Study On The Synergistic Mechanism Of Bran Fried Atractylodes Rhizoma In The Prevention And Treatment Of Colitis Based On Microflora-metabolism-inflammation

1 OBJECTIVEAtractylodes rhizome is a traditional Chinese medicine commonly used in clinical treatment of spleen and stomach diseases.The commonly used herbal pieces in clinical practice are Atractylodes and bran fried Atractylodes.Raw Atractylodes has strong dryness and is good at drying,while bran-fried Atractylodes has moderate dryness and is good at strengthening the spleen.At present,the research of raw and bran fried atractylodes mainly focuses on the regulation of the gastrointestinal tract on the model of spleen deficiency in traditional Chinese medicine,and there is little research on the efficacy of the gastrointestinal disease model of Western medicine corresponding to the pathogenesis of spleen deficiency.The purpose of this paper is to use DSS-induced colitis mice as a model,based on the regulatory function of Atractylodes Atractylodis on the gastrointestinal tract,by comparing the efficacy differences of raw and alcoholic extracts of Atractylodes glutinosa on colitis,and to further explore the effects of Atractylodes Rhizoma and its processed products on colitis.The potential therapeutic effect of colitis lays the foundation for the innovative application of Atractylodes Rhizoma in modern clinical practice,and provides a new strategy for the treatment of colitis.At the same time,the processing and synergistic mechanism of bran-fried Atractylodes Rhizoma in the prevention and treatment of colitis was explained from the aspects of the regulation of intestinal flora,the regulation of body metabolism and the screening of anti-inflammatory components,which provided a theoretical basis for the clinical application of processed Atractylodes Rhizoma products.2 METHODS2.1 Comparative study on pharmacodynamics of bran fried Atractylodes and raw Atractylodes in the prevention and treatment of DSS-induced ulcerative colitis60 BALB/c mice were randomly divided into normal group,model group,raw Atractylodes group,bran fried Atractylodes group and sulfasalazine group.Body weight change,DAI score,colon length,HE pathological structure,AB-PAS staining,MUC2 protein,tight junction protein(ZO-1,Occludin),pro-inflammatory factors(TNF-α,IL-6,IL-1β),macrophage activation markers(F4/80,iNOS)were used as detection indicators in the DSS-induced colitis model to compare the efficacy of raw Atractylodes Rhizoma ethanol extract and bran fried Atractylodes Rhizoma ethanol extract in preventing and treating colitis.2.2 Study on the synergistic mechanism of bran fried Atractylodes in the prevention and treatment of ulcerative colon based on flora regulationThe 16S rRNA technology was used to sequence the intestinal flora in the feces of the mice in the normal group,model group,raw Atractylodes Rhizoma group,and bran fried Atractylodes group,and the a diversity,βdiversity and species composition differences of the intestinal flora of the mice in each group were analyzed,comparing the regulatory effects of bran fried Atractylodes and raw Atractylodes on the intestinal flora of mice with DSS-induced colitis.2.3 Study on the synergistic mechanism of bran fried Atractylodes in the prevention and treatment of ulcerative colon based on metabolic regulationMetabonomics technology was used to detect the endogenous metabolites in the serum of mice in normal group,model group,raw Atractylodes group and bran fried Atractylodes group.Through t-test and OPLS-DA analysis,the serum differential metabolites between colitis mice and normal mice were screened under the limited conditions of P<0.05 and VIP>1,and then through statistical analysis,the differential metabolites that could be regulated by raw Atractylodes and bran fried Atractylodes were determined,and to compare the regulatory effects of raw Atractylodes and bran Atractylodes on serum metabolites in DSS induced colitis mice.After determining the regulated metabolites,Pearson analysis was used to analyze the correlation between intestinal flora,differential metabolites,body weight and colon length of mice with colitis regulated by raw and bran Atractylodes.2.4 Comparison of anti-inflammatory activities of main common com ponents of raw Atractylodes and bran Fried Atractylodes and their efficacy in preventing and treating colitisUsing LPS-induced RAW264.7 cell inflammation model,with TNF-α,IL-6,IL-1 β as indicators,the anti-inflammatory activities of the two components with the highest content in raw and bran fried atractylodes,atractylodin and β-eudesmol were compared.Pan-antibody protein detection and co-immunoprecipitation were used to detect the effect of atractylodin on the malonylation of