Interaction Between Trimethylamine Oxide And Angiotensin Ⅱ Type 1 Receptor In Atherosclerosis

Objective:To investigate the interaction between tmao and Angiotensin II type 1receptor（AT1R）in atherosclerosis。Methods:1.Part 1:17 6-week-old Apo E-/-mice were randomly divided into two groups:Control group（n=8）and telmisartan treatment group（n=9）;All mice were fed with high fat diet,and the treatment group was given telmisartan 10mg·kg^-1·d^-1.After 12 weeks of breeding,blood was collected from the orbital sinus,and blood lipids were detected by fully automatic enzyme colorimetry,and the content of TMAO in plasma was determined by high performance liquid chromatography-tandem mass spectrometry;Detection of the degree of atherosclerosis:Oil red"O"staining was used to determine plaque area in aortic root;Immunohistochemistry was used to detect the expression of inflammatory factors IL-6 and MCP-1 in the plaque;16S-r RNA sequencing analysis was performed to determine the intestinal flora structure of the two groups of colonic stool.2.Part two:Twenty-one 6-week Apolipoprotein E gene knockout（Apoe-/-）mice were randomly divided into control groups（control,CTL）,high-oxidized trimethylamine（TMAO）,TMAO+Telmisartan（TLM）intervention group,fed with ordinary food,1%choline,1%choline+telmisartan（10mg·kg-1·d-1 gavage）respectively.All mice were kept for 12 weeks,and tail blood pressure and body weight were measured regularly;After anesthesia with 3%chloral hydrate solution,venous blood was collected in the orbital sinus to detect the blood lipid and plasma TMAO content.Oil red staining was used to detect the area of atherosclerotic plaque in the aortic root and the expression of inflammatory factor IL-6 and MCP-1 in the plaque was detected by immunohistochemical method.The molecular docking method was used to determine whether the TMAO ligand and AT1 receptor could bind stably and whether there was any interaction between them.Histological level:Western Blot was used to detect the AT1R expression in the whole aorta and the phosphorylation level of Extracellular regulated Protein kinases（erk1/2）downstream of Protein kinase C（PKC）to determine,To determine whether TMAO can activate AT1R signaling pathways.Cytological level:Construct a recombinant plasmid containing the AT1R target gene,transfect HEK-293T cells,add TMAO（200umol/l）to stimulate the cells,and observe whether TMAO can increase the phosphorylation levels of PKC and ERK1/2 at different time points（0,5,10,15,20,30,60min）and the role of telmisartan in this process.Results:Part one:Part 1:Compared with the control group,the telmisartan treatment group reduced the abundance and proportion of the six intestinal flora（Anaeroplasiaceae,Bacteroideaceae,Clostridiaceae,Lachnospiraceae,Ruminoccaceae and Proteus）involved in the production of TMAO and accompanied by a decrease in TMAO plasma levels（P=0.023）.And to some extent,it improved the atherosclerosis of Apoe-/-mice fed high-fat diet（P<0.01）,and reduced the expression of inflammatory factors IL-6 and MCP-1 in plaques（P<0.01）.Part two:1.The results of molecular docking indicated that TMAO was deeply embedded in the AT1R protein molecule by means of hydrogen bond and cationic-pi bond,and its stable binding indirectly confirmed the interaction between the two molecule.2.Compared with the control group:the aortic root plaque area increased in the TMAO group（P=0.0468）and the plasma TMAO content also increased（P=0.002）;compared with the TMAO group:the aortic root plaque area（P=0.0092）and plasma TMAO content（P=0.030）decreased in the TMAO+TLM group.These results showed that 1%choline could significantly increase the plasma TMAO content,and the modeling was successful.At the same time,it has been verified that Telmisartan can reduce the TMAO plasma content;TLM could also improve atherosclerosis aggravated by high plasma concentrations of TMAO.3.In terms of blood lipids,the HDL-C content in the control group was significantly higher than that in the TMAO group and the TMAO+TLM group.There was no difference in triglyceride,low density lipoprotein,and total cholesterol among the three groups.4.Western Blot detection of protein expression in aortic tissue of mice:Compared with the CTL group:AT1R（P=0.001）expression,phosphorylated extracellular regulatory protein kinases（p-ERK1/2）（P-ERK1/2）（P=0.019）,Phosphorylated protein kinase C（p-PKC）（P=0.027）expression level increased in TMAO group;compared with TMAO group:AT1R,p-ERK1/2,phosphorylated protein kinase C(Phosphorylated protein kinase（p-PKC）（P=0.009,P=0.031,P=0.019）decreased expression levels TMAO+TLM group,indicating that TMAO has the effect of increasing AT1R expression in mouse aortic tissues and activating its signaling pathway.5.The AT1R eukaryotic expression plasmid was constructed and transfected into HEK293T cells.After TMAO（200umol/l）was added to stimulate cells,ERK1/2 phosphorylation levels increased significantly at 15min and 30min（*P=0.021,**P=0.037）,and PKC Phosphorylation levels increased significantly at 10min and 15min（*P=0.046,**P=0.01）;after TLM（1umol/l）intervention,ERK1/2 and PKC phosphorylation levels were not different from the control group（P>0.05）.This result further supports the role of TMAO in activating AT1R.Conclusion:1.The mechanism of the aggravation of atherosclerosis by TMAO may be related to the activation of the AT1R signaling pathway by TMAO.2.Mechanism of angiotensin receptor blockers to reduce atherosclerosis:It is possible to reduce TMAO plasma content by changing the composition of intestinal flora;block TMAO from binding to AT1R.