The Research On Factors Of Affecting Apolipoprotein M Gene Expression And Its Serum Level Relating To Coronary Artery Disease

Chapter One The expression of apolipoprotein M and the effect of Rosiglitazone in Diabetic RatsBackgroundApolipoprotein M(apoM),a member of lipocalin super-family,is a recently discovered apolipoprotein.It is associated mainly with high-density lipoprotein(HDL)in plasma and is also present in triglyceride-rich lipoprotein(TGRLP)and low-density lipoprotein(LDL). It was observed in animal experiments that mice with apoM deficiency showed impaired preβ-HDL formation and abnormally large particulate HDL appearance;and further adenovirus mediated over expression in the liver of apoM in LDL-recepter knock-out mice protected against atherosclerosis.The results showed that apoM may regulate the metabolism of HDL and may have protective effect on atherosclerosis. Up to now the physiological function,the clinical significance and the mechanism of antiatherosclerosis effect are still unknown,although apoM cDNA has been cloned and sequenced in 1999 and we have done some initial study of it.Now it is demonstrated that the disorder of glycometabolism promotes the development of atherosclerosis and diabetes mellitus has been regarded as the equal danger disease of coronary heart disease.PPARγ,a ligand-inducible transcription factor, may increase the expression of ApoA1 and HDL and may promote the counter transport of cholesterol.Whether it can effect apoM expression and further regulate the metabolism of HDL in Diabetes mellistus are still unknown.ObjectiveThe aim of this study was to investigate the expression of plasma apoM and the apoM mRNA expression in the liver,kidney and adipose tissue by the intervention of rosiglitazone in the type 2 diabetic rats;and to explore the underlying mechanism of the effection and the regulation of apoM.MethodsForty male healthy SD rats were chosen as study objects.And establish the animal model of four groups which are control group,high fat chow group(HF group),diabetes mellitus group(DM group)and diabetes mellitus with rosiglitazone inter-vention group(RSG group). Fasting blood glucose(FBG),fasting insulin(FINS),triglyceride(TG) and total cholesterol(TC)were measured at the beginning of the study. The diabetic rats' model was established in accordance with the method of Zhou etc,which was feeding high fat chow and intraperitoneal injection of streprozotocin.And then the diabetic rats were randomly treated with rosiglitazone by daily gavage for 8 weeks.All the rats were killed at the fifteenth week,and collect the blood preparation and tissue preparations the liver,kidney,and adipose.Then examine the level of FBG,FINS,TG and TC,and measure the level of apoM in serum by enzyme linked immunosorbent assay(ELISA),reverse transacription polymerase chain reaction(RT-PCR)was used to apoM mRNA expression in the liver,kindney and adipose tissue in diabetic rats.Results1.Compared to control group and HF group,serum apoM concentrations in DM group were decreased significantly;and serum apoM concentrations were decresed by 65.4%in DM group compared to control group.Whereas compared to DM group,the concentrations in RSG group were increased.All the differences have statistical significance(P＜0.05).2.ApoM mRNA was detected in all the liver,kindney and adipose tissue. The expression in liver was largest,in kidney was larger and in adipose tissue was extremely fewest(all the P＜0.05).And apoM mRNA levels in liver was decreased by 41.4%and apoM mRNA in kidney was decresed by 22.9%in DM group compared to control group.And compared to DM group,apoM mRNA in liver,kidney and adipose tissue of RSG groups was all increased markedly(all the P＜0.05). 3.The level of serum apoM negtive correlated with TG(r=-0.466, P=0.011),TC(r=-0.568,P=0.001),FBS(r=-0.371,P=0.000),FINS (r=-0.768,P=0.048)significantly.ConclusionThe level of serum apoM and apoM mRNA in the liver,kidney and adipose tissue in diabetic rats was all decreased,and rosiglitazone up-regulated all the level;And the level of serum apoM negtive correlated with TG,TC,FBS and FINS significantly.It is indicated that ApoM may participate the glycose and lipid metabolism. Chapter Two The Expressions of PPARα,PPARγand their Correlations with Apolipoprotein M in Liver,Kidney and Adipose Tissue of Diabetic RatsBackgroundIt is considered that apoM may play an important role in the metabolism of high-density lipoprotein(HDL)and may have protective effect on atherosclerosis.Peroxisome Proliferator-Activated Receptor(PPAR),the new member of the nuclear hormone receptor super family,show their biologic effect in the metabolisms of adipose cells and in the development of atherosclerosis by binding specific ligands The insulin-sensitizing thrazolidinedion—rosiglitazone,the artificial ligand of PPARγ,can effect by activating PPARγ.Our previous study found that: the level of serum apoM and apoM mRNA in the tissues was all decreased;and rosiglitazone up-regulated all the levels.However,the action mechanism of apoM whether or not correlates PPARγ? How about the expression of PPARs in diabetic rats? And how about the effect of PPARγ,ligand on apoM and PPARγ? Now there have not been reports about these questions.ObjectiveThe aim of this study was to investigate the expression of PPARαand PPARγin diabetic rats and the correlations with apoM;and to explore the effect of PPARγligand on these expressions.MethodsForty male healthy SD rats were chosen as study objects.And establish the animal model of four groups which are control group,high fat chow group(HF group),diabetes mellitus group(DM group)and diabetes mellitus with rosiglitazone inter-vention group(RSG group)(the same to Chapter One).All the rats were killed at the fifteenth week,and collect the blood preparation and tissue preparations the liver,kidney,and adipose.Reverse transacription polymerase chain reaction(RT-PCR)was used to measure the expressions of apoM(the same to Chapter One), PPARαMrna,and PPARγmRNA in liver,kindney and adipose tissue of diabetic rats.Analyze