Exploring The Biological Basis Of Atrophic Gastritis With Spleen-stomach Damp-heat Syndrome Based On ?-catenin Mediated Pyroptosis Mechanism And Multi-omics Sequencing

Objective1.The biological mechanism of CAG spleen-stomach damp-heat syndrome was preliminarily obtained by detecting the expression of ?-catenin and cell death-related factors NLRP3,Caspase-1,ASC,GSDMD and IL-1? in rat models with Chronic atrophic gastritis(CAG)spleen-stomach damp-heat syndrome.Through the intervention of Qinghua Yin,reverse verification was carried out with the help of the thinking of "prescription test syndrome"to explain the biological mechanism of CAG spleen-stomach damp-heat syndrome.2.The omics technology based on the theory of systems biology is applied to the study of the biological mechanism of TCM syndromes.The biological mechanism of CAG spleen-stomach damp-heat syndrome was studied from the intestinal microbiome and metabolome by detecting the fecal intestinal flora and metabolites of rats with CAG spleen and stomach damp-heat syndrome.MethodsThe male Wistar rats were randomly divided into two groups:control group(C,n=8)and model establishing group.In the model establishing group,the rat model o f CAG spleen-stomach damp-heat syndrome was established by "free drinking of 1-methyl-3-nitro-1-nitrosoguanidine+fasting administration of ethanol+abnormal feed ing of hunger and satiety" combined with "high-fat and high-sugar diet+artificial c limate box".After successful establishment of the model,model establishing group r ats were randomly divided into model group(M,n=8),Qinghuayin group(QHY,n=8),LiCl group(n=8),XAV939 group(n=8),Vitacoenzyme group(n=8),model+antibiotic group(M?ATB,n=8),Qinghuayin+antibiotic group(QHY?ATB,n=8)an d Vitacoenzyme+antibiotic group(V?ATB,n=8).The corresponding drug interven tion was subsequently given for 30 days.After that,the gastric tissue,blood and in testinal contents of the rats were taken.The pathological changes of gastric mucosa of rats in each group were observed by HE staining.The expression levels of ?-cat enin,NLRP3,Caspase-1,ASC,GSDMD and IL-1 ? in gastric tissue of rats were d etected by Western blotting and qPCR methods.The levels of serum IL-6 and IL-18 were detected by ELISA method,and the expression of PCNA and ?-catenin in ga stric mucosa was detected by immunohistochemical method.At the same time,16s rRNA and UHPLC-QTOFMS techniques were used to detect the changes of intestinal microflora and metabolites in rats.ResultsThe gastric tissue and serum of rats in group C,M,QHY,LiCl and XAV939 were detected by WB,qPCR,immunohistochemistry and Elisa.?Gastric tissue-relat ed protein expression:compared with group C,the expression of ?-catenin,NLRP3,Caspase-1 and IL-1? protein in group M was significantly higher than that in grou p C(P<0.01).The expression of GSDMD protein in M group was significantly 1 ower than that in group C(P<0.05).Compared with group M,the expressions of?-catenin,NLRP3,Caspase-1 and IL-1? in QHY group and XAV939 group were si gnificantly decreased(P<0.05,0.01).The protein expression of ?-catenin,NLRP3,ASC,Caspase-1 and IL-1? in LiCl group was further increased,and the expression of ?-catenin and ASC protein was significantly higher than that in group M(P<0.01,0.01).The expression of GSDMD protein in QHY group and XAV939 group in creased significantly(P<0.05,0.01),while the expression of GSDMD protein decre ased in LiCl group,but the difference was not statistically significant.?The expres sion of mRNA in gastric tissue:compared with group C,the expression of ?-cateni n,NLRP3,ASC,Caspase-1 and IL-1? mRNA in group M was significantly higher than that in group C(P<0.01).The expression of GSDMD mRNA in);M group was significantly lower than that in group C(P<0.05).Compared with group M,the expressions of ?