| Background:Hypertension is one of the most common cardiovascular diseases,and it is also the main risk factor of atherosclerosis and death caused by cardiovascular and cerebrovascular diseases.Epidemiological studies have shown that unhealthy diet and lifestyle can lead to abnormal blood pressure regulation,mainly manifested as elevated blood pressure.The human body is in a state of high blood pressure will lead to vascular wall remodelling and dysfunction,including endothelial dysfunction and vascular rigidity.Intestinal microbial metabolism plays an increasingly important role in cardiac homeostasis.Dietary choline,which is found in fish,eggs,grains,dairy products,and meat products,is converted to trimethylamine by lyases of gut flora,which is then metabolized by the liver to produce trimethylamine oxide(TMAO).Numerous studies have shown that TMAO,a choline metabolite,is closely related to the occurrence of cardiovascular diseases,which provides new opportunities for the prevention and treatment of cardiovascular diseases.However,due to the rapid metabolism and excretion of TMAO,the large differences among individuals of different ages,and the fact that different kinds of choline-derived foods do not affect the fasting plasma TMAO concentration,TMAO may not be suitable as a transient biomarker,and the biological significance of single evaluation of TMAO body fluid level at a certain time may not be comprehensive.Furthermore,a few studies have suggested that TMAO may have beneficial cardiovascular and metabolic effects.Therefore,the clinical significance of TMAO in cardiometabolic diseases and its pathogenesis need to be further evaluated.The pathogenesis of TMAO on cardiovascular diseases includes platelet hyperreactivity,decreasedβ-oxidation of fatty acids in cardiomyocytes,altered cholesterol and sterol metabolism,and increased inflammatory reaction and reactive oxygen species production in vascular wall.However,some studies suggest that the increase in plasma TMAO in cardiovascular disease may be similar to the increase in plasma B-type natriuretic peptide levels,both as a cardiovascular risk marker and as a compensatory response to the beneficial effects of pressure/volume overload on the heart.However,there is still a lack of systematic and comprehensive studies on the regulatory role of TMAO in hypertension.This study is a comprehensive study combining clinical population data and cell animal experiments.Firstly,the relationship between total intake of choline,the main precursor nutrient of TMAO,and hypertension and blood pressure was explored by using large-scale population nutrition survey data.Then,the potential mechanism of TMAO on blood pressure regulation and vascular disease was explored by combining aortic transcriptomics.Meanwhile,the regulation of TMAO on fat metabolism in hypertensive rats was further revealed by observing the status of visceral fat metabolism and fatty acid metabolomics.Finally,combined with visceral adipose tissue transcriptomics and cell model to reveal the potential molecular mechanism of TMAO regulation of fat metabolism in hypertensive rats.so as to deepen that understand of the complex regulation network between TMAO and hypertension body,and provide new evidences for elucidate the potential mechanism of TMAO on cardiovascular and metabolic regulation of hypertension body.Chapter Ⅰ Investigation on the relationship between the intake of choline,the pro-nutrient of TMAO,and hypertension in populationObjective:To explore the association between total intake of the TMAO pro-nutrient choline and hypertension in a population.The association of total choline intake with systolic and diastolic blood pressure was further explored after excluding people who were being treated for hypertension.To provide clinical evidence for the role of TMAO,the intestinal metabolite of choline,in the pathogenesis of hypertension and the regulation of blood pressure.Methods:1.A statistical analysis of 25890 participants in United States National Health and Nutrition Examination Survey from 2007 to 2018 was conducted using R software and logistic regression model.Total choline intake was divided into five levels(Q1 to Q5 from low to high)according to the quintile(Q)to explore the correlation between hypertension and total choline(TC)intake.2.Multiple linear regression models were used to analyse the relationship between systolic blood pressure(SBP)and diastolic blood pressure(DBP)and TC intake in the remaining 17296participants,excluding those who were receiving antihypertensive medication.3.Subgroup analyses were performed to determine whether the relationship between TC and blood pressure varied with age,sex,body mass index(BMI),estimated glomerular filtration rate(e GFR),and smoking status,and to test for interactions.Results:1.In regression model I without adjusting for any covariates,odds ratios(ORs)for hypertension were significantly inversely correlated with the upper range of TC levels(Q3,Q4,and Q5)(P<0.001),especially in the Q5 range(OR:0.81,95%CI:0.75,0.87,P<0.001)。