Research On The Mechanism Of Atractylodes Glutinous Frying Based On The Intestinal Flora And The Co-metabolic Regulation Of The Flora-host

Purpose: To compare the pharmacodynamic difference of raw and bran-fried Atractylodis Rhizoma(AR)on the spleen-deficiency rats,and on this basis,to compare the regulatory effect of raw and bran-fried AR on the gut flora of the spleen-deficiency rats,to compare the effect of raw and bran-fried AR on the fecal metabolism spectrum of the spleen-deficiency rats,and to compare the effect of raw and bran-fried AR on the metabolism of short chain fatty acids(SCFAs)in the feces of the spleen-deficiency rats.From the perspective of regulating the gut flora and the metabolism of gut flora-host,the mechanism of AR against spleen-deficiency and the mechanism by which bran-fried AR enhanced the therapeutic effect were discussed.Material and method: 1.Forty male Sprague Dawley rats were randomly divided equally into 4 groups: control group(CG),model group(MG),raw AR group(RA)and bran-fried AR group(BFA).The model of spleen-deficiency was duplicated by compound factor method,which was composed of poor diet,excessive fatigue and severe diarrhea caused by bitter and cold Chinese medicine.Then,raw and bran-fried AR powder suspension were gavaged for seven consecutive days to treat spleen-deficiency.The serum was collected for the determination of tumor necrosis factor-?(TNF-?),interleukin 6(IL-6),immunoglobulin G(Ig G),somatostatin(SS),motilin(MTL),gastrin(GAS),amylase(AMS),trypsin(TRY),Na+-K+-ATPase by Enzyme-linked immunosorbent assay(ELISA).2.The intestinal microbiota composition was measured by 16 S r RNA gene Illumina Mi Seq sequencing(V3-V4 region).LEf Se difference test was used to seek the bacteria closely related to spleen-deficiency,which were going to be analyzed for the correlation with serum biochemical indexes.The modulatory effect of raw and bran-fried AR on these bacteria were compared.3.A fecal metabonomics method of raw and bran-fried AR intervention on spleen-deficiency rats was established by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS).Multiple pattern recognition was used to analyze the metabolite data of each group and further identify the biomarkers of spleen-deficiency.The modulatory effect of raw and bran-fried AR on these biomarkers were compared.The correlation between intestinal microbiota and fecal biomarkers was analyzed by SPSS.4.A rapid method was established for determining 7 SCFAs in rat feces by gas chromatography-mass spectrometer(GC-MS)and was applied to analyze the metabolic effect caused by raw and bran-fried AR on SCFAs in spleen-deficiency rats.The modulatory effect of raw and bran-fried AR on these SCFAs were compared.The correlation between intestinal microbiota and SCFAs was analyzed by SPSS.Results: 1.Raw and bran-fried AR can decrease the level of TNF-?,IL-6,Ig G,SS and increase the level of MTL,GAS,AMS,TRY,Na+-K+-ATPase in the serum of spleen-deficiency rats,and bran-fried AR was superior to raw AR.Among them,bran-fried AR was significantly superior to raw AR in the regulation of MTL,GAS,SS,AMS and TRY.2.The composition of intestinal microbiota of spleen-deficiency rats has changed significantly compared with healthy rats and tented to recover to the normal level after the treatment with raw and bran-fried AR.Nine bacteria closely related to spleen-deficiency were identified at genus level.Among them,the modulatory effect between raw and bran-fried AR was different.Raw AR was better than bran-fried AR in the regulating of Parasutterella,and bran-fried AR was better than raw AR in the regulating of Bacteroides,Escherichia-Shigella,Phascolarctobacterium,Incertae-Sedis(Defluviitaleaceae Family)and Incertae-Sedis(Erysipelotrichaceae Family).The correlation analysis revealed that the abundance of intestinal microbiota were closely related to the secretion and expression of cytokines and gastrointestinal hormones.They may interact to regulate immune function and gastrointestinal function.3.Twenty-two biomarkers of spleen-deficiency were identified under the positive and negative ion mode.Through the analysis of metabolic pathway,6 significant changes of metabolic pathway were found,5 of which were both improved by raw and bran-fried AR.Especially bran-fried AR was superior to raw AR in the improvement of tyrosine metabolism and purine metabolism.The correlation analysis between intestinal microbiota and metabolic biomarkers showed that the expression of metabolites was closely related to the structure of gut flora,that is,the structure of gut flora was disordered in the state of spleen-deficiency and thus the metabolism of gut flora-host was disturbed.4.Seven SCFAs had a good separation under the analysis method,and had a good linear relationship within their respective determination range(r > 0.995).This method had a good precision,stability and extraction recovery rate,and the matrix effect was within the acceptable range.Raw and bran-fried AR can significantly decrease the levels of acetic acid and propionic acid,and significantly increase the level of hexanoic acid in the feces of spleen-deficiency rats,and bran-fried AR was significantly superior to raw AR.The correlation between intestinal microbiota and SCFAs showed that the metabolism of SCFAs was closely related to the intestinal flora,that is,the structure of gut flora was disordered in the state of spleen-deficiency and thus the metabolism of SCFAs was disturbed.Conclusion: 1.One of the mechanisms by which raw and bran-fried AR act against spleen-deficiency may be that they can down-regulate inflammatory factors,enhance immune function and improve gastrointestinal digestion,absorption and water-liquid conversion function.One of the mechanisms by which bran-fried AR enhanced the therapeutic effect may be that the effect of bran-fried AR on improving gastrointestinal digestion and absorption function was significantly superior to that of raw AR.2.One of the mechanisms by which raw and bran-fried AR treat spleen-deficiency may be related to the modulation of intestinal microbiota.The mechanism by which bran-fried AR enhanced the therapeutic effect could be associated with the better regulation on Bacteroides,Escherichia-Shigella,Phascolarctobacterium,Incertae-Sedis(Defluviitaleaceae Family)and Incertae-Sedis(Erysipelotrichaceae Family).3.One of the mechanisms by which raw and bran-fried AR treat spleen-deficiency may be related to the modulation of gut flora and host co-metabolism.One of the mechanisms by which bran-fried AR enhanced the therapeutic effect may be related to the better improvement of tyrosine metabolism and purine metabolism.4.The method established for simultaneously determining 7 SCFAs in rat feces by GC-MS was simple and accurate with short analysis time,high sensitivity and satisfactory recovery,which can meet the quantitative requirements of SCFAs in rat feces.The mechanism by which raw and bran-fried AR treated spleen-deficiency may be related to the improvement of metabolism of SCFAs.The mechanism by which bran-fried AR enhanced the therapeutic effect could be associated with the better regulation on acetic acid,propionic acid and hexanoic acid.5.The results of correlation analysis suggested that raw and bran-fried AR may improvem the gut flora and host co-metabolism and SCFAs metabolism by regulating gut-flora,so as to improve the gastrointestinal dysfunction in the state of spleen-deficiency.The better regulatory effect of bran-fried AR may be the mechanism by which bran-fried AR enhanced the therapeutic effect of spleen-strengthening.