bacteroidetes and Firmicutes in the modeling and treatment stages.main.(5)The main species changed at the genus level:Muribaculaceae was the most abundant in the three stages of group B;Muribaculaceae was the most abundant(36.47%)in the normal stage of the SQ group,and Lactobacillus was abundant in the modeling stage(14.68%)and the treatment stage(42.40%).The genus Muribaculaceae in the PH group had the highest abundance in the normal stage(34.33%)and the treatment stage(27.37%),and the genus Romboutsia had the highest abundance in the modeling stage(18.25%).The treatment stage(23.29%)had the highest abundance,and the Lachnospiraceae genus had the highest abundance in the modeling stage(13.26%).The SUC group had the highest abundance of Muribaculaceae in the normal stage(35.98%)and treatment stage(26.40%),and Escherichia-Shigella had the highest abundance.The mode stage(12.45%)has the highest abundance.From the above,it can be seen that the polysaccharide of Shenling Baizhusan can increase the species abundance and diversity of the total intestinal flora in rats with spleen-qi deficiency syndrome,and mainly increase the abundance of Muribaculaceae.3.Changes of intestinal butyric acid-producing bacteria in rats with spleen-qi deficiency syndrome:The SQ group,PH group,D group and SUC group were amplified with the butyryl Co A-Co A transferase gene as the molecular marker,and 300 sequences were obtained,which were determined as the diversity results of butyric acid-producing bacteria.The number of OTUs in the SQ group decreased to 33%of the normal stage(9)in the modeling stage(9),and returned to 44.44%in the treatment stage(4);the number of OTUs in the PH group was in the modeling stage(4)Reduced to 44.44%of the normal stage(9),and recovered to 77.78%in the treatment stage(7);group D,the number of OTUs,the modeling stage(3)was reduced from the normal stage(8)to 37.50%%,the treatment stage(5)returned to the normal stage 62.50%;the number of OTUs in the SUC group,the modeling stage(4)was reduced to 44.44%from the normal stage(9),and the treatment stage(5)returned to the normal stage 55.56%.Butyric acid-producing bacteria were present in all stages of the four groups of rats,all of which were butyric acid-producing bacteria of Firmicutes.Compared with the modeling stage,the main changes of intestinal butyric acid-producing bacteria in the rats in the treatment stage were as follows:the abundance of Clostridium and Eubacterium in the SQ group was slightly decreased;the abundance of Roseburia was significantly increased in the PH group.high,the abundance of Clostridium was significantly decreased;the abundance of Roseburia and Eubacterium in group D increased,and the abundance of Clostridium decreased;in SUC group,the abundance of Clostridium increased,and the abundance of Eubacterium decreased.The phylogenetic tree shows that all sample sequences can be assigned to 9 clusters.The percentage analysis of the clusters in each stage of Shenling Baizhusan polysaccharide treatment of spleen-qi deficiency rats shows that the diversity of butyric acid-producing bacteria in PH3 is lower than that in PH1.However,compared with PH2,the diversity increased and the structure of butyric acid-producing bacteria changed.It can be seen that Shenlingbaizhusan polysaccharide can increase the abundance and diversity of intestinal butyric acid-producing bacteria in rats with spleen-qi deficiency syndrome,mainly increase the abundance of Roseburia and decrease the abundance of Clostridium.4.Changes of intestinal propionic acid-producing bacteria in rats with spleen-qi deficiency syndrome:In the PCR amplification of propionic acid-producing bacteria mmd A,lcd A,pdu P,and pdu Q,only the mmd A gene and pdu P gene were successfully amplified.The mmd A gene and pdu P gene were used as molecular markers to detect the changes of propionic acid-producing bacteria in the rat intestine.Obtained by sequencing:SQ group obtained 7 sequences,PH group PH1,PH2,PH3 obtained 37,47,21 sequences;D group D1,D2,D3 obtained 50,50,29sequences,SUC group SUC1,SUC2 97,15 and 55 sequences were obtained from SUC3,and the measured sequences were all determined as the results of the diversity of propionic acid-producing bacteria.The propionic acid-producing bacteria group existed in each stage of the treatment group.The normal stage was dominated by the Bacteroidetes propionic acid-producing bacteria group,accounting for about 75.54%,and the modelling stage was the Bacteroidetes phylum propionic acid-producing bacteria group;In the treatment stage,the proportion of propionic acid-producing bacteria in the phylum Bacteroides accounted for about 51.70%,and the proportion of propionic acid-producing bacteria in the