the correlations between the levels of apoM mRNA and PPARαmRNA,PPARγmRNA in liver,kindney and adipose tissue respectively.Results1.The level of PPARαmRNA in liver was the largest,and in kidney was larger;meanwhile in adipose tissue was the fewest(all the P＜0.05). Compared to control group and HF group,PPARαmRNA level of liver in DM group was markedly decreased(all the P＜0.05);and compared to DM group,the level of liver in RSG group was markedly increased(P＜0.05).2.The level of PPARγ,mRNA in adipose tissue was larger than the level in the liver and the kidney obviously(all the P＜0.05).Compared to control group and HF group,PPARγmRNA level of liver and adipose tissue in DM group was both increased(all the P＜0.05);and compared to DM group,the level in RSG group was obviously increased(P＜0.05). But the expression in the kidney is different;Compare to control group and HF group,the level in DM group was decreased obviously(all the P＜0.05);and compare to DM group,the level in RSG group was obviously increased(P＜0.05).3.ApoM mRNA level had no relationship with PPARαmRNA level in liver,kidney and adipose tissue;while apoM mRNA level correlated PPARγ,mRNA level obviously.ApoM mRNA level negative correlated the PPARγmRNA level obviously in liver and adipose tissue(both the P＜0.01)and positive correlated obviously in kidney(P＜0.01).ConclusionsPPARαmRNA level in liver is the largest;while PPARγ,mRNA level in adipose tissue is much larger.PPARγagonist could effect the expressions of PPARαmRNA and PPARγmRNA in diabetic rats.The expression of ApoM mRNA correlates the expression of PPARγmRNA obviously.It is indicated that apoM may take part in the glycose and lipid metabolism by the regulation of PPARγ. Chapter Three The research between of Apolipoprotein M and Coronary Atherosclerosis DiseaseBackgroundDyslipidemia not only has a close relationship with the development of atherosclerosis,but also plays an important role in the acute events of coronary heart disease(coronary sudden death,acute myocardial infarction and unstable angina pectoris).As an important part of lipoprotein,apolipoproteins take part in the metabolism and regulation of blood lipid;and the abnormality of apolipoprotein correlate the development of coronary heart disease closely.It is demonstrated that low level in high-density lipoprotein(HDL)-C and high level in LDL-C and TG may raise the risk of getting cardiovascular diseases;and HDL is widely accepted as a good part in protecting from atherosclerosis. Apolipoprotein M is a recently discovered human apolipoprotein that is associated mainly with HDL in human plasma;it is considered that apoM may play an important role in the metabolism of HDL and may have protective effect on atherosclerosis.However,the physiological function of apoM and the relationship between apoM and cardiovascular diseases have not been demonstrated clearly.Untill now,there is no report on the research of apoM and the degree of different atherosclerotic lesions.ObjectiveThe aim of the study is to investigate the relationship between apoM and coronary atherosclerotic lesion;and further to reveal the physiologic function of apoM and the clinical significance of its concentration in human serum.MethodsSeventy-three patients with coronary heart disease(CHD group)and 47 subjects without CHD(control group)confirmed by coronary angiography were studied.The patients with CHD were divided into two groups,including acute coronary syndrome(ACS)group and stable angina(SA)group according different clinic type of coronary heart disease;evaluate the extent of coronary atherosclerotic lesion according to the number of diseased coronary artery,the severity of coronary stenosis estimated by Gensini scores and the category of coronary artery lesion of type A lesion,type B lesion and type C lesion by ACC/AHA(The American College of Cardiology/American Heart Association).The serum apoM and leptin levels were measured by enzyme linked immunosorbent assay(ELISA);and the serum apoM levels of different clinic types and different coronary atherosclerotic lesion extent were compared;then the correlations of serum apoM level with serum leptin,HDL-C,apoAI,LDL-C,LP(a),FFA and apoB level were analyzed respectively.Results1.Compared to controls,the serum level of apoM was significantly lower in CHD group(P＜0.05);while the serum level of leptin was significantly higher in CHD group(P＜0.05).2.The serum level of apoM was significantly lower in ACS group than in SA group and controls,and the difference had statistical significance(P＜0.05).3.The serum level of apoM in CHD group correlated with the number of diseased coronary artery,the severity of coronary stenosis and the category of coronary artery lesion.Serum apoM level in multiple-branch lesions group was significantly lower than in single-branch lesion group and in two-branch lesion group(P＜0.05);Serum apoM level in midrange narrow group was significantly lower than in the gently narrow group(P＜0.05);the serum level of apoM in the serious narrow group was both lower than in gently and midrange narrow groups(both P＜0.05);the serum level of apoM in type C lesion group was both lower than in type A and type B lesion groups(both P＜0.05).4.The serum level of apoM negatively correlated with the number of diseased coronary artery,the category of coronary artery lesion and Gensini scores(all the P＜0.05),and the serum level of apoM positively correlated with Leptin,HDL-C,HDL/CHOL,meanwhile negatively correlated with LDL-C and FFA(all the P＜0.05).Multiple linear stepwise regression analysis showed that serum apoM level correlated serum leptin level most closely.Conclusion:The serum level of apoM in coronary atherosclerosis patients is significantly lower and the level correlates with the clinic types of CHD and the extent of coronary atherosclerotic lesion;the serum level of apoM positively correlates with leptin,HDL-C,HDL-C/TC,meanwhile negatively correlates with LDL-C,FFA.All of the correlations indicate apoM takes part in the lipid metabolism and may have protective effect in atherosclerosis.