| Study BackgroundWith the improvement of people’s life quality and the rapidly aging population, the morbidity and mortality of cardiovascular and cerebrovascular diseases are increasing fast. Atherosclerosis (AS) is important pathological basis of coronary heart disease a myocardial ischemic disease cause by Coronary atherosclerosis. Dyslipidemia is one of the most important factors for atherosclerosis and it come with the formation, progress and deterioration of atherosclerosis. Almost all epidemiological studies had demonstrated a positive relationship between total cholesterol and low-density lipoprotein cholesterol whit mortality of cardiovascular. LDL-C level is the most important pathological changes of atherosclerotic risk factor. Patients can benefit from lowering LDL-C treatment through Statins. We found that patients’LDL-C level receiver desired level can reduce cardiovascular events by30%, but remain70%cardiovascular events would be happened. So we believe that high density lipoprotein (HDL) was a therapy target in atherosclerosis prevention.High-density lipoprotein cholesterol is another important lipoprotein which have Atheroprotective property. Almost all epidemiological studies had demonstrate an inverse relationship between plasma levels of HDL-C and cardiovascular atherosclerotic disease. The Framingham study found people of HDL-C levels of <35mg/dl have4.1times mortality rate than people of HDL-C levels of>55mg/dl Franceschini showed that for every1mg/dl increase in HDL-C, the predicted incidence of coronary events decreases by2%in men and3%in women. The major cardiovascular protective effects of HDL function may be attributed to its role in reverse cholesterol transport (RCT), anti-oxidant and anti-inflammation and so on.However, several lines of evidence indicate that the relationship between HDL and CAD risk is more complex and extends beyond the serum HDL-C levels. Torcetrapib, a potent CETP inhibitor, markedly increased the plasma concentration of HDL-C, but the risks of deaths and cardiac events in patients receiving tocetrapib had been increased simultaneously. The Milano people who carry the apolipoprotein A-I Milano mutant have very low serum HDL-C level while show very low incidence of CAD. Our results support the theory that not all HDL possess atheroprotective properties. Some HDL is dysfunctional or pro-inflammatory. Surem HDL-C level is not equivalent to HDL function.The proposed atheroprotective properties of HDL are multifaceted, including Reverse Cholesterol Transport and cholesterol efflux capacity, anti-oxidative and anti-inflammatory activities. The most important theory revolves around the role of HDL in macrophage reverse cholesterol rransport (RCT), in which excess cholesterol is effluxed to HDL and ultimately returned to the liver for metabolism by receptor of ATP binding cassette transporter A1(ABCA1) and Scavenger receptor class B type I (SR-BI).Structural modification and composition alteration of HDL may lead to HDL loss of normal biological function, and HDL-C is normal which still failed to inhibit atherosclerosis. The most important theory revolved around the role of HDL function is RCT, in which excess cholesterol effluxes to HDL and ultimately returned to the liver for metabolism. The process of RCT is extremely complicated:At first, cholesterol efflux from macrophages and then lipid-poor ApoA-I quickly acquires it via the ABCA1transporter. Lipidation of the lipid-poor ApoA-I and cholesterol complex generates nascent (pre-β) HDL. Subsequently, lecithin cholesterol acyl transferase (LCAT) mediated cholesterol esterification generates small HDL3particles; small HDL3can be converted to large mature HDL2in turn upon CETP [20]. At last, these mature HDL2transfer its cholesterol to the liver directly via SR-BI and subsequently excrete cholesterol through the bile. The process of RCT is extremely complex and ApoA-I, LCAT and CETP play an important role in this process.HDL has anti-inflammatory and anti-antioxidant property, which play an important role in AS protecting. HDL plays an important role in protecting against LDL oxidative modification, the enzyme PON1and ApoA-I contribute the key role to the antioxidative effects of HDL. MPO, LPO, SOD can change the structure of HDL which would oxidative modification HDL in to ox-HDL, and thus may reduce property of anti-inflammatory and anti-antioxidant in HDL, even