| Objective:In this study,mice given a high-fat diet were utilized to create a severe acute pancreatitis(SAP)model by injecting 20% L-arginine intraperitoneally.Technical approaches such as 16 s r DNA high-throughput sequencing and GC-MS metabolomics were utilized to explore the relationship between a high-fat diet,gut microbiota,metabolites,and SAP.On this foundation,the mechanism of Dahuang Fuzi decoction in the treatment of hyperlipidaemic severe acute pancreatitis(HLSAP)from the standpoint of the intestinal microbes and metabolites was investigated.Material and method:1.1 Animal groupingNinety-six C57BL/6 mice were randomly divided into two groups,high-fat diet group(n=48)and normal diet group(n=48).The high-fat diet group were divided into control group(HFD),Dahuang Fuzi Decoction control group(HFD-D),model group(HFD-M)and treatment group(HFD-M-D).The normal diet group were also divided into control group(ND),Dahuang Fuzi Decoction control group(ND-D),model group(ND-M)and treatment group(ND-M-D).The mice were fed with high-fat diet or normal diet for 8 weeks,and the diet,water intake,activity,hair,stool and urine were all monitored.1.2 Animal-model preparation and sample collectionThe SAP model was established by administering intraperitoneal injections of 20% Larginine to both the model and treatment groups.After successful SAP preparation,the Dahuang Fuzi Decoction control and treatment groups were received Dahuang Fuzi Decoction by gavage every 12 hours.ELISA was used at the end of the experiment to quantify serum amylase,endotoxin,blood lipids(TG,TC,LDL,HDL),and inflammatory factors(TNF-,IL-6,IL-1β).Routine pathological sections were obtained from the pancreas,lung,ileum,and colon.The contents of the ileum and colon were deposited in frozen storage tubes for GC-MS metabolomics investigation.The intestinal contents were rinsed away with normal saline,andthe surface layer of the intestinal mucosa was gently scraped with a cotton swab and deposited in cryopreserved tubes for microbiota investigation.l6 s r DNA sequencing was utilized to detect changes in intestinal microbiota in mice’s ileum and colon mucosa.Results:Thesis I The establishment of SAP model in C57BL/6 mice and the intervention effect of Dahuang Fuzi DecoctionThe experimental period was 8 weeks.The body weight of the HFD group was significantly higher than that of the normal diet fed ND group at 4,6,and 8 weeks(P < 0.001).The serum levels of total cholesterol(TC),triglyceride(TG)and low-density lipoprotein(LDL)in the HFD group were significantly higher than those in the ND group(P < 0.01).The highdensity lipoprotein(HDL)was lower than that of the normal diet group(P < 0.05).Serum AMS,pathological scores of the pancreas,lung,ileum and colon,LPS,TNF-α,IL-1β and IL-6 in NDM group were significantly higher than those in ND and ND-D group(P < 0.01).The above indexes in group of HFD-M were also higher than those in group of HFD and HFD-D(P < 0.01).After Dahuang Fuzi Decoction treatment,all parameters in the ND-M-D and HFD-M-D groups were significantly decreased(P < 0.001).In addition,the serum levels of LPS,TNF-α,IL-1β and IL-6 in HFD-M group were significantly higher than those in ND-M group(P < 0.05).Thesis II The changes mucosa-associated microbiota in ileum and colon of HLSAP mice and the intervention effect of Dahuang Fuzi DecoctionAfter 16 s high-throughput sequencing data optimization,a total of 5717346 optimized effective sequences were obtained from 96 samples.After OTU clustering and smoothing,22 bacterial phyla,36 classes,76 orders,145 families,355 genera,and 594 species were obtained.The dilution curve showed that the sequencing depth was sufficient.First,the high-fat diet caused significant changes in the microbiota.At the phylum level,compared with the ND group,the HFD group had a decrease in Bacteroidetes,an increase in Firmicutes,and a significant increase in the Firmicutes/Bacteroidetes(F/B)ratio in both ileum and colon segments.At the genus level,Faecalibaculum was significantly increased in ileum segment,and norank_f_Bacteroidales_S24-7_group was significantly decreased in colonsegment.Significant dysbiosis of microbiota was observed in ileum and colon of HLSAP mice.In the ileum,there were a significant decrease in Bacteroidetes,an increase in F/B ratio and an increase in Escherichia-Shigella were observed.The colonic segment was mainly characterized by a significant increase in Proteobacteria.After treatment with Dahuang Fuzi Decoction,Escherichia-Shigella were significantly decreased,while unclassified_f_Lachnospiraceae and Akkermansia were significantly increased.The colonic segment showed a significant decrease in Proteobacteria,accompanied by an increase in Blautia abundance.The changes of microbiota at phylum and genus levels in SAP were basically consistent with those in HLSAP under normal diet intervention.Compared with SAP,in both ileum and colon segments,Proteobacteria and Escherichia Shigella were significantly