phylum Firmicutes accounted for about 48.30%.Compared with the modeling stage,the main changes of intestinal propionic acid-producing bacteria in each group of rats in the treatment stage were as follows:the abundance of Barnesiella and Alistipes in the PH group increased;the abundance of Phocaeicola and Barnesiella in the D group increased In SUC group,the abundance of Blautia increased,while the abundance of Alistipes and Bacteroides decreased.The phylogenetic tree shows that all sample sequences can be assigned to 8 clusters.The percentage analysis of the clusters at each stage of Shenlingbaizhusan polysaccharide treatment of spleen-qi deficiency rats shows that:PH1 has a lower diversity of propionic acid-producing bacteria than PH2.,compared with PH3,the diversity did not change significantly,and the structure of propionic acid-producing bacteria changed.5.Changes of intestinal short-chain fatty acids content in rats with spleen-qi deficiency syndrome during the treatment stage:The three highest content of 8 short-chain fatty acids in group B were acetic acid(36.79±5.39μg/m L),propionic acid(20.09±0.46μg/m L),butyric acid(25.97±2.26μg/m L);Compared with the control group,the contents of acetic acid,propionic acid and butyric acid in the SQ group were significantly decreased;in each treatment group,acetic acid(31.07±4.34μg/m L),propionic acid(18.66±1.97μg/m L),butyric acid(23.48±4.18μg/m L)in the PH group was the highest,which was significantly higher than that in the SQ group;the content of butyric acid in each group from high to low was as follows:group B(25.97±2.26μg/m L)>PH group(23.48±4.18μg/m L)>SUC group(22.96±3.59μg/m L)>D group(21.90±10.52μg/m L)>SQ group(20.62±3.13μg/m L);the average content of propionic acid in each group from high to low:group B(20.09±0.46μg/m L)>PH group(18.66±1.97μg/m L)>D group(17.37±2.72μg/m L)>SUC group(16.18±2.98μg/m L)>SQ group(12.48±5.90μg/m L)).It can be seen that Shenling Baizhusan polysaccharide can promote the production of butyric acid and propionic acid in the intestine of rats with spleen-qi deficiency syndrome,which is beneficial to restore the intestinal microecological balance.6.In vitro carbon source experiment:The maximum bacterial growth of mixed bacteria Clostridium butyricum and Clostridium sp.YT2 in Shenlingbaizhusan polysaccharide as the sole carbon source medium((0.97±0.006)×10~9CFU/m L)was higher than that in BHI medium((0.87±0.013)×10~9CFU/m L),Shenling Baizhusan polysaccharide can promote the growth of mixed bacteria;the mixed bacteria reached the maximum growth at the 10th hour in BHI medium,and Shenling Baizhusan polysaccharide was the only carbon source medium in the medium The maximum growth rate was reached at the 36th hour,and the growth rate of mixed bacteria in BHI medium was faster than that of Shenling Baizhusan polysaccharide as the sole carbon source medium;Shenling Baizhusan polysaccharide was the highest concentration of butyric acid produced in the sole carbon source medium(3142.88±28.47μg/m L)higher than the highest concentration of butyric acid in BHI medium(1658.35±31.09μg/m L),Shenling Baizhusan polysaccharide can promote the production of butyrate.The mechanism of Shenling Baizhusan polysaccharide regulating butyrate-producing bacteria in the intestine may be that the polysaccharide components are first degraded into monosaccharides by Firmicutes YT2 with polysaccharide utilization sites,and then the monosaccharides are catabolized by butyric acid-producing bacteria into dimethicone.Acid,increase the production of butyric acid for the body to absorb and utilize to achieve therapeutic effect.Conclusion:In this paper,through the treatment of animal models of spleen-qi deficiency syndrome and in vitro carbon source experiments,the micro-ecological mechanism of Shenling Baizhusan polysaccharide in regulating spleen-qi deficiency syndrome was studied.Experiments found that Shenling Baizhusan could effectively regulate the abundance and diversity of total intestinal flora and intestinal short-chain fatty acid-producing bacteria(mainly butyric acid-producing bacteria)in rats with spleen-qi deficiency syndrome,and increase the probiotics.Among them,Shenling Baizhusan polysaccharide not only has the advantage of obviously regulating the flora,but also can promote the growth of butyric acid-producing bacteria and increase the content of butyric acid.The results of this study suggest that Shenling Baizhusan polysaccharide is the most important active ingredient in regulating intestinal flora in rats with spleen-qi deficiency syndrome,which can provide a reference for the clinical pharmacology of Shenling Baizhusan in the treatment of spleen-qi deficiency syndrome. |
PDF Full Text Request