Study On The Level Of Soluble Epoxide HydrolAse In Peripheral Blood Of Atherosclerosis Patients And Its Function On Cholesterol Metabolism

Part ? The expression level of serum soluble epoxide hydrolase in patients with atherosclerosisBackground In recent years, the medical level of our country has made great progress, but atherosclerosis and its related complications are still one of the main causes of the threat to human health. Atherosclerosis(AS) is a common cause of atherosclerosis.Atherosclerosis is an inflammatory disease, systemic and / or local inflammation is involved in the whole process from the initiation of atherosclerosis to plaque rupture.Many factors of atherosclerosis such as oxidative stress, advanced glycation end products, hypertension and smoking can lead to autophagy of vascular cells, thereby accelerating vascular lipid deposition, mononuclear cells to endothelial adhesion and migration into Intima into macrophages. Macrophages also secrete various proteolytic enzymes such as collagenase and extracellular matrix metalloproteinase and collagen degradation of fibrous cap, lead to plaque instability and rupture, resulting in severe clinical consequences such as myocardial infarction and cerebral infarction.In a variety of human protein degrading enzymes, epoxide hydrolase may play a role in the regulation of atherosclerotic plaque. Epoxide hydrolase is widely distributed in the animal world(including humans), in the liver, epoxide hydrolase is mainly distributed in the endoplasmic reticulum, recent studies have shown that it is also distributed in the liver cell membrane, cytoplasm, and the epoxide hydrolase body, lysosomes and mitochondria in the absence of. Epoxide hydrolase exists in many kinds of isozyme, the relative molecular weight of monomeric enzyme for48k-54 k, hemoglobin and flavin cofactor do not. Epoxide hydrolase can also catalyze endogenous and exogenous epoxides, the rate of endogenous epoxide than exogenous epoxides. Because epoxide hydrolases play a role in the formation of carcinogens, so it is extensively used as early markers of hepatocellular carcinoma. Soluble epoxide hydrolase(soluble epoxide, hydrolase, s EH) play an important role in the regulation of epoxide metabolism, its main function is to degrade epoxyeicosatrienoic,acids(EETs) and its biological activity. Normally, EETs is a metabolic product formed by the presence of a large number of P450(acid arachidonic, AA), which is formed by the presence of a large number of cells. The biological function of EETs complex in tumor is regulating cell migration and metastasis in cardiovascular disease.EETs can inhibit s EH, and inhibits platelet aggregation and reduce inflammation,atherosclerotic plaques formation and rupture of atherosclerotic infarction area, which promotes angiogenesis and other biological activity, therefore, in recent years, and the s EH also deemed as inhibitors of atherosclerosis, coronary heart disease and the main target in the treatment of hyperlipidemia and other diseases. In this study, we further explore the important role of s EH in the development of atherosclerosis, in order to provide a rich theoretical basis for s EH as a therapeutic target for atherosclerosis.Objective To detect soluble epoxide hydrolase in Chinese patients with atherosclerosis, to investigate the correlation of serum lipid level in patients with atherosclerosis and peripheral blood levels of soluble epoxide hydrolase.Method(1) According to the standard of clinical diagnosis of atherosclerosis, the patients with atherosclerosis and CHD were randomly collected in our outpatients in our hospital. During the same period, the ang and sex ratio matched healthy control were sellected from physical examination center in our hospital.(2) Review the literature and to carry out a survey questionnaire design.The biochemical examination and echocardiography indicators was collected.All the enrolled patients and healthy control signed informed consent.(3) Peripheral venous blood was collected, and the upper serum samples were collected at 4oC to detect the s EH levels in peripheral blood of patients and healthy controls, and the statistical analysis was performed.(4) Spearman correlation analysis was used to further analyze the correlation between s EH levels and serum markers and echocardiography in patients.Results(1) From January 2014 to June 2015, according to AHA diagnostic criteria of atherosclerosis, 214 cases of patients with atherosclerotic arteriosclerosis screening treatment in our hospital, 138 cases of patients with coronary heart disease, screening and earlier treatment in our hospital health examination center,182 healthy adult age and gender matched.(2) Groups of patients with age and gender matched, this equilibrium test showed that the age group(P=0.231>0.05), gender(P=0.844>0.05) between the two groups were not statistically significant, age and gender balanced groups were comparable.