-catenin,NLRP3,ASC,Caspase-1 and IL-1 ? mRNA in QHY g roup and XAV939 group were significantly decreased(P<0.05,0.01).The expressio n of ?-catenin,NLRP3,ASC,Caspase-1 and IL-1? mRNA in LiCl group was furt her increased,but the expression of ?-catenin,NLRP3,Caspase-1 and IL-1? mRN A was significantly higher than that in group M(P<0.05,0.01).The expression of GSDMD mRNA in QHY group and XAV939 group increased significantly(P<0.05,0.01),and the expression of GSDMD mRNA in LiCl group further decreased(P<0.01,0.01).?The expression of ?-catenin and PCNA in gastric mucosa of rats:co mpared with group C,the levels of ?-catenin and PCNA in gastric tissue of rats in group M were significantly higher than those in group C,and the pathological chan ges of gastric mucosa were obvious.Compared with group M,the levels of ?-cateni n and PCNA in gastric tissue of rats in QHY group and XAV939 group were signif icantly decreased(P<0.01).The expression of ?-catenin and PCNA in gastric tissu e of rats in),LiCl group was significantly higher than that in group M(P<0.01).?The levels of serum IL-6 and IL-18:compared with group C,the expression of serum IL-6 and IL-18 in group M increased significantly,while the expression of se rum IL-6 and IL-18 in QHY group and XAV939 group decreased significantly comp ared with group M,and the expression of serum IL-6 and IL-18 in LiCl group incr eased significantly(P<0.01).The composition and abundance of microflora in intestinal contents of C group,M group,QHY group,V group,M?ATB group,QHY?ATB group and V?ATB gro up were analyzed.An overview of the five levels of flora from phylum to genus:At the phylum classification level,the intestinal flora of all samples of rats is mainl y composed of Firmicutes,Proteobacteria,and Bacteroidetes,Verrucomicrobia,Actino bacteria,Saccharibacteria 6 categories,of which Firmicutes is the most dominant,ac counting for 85.1%?90.5%in the treatment without antibiotics.At the class level,the rat intestinal flora consists of Clostridia,Erysipelotrichia,Bacteroidia,Bacilli,Ga mmaproteobacteria,Delta-Proteobacteria,Verrucomicrobiae,Coriobacteriia,?-Proteobac teria,Negativicutes and other 10 major classes.At the order level,the rat intestinal flora is mainly composed of Clostridiales,Erysipelotrichales,Bacteroidales,Lactobaci llales,Enterobacteriales,Desulfurization It is composed of Desulfovibrionales,Verruc omicrobiales,Coriobacteriales and Burkholderiales.At the family level,the intestinal flora of rats in all samples is mainly composed of Ruminococcaceae,Erysipelotricha ceae,Lachnospiraceae,Enterobacteriaceae,and Bacteroides Bacteroidaceae,Lactobacil laceae,Desulfovibrionaceae,Peptococcaceae,Family???,Verrucomicrobiaceae,Christ ensenellaceae,Christensenellaceae,Bacteroidales?S24-7?group,Coriobacteriaceae,Alca ligenaceae,Enterococcaceae and other 15 major families.At the genus level,the ge nera involved are richer,including Allobaculum,Unidentified,Bacteroides,Lactobacil lus,Rumbutzia,Eubacterium?coprostanoligenes?group,Akkermansia,Faecalibacterium,Roseburia,Blautia and more than 30 species.Through screening,the distribution re sults of the flora were further screened out from five levels of phyla to genus.Gro up C contains Clostridia,Clostridiales,Ruminococcaceae,The relative abundance of 25 microbial information such as Reptostreptococcaceae,Romboutia,Ruminococcacea e?UCG-005,and Turicibacter is higher than that of other groups;group M includes Lachnospiraceae,cloth Blautia,Gammaproteobacteria,Enterobacteriaceae,Enterobacter iales,Eschscherichia?coli,Eschscherichia?shigella,Proteobacteria,Eubacterium?nodatiu m?group and other 34 microbial information relative