In Model II,only TC levels in the Q3 range were negatively associated with hypertension(OR:0.90,95%CI:0.82,0.99,P=0.033)。After adding more covariate-adjusted models(Model III),TC levels were not significantly associated with hypertension.2.Multivariate-adjusted linear regression analysis of TC intake and blood pressure values showed that TC intake was negatively associated with SBP,and TC was not associated with DBP.In regression model I,it was found that TC intake was positively correlated with DBP.When the TC-DBP association model was adjusted for age,sex,and ethnicity(model II),TC intake was positively associated with DBP in the Q4 and Q5 range of TC(Q4,β=0.575,SE=0.28,P=0.0401;Q5,β=0.62,SE=0.287,P=0.031)。TC was negatively correlated with SBP.In Model III,when all covariates were adjusted,there was no significant association between TC and DBP,while TC remained significantly negatively associated with SBP,especially in Q4and Q5(β=-1.084,SE=0.429,P=0.012;β=-1.308,SE=0.59,P=0.027)。In addition,the negative trend with systolic blood pressure increased with the increase of TC level(P=0.014).In analyses that did not exclude participants on antihypertensive therapy,linear regression analyses of TC and systolic blood pressure also showed a negative association between TC and SBP in each quintile of Models II and III,with an increasing negative trend with increasing TC levels(P=0.016 and P=0.004).3.There was a significant interaction with age,BMI,and e GFR in the relationship between TC levels and systolic blood pressure(P=0.001,0.015,and 0.001,respectively).In people aged≥60 years,higher total choline intake was more likely to be associated with lower SBP.The negative association between total choline intake and SBP was stronger in participants with BMI 25 kg/m~2 and e GFR 90 mg/min/1.73m~2.Conclusion:1.Large population-based data from the 2007-2018 National Health and Nutrition Examination Survey confirmed no significant association between TC intake and the prevalence of hypertension.It is suggested that high TC intake may not be a risk factor for hypertension.This will provide important evidence for the study of the relationship between TMAO and hypertension.2.High levels of TC intake were negatively associated with SBP,which was influenced by age,BMI,and e GFR.It is suggested that choline and its metabolites may help to reduce SBP.This study will provide important hints and evidence for the study of the mechanism of TMAO on blood pressure regulation.Chapter 2 Effects of TMAO on cardiovascular system in spontaneously hypertensive rats Objective:To investigate the effects of long-term supplementation of different levels of TMAO on the cardiovascular system in spontaneously hypertensive rats(SHR),in order to further understand its role in the development of hypertension,and to provide experimental evidence for clarifying the role and potential mechanism of TMAO in blood pressure regulation and vascular lesions.Methods:1.Spontaneously hypertensive rats(SHR)were fed with drinking water containing 333mg/L or1 g/L TMAO and normal diet for 22 weeks.Grouping:Wistar-Kyoto(WKY)rats fed normally served as normal control group,SHR fed normally served as disease control group,SHR fed with drinking water containing 333mg/L TMAO served as low concentration intervention group,and SHR fed with drinking water containing 1 g/L TMAO served as high concentration intervention group.2.Systolic blood pressure,diastolic blood pressure,body weight,24-hour water intake,urine output and food intake were measured.3.Heart rate(HR),cardiac output(CO),stroke volume(SV),fractional shortening(FS),ejection fraction(EF),left ventricular outflow tract(LVOT),aortic root(Ao Root),left ventricular anterior wall thickness in diastole(LVAWd),left ventricular anterior wall thickness in systole(LVAWs)and left atrial size(LA)were measured by echocardiography to evaluate cardiac function.4.Plasma urea,creatinine,sodium,potassium and urinary sodium and potassium were measured by automatic chemical analyser.5.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of angiotensin II(Ang II),antidiuretic hormone(ADH),aldosterone(Aldo)and N-terminal pro-brain natriuretic peptide(N-terminal pro-brain natriuretic peptide)in plasma of rats after TMAO intervention.6.The levels of TMAO,neurotransmitters and their metabolites in rat plasma were detected by liquid chromatography tandem mass spectrometry.7.The heart,kidney and aorta tissues were collected and made into frozen sections.Masson staining and Von kossa staining were performed to evaluate the pathological changes such as fibre accumulation and calcium salt precipitation in rats’organs.Oil red O staining was performed to evaluate the degree of intimal fat accumulation in aorta tissues.Results:1.The plasma level of TMAO in SHR treated with low concentration of TMAO was significantly higher than that in SHR control group(P<0.0001).Plasma TMAO levels in SHRs treated with high TMAO concentrations increased 3-fold compared with those