(3) We conducted a significant test of the correlation between the groups of patients and healthy controls, to determine the cause of the disease closely related factors.Because there are more variables, we first use single factor analysis, we found that 7 of the 16 suspicious factors are related to the study of the disease. Because the value of blood sugar can not be used in two categories, so we use a quantitative analysis of the above indicators. The education level and occupation belong to the grade classification index, and the rank sum test is adopted. Table1-5 shows that: smoking(P<0.001), total cholesterol(P=0.004<0.05), triglyceride(P=0.021<0.05), low density lipoprotein(P<0.001), essential hypertension(P<0.001) is statistically significant.(4) We use ELISA detection of three groups of patients with peripheral blood s EH expression level, results showed that, compared with the healthy control group(0.21±0.02) ng/ml compared to AS group(128.48±19.42)ng/ml and CHD group(131.21±18.52)ng/ml, s EH level in peripheral blood of patients with ng/ml significantly increased, the difference was statistically significant(F=28.32,P=0.001), AS group and CHD group of patients, no statistically significant differences(F=1.87, T=0.33).(5) Analysis and statistics of patients in the three group echocardiography index found that patients in the CHD group,EF was(45.4±4.6)% which was significantly lower than the pure AS patients(58.3±8.5)% and healthy controls(60.9±4.2)%, and left ventricular end diastolic diameter(LVEDD) in CHD patients was(58.7±3.5)mm and AS(58.2±2.8)mm which were obviously higher than that of healthy controls(52.3±5.3)mm, the difference was statistically significant(P<0.05).(6) We further use the homogeneity test of variance, blood glucose and other data for statistical analysis. The numerical results was input SPSS 20 statistical software(see table 1-8), we finally found that the sedentary time, fasting blood glucose levels were not statistically significant, only the daily physical activity time(P=0.001<0.05) is AS related factors. The education level of non two classification, but with this classification, using rank sum test, see table 1-9,which shows that: Z=1.839<1.96, P=0.068>0.05. No significant difference was found, which indicates that education is not AS related factors.(7) Rank test was further used on analysis the professional nature, the results show that P<0.001, which shows that the difference between the two groups is significant, which indicated that the occupation is one of the AS related factors.(8) After the significant test, 6 related risk factors were identified as AS. In order to further explore the correlation factors and AS, the correction of confounding factors will be included in a variety of factors analysis, and ultimately into the non conditional Logistic regression model. Back method was used to screen one by one, the probability of the selection criteria was 0.05, excluding the standard 0.05.The results showed that smoking, high blood pressure, occupation, low density lipoprotein cholesterol and total cholesterol were statistically significant(P<0.05).(9) The s EH was deemed as independent variables and other related risk factors as dependent variables, statistical analysis of the data,we finally found the correlation between the use of Spearman correlation analysis,which showed that s EH was negatively correlated with high density lipoprotein rs=-1.18, y=-1.18x-12.381 and associated with low density lipoprotein level was positively related.The coefficient rs=0.24, the equation was y=0.24x+1.398,which has a significant differences(P<0.05).(10)Spearman correlation was found between s EH and echocardiography in the peripheral blood of the patients in the group,we finally found that in CHD patients,s EH levels were negatively correlated with ejection fraction EF.The correlation coefficient of rs=-0.12, y(EF) =-0.12x+9.343. LVEDD was positively correlated with left ventricular end diastolic diameter, correlation coefficient rs=0.16,y(LVEDD) =0.16x-2.587.We believe that monitoring of peripheral blood s EH levels has a good effect on cardiac remodeling in patients with cardiac remodeling.Conclusion(1) Smoking, total cholesterol, triglyceride, low density lipoprotein cholesterol,hypertension, daily physical activity time are the risk factors of AS. In the course of clinical treatment and prevention of AS should be in drug treatment at the same time, improve the level of risk factors for patients.