to.produce more ox-LDL.The role of endothelium protection is an important function of HDL. On one hand, HDL inhibits monocyte adhesion and migration to the vessel wall; stimulate the repair and proliferation of endothelial cells, inhibited proliferation of vascufar smooth muscle cells by growth factor. On the other hand, HDL can promote endothelial cells producing the L-arginine and NO by-the NOS catalytic to elevated vascular endothelial function, can also prevent endothelial dysfunction by inhibit the aggregation and adhesion of platelets. HDL play a important role in the protecting endothelial function can by eNOS.Lipoprotein-associated phospholipase A2(LP-PLA2) is a newly discovered closely related with atherosclerosis. It hydrolyzed ox-LDL into a large number of lysolecithin and free oxidation of fatty acids. The function HDL can bind Lp-PLA2; reduce the free amount of Lp-PLA2and the formation of thrombus.High homocysteine causes many kinds of diseases, especially increase the risk of cardiovascular and cerebrovascular events. The synthesis and metabolism of homocysteine are maintaining homeostasis, higher than15.0μmol/L called high hyperhomocysteinemia (HHcy). Hhcy is a new independent risk factor of AS including hypertension, hyperlipidemia, smoking, obesity, cardiovascular and cerebrovascular disease, which was a positive relationship between Hcy levels and cardiovascular risk. The plasma Hcy escalate5umol/L can increase ischemic heart disease by32%and stroke by59%; Plasma Hcy reduced3umol/L, the risk of ischemic heart disease can be reduced by11to20%, and the risk of stroke reduced by15-33%. Total plasma Hey level is increased by5umol/L is equivalent to the the total cholesterol each elevated a20mg/dL risk, and therefore to be an independent risk factor for coronary heart disease.Dyslipidemia is the basis of atherosclerosis. Relationship of Hey and dyslipidemia are unclear. HHcy slows down LDL metabolism and elevated levels of LDL-C. Hey thiolated low density lipoprotein into Hcy-LDL complexes, which cannot be macrophage phagocytosis, increased intracellular degradation, caused by the intracellular accumulation of cholesterol. HHcy can autoxidation to produce large amounts of peroxide, causing protein damage, which produce large amounts of Ox-LDL, exacerbated atherosclerosis progression.High-density lipoprotein is cardiovascular protective factor, but its relationship with Hey is no clear now. A research found that Oral folic acid to reduce high Hey can be improved PON1activity in the HDL particles, thereby reducing the content of diabetes Ox-LDL and alleviate the progression of atherosclerosis. PON1is one of the HDL enzymes; PON1can not reflect the HDL function. HHcy may affect HDL function through more mechanism.ObjectivesThe study was to investigate the relationship between HCY and MTHFR gene and atherosclerosis in in Patients with Medium HDL level, explore the relevance of HCY with coronary artery disease severity. We investigated the impact of HHcy to the RCT function of HDL by detecting the amount of Apo-AI, of LCAT, CETP in HDL particles. We also investigated the anti-inflammatory and anti-antioxidant function of HDL by detecting the PONl and MPO activity, the amount of Apo-AI, of LCAT, CETP in HDL particles.Methods1. Patient inclusion criteria, grouping and exclusion criteriaWe collect the patient’s history of smoking, hypertension, diabetes, family history of coronary heart disease and other past medical history. We divided patients into two groups according HCY levels the HHcy group was defined HCY level above15.0μmol/L and the control group was below15.0μmol/L (guideline of ACC). Both two group HDL level was normal (40mg/dL <HDL-C <60mg/dL). All patients signed informed consent, who exit experiment without obtaining the informed consent. Exclusion criteria:1, chronic renal insufficiency patients, patients receiving kidney transplants;2, hyperthyroidism or thyroid dysfunction;3, impaired liver function or pre-existing liver disease patients;4, pernicious anemia, acute leukemia, cancer;5, the currentor recent oral methotrexate, theophylline, B vitamins and folic acid in patients.2. Serum lipid analysisBlood lipid analysis was performed at the beginning of the experiment. Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (HDL-C) concentrations were measured by using an automated biochemical analyzer.3. HCY analysisHCY analyses were using Agilentl100liquid chromatography, respectively determine by two people in the same day. Of variation of3.7%, inter-assay variation was4.1%; deviation of measurement results of the quality control samples of low-level point of less than20%deviation of the measurement results, a high level point of less than15%; the sample twice determination error does not exceed15%; if the error is greater than15%is re-measured, until the error is less than15%.4. MTHFR gene analysisMTHFR was test by PCR-RFLP, and through2%agarose gel electrophoresis to separate the different length fragments5. Coronary lesionsCoronary angiography was carry by Philip H3000. Coronary lesions were divided into single, double and multi-vessel disease. Gensini score system quantitates assessment of severity of vessel:narrow <25%1score,25%-49%2score,50%-74%4score,75%-90%8score,91%-99%16score,100%32score.6. The separation of high-density lipoproteinHDL separated by density gradient centrifugation. BCA assay was use to analysis the HDL particles concentration7. Assessment of the RCT function of HDLLCAT and CETP were assayed using Elisa.8. Assessment of the anti-inflammatory and anti-antioxidant function of HDLPON1, MPO, LPO, SOD in HDL particles was assayed using kits according to the manufacturer’s instructions. eNOS and Lp-PLA2were assayed using Elisa.Results1.There were no differences in Age(χ2=0.407, P=0.685), sex (χ2=0.003, P=0.959), hypertension(χ2=0.421,P=0.517), diabetes(χ2=0.778, P=0.378), smoking (χ2=1.014, P=0.314), TC (t=0.534, P=0.595)、TG (t=0.457, P=0.649) HDL-C (t=1.347, P=0.181)、LDL-C (t=0.650, P=0.517)、Apo-B (t=1.426, P=0.147)、FBG (t=0.687, P=0.494)、PBG (t=0.484, P=0.630)、Cr (t=0.301, P=0.764)、BUN (t=0.443, P=0.659)、ALT (t=0.716, P=0.476)、AST (t=1.725, P=0.088) in two group,2. The Coronary lesions were Serious in HHCY group than control group (χ2=17.731, P=0.000).The Gensini score was higher in HHCY group than control group (t=4.222, P=0.000)3. There were significant differences in two group in MTHFR gene (χ2=7.463, P=0.024). HCY levels were positively correlated with MTHFR (r=0.258, P=0.014), Coronary lesions(r=0.438, P=0.000) and Gensini score(r=0.641, P=0.000). MTHFR gene were positively correlated with Coronary lesions(r=0.600, P=0.000)and Gensini score(r=0.621, P=0.000).Finally, we take multiple linear regression analysis our data, we found that Gensini score were closed correlated with MTHFR gene (t=7.851, P=0.000), HCY (t=5.805, P=0.000)、TC (t=2.748, P=0.007)、Apo-AI (t=-2.253, P=0.027), the model was meaning full (F=45.923, P=0.000)4. Conpared with control group, Apo-AI (1.27±0.12mmol/L vs1.21±0.13mmol/L,P=0.025), LCAT (1029.02Q133.88U/mg vs913.20±117.42U/mg, P=0.000) and CETP (34.70±4.72ug/mg vs26.33±4.13ug/mg,P=0.000) was significantly lower in HHCY group.5. Conpared with control group, PON1(450.55±48.55U/ml vs206.48±75.02U/ml, P=0.000) and Lp-LPA2(37.35±8.85ng/mg vs31.80±6.91ng/mg, P=0.001) was significantly lower in HHCY group.6. Conpared with control group, MPO (3.15±1.49U/L vs5.02±2.02U/L, P=0.000) and LPO (0.82±0.09umol/gprot vs0.93±0.9umol/gprot, P=0.000) was significantly higher in HHCY group.7. There were no differences in eNOS and SOD levels.Conclusions1. HHCY group coronary lesions were serious than control group, and Gensini score were higher than control group. The HCY level was positive correlated with Gensini score.2. MTHFR gene in HHCY group was significant differences with control group. The MTHFR gene was positive correlated with Gensini score and coronary lesions. High Hcy may be an independent risk factor for coronary heart disease.3. HHCY may reduced the RCT function of HDL through decreased. Apo-AI, LCAT, CETP in HDL particles.4. HHCY reduced the anti-inflammatory and anti-antioxidant function of HDL through decreased PON1and Lp-PLA2in HDL particles.5. HHCY reduced the anti-inflammatory and anti-antioxidant function of HDL through increased MPO and LPO in HDL particles.6. HHCY were no effect of SOD and eNOS of HDL, the result need more research to confirm.7. All of above, High Hcy may be an independent risk factor for coronary heart disease. HHCY reduce the RCT, anti-inflammatory and anti-antioxidant function of HDL to aggravate atherosclerosis. Reduce plasma homocysteine levels may improve the function of HDL, and reduce Hcy may become one of the targets for the treatment of atherosclerosis. |
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