| Purpose:Based on the theoretical basis of Atherosclerosis(AS)caused by "phlegm,blood stasis and poison",establishing the rabbits model of AS.Observing the changes of the general condition,blood lipid level and blood viscosity,common carotid artery tissue structure,intestinal flora structure characteristics,host plasma differential metabolites,and the correlation between bacterial flora and host metabolites of rabbits.Study the influence of electroacupuncture intervention on AS-related host metabolites and metabolic pathways,and explore possible ways of electroacupuncture prevention and intervention against AS.Material and method:1.Modeling and intervention:according to the completely random number table,24 New Zealand rabbits were fed for one week adaptively and,then,randomly divided into three groups,named the blank control group,the modeling group,and the electroacupuncture preventive with cure group.There were 6 rabbits in the blank control group,12 in the model group and 6 in the electroacupuncture preventive with cure group.High fat diet and common carotid artery balloon injury were used to establish AS rabbit model(The evaluation of modeling was completed in the stage of preparatory experiment).After successfully modeling,the models were randomly divided into two groups:the model group and the treatment group,with 6 rabbits in each group.Blank group(A)was fed with standard rabbit feed during the whole experiment,without intervention factors,but tied and restricted exercise;Model control group(B)was feed with high-fat diet after modeling,without any intervention,but tied and restricted exercise;Treatment group(C)was given electroacupuncture intervention at Neiguan,Guanyuan and Zusanli Points,once a day,20 times a time,6 days as a course of treatment,with a day off between courses,which last for 4 courses.Before modeling,the electroacupuncture preventive and cure group(D)was intervened Neiguan,Guanyuan and Zusanli points by electroacupuncture for 3 courses;After modeling,group D was intervened in the same points by electroacupuncture for 4 courses,again.During the courses,C and D were restricted exercise.2.Index detection:The general health status of rabbits was evaluated according to the existing scales before modeling.After modeling and intervention,the general health status was evaluated again.The contents of serum TC,TG,LDL-C and HDL-C were detected by Elisa kits.The whole blood viscosity(high shear,middle shear,low shear)and capillary plasma viscosity were detected by automatic blood rheometer.The lipid plaque deposition on the inner wall of blood vessels was observed with oil red O staining method.The blood lipid plaque deposition was observed by HE staining method under microscope.The changes of intestinal flora abundance and diversity were observed by 16S rRNA high-throughput gene sequencing technology,and the metabolites in plasma were detected by ultra-performance liquid chromatography tandem mass spectrometry.3.Conjoint analysis:Integrate the data of microbiomics and non-target metabonomics for correlation analysis,further study the the electroacupuncture intervention regulating intestinal flora related host metabolites to achieve anti-atheroscerosis effect,and explore the possible ways.Results:Paper 1:The results of general condition comparison showed that there were no significant difference between groups before modeling and intervention(P>0.05).After modeling and intervention,the score of group B was the highest,but that of group C and D were closer to group A.The results of serum lipid tests showed that compared with group A,the contents of serum TG,TC and LDL-C in group B were significantly increased(P<0.01),and the content of HDL-C was significantly decreased(P<0.01).Compared with group B,the contents of serum TG,TC,LDL-C in group C and D were significantly decreased(P<0.01),and the content of HDL-C