(2) s EH expression level in AS and CHD patients was increased, and the level of serum low density lipoprotein cholesterol was positively correlated with the serum high density lipoprotein cholesterol level was negatively correlated.In addition, in patients with coronary heart disease, its expression level was negatively correlated with ejection fraction EF, and left ventricular end diastolic diameter was negatively correlated, can occur in clinical prompt cardiac remodeling in atherosclerotic heart disease.Part ? Study of Soluble Epoxide Hydrolase mutants on cellular cholesterol metabolismBackground In the body, the soluble epoxide hydrolase gene is encoded by EPHX2, which is located on human chromosome eighth, encoding 555 amino acids. In the biochemical structure, the gene encoding protein contains an N terminal phosphatase domain and C terminal soluble epoxide hydrolase domain.The two structures are combined to form a stable existence in the form of two polymer.The N terminal usually has phosphatase activity. Under normal circumstances, the level of serum cholesterol and the process of cell signal transduction can be regulated by the phosphorylation.The C terminal is soluble epoxide hydrolase domain, this part can catalyze the lipid signaling molecule four arachidonic acid epoxy compounds, through a combination of a water molecule, forming diol structure degradation and destruction, so that the level of serum ETTs can be controlled.In some special cases, such as inflammation,effects of changes in the external environment or genetic factors, abnormal overexpression level of EPHX2 can contribute a large number of transcription into soluble epoxide hydrolase, the relative balance of the level of ETTs in vivo is damaged, and ETTs in human tissues may play a regulatory role, such as regulation of vasoconstriction and vasodilatation, regulation regulation of renal blood flow, renal tubular filtration and reabsorption function. Vascular homeostasis is influenced and regulated by endothelial and vascular smooth muscle cell proliferation and migration,while s EH seems to play an important role in these biological processes. EETs can promote endothelial cell proliferation, migration and angiogenesis, the epoxide can promote the proliferation of human cells in mice and the effects of, overexpression of EETs or CYP2 C oxidase can induce the proliferation of cells, which may be associated with the two kinds of cell signaling pathway activation; p38 mitogen activated protein kinase(MAPK) pathway and PI3K-AKT signal pathway of(PI3K/Akt). 11,12-EET activates MAPK, and up regulates D cyclin and Akt,phosphorylated FOXO factor, and reduces the activity of protein in the endothelial cell cycle protein dependent kinase inhibitor p27kip1. A recent study reported that11,12-EET mediated cell proliferation, migration and tube formation in human umbilical vein endothelial cells(HUVEC) in the experiment has been proved to be activation of sphingosine kinase 1(SK1) which promotes sphingosine 1-phosphate synthesis of S1 P. On the other hand, it can act on vascular smooth muscle cells and play the role of anti migration. 11,12-EET and 14,15-EET can suppress mild aortic smooth muscle cell migration, as of platelet-derived growth factor and CYP2 J epoxygenase overexpression or inhibition of s EH, which can reduce cell proliferation and migration. Activating c AMP was dependent on PKA signaling pathway and decreased D cyclin levels plays a role on cell migration in EETs activation and s EHIs in vascular endothelialcells. Although the results of this study showed that s EHIs has a certain effect on vascular smooth muscle cell migration, but there are also studies reported that the role of s EHIs in vascular smooth muscle cell proliferation is not clear. More importantly, in vivo experimental,in subcutaneous sponge model, EETs can stimulate and induce angiogenesis by inhibiting s EH,by which promoting angiogenesis and neovascularization. It shows that the role of EETs and s EHIs in the proliferation of endothelial and vascular smooth muscle cells and migration, this pathway plays an important role in angiogenesis, atherosclerosis and other cardiovascular diseases, and could be used as potential drug targets.Thus, we thought that EETs plays an important role in the field of cardiovascular disease, therefore, in patients with cardiovascular disease, to protect the level of EETs in the body and reduce the level of serum soluble epoxide hydrolase may play a certain role in anti-atherosclerosis. In the last part of the study, we have found that in patients with atherosclerosis and coronary heart disease, peripheral blood soluble epoxide hydrolase levels can be significantly increased, and positively correlated with peripheral low density lipoprotein cholesterol, and high density lipoprotein and negatively related to the phenomenon. However, the EPHX2 gene encoding soluble epoxide hydrolase in lipid metabolism and maintain the biological role of intracellular LDL and HDL equilibrium is not clear. Because of the special role of EPHX2 in lipid metabolism, such as intracellular EPHX2 gene knockout cell normal growth due to the use of si RNA, in the in vivo study, EPHX2 gene knockout, due to cholesterol and lipid metabolism abnormalities, usually knockout mice survival time is short. This has also become a bottleneck in the current in vitro studies. But it also reminds us that whether the gene editing method, design a vector of EPHX2 mutation, which can not only ensure the normal growth of cells and animal experiment, and to explore and in-depth study of the function of EPHX2 gene may play a certain advantage.Objective To investigate the effects of soluble epoxide hydrolase gene mutant on cellular cholesterol metabolism.Method(1) Mutant plasmid was constructed and sequencing was verified according to the wild-type plasmid WT in vitro, then they were transfected into 293 T cell line.