| Objective:To study the characteristics and changes of oral flora abundance and composition in atherosclerotic population and their correlation with carotid ultrasound results and blood biochemical indexes,to explore the feasibility of early diagnosis of atherosclerosis and adjustment of oral flora disorder in the treatment of atherosclerosis through oral flora;to collect TCM Syndromes of atherosclerotic population,summarize their syndrome differentiation and typing,for a better system Make clinical diagnosis and treatment plan to provide reference.In the part of experimental study,we observed the effect of Eucommia ulmoides Oliv on intestinal flora and its metabolites in atherosclerotic mice,evaluated the correlation between the change of intestinal flora level and atherosclerosis metabolites and inflammatory cytokines after Eucommia ulmoides Oliv intervention,and determined the target of Eucommia ulmoides Oliv in improving atherosclerosis.Through the fecal bacteria transplantation experiment to further clarify and verify the role of Eucommia ulmoides Oliv in the pathological process of atherosclerosis by adjusting the intestinal flora,so as to provide experimental support for Eucommia ulmoides Oliv to prevent and treat atherosclerosis from the perspective of flora.Methods:1.Comparative analysis of oral flora abundance and composition of atherosclerotic and non atherosclerotic population:the patients who were hospitalized in the cardiology department of our hospital from August 2018 to August 2019 were selected in order,as well as the healthy volunteers who came to the physical examination center of our hospital for physical examination.Finally,30 patients with atherosclerosis were selected as the as group.30 non atherosclerotic patients as non as group.The general data of the two groups were collected,including the results of carotid ultrasound,blood biochemical test results of blood lipids,glycosylated hemoglobin,homocysteine and various syndromes.Swabs in the culture tube were used to wipe the maxillary,lingual,pharyngeal and buccal parts to obtain oral flora.The oral swabs were sequenced with 16S rRNA amplification.The abundance and structural changes of oral flora in the experimental group and the control group were compared and analyzed.2.The effect of Eucommia ulmoides Oliv on the intestinal flora and metabolites of ApoE-/-mice:30 SPF grade ApoE-/-six week male mice were randomly divided into three groups(n=10):experimental group,western medicine group,model group;10 SPF grade C57BL/6J mice of the same week age were used as the normal control group.After one week of adaptive feeding,the atherosclerotic model was induced by high-fat diet for12 weeks.From the 13th week,the corresponding drugs were gavaged into the stomach,among which the experimental group was gavaged with eucommia Decoction free granule water solution,the gavage dose was 0.046g/kg/d;the western group was gavaged with rosuvastatin calcium tablet water solution,the gavage dose was 0.45mg/kg/d,the model group and the control group were gavaged with the same amount of normal saline for 8weeks.At the end of gavage,blood was collected from inferior vena cava,serum was separated,and serum TMAO and related metabolites were detected by LC-MS;aorta and ileum were separated from ice,and tissue morphology was observed by HE staining,and active vein inflammatory cytokine vascular cell adhesion molecule-1(VCAM-1)and monocyte chemoattractant protein-1(MCP)were observed by immunohistochemistry staining-1)The expression of cd-68 antigen,IL-6,ifn-y and TGF-?1 in ileum were detected and analyzed.3.Fecal bacteria transplantation experiment:30 SPF ApoE-/-six week male mice were selected and randomly divided into three groups(n=10):apo E(Eucommia ulmoides)group,ApoE(C57BL/6J)group and ApoE(normal saline)group.After one week of general feed adaptive feeding,high fat feed was given until the end of the experiment.The faeces of ApoE-/-mice and C57BL/6J mice were collected from Eucommia ulmoides Oliv.The faeces of ApoE-/-mice fed with eucommia ulmoides Oliv were transplanted to apo E(Eucommia ulmoides Oliv.)group and C57BL/6J mice fed with normal saline to apo E(C57BL/6J)group.At the same time,the faeces of ApoE-/-mice fed with normal saline to apo E(normal saline)group were respectively transplanted to ApoE-/-mice in each group via the stomach for 8 weeks.At the end of gavage,the blood of inferior vena cava was