Role Of Trimethylamine N-oxide In Atherosclerotic Progression And The Modulative Effects Of Eliminating Sputum And Removing Stasis Formulation

IntroductionAtherosclerosis(AS),is the basis of pathological changes of coronary heart disease,stroke and other cardiovascular diseases,has recently surpassed the cancer and become the leading cause of mortality and disability in China.The pathophysiology mechanism and prevention of AS are the most critical subject.Atherosclerosis(AS),characterized by excessive inflammatory cell infiltration and lipid deposition in medium-sized artery wal.Now we believe that the intestinal floras are related to the occurrence and development of atherosclerosis.A meta-organismal pathway was elucidated involving gut microbiota-dependent formation of TMA and host hepatic flavin monooxygenase 3-dependent(FMO3-dependent)formation of TMA-N-oxideTrimethylamine N-oxide(TMAO),a metabolite shown to be both mechanistically linked to atherosclerosis and whose levels are strongly linked to cardiovascular disease(CVD)risks.TMAO is considered as possible causative factor of atherosclerosis.Gradually.To date,great strides have been made by traditional Chinese medicine.Studies have revealed that there is significantly formation of blood stasis during the plaque rupture accompanying with thrombus,however,the most commonly phenomenon is phlegm during the early period of plaque formation with lipid-rich soft plaque.To focus on the treatment of the balance between ‘phlegm’ and ‘blood stasis’,we reported that the aggressive treatment strategy,involving inhibition the progression of early stage plaques by eliminating sputum and removing stasis formulation instead of getting rid of blood stasis after plaque rupture might benefit the clinical patients.Therefore,a new decoction based on the most famous decoction named Gualouxiebai was formed,mentioned by Zhongjing Zhang(the famous doctor in Han Dynasty)in the book ‘Golden Chamber’.Previous studies have confirmed that the new release drug could improve the intestinal flora disorders in patients with coronary heart disease and animal models,and decline of plaques burden in animal models.Serial experiments will be done to firstly observe the correlation of serum TMAO concentrations and AS severities among clinical patients,furthermore to validate explore the potential effects of a new prescription intervention in animal models,and at last to investigate the possible mechanism of new drugs’ anti-atheroscelerotic effects with human umbilical vein endothelial cell model.ObjectivesTo clarify the pathogenic role of TMAO in atherosclerosis,eliminating sputum and removing stasis formulation,can improve lipid metabolism disorder to anti-atherosclerosis by modulating intestinal flora.And endothelial function.MethodsⅠPartTo observe the relationship of the concentration of TMAO with the lipid metabolism and vascular endothelial function in the patients with different degree atherosclerosis.We selected patients with coronary heart disease hospitalized in the Cardiovascular Department,Changzheng Hospital from May 2015 to November 2015.They underwent selective coronary angiography and color Doppler ultrasound detection of carotid IMT,and brachial artery flow mediated dilation(FMD).According to the Gensini score the patients were divided into normal control group(n=16),mild atherosclerosis group(n=18),moderate atherosclerosis group(n=21)and severe atherosclerosis group(n=19).All patients were collected blood samples for blood lipids(TG,TC,LDL-C and HDL-C);the content of ADMA in plasma were determinated by High performance liquid chromatography(HPLC);s VCAM-1were measured by double antibody sandwich ABC-ELISA;the thickness of intima-media of carotid(IMT)and brachial artery flow mediated vasodilation(flow-mediated dilation,FMD)were measured by ultrasound,At the same time,specimens of peripheral blood progenitor cells were analyzed by flow cytometry.All the clinical indicators were statistically analyzed.Ⅱ PartIn LDLr-/-mouse model of atherosclerosis,we observed the relationship the TMAO with lipid metabolism,vascular endothelial function and vascular plaque changed to compare anti atherosclerosis effect of new prescription and atorvastatin.A total of 24 mice with SPF grade male LDLR knockout(LDLr-/-)C57BL,were adaptive fed after 2 weeks for high fat diet and left renal artery current limiting operation.They were prepared the atherosclerosis model to be fed for 8 weeks again,then were given new prescription and atorvastatin treatment.The mice were randomly divided into 4 groups:(1)blank control group(n=6),normal diet,sham operation,distilled water(10 ml/kg*d)by gavage;(2)the model control group(n=6),high fat diet,the left renal artery flow limiting operation,distilled water irrigation(10 ml/kg*d)stomach;(3)new drug group(n=6),high fat diet,left renal artery flow limiting operation,new medicine(0.23 g/kg*d)by gavage;(4)atorvastatin group(n=6),high fat diet,left renal artery limiting operation,atorvastatin(10 ml/kg*d)by gavage.Each group received intervention for 8 weeks.The mice were killed after anesthesia.Aortic initial sections were prepared,plaque stability was analyzed by immunohistochemical technique,blood lipid of mice(TG,TC,LDL-C and HDL-C)were detected,serum TMAO was determinated by high performance liquid chromatography tandem mass spectrometry,vascular endothelial function related factors(NO,SOD and s VCAM-1)and progenitor cells in the bone marrow were detected,finally the results of two interventions were compared.