in C and D were significantly increased(P<0.01);But there were differences between group C and group D:TG(P<0.01),TC(P<0.05)and LDL-C(P<0.05)in group D were lower than those in group C.The results of hemorheology showed that compared with group A,the whole blood viscosity(low shear,middle shear,high shear)and plasma viscosity in group B were significantly increased(P<0.01).Compared with group B,the whole blood viscosity(low shear,medium shear,high shear)and plasma viscosity in group C and D were significantly decreased(P<0.01).There was no significant difference among groups A,C and D(P>0.05).Paper 2:After oil red O staining,the carotid artery endothelial tissue in group A was normal,in which the inner surface of the common carotid artery was smooth,and there was no lipid deposition on the endothelium;After AS modeling,the endothelium of the common carotid artery in group B was rough and uneven,with a large amount of orange red lipid attached on the surface of the endothelium;After the intervention of electroacupuncture,the endothelium of the common carotid artery in group C was still rough and uneven,and there was orange red lipid adhesion on the surface of the endothelium.Under the intervention of electroacupuncture,the endothelium of common carotid artery in group D was smoother than that in group B.Although there was a small amount of orange red lipid adhesion in the local area,it was significantly less than that in group B and better than that in group C.The results of HE staining section of common carotid artery under microscope showed that the vascular wall structure of common carotid artery in group A was clear,and the intima was intact,and the vascular smooth muscle cells were arranged in order.The carotid artery tissue of group B was in the lipid phase of early AS,and its intima was damaged.A large number of foam cells accumulated beneath the intima,and a small lipid pool has been formed in the smooth muscle layer,which destroyed the intimal layer and vascular wall structure.In group C,after electroacupuncture,there was still a small amount of foam cells in the common carotid artery.Compared with group B,there was a significant decreasing in foam cells.In group D,under the intervention of electroacupuncture,only a small amount of foam cells were seen under the common carotid artery,and the improvement was better than that in group C.Thesis 3:16S rRNA high-throughput gene sequencing results showed that compared with group A.After AS modeling,the Chaol index and Shannon index of group B were significantly decreased(P<0.01).But compared with group B,the Chaol index of group C and D was significantly increased(P<0.05),and the Shannon index was significantly increased(P<0.01),too.In terms of family level,the relative abundance of microbial community was different among groups.Further NMDS analysis at genus level showed that the spatial relationship of each sample in group C and D were closer to group A than that in group B.At the genus level,there were 6 species in the top 20 genus with relative abundance difference showing significant.Compared with group A,the levels of Coprococcus,Turricibacter,Dorea,Roseburia and Bifidobacterium in group B were decreased(P<0.05),while Phascolarctobacterium was increased(P<0.05).And compared with group B,Coprococcus,Turricibacter,Roseburia,Bifidobacterium in group C were increased(P<0.05),while Phascolarctobacterium was decreased(P<0.05).Coprococcus,Turricibacter,Dorea,Roseburia,Bifidobacterium in group D were increased(P<0.05),however,Phascolarctobacterium was decreased(P<0.05).Due to the influence of as modeling and interventions,the intestinal flora may involve 12 secondary metabolic functions,including Xenobiotic Biodegradation and Metabolism,Nucleotide Metabolism,Metabolism of Terpenoids and Plyketides,Metabolism of other Amino acid,Metabolism of Cofctors and Vitamin,Lipid