(2) Western blot was used for the EPHX2 protein expression.(3) The soluble epoxide hydrolase activity kit for detection of EPHX2 activity.(4) Flow cytometry was used for the detection of EPHX2 mutant on the effect of LDLR internalizing LDL.(5) EPHX2 mutant on the effect of LDLR bingding LDL was detected by flow cytometry.(6) The invasion ability of 293 T cells was detected by Transwell assay, and the cell migration ability was detected by scratch test.(7) PKA were detected by western blot and the data was statistically analysised.Results(1) The wild-type EPHX2 plasmid was used as template to amplify, the product was transformed to E.coli cells, and the plasmid was amplified and extracted. Finally,it was confirmed that the clones were selected as target genes.(2) The soluble epoxide hydrolase molecular weight is about 62 KDa, and the GFP tag protein molecular weight is about 28 KDa, so the EPHX2-GFP fusion protein had a molecular weight about 90 KDa.In the adjustment of GAPDH favorable circumstances, namely the normal group without any treatment of the cells.Because there is no GFP tag, so when the label antibody detected by GFP was not observed in EPHX2-GFP fusion the presence of protein, while the wild type and mutant plasmid is convenient observation with GFP tag, so at about 90 KDa is observed at the presence of the fusion protein.(3) The enzyme activity in normal cell,EPHX2 was used as the enzyme activity in the cell. By transient transfection of wild-type and mutant plasmid EPHX2,overexpression of EPHX2 protein can be detected by transfection of wild-type and mutant plasmid EPHX2, the total enzyme activity of cells was significantly higher than the non transfection group, transfected with mutant EPHX2 group of soluble epoxide hydrolase activity was significantly lower than the wild type(**P<0.01).(4) Normal cells, transfection of wild-type and mutant plasmid cells were plated on6-well plates, 1.5 g/ml Dil-LDL antibody was incubated for 4 hours at 37 DEG C,the ability to detect cell LDLR endocytosis of LDL by flow cytometry. The results showed that EPHX2 significantly influenced the function of LDLR in LDL, and the LDL ability of LDLR was decreased by 25.3% compared with the wild type,which was statistically significant(P<0.05).(5) Cells was cultrured in six well plate, 4o C Dil-LDL antibody were incubated for 4h,flow cytometry EPHX2 binding ability of LDL to LDLR.The results showed that EPHX2 can significantly affect the LDLR binding LDL function, LDLR combined with LDL ability is relatively wild type decreased by 19.5%, with statistical difference(P<0.05).(6) After 293 T cells were transferred into the wild and mutant plasmids,by continuous cultivation of 48 h in the incubation box of 37 o C and 5%CO2, we found that, compared with the WT group, the proliferation rate of 293 T cells in the mutant group was significantly higher than that in the control group, and the difference was statistically significant(P<0.05). Cells were determined by Transwell invasion, the invasion ability of R287 Q cells was significantly higher than that of WT group and CON group, the difference was statistically significant(P<0.05), suggesting that 293 T mutant cell invasion ability.(7) After EPHX2 wild-type and mutant plasmids were detected, the cell cycle was found. Compared with the WT group, the number of S cells in WT group was significantly smaller than that in R287 Q group, and the difference was statistically significant(P<0.05).(8) The Transwell method was used to detect the migration ability in R287 Q and WT,CON cells,compared with 0h, three groups of cell scratch distance was significantly reduced, but the R287 Q group reduced significantly less than CON and WT group, 48 h R287Q group significantly enhanced the ability of cell migration, the difference was statistically significant(P<0.05).lipoprotein cholesterol and total cholesterol were statistically significant(P<0.05).(9) To further detect the expression level of PKA signaling pathway in the three groups of cells,compared with group WT and group CON, the expression level of R287 Q in PKA group was significantly lower, suggesting that EPHX2 gene may play a regulatory role in the PKA signaling pathway.Conclusion The mutant EPHX2 gene decreased soluble epoxide hydrolase activity, and reduced the cells LDLR to LDL endocytosis and binding function.