Ⅲ PartHuman umbilical vein endothelial cells(human Umbical vein endothelial cells strain,HUVEC-12)were from the Research Institute at the Fudan University School of medicine and finished passage and resuscitation in vitro.Indirect immunofluorescence staining was observed and identificated under light microscope.After establishing the TMAO injury model of HUVECs in MTT method With the concentration of TMAO 0,10,250,50,100,150 and 200 μmol/L,respectively,OD value was observed to evaluate the intervention effect by the cells survival rate of 248 h To evaluate the intervention effect of MTT method on the TMAO damage model of new drugs,HUVECs stimulated TMAO were added to 0,10,25,50,100,150 μg/ml new drug treatment,48 hours of the survival rate were observated.Given low to high concentration of new prescriptions intervention TMAO damage model,RT-PCR method to measure m RNA culture and s ICAM-1 m RNA expression was in NOS,Western Bloting detect s ICAM-1 and e NOS expression.ResultsⅠPart1,Compared with the normal control group,there were abnormal metabolism of lipid in different degree of atherosclerosis group.Triglyceride(TC),total cholesterol(TG)and low density lipoprotein cholesterol(LDL-C)decreased significantly(P<0.05),and high density lipoprotein cholesterol(HDL-C)increases significantly higher(P<0.05).2,Compared with the normal control group,the levels of serum NO and SOD were significantly decreased(P<0.05),and serum ADMA and s ICAM-1 levels were significantly higher(P<0.05)in the patients with different degrees of atherosclerosis.3,Compared with the normal control group,the serum levels of TMAO were significantly increased in patients with different degrees of atherosclerosis(P<0.05),IMT was significantly elevated(P<0.05),and FMD was significantly lower(P<0.05),progenitor cell in peripheral blood levels decreased significantly(P<0.05).4,Serum levels of TMAO and Gensini score was positive correlation,Pearson’s correlation coefficient was 0.839 respectively(P < 0.0001);and LDL-C were positively correlated,the Pearson correlation coefficient for 0.5616(P < 0.0001).Serum levels of TMAO and NO showed negative correlation,the Pearson correlation coefficient for-0.885(P < 0.0001);and SOD showed a negative correlation,Pearson correlation coefficient for-0.717(P < 0.0001);and ADMA was positively correlated,the Pearson correlation coefficient to 0.860(P < 0.0001);and s VCAM-1 were positively correlated,the Pearson correlation coefficient is 0.795(P < 0.0001).5,The level of serum TMAO and IMT was positively correlated,the correlation coefficient of Pearson was 0.623(P <0.0001);and FMD was negatively correlated,and the correlation coefficient of Pearson was-0.726(P <0.0001),EPCs was negatively correlated,and the correlation coefficient of Pearson was-0.726(P <0.05).6.Serum TMAO > 8 μmol/L is a risk factor for atherosclerosis.Ⅱ Part1,Compared with the blank control group,serum TC,TG,LDL-C were significantly increased,HDL-C significantly decreased,plasma TMAO was significantly increased,NO and SOD levels were significantly decreased,s VCAM-1 levels were significantly higher(P <0.05)in model control group.2,Compared with model control group,the lipid metabolism was significantly improved in mice in the new drug intervention group and atorvastatin group(P < 0.05),the endothelial function was significantly improved in two groups(P < 0.05).TMAO concentrations were significantly reduced(P < 0.05)in the new drug intervention group,yet TMAO concentration was no changed in atorvastatin group.The new prescription compared with the model group significantly increased endothelial progenitor cells in the bone marrow,which is more effective than atorvastatin(P < 0.05).3,Compared with the Atorvastatin intervention group,there was no significant difference in the improvement of lipid metabolism and endothelial function in mice in the new drug group,and the intervention of the new prescription could significantly reduce the TMAO.4,Compared with model control group,the ratio of the aortic intima / media thickness decreased significantly,macrophages in the aortic atherosclerotic plaque and lipid content decreased significantly(P < 0.05),and smooth muscle cells and collagen content were higher(P < 0.05);vulnerable plaque index also decreased significantly(P < 0.05)in the new drug intervention group.But there was no significant difference between the atorvastatin group and the new drug intervention group(P < 0.05).Ⅲ Part1,Human umbilical vein endothelial cells(HUVECs)growth is good.By indirect immunofluorescence identification of factor VIII related antigen,the cultured cells can express factor VIII related antigen with high density.Under the fluorescence microscope,there was yellow green fluorescence in the cytoplasm of HUVEC cells.2,The establishment of TMAO inhibition HUVECs model: the concentration of 0,10,25,50,100,150 and 200 μmol/L TMAO effected on the HUVECs,respectively.Its survival rate curve showed that 100μmol/L TMAO can be used as a suitable concentration of stimulating HUVECs for 48 hours.3,100 μmol/LTMAO has stimulated HUVECs for 48 hours,then the different concentrations of new drug intervention were given.25μg/ml-100μg/ml can decrease the damage of the TMAO.50 μg/ml can be used as the appropriate concentration in the low-moderate new cell experiment.4,In the TMAO damage model,25μg/ml,50μg/ml and 100μg/ml new prescription can increase expression of e NOS and decrese the expression of s ICAM-1 in HUVECsConclusionClinical proof showed that the level of serum TMAO was correlated with the severity of atherosclerosis and the function of vascular endothelium.,Serum TMAO > 8 μmol/L is a risk factor for atherosclerosis.In atherosclerosis mice model,the new medicine not only has the similar effect of lipid metabolism regulation,improving endothelial function and other anti atherosclerosis effect as atorvastatin,but also can reduce the level of TMAO,escalate endothelial progenitor cells in the bone marrow.Cell experiments confirmed that the new prescription can reduce the toxic effect of TMAO on endothelial cells.The new recipe for improving TMAO may be one of the possible mechanisms of its anti atherosclerosis.