Metabolism,Glycan Biosynthesis and Metabolism,Enzyme Families,Enzyme Metabolism,Carbohyrate Metabolism,and Amino acid Metabolism.Paper 4:The results of three-dimensional PCA analysis showed that there were differences in plasma metabolites between groups.The degree of separation between group A and B were the most obvious,while the three-dimensional space distance between group C,D and A was closer.The metabolites were identified by Agilent Mass Profiler Professional 12.6 software,and 221 kinds of plasma metabolites showed difference with statistical significance.Compared with group A,153 kinds of plasma metabolites were up-regulated and 69 kinds were down-regulated in group B.Compared with group B,86 kinds of plasma metabolites were up-regulated and 135 kinds were down-regulated in group C,while 112 kinds of plasma metabolites were up-regulated and 109 kinds were down-regulated in Group D.Compared with group B,there were 164 plasma differential metabolites in group C or D,however,as the same change trend as group A,among which there were 129 in group C and 107 in Group D.A preliminary comparison of NCBI,KEGG,HMBD and METLIN databases showed that at least 89 of the 164 metabolites mentioned above might be related to the occurrence and development of AS.At the same time,Toluene degradation,Cholesterol metabolism,2-Oxocarboxylic acid metabolism,Aminobenzoate degradation,Arachidonic acid metabolism,Tryptophan metabolism,Bile secretion,Primary bile acid biosynthesis,Secondary bile acid biosynthesis,Metabolic pathways,Microbial metabolism in diverse environments,Biosynthesis of secondary metabolites,Inflammatory mediator regulation of TRP channels,Pentose and glucuronate interconversions were also relating with AS.Paper 5:The omics association analysis showed that the changes of intestinal flora were related to the changes of metabolites in host.Coprococcus was negatively correlated with 5(6)-EpETrE-EA,Luteolin 7,3'-dimethyl ether 5-glucoside,Gabacaline,2-glyceryl-PGF2?,phorbol 12-tiglate 13-decanoate,lys lys Trp(P<0.05).And positively correlated with LysoPE(0:0/16:0),Xeniasterol-b,antiogenin,Arg Ile Pro,4-Methyl-3-oxoadipate-enol-lactone and capillarin(P<0.05).Turicibacter and PG(O-16:0/20:2(11Z,14Z)),Orthothymotinic Acid,Diisobutyl phthalate,13(S)-HODE,Ifosfamide mustard,Ornaline,5-Chloro-1-piperidin-4-y1-1,3-dihydrobenzimidazol-2-one;R29676,L-Djenkolic acid,2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene(Compound A)were negatively correlated(P<0.05),while m-Trifluoromethylhippuric acid,6-Hydroxykynurenic acid,Chenodeoxycholic acid glycine conjugate,(-)-Salvisyriacolide,PS(O-18:0/22:4(7Z,10Z,13Z,16Z)),k-Strophanthin-beta,PS(O-18:0/20:3(8Z,11Z,14Z)),Rifacycin W,26:5(11Z,14Z,17Z,20Z,23Z),2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene(Compound A)was positively correlated with Turicibacter(P<0.05).Roseburia was negatively related with 4-Sulfobenzyl alcohol,11(R)-HETE,Indoxyl sulfuric acid,9,10-Epoxy-18-hydroxy stearate,N-Hydroxy-L-phenylalanine,12-hydroxy-5,8,10-heptadecatrienoic acid Related(P<0.05),while Kentsin,Risperidone,His Asn Cys,3a,7b,12a-Trihydroxyoxocholanyl-Glycine,3-oxoglycyrrhetinic acid,Stigmaellin A,p-Hydrotiaprofenic acid,Gln Thr lle,(4S,5S)-4,5-Dihydroxy-2,6-dioxohexanoate,Patulin,Pantothenic Acid,2,4-Dinitrophenol,PI(19:1(9Z)/18:2(9Z,12Z)),Ursodeoxycholic acid 3-sulfate,26-hydroxycholesterol 3-sulfate,18-acetoxy-25-hydroxyvitamin D3,Norselic acid E,Hydrocortisone cypionate were positively correlated with Roseburia(P<0.05).Bifidobacterium and p-Cresol glucuronide,Tolrestat,PE(20:2(11Z,14Z)/0:0),Miraxanthin-II,Proscillaridin A,N-[(3a,5b,7b)-7-hydroxy-24-oxo-3-(sulfoxy)cholan-24-y]-Glycine and N-depyridomethyl-lndinavir were negatively correlated(P<0.05).But positively correlated with PS(20:2(11Z,14Z)/0:0)(P<0.05).Dorea and OA-6129E,N-[(3a,5b,7b)-7-hydroxy-24-oxo-3-(sulfoxy)cholan-24-y]-Glycine,3-(3-Indolyl)-2-oxopropanoic