treated with low TMAO concentrations(P<0.0001).There were no statistically significant differences in food intake and body weight between groups.However,urine volume levels were significantly increased in both low and high TMAO intervention groups compared to SHR controls(P=0.0166 and P=0.0268).With the increase of TMAO dose,diastolic blood pressure tended to decrease but not statistically significant(P=0.1775).The systolic blood pressure of SHR treated with high concentration of TMAO was significantly lower than that of SHR control group(P=0.0013).2.Basic echocardiographic parameters,including heart rate,cardiac output,stroke volume,fractional shortening,and ejection fraction,were not statistically different between groups.Compared with SHR control group,some cardiac structural parameters(LVOT,Ao Root,LVAWd and LVAWs)were significantly decreased after high concentration TMAO treatment(P=0.0365,P=0.0368,P=0.0119 and P=0.0101).However,there was no significant difference in LA level between SHR treated with high concentration of TMAO(P=0.0563).3.There was no significant difference in plasma urea and creatinine among the groups.The plasma sodium concentration of SHR rats in the high concentration TMAO intervention group was significantly decreased(P=0.0113),but the plasma potassium concentration was not significantly different.The results of 24-hour urinary electrolyte excretion showed that the urinary sodium and potassium excretion of SHR were increased after high concentration TMAO intervention(P=0.0086 and P=0.0481).TMAO treatment did not change the secretion of angiotensin II,but decreased the secretion of antidiuretic hormone(P=0.0012),aldosterone(P=0.0457),and the secretion of N-terminal pro-brain natriuretic peptide(P=0.0543).4.In SHR,the plasma concentration of glutamine was significantly increased by long-term high concentration of TMAO(P=0.0037),but the other 19 neurotransmitters and their metabolites had no statistical difference.5.The pathological examination of left ventricular heart,aorta and kidney showed that the myocardial fibroplasia and calcium deposition were the most significant in SHR control group,while the myocardial fibroplasia(P=0.0096)and calcium deposition(P=0.0024)were significantly reduced in SHR treated with high concentration of TMAO.In the histological sections of kidney and aorta,there was no significant difference in the proportion of pathological staining positive area among groups.Conclusion:1.Long-term high-concentration TMAO intervention on SHR can promote sodium excretion of diuretic effect and has no effect on renal function,while significantly reducing systolic blood pressure and improve some cardiac ultrasound indicators.This will provide important animal experimental evidence for the effect of TMAO on blood pressure regulation and its mechanism.2.Long-term high-concentration TMAO intervention did not produce adverse cardiovascular effects on SHR.On the contrary,TMAO reduced myocardial fibrosis and calcium deposition in SHR,suggesting that TMAO may play a role in relieving cardiac fibrosis,which may be related to its diuretic effect.Meanwhile,no significant pathological changes were found in aorta,suggesting that long-term TMAO supplementation alone did not affect vascular fibrosis,calcification or lipid deposition in SHR.Chapter 3 Effects of TMAO on transcriptomics of aorta in spontaneously hypertensive ratsObjective:Multi-dimensional bioinformatics analysis was used to study the changes of SHR aorta transcriptomics,further explore the potential mechanism of TMAO on SHR blood pressure regulation and vascular lesions,and clarify whether TMAO will affect the biological processes and signal pathways related to atherosclerosis or inflammation in SHR.Methods:1.The aortic tissue isolated from rats in Chapter 2 was used as the research object.Transcriptome sequencing was used to detect the gene expression level of aortic tissue.DESeq2toolkit was used to analyse the differentially expressed genes in aorta of SHR after high concentration TMAO intervention.Gene Ontology(GO)and Kyoto Encyclopedia of genes and genomes databases(KEGG)enrichment analysis were performed by cluster Profiler package.2.Weighted correlation network analysis(WGCNA)was constructed to analyse the relationship between gene modules and phenotypes after TMAO intervention.3.The abundance of 36 immune cells was estimated based on immune microenvironment analysis of transcriptome data of rat aortic tissue to assess the status of aortic immune cell infiltration.Results:1.Among the differentially expressed genes in aorta between SHR and WKY,619 up-regulated genes were down-regulated and 250 down-regulated genes were up-regulated after TMAO treatment.These genes were mainly enriched in insulin and lipid metabolism pathways,including thyroid hormone signalling pathway,glucagon signalling pathway,regulation of adipocyte lipolysis,synthesis of thyroid hormone,insulin resistance,insulin signalling pathway,adenosine-3’,5’-cyclic monophosphate signalling pathway,and