extracted,the serum was separated,the level of serum TMAO and related metabolites were detected by LC-MS target metabolome,and the feces were collected for16S rRNA amplification.Results:1.Comparative analysis of oral flora abundance and composition between atherosclerotic group and non atherosclerotic group:there was no significant difference in age and gender between the two groups(P>0.05),and there was comparability between the two groups;color Doppler ultrasound of carotid artery in as group:IMT average 1.31±0.17mm,including minimum 0.9mm,maximum 1.6mm,maximum plaque area average 32.65±20.99mm2,of which the minimum value is 9.69mm2 and the maximum value is 76.14mm2;the levels of triglyceride,low-density lipoprotein and homocysteine in as group are significantly higher than those in non as group(P There were 5 cases of syndrome,3 cases of deficiency of kidney qi+phlegm obstruction,3 cases of excess of phlegm heat,2 cases of stagnation of Qi and blood stasis,1 case of deficiency of liver and kidney Yin+phlegm heat,1 case of deficiency of kidney yin and Yang,1 case of deficiency of kidney yin and hyperactivity of fire.The total proportion of cases with deficiency of kidney was 60%.Compared with non as group,the diversity of oral flora in as group was significantly reduced(Shannon index p=0.038,Simpson index p=0.014),and the richness was not significantly different(AC index p=0.038,Simpson index p=0.014)E index p=0.685,Chao1 index p=0.572);the sequence levels of two groups of oral microflora were mainly divided into the following five phylum:sclerectophy,Bacteroides,proteus phylum,actinomycete,fusobacteria;compared with non as group,the abundance of Bacteroides in as group was significantly reduced(P<0.05),while that of sclerectophy was significantly increased(P<0.05),proteus phylum,Fusobacterium,actinomycete There was no significant difference in the abundance of the three phylum(P>0.05);the sequence level of oral flora in the two groups was mainly divided into the following nine genera:Streptococcus,Neisseria,veronococcus,actinomycetes,fusobacteria,carbon dioxide phagocyte,granulosa Streptococcus,macroglobulus and twinning cocci;compared with non as group,Streptococcus and actinomycetes in as group There was a significant positive correlation between the abundance of Streptococcus and the maximum plaque area,total cholesterol level(P<0.05),There was a significant negative correlation between HDL and HDL(P<0.05).2.Effects of Eucommia ulmoides Oliv on intestinal flora and its metabolites in ApoE-/-mice:compared with the control group,VCAM-1,cd-68,ifn-y and IL-6 in the model group were significantly increased(P<0.05),TGF-?1 in the ileum was significantly decreased(P<0.05),and VCAM-1 and IL-6 in the experimental group were significantly decreased compared with the model group Compared with the model group,the expression of VCAM-1 and IL-6 in the aorta and ileum of the western medicine group treated with rosuvastatin calcium tablet significantly decreased(P<0.05);the TMAO level of the model group was significantly higher than that of the control group(P<0.05),and the TMAO level of the experimental group and the western medicine group was significantly lower than that of the model group(P<0.05)Compared with the control group,the model group had no significant difference in intestinal flora richness(ACE index p=0.173,Chao1 index p=0.176),and the diversity was significantly reduced(Shannon index p=0.006,Simpson index p=0.009);compared with the model group,the experimental group had significantly higher intestinal flora richness and diversity(ACE index p=0.000,Chao1 index p=0.000,Shannon index p=0.005,Simpson index p=0.005)009),compared with the model group,there was no significant difference in the richness and diversity of intestinal flora in the western medicine group(ACE index p=0.090,pao1index p=0.153,Shannon index p=0.306,Simpson index p=0.313);the sequence level of fecal flora in the four groups of mice mainly belonged to the following four phylum:phylum,Bacteroides,Proteus and actinomycetes;the abundance of phylum in the model group The abundance of Bacteroides in the experimental group and Western medicine group was significantly higher than that in the model group(P There was no significant difference in abundance(P>0.05);the four groups of mice fecal flora were mainly divided into the following 10 genera:Dunaliella,fecal