GAPDH.Using DSS-induced colitis mouse model,40 mice were randomly divided into normal group,model group,atractylodin 10 mg/kg group,atractylodin 20 mg/kg group,and sulfasalazine group.Body weight change,DAI score,colon length,HE pathological structure,AB-PAS staining,MUC2 protein,tight junction protein(ZO-1,Occludin),pro-inflammatory factors(TNF-α,IL-6,IL-1β),macrophage activation markers Drugs(F4/80,iNOS)and macrophage activation pathway(MAPK pathway)protein were used as detection indicators to evaluate the efficacy of atractylodin in preventing and treating DSS-induced colitis.2.5 Study on the synergistic mechanism of bran fried Atractylodes in the prevention and treatment of colitis based on incremental ingredientsUsing the LPS-induced RAW264.7 cell inflammation model,with TNF-α,IL-6,IL-1β as indicators,the anti-inflammatory activities of the main incremental components(Atractylenolide I,Atractylenolide II,Atractyloside A,5-Hy droxymethy lfurfural)of Atractylodes Rhizoma after stir-frying were compared.Using DSS-induced colitis mouse model,60 mice were randomly divided into normal group,model group,Atractylenolide I(25 mg/kg)group,Atractylenolide I(50 mg/kg)group,sulfasalazine(SASP)group.Body weight,DAI score,colon length,HE pathological structure,AB-PAS staining,MUC2 protein,tight junction protein(ZO-1,Occludin),pro-inflammatory factors(TNF-α,IL-6,IL-1β),macrophage Phage activation markers(F4/80,iNOS)were used as detection indicators to evaluate the efficacy of Atractylenolide I in preventing and treating DSS-induced colitis.16S rRNA gut microbiota sequencing combined with metabolomic analysis,transcriptome sequencing analysis,and network analysis were used to reveal its mechanism of action.Using DSS-induced colitis mouse model,36 mice were randomly divided into the normal group,the DSS group,the DSS+scrambled plasmid group,the DSS+SPHK1 plasmid+B4GALT2 plasmid group,the DSS+scrambled plasmid+AT-1(50 mg/kg)group,and the DSS+SPHK1 plasmid+B4GALT2 plasmid+AT-1(50 mg/kg)group.To verify the effect of overexpression of SPHK1 and B4GALT2 genes on the efficacy of Atractylenolide I.3 RESULTS3.1 Comparative study on pharmacodynamics of bran fried Atractylodes and raw Atractylodes in the prevention and treatment of DSS-induced ulcerative colitisIn the DSS-induced ulcerative colitis mouse model,both bran fried Atractylodes and raw Atractylodes could improve weight loss,DAI score,shortening of colon length,deletion of goblet cells,and colonic pathological changes to varying degrees,inhibiting the reduction of MUC2,tight junction protein ZO-1 and Occludin,inhibiting the production of inflammatory factors TNF-α,IL-6 and IL-1 β in colon and the activation of pro-inflammatory macrophages,and the effect of bran fried Atractylodes was significantly better than that of raw Atractylodes.3.2 Study on the synergistic mechanism of bran fried Atractylodes in the prevention and treatment of ulcerative colon based on flora regulationBoth bran fried Atractylodes and raw Atractylodes could adjust the a diversity and β diversity of the intestinal flora of colitis mice to varying degrees.At the same time,they could also increase the abundance of some beneficial bacteria such as Lactobacillus,and reduce some harmful bacteria.Such as the abundance of Bacteroides,Parabacteroides,Clostridium,Turicibacter.And the regulation effect of bran fried Atractylodes is obviously better than that of raw Atractylodes.3.3 Study on the synergistic mechanism of bran fried Atractylodes in the prevention and treatment of ulcerative colon based on metabolic regulationRaw and bran fried Atractylodes can significantly improve the disorder of serum metabolites in DSS-induced colitis.Raw Atractylodes mainly regulates serine,glutamic acid,and cholesterol metabolism,while bran fried Atractylodes can not only regulate serine,glutamic acid,and cholesterol metabolism,but also regulates metabolism of alanine,proline,threonine,hypoxanthine,and sorbitan.The results indicated that bran fried Atractylodes had better regulation effect on serum metabolites in DSS-induced colitis than raw Atractylodes.Pearson analysis showed that the abundance of beneficial bacteria was positively correlated with the content of alanine,proline,serine,threonine,glutamic acid,and sorbitan,and negatively correlated with cholesterol content;the harmful bacteria Bacteroides,Parabacteroides,Clostridium,Turicibacter were positively correlated with cholesterol content,and negatively correlated with alanine,proline,serine,threonine,glutamic acid,hypoxanthine,and sorbitan;The body weight of mice was positively correlated with the content of alanine,proline,serine,threonine,glutamic acid,hypoxanthine and sorbitan,and negatively correlated with the content of cholesterol.The