acid were negatively correlated(P<0.05).But positively correlated with Enalkiren,MEDIC A 16,Formoterol,5,4'-Dihydroxy-7'-methoxy-8-methylflavan,Trinexapac-ethyl,3,5-Dinitroguaiacol(P<0.05)).There were similarities and differences in the effects of electroacupuncture treatment intervention and electroacupuncture preventive with treatment intervention on intestinal flora and host metabolism.The similatrities of these two interventions was reflect at raising the relative abundance of intestinal flora with protective effect on the occurrence and development of AS lesions,such as Coprococcus,Turicibacer,Roseburia,Bifidobacterium.And down regulated the relative abundance of intestinal flora that could promoting AS,such as Phascolarctobacterum.Accordingly,these two interventions could also regulate the metabolites in host through the intestinal flora,such as regulating Turicibacer to raise PG(O-16:0/20:2(11Z,14Z)),Chenodeoxycholic acid glycine conjugate,PS(O-18:0/22:4(7Z,10Z,13Z,16Z))?PS(O-18:0/20:3(8Z,11Z,14Z)),and to down Ifosfamide mustard,Ornaline,L-Djenkolic acid.The other example was that regulating Bifidobacterium to lower p-Cresol glucuronide,Tolrestat,Proscillaridin A and Miraxanthin-?,while regulating Phascolarctobacterium to lower PA(P-16:0/15:1(9Z))and 3S-hydroxydodecanoic acid.In the other hand,the differences between two interventions were reflected with the ability to regulate bacterial flora and metabolite.Electroacupuncture treatment may lower the metabolites,which could promote AS,through different intestinal flora.For examples,electroacupuncture treatmen could regulate Coprococcus to lower 5(6)-EpETrE-EA,Gabacaline,2-glyceryl-PGF2 ?,phorbol 12-tiglate 13-decanoate,and could regulate Roseburia to lower 11(R)-HETE,Indoxyl sulfuric acid,N-Hydroxy-L-phenylalanine,12-hydroxy-5,8,10-heptadecatrienoic acid,2,4-Dinitrophenol,while regulate Roseburia to raise LysoPE(0:0/16:0),Xeniasterol-b,antiogenin and 4-Methyl-3-oxoadipate-enol-lactone.Another part,electroacupuncture preventic and cure intervine could not only lower the metabolites which could promot AS,but also raise the metabolites that could prevent AS through intestinal flora.This intervention could ragulate Coprococcusto lower phorbol 12-tiglate 13-decanoate,regulate Rosebuira to lower 4-Sulfobenzyl alcohol,11(R)-HETE,Indoxylsulfuric acid,9,10-Epoxy-18-hydroxystearate,N-Hydroxy-L-phenylalanine,12-hydroxy-5,8,10-heptadecatrienoic acid,and reculate Coprococcu to raise Luteolin 7,3'-dimethyl ether 5-glucoside,lys lys Trp,LysoPE(0:0/16:0),Xeniasterol-b,antiogenin and 4-Methyl-3-oxoadipate-enol-lactone,and regulate Roseburia to raise Pantothenic Acid,PI(19:1(9Z)/18:2(9Z,12Z)),Ursodeoxycholic acid 3-sulfate,26-hydroxycholesterol 3-sulfate,18-acetoxy-25-hydroxyvitamin D3,Norselic acid E,Hydrocortisone cypionate,and could regulate Dorea to raise Enalkiren,MEDICA 16,Formoterol.Conclusion:1.Electroacupuncture combined with intervention can effectively improve the blood lipid and hemodynamic abnormalities of AS animals.2.Electroacupuncture combined with intervention can effectively inhibit intimal injury,foam cells and atheromatous plaque formation.3.Electroacupuncture combined with intervention can change the structure of intestinal flora(diversity and abundance of specific bacteria)in AS animals.4.Electroacupuncture combined with intervention can affect the host metabolites of atherosclerotic animals,and 89 of which are closely related to AS.5.The combination effect of electroacupuncture may be achieved by regulating the relative abundances of Coprococcus,Turicibacer,Roseburia,Bifidobacterium,Phascolarctobacterium and Dorea,and up regulating the host metabolites with therapeutic and preventive effects,down regulating the host metabolites with promoting effects,and improving the host lipid metabolism and amino acid metabolism. |
PDF Full Text Request