abundance is higher than other groups;QHY group includes Erysipelotrichaceae,Erysipelotrichales.The relative abu ndance of 18 microbial information,such as Erysipelotrichia,Allobaculum,and Lact obacillus?acidophilus is higher than that of other groups;group V includes Lactobac illales,Bacilli,Lactobacillaceae,Lactobacillus,Lachnospiraceae?NK4A136?group,Fir micutes,Eubacterium?coprostanoligenes?group,Ruminococcaceae?Veillonae?Veillon.T he relative abundance of 29 microbial information including Quinella,Selenomonadal es,Negativicutes,Ruminococcaceae?UCG-014 is higher than that of other groups.In this study,Lactobacillus was strongly positively correlated with Ruminococcaceae?U CG?005 and Christensenellaceae?R7 abundance,and negatively correlated with Subd oligranulumum,Klebsiella and Lachnoclostridium.Bacteroides is positively correlated with Klebsiella,Akkermansia and subdoligranulum,and negatively correlated with E ubacterium?coprostanoligenes?group and Laospirillaceae NK4A136 group.Gallophilia and Subdoligranulum,Akkermansia and Shigella show a strong negative correlation.In addition,Broutella is positively correlated with Ruminococcaceae?UCG?014,Rumi nococcaceae NK4A214 group,Laospirillaceae NK4A136 group and Eubacterium?copr ostanoligenes?group,and negatively correlated-with Klebsiella,Akkermansia and Sub doligranulum.Akkermansia is positively correlated with Bileophilus,Subdoligranulum,and Morganella,and negatively correlated with multiple genera of Rumenococcus a nd Laurespirillum.The metabolites of intestinal contents in group C,group M,group QHY and gr oup V were detected.The metabolic pathway enrichment map shows that CAG splee n-stomach damp-heat syndrome is mainly involved in phenylalanine,tyrosine and try ptophan biosynthesis,linoleic acid metabolism,ketone body synthesis and degradatio n,arginine biosynthesis,D-glutamine and D-glutamate metabolism,phenylalanine met abolism,arginine and proline metabolism,pyrimidine metabolism,taurine and taurine metabolism,aminoacyl-tRNA biosynthesis,butyrate metabolism.Histidine metabolism and other pathways affect amino acid metabolism,fatty acid metabolism,pyrimidin e metabolism and sugar metabolism in rats.A total of 34 kinds of specific differenti al metabolites were screened from rat fecal samples.The contents of L-tryptophan(P 1),L-phenylalanine,L-valine,L-leucine and L-serine in fecal metabolites of rats wit h CAG spleen-stomach damp-heat syndrome were significantly increased.N-acetylputr escine.?-alanine,L-glutamine 5-phosphate,uracil,L-alanine,L-glutamine,N(?)-hy droxyarginine,L-citrulline,phenylacetyl glycine,N-acetyl-L-glutamic acid,L-methioni ne,L-proline,L-glutamic acid,L-tyrosine,N-methyl-L-histidine,2-hydroxyphenylaceti c acid,4-guanidinic acid,The contents of 4-acetylaminobutyric acid,hippuric acid,ta urine,taurine cholate,cytidine diphosphate,deoxycytidine,thymine,cytidine,linoleat e,imidazole-4-acetate,acetoacetate and N-(L-arginine)succinate decreased significantl y.It is confirmed that there are abnormal metabolism of amino acids,nucleic acids and sugars in rats with CAG spleen-stomach damp-heat syndrome,which involves t he changes of various physiological functions.Conclusion1.One of the vital biological mechanisms of CAG spleen-stomach damp-heat syndrome:?-catenin can mediate gastric pyroptosis,promote cell proliferation and trigger CAG inflammation.2.The occurrence of CAG spleen-stomach damp-heat syndrome is closely related to the intestinal flora,involving changes in the composition and the abundance of related intestinal flora.3.The occurrence of CAG spleen-stomach damp-heat syndrome is related to the differential metabolites of intestinal contents,involving 12 related metabolic pathways and 34 metabolites.