colon length of mice was positively correlated with the content of proline,serine,threonine,hypoxanthine and sorbitan,and negatively correlated with the content of cholesterol.It shows that the improvement of pathological symptoms by raw and bran fried atractylodes is closely related to the regulation of intestinal flora and metabolites.3.4 Comparison of anti-inflammatory activities of main common com ponents of raw Atractylodes and bran Fried Atractylodes and their efficacy in preventing and treating colitisAtractylodin can significantly inhibit the protein expression of TNF-α,IL-6 and IL-1β and the mRNA levels of IL-6 and IL-1 β,but cannot inhibit the mRNA level of TNF-α.The anti-inflammatory effect of equal dose ofβ-eudesmol was weaker than that of atractylodin.Atractylodin can bind to the rate-limiting enzyme GAPDH in the glycolytic pathway of macrophages,inhibit the post-malonylation modification of GAPDH,and inhibit the translation of TNF-α.At the same time,atractylodin can inhibit the enzymatic activity of GAPDH,inhibit the production of lactate,the final product of glycolysis in macrophages,thereby inhibiting the inflammatory response.Atractylodin can significantly improve DSS-induced colitis mice weight loss,diarrhea,blood in the stool,shortening of the colon,destruction of colon histopathological structure,loss of goblet cells,inhibition of colon tissue mucin MUC2,tight junction protein(ZO-1,occludin),inhibits the activation of MAPK pathway,inhibits the activation of macrophages,and finally inhibits the secretion of pro-inflammatory factors(TNF-α,IL-6,IL-1β)in colon tissue.It indicated that atractylodin had a good preventive effect on DSS-induced colitis.3.5 Study on the synergistic mechanism of bran fried Atractylodes in the prevention and treatment of colitis based on incremental ingredientsAtractylenolide I can significantly inhibit the mRNA level and protein expression of TNF-α,IL-6 and IL-1β.However,the anti-inflammatory effects of equal doses of Atractylenolide Ⅱ,Atractyloside A and 5-Hydroxymethylfurfural were weaker than that of Atractylenolide Ⅰ.Atractylenolide Ⅰ can significantly improve body weight loss,diarrhea,blood in the stool,shortening of colon,deletion of colonic goblet cells,reduction of mucoprotein MUC2,reduction of tight junction proteins(zo-1,occludin),Secretion of colonic proinflammatory factors(TNF-α,IL-6,IL-1β)and activation of macrophages.The results of 16S rRNA sequencing indicated that Atractylenolide Ⅰ could regulate the diversity and richness of intestinal flora in mice with colitis.Metabolomics studies have shown that Atractylenolide Ⅰ mainly affects the metabolism and absorption of fructose and galactose in mice with colitis.Colon tissue transcriptomics combined with metabolite network association analysis showed that two genes related to fructose and galactose metabolism regulated by Atractylenolide Ⅰ were SPHK1 and B4GALT2.WB and immunohistochemical results showed that Atractylenolide Ⅰ could significantly inhibit the abnormal increase of SPHK1 and B4GALT2 in colon of mice with colitis.At the same time,AtractylenolideⅠ can inhibit the activation of PI3K-AKT pathway.SPHK1 and B4GALT2 overexpression plasmids could reverse the therapeutic effect of Atractylenolide Ⅰ on colitis mice.4 CONCLUSIONThis study clarified the preventive and therapeutic effects of raw and bran fried Atractylodes on DSS-induced colitis,and proved the preventive and therapeutic advantages of bran fried Atractylodes.At the same time,it is explained that the reasons for the synergistic effect of bran fried Atractylodes in the prevention and treatment of DSS-induced colitis in mice are as follows:the ability of bran fried Atractylodes to regulate the intestinal flora of mice with colitis is better than that of raw Atractylodes,the metabolic regulation ability of bran fried Atractylodes is better than that of raw Atractylodes,and the incremental ingredients(Atractylenolide I)of bran fried Atractylodes has a better improvement effect on colitis mice.It was preliminarily clarified that the main component of raw and bran Atractylodes in the prevention and treatment of DSS-induced colitis in mice is Atractylodin.At the same time,it was revealed that the mechanism of the incremental ingredients(Atractylenolide I)in preventing and treating colitis is to regulate the composition of the intestinal flora by targeting SPHK1 and B4GALT2 to regulate the metabolism of fructose and galactose,and to inhibit the inflammatory response by regulating the SPHK1/PI3K/AKT axis.This study provides a reference for the clinical application of Atractylodes Rhizoma in the treatment of colitis,and provides a new basis for the processing and synergistic mechanism of bran fried Atractylodes.