higher in HLSAP mice.And the abundance of Faecalibacterium(ileum)and Desulfurvibrio(colon)was higher in HLSAP.Spearman correlation analysis showed that Bacteroidetes were negatively correlated with LPS and Proteobacteria were positively correlated with LPS at the phylum level.At the genus level,norank_f_Bacteroidales_S24-7_group,unclassified_f_Lachnospiraceae were negatively correlated with LPS,Escherichia Shigella,Enterococcus,Faecalibaculum was positively correlated with LPS.Thesis III The changes of metabolites in ileum and colon of HLSAP mice and the intervention effect of Dahuang Fuzi DecoctionA total of 224 metabolites were obtained by GC-MS metabolomics,which were annotated to 91 compounds by KEGG compound classification and involved in 39 metabolic pathways.After sample comparison analysis,KEGG compound and functional pathway analysis,differential metabolites screening,and KEGG enrichment pathway analysis,three differential metabolites were involved in the ileal segment between the IHFD-M group and IHFD group.Stearic acid and linoleic acid were up-regulated and glycerol was down-regulated in the model group.Compared with IHFD-M group,myristic acid was the differential metabolite involved and its expression was down-regulated in IHFD-M-D group.Compared with CHFD group,atotal of 6 differential metabolites were involved in CHFD-M group,among which uracil,cholesterol,6-phosphogluconic acid were up-regulated,stearic acid,maltose,linoleic acid were down-regulated.Fumaric acid and 6-phosphogluconic acid were involved in the enriched pathway between CHFD-M-D group and CHFD-M group.Compared with IND group,eight differential metabolites were identified in IND-M group,among which glycerol,galactol and fructose were up-regulated,xanthine,Myo-inositol,LMalic acid,L-glutamic acid and arachidonic acid were down-regulated.Eight differential metabolites were identified between IND-M-D group and IND-M group,including palmitic acid,uracil,stearic acid,linoleic acid,L-glutamic acid,glycolamine,creatine and arachidonic acid,among which palmitic acid was down-regulated.In colon segment,CND-M group was compared with CND group,and 13 differential metabolites were matched.Among them,sucrose,succinic acid,Myo-inositol,L-Malic acid,and gallol were up-regulated,while stearic acid,squalene,palmitic acid,myristic acid,linoleic acid,hydrocinnamic acid,glycerol,and ethanolamine were down-regulated.When CND-M-D group compared with CND-M group,five differential metabolites were matched,among which palmitoleic acid,myristic acid,ethanolamine,3-hydroxybutyric acid were up-regulated and sucrose was down-regulated.Comparison of the two diets in the ileum segment,high-fat diet up-regulated the expression of myristic acid,tetacanoic acid,glycerol,pectate acid,arachidic acid,and downregulated stearic acid,L-Malic acid,linoleic acid.In the colonic segment,high-fat diet upregulated palmienoic acid,myristic acid,Myo-inositol,tetranecanoic acid,pectate acid and arachidic acid.Taurine,succinic acid,oxalic acid,α-tocopherol were down-regulated.In the ileum,four differential metabolites were finally matched between the IHFD-M group and the IND-M group,among which palmitoleic acid,palmitic acid,myristic acid were up-regulated,and gallol was down-regulated.Compared with CND-M group,CHFD-M group matched 13 differential metabolites,of which 6 were up-regulated,including squalene,palmitoleic acid,myristic acid,tetacanoic acid,pectic acid,araconic acid,and 7 were downregulated,including sucrose,succinic acid,stearic acid,L-Malic acid,linoleic acid,galactomol,24,24,24.25-dihydrolanosterol.Conclusions:1 Dahuang Fuzi Decoction can significantly reduce the serum AMS,improve the pathological scores of pancreas,lung,ileum and colon,and reduce the levels of LPS and inflammatory markers in HLSAP mice.2 The high-fat diet affects the formation of gut microbiota and intestinal metabolites before SAP,and the changes in specific microbiota and metabolites have a common trend with the changes in HLSAP.Proteobacteria and Escherichia Shigella in ileum and colon segments of HLSAP were significantly higher than those of SAP.Meanwhile,Faecalibacterium(ileum)and Desulfurvibrio(colon)were more abundant in HLSAP.In terms of metabolites,both had a significant increase in myristic acid.This may be a potential marker for the severity of HLSAP compared with other types of SAP.3 The therapeutic effect of Dahuang Fuzi Decoction on HLSAP is mostly evident in changing the structure of bacterial community,decreasing the abundance of Proteobacteria,Escherichia Shigella and Desulfovibrio,and increasing the abundance of probiotics,such as norank_f_Bacteroidales_S24-7_group,unclassified_f_Lachnospiraceae,Akkermansia(ileum),and Blautia(colon).4 In terms of metabolites,Dahuang Fuzi Decoction may play a therapeutic role by regulating endocannabinoid signaling and regulating the production of myristic acid and fumaric acid. |
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