cyclic guanosine monophosphate-protein kinase G signalling pathway,but no significant immune inflammation-related pathways were found.2.In the weighted gene co-expression network,three key gene modules were identified to be significantly associated with the phenotypic characteristics of SHR after high concentration TMAO intervention.Key gene modules are mainly closely related to biological processes such as lipid metabolism regulation and apoptosis autophagy,including:negatively regulating the remodelling of very low-density lipoprotein particles,cholesterol transfer,lipoprotein breakdown and lipid metabolism,protein folding,apoptosis and nuclear autophagy,but no significant immune inflammation-related pathway enrichment was found.3.Compared with SHR control group,there was no significant difference in the abundance of36 kinds of immune cells in aorta of high concentration TMAO group.Conclusion:TMAO may have a potential role in regulating insulin secretion and lipid metabolism in SHR,but the experimental results in the previous chapter have shown that lipid deposition in aortic intima is not affected by TMAO,suggesting that TMAO may have a regulatory effect on lipid metabolism in other organs or tissues of SHR.Transcriptome analysis did not show inflammatory cell infiltration and changes in immune inflammation-related signalling pathways in aorta,suggesting that TMAO supplementation alone may not lead to vascular inflammation.Chapter 4 Effect of TMAO on Visceral adipose tissue in spontaneously hypertensive rats Objective:To observe the effects of TMAO on insulin secretion and visceral adipose tissue metabolism in SHR,and to reveal the potential effects of TMAO intervention on the pathogenesis of hypertension and body metabolism.To provide experimental evidence for the role of TMAO in visceral fat metabolism in hypertension.Methods:1.Animal intervention methods are the same as those in Chapter II.After intervention,oral glucose tolerance test(OGTT)was performed and plasma was collected.Plasma triglyceride(TG)and total cholesterol(CHO)were detected by automatic biochemical analyser.2.Plasma levels of IL-6,IL-1β,TNF-α,MCP-1,leptin and insulin were measured by ELISA.3.The white adipose tissue of abdominal omentum was collected and the level of angiotensinogen(AGT)was detected by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin and Masson staining were performed,and the size of adipocytes and the proportion of fibrous tissue were analysed by Image J software.4.The levels of 40 medium and long chain fatty acids in visceral adipose tissue were determined by gas chromatography tandem mass spectrometry.Results:1.The serum insulin level of SHR was significantly higher in the 1g/L TMAO group(P=0.0260).Compared with SHR pure water group,TMAO treated SHR showed a slight decrease in fasting blood glucose level,but the difference was not statistically significant(P=0.2453).The results of oral glucose tolerance test showed that although insulin increased,glucose tolerance was not impaired,and the area under the curve of glucose tolerance test tended to decrease,but the difference was not statistically significant(P=0.0711).2.In SHR treated with 1 g/L TMAO,plasma CHO did not change significantly,while TG decreased significantly(P=0.0318)and plasma IL-6 increased significantly(P=0.0152).The levels of other adipocytokines were not statistically different.TMAO was not found to modulate the increased ATG secretion from visceral adipose tissue.3.The visceral adipocytes of SHR treated with 1 g/L TMAO decreased significantly(P<0.0001).In addition,the effect of TMAO on the secretion of adipose collagen fibbers was not found.4.Compared with WKY rats,16 kinds of medium and long chain fatty acids were significantly decreased in SHR water group,including 8 saturated fatty acids(undecanoate,pentadecanoate,heptadecanoate,arachidonate,heneicosanoate,behenate,tricostate,and tetracosanoate),2 monounsaturated fatty acids(cis-11-eicosenoic acid and cis-15-tetracosenoate)and 6 polyunsaturated fatty acids(linoleate,linolenate,cis-11,14,17-ricosatrienoic acid,cis-5,8,11,14,17-ricosapentaenoic acid,docosatetraenoate and docosapentaenoic acid(C22:5N3)].However,1 g/L TMAO had effect on that content of cis-15-tetracosenoate and four polyunsaturate fatty acids[cis-11,14,17-ricosatrienoic acid,docosatetraenoate,docosapentaenoate n-3(C22:5N3),and docosapentaenoate n-6(C22:5N6)]was significantly higher.Conclusion:1.TMAO can increase insulin secretion and there is a tendency to improve their glucose tolerance.TMAO can decrease plasma TG level and visceral adipocyte volume in SHR.These results suggest that TMAO may play an active role in lipid metabolism of SHR.2.TMAO did not regulate the secretion of AGT in visceral adipose tissue,suggesting that the hypotensive effect of TMAO on SHR may not be related to the secretion of AGT in visceral adipose tissue.3.TMAO can partly improve the metabolic disorder of unsaturated fatty acids in SHR,which will provide a new clue for the study of the role of TMAO in