bacteria,Prevotella,Bacteroides,Lactobacillus,Staphylococcus,Helicobacter,odoribacter Compared with the control group,the abundance of Spirillum,Staphylococcus and Ramulus in the model group increased significantly(P<0.05),while that of Lactobacillus,faecalis,Bacteroides,odoribacter and Acinetobacter decreased significantly(P<0.05).Compared with the model group,the abundance of Helicobacter and Staphylococcus in the experimental group decreased significantly(P<0.05),and that of Lactobacillus,faecalis,Bacteroides and odoribacter in the experimental group increased significantly(P<0.05);compared with the model group,the abundance of Staphylococcus and Helicobacter in the western medicine group decreased significantly(P<0.05),and that of faecalis,Lactobacillus,odoribacter and Acinetobacter in the western medicine group decreased significantly(P<0.05)The abundance of Staphylococcus was positively correlated with the level of IL-6 and TMAO(P<0.05).The abundance of Bacteroides was negatively correlated with the level of TMAO and VCAM-1(P<0.05).The abundance of faecalis was negatively correlated with the level of IL-6(P<0.05).3.Fecal bacteria transplantation experiment:the TMAO level of apo E(normal saline)group was significantly higher than that of apo E(C57BL/6J)group and apo E(Eucommia)group(P<0.05),the TMAO level of apo E(C57BL/6J)group and apo E(Eucommia)group had no significant difference(P>0.05);the intestinal flora richness of apo E(normal saline)group was significantly lower than that of apo E(C57BL/6J)group(ACE index p=0.010,Chao1 refers to The number P=0.008),compared with APOE(normal saline)group,the intestinal flora richness of mice in ApoE(Eucommia ulmoides Oliv)group increased significantly(ACE index p=0.023,Chao1 index p=0.028);the intestinal flora diversity of mice in ApoE(normal saline)group was significantly lower than that in ApoE(C57BL/6J)group(Shannon index p=0.025,Simpson index p=0.000),compared with APOE(normal saline)group,the intestinal flora diversity of mice in ApoE(normal saline)group was significantly lower than that in ApoE(C57BL/6J)group(Shannon index p=0.025,Simpson index p=0.000)The diversity of intestinal microflora in three groups of mice was significantly increased(Shannon index p=0.031,Simpson index p=0.015);the sequence levels of fecal microflora in three groups of mice were mainly divided into the following four phylum:Bacteroides,phylum sclerecta,verrucosa microflora,proteus phylum;the abundance of Bacteroides in ApoE(Eucommia)group and apo E(C57BL/6J)group was significantly higher than that in ApoE group(P<0.05),and phylum sclerecta was abundant There was no significant difference(P>0.05)in the abundance of verruca microflora and Proteus microflora in ApoE group(P<0.05).The sequence level of fecal microflora in three groups of mice mainly belonged to the following 8 genera:Dunaliella,Bacteroides,Eichmann,Pseudomonas,Staphylococcus,Helicobacter,pseudoprevotella,erysipe Compared with APOE(normal saline)group,the abundance of Staphylococcus and Pseudomonas in ApoE(C57BL/6J)group was significantly lower(P<0.05),and the abundance of Pseudomonas and Pseudomonas was significantly higher(P<0.05);compared with APOE(normal saline)group,the abundance of Staphylococcus and erysipelatococcus in apo E(Eucommia)group was significantly higher(P<0.05)The abundance of Bacteroides and Eichmann significantly increased(P<0.05).Conclusion:The TCM syndrome differentiation of atherosclerotic population is mainly based on kidney deficiency syndrome,which suggests that tonifying kidney and pulse is an important link in the prevention and treatment of atherosclerosis;the diversity of oral bacteria in atherosclerotic population is significantly lower than that in non atherosclerotic population,and there are structural differences.The increase of Streptococcus and veronococcus in oral bacteria can be used as the characteristic biological indicators of atherosclerosis,It can provide new ideas and research directions for the clinical prevention and treatment of atherosclerosis;Eucommia can effectively regulate the diversity and composition of intestinal flora,affect its metabolites,reduce the level of blood TMAO,reduce the expression of VCAM-1 factor in aorta and IL-6 factor in ileum,and then affect the process of atherosclerosis;Eucommia can improve the role of atherosclerosis The metabolized products of intestinal flora were affected by the transfer of fecal bacteria. |
PDF Full Text Request