fatty acid metabolism in hypertensive organisms.Chapter 5 Mechanism of TMAO on adipose tissue Objective:To analyse the changes of visceral adipose transcriptome and to reveal the potential molecular biological mechanism of TMAO regulating lipid metabolism in SHR visceral adipocytes by cell model.Methods:1.The rat visceral adipose tissue isolated in chapter 4 was used as the research object.The gene expression level of visceral adipose tissue was detected by transcriptome sequencing,the differentially expressed genes after high concentration TMAO intervention were analysed by DESeq2 toolkit,and GO and KEGG enrichment were analysed by cluster Profiler.2.A weighted gene co-expression network was constructed to analyse the relationship between gene modules and phenotypes after TMAO intervention.Enrichment analysis of related signalling pathways and mapping visualization in KEGG pathway map were performed.3.Using 3T3-L1 preadipocytes as the research object,the lipid accumulation cell model was constructed by using free fatty acids(palmitic acid and oleic acid).The cell viability and the expression of apoptosis-related genes were detected by CCK and polymerase chain reaction after TMAO intervention,and the optimal intervention concentration of TMAO was determined.4.Pik3r3 was overexpressed by plasmid transfection of 3T3-L1 preadipocytes.Cells were divided into normal culture control group,free fatty acid induction group,TMAO+free fatty acid induction group,Pik3r3 overexpression+TMAO+free fatty acid induction group.5.Oil red O staining was used to detect the lipid accumulation of the cell models.6.The expression of PIK3R3,Lamp1,Lamp2,Ctss,Ctsc,p-PI3K,p-AKT(Ser473 and Thr308),p-p70S6K and LC3 protein was detected by western blot.Results:1.Among the differentially expressed genes in visceral adipose tissue between SHR pure water group and WKY group,40 up-regulated genes were significantly down-regulated after TMAO intervention,and 33 down-regulated genes were significantly up-regulated after TMAO intervention.These genes are mainly enriched in phagocytosis,pattern recognition receptor signalling pathway and aldosterone synthesis and secretion signalling pathway.2.In the weighted gene co-expression network,three key gene modules were identified that were significantly associated with phenotypic characteristics of SHR after TMAO intervention.The key gene modules are mainly enriched in phagosomes,lysosomes and fatty acid metabolism signal pathways.Visual enrichment analysis of upstream signalling pathways related to phagosomes and lysosomes showed that compared with SHR pure water group,PI3K/AKT signalling pathway showed an overall down-regulation trend after TMAO intervention,among which Pik3r3,a regulatory subunit of PI3K,was significantly down-regulated(adjusted P=0.0098,log2 fold change=-2.1689).3.TMAO significantly attenuated free fatty acid-induced lipid accumulation in 3T3-L1preadipocytes,with a significant decrease in oil red O staining(P=0.0063).Overexpression of Pik3r3 counteracted the effect of TMAO,and the intensity of oil red O staining(P=0.0016)were significantly enhanced.4.TMAO can significantly increase the lysosomal marker Lamp1 of 3T3-L1 cells induced by free fatty acids(P=0.0032),Lamp2(P=0.0061)and Ctss(P=0.0013),while Pik3r3 expression was inhibited by TMAO(P=0.0048),while Akt phosphorylation at threonine 308(P=0.0492),serine 473(P=0.0057)and p70S6K(P=0.0140)were also inhibited by TMAO.Autophagy marker LC3II/I ratio was significantly increased after TMAO intervention(P=0.0095).Upon overexpression of Pik3r3,the effect of TMAO was counteracted.Conclusion:The regulation mechanism of TMAO on adipose tissue metabolism is related to lysosome.TMAO can inhibit the Akt pathway and activate lysosomal autophagy by inhibiting the regulatory subunit PI3KR3 of PI3K,thus reducing lipid accumulation induced by free fatty acids.This study will provide new experimental evidence for elucidating the potential mechanism of TMAO regulation on lipid metabolism.In conclusion,the results of large-scale population clinical data and experimental animal models in this study consistently suggest that TMAO supplementation may not be associated with the onset of hypertension and that TMAO may have a positive effect in reducing systolic blood pressure.Meanwhile,the animal and cell experiments in this study also revealed the positive role of TMAO in regulating lipid metabolism in visceral adipose tissue and the possible molecular regulation mechanism.This study provides new evidence for clarifying the potential mechanism of TMAO on cardiovascular and lipid metabolism regulation in hypertensive organisms,and provides important scientific basis for deepening the understanding of the complex regulatory network between TMAO on hypertension and visceral fat metabolism,which is expected to enrich the mechanism theory of TMAO on cardiovascular metabolic diseases and provide new ideas for disease warning or targeted therapy in the future. |
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