| BackgroundAt present, about20million people die of acute cardiovascular events and more than half of them die of coronary heart disease(CHD) each year in the worldwide. With the development of living style and the increasing living pressure, many characteristics of CHD such as the younger tendency of the sufferers, the high rate of morbidity, disability and mortality are beginning to surface. Therefore, the early diagnosis and treatment of CHD are becoming more and more important. Now, the exploration of early warning indicator to prevent the progress of CHD is a hot spot in CHD research.Coronary artery atherosclerotic plaque formation is the pathophysiological basis of coronary heart disease, whose stability determines the onset of acute cardiovascular events. Atherosclerotic plaque stability depends on the support of the extracellular matrix, of which the most important ingredients are type I, type II and type III collagen. Collagen metabolism is mainly composed of collagen synthesis and degradation. Prolyl-4-hydroxylase(P4H) is the rate-limiting enzyme of collagen synthesis which consists of two a-subunits (P4Ha1, P4Ha2) and two β-subunits (P4Hβ1, P4Hβ2). The overexpression of P4H will lead to an increase in collagen synthesis and correspondingly the inhibition of P4H generation can lead to instability and degradation of collagen. Our previous studies found that:by means of the ASK1-MKK4-JNKl-NonO signal pathway, TNF-a inhibits the expression of P4Hal, thereby inhibits collagen synthesis; more than60%inhibition of TNF-a to P4Hα1mRNA disappeared after NonO protein being knocked out,. These indicate that NONO protein plays an important role in the collagen synthesis process, and may be involved in the regulation of plaque vulnerability.In this study, by analyzing NONO protein expression level in the stable angina pectoris(SAP)group,unstable angina(UA) group and acute myocardial infarction(AMI) group, we aimed to elucidate its role in the onset of CHD, and thus to evaluate its clinical value as early warning indicators by analyzing differential expression in different patients.Purpose1. To evaluate the feasibility of NONO protein as early warning indicator by analyzing its differential expression in different CHD groups.2. To elucidate the roles of NONO protein in the promoting atherosclerotic plaque vulnerability.Methods1. Collection of clinical data120Peripheral blood samples were collected from90patients and30normal persons, of which72cases are males and48are females. Aged38to73years old, averaged55years old, they were divided into four groups:normal group, acute myocardial infarction (AMI) group, unstable angina pectoris (UAP) group and stable angina pectoris (SAP) group. Each group includes30cases. Inclusion criteria: normal group cases were ruled out coronary heart disease by examination and coronary artery CT scan. We grouped CHD cases according to the diagnostic criteria of World Health Organization (WHO).2. The separation of blood mononuclear cells10ml elbow venous blood of each case were collected into EDTA-tubes, then isolated by equivalent volume of lymphocyte separation medium. Subsequently, cultured for2-3hours in the training box,80%adherent cells observed under microscope were mononuclear cells.3. Immunofluorescence stainingWe added primary antibody and fluorescently-labeled secondary antibody to the isolated monocytes, then observed the location of the NONO protein in the cells under the laser scanning confocal microscope.4. Quantitative Real-Time PCRWe used RNA extraction, reverse transcription, and quantitative real-time PCR to detect the expression of NONO protein at the mRNA level.5. Western blot Through the extraction of proteins, gel electrophoresis, transmembrane, marking a primary and secondary antibodies, color and other steps to observe the expression of the NONO protein.6. Statistical analysisAll data are presented as mean±SEM. We used the SPSS13.0to carry out the statistical analyses. The variance analysis, pairwise comparison q test were used. P<0.05was considered statistically significant.Results1. Collection of normal subjects and patients with blood glucose, blood lipids, blood pressure, serum creatine kinase, smoking status and other information. Enzymes in the serum of patients with acute myocardial infarction were significantly higher index; four groups of patients with a history of smoking was no significant difference; compared with the normal group, triglycerides and LDL was significantly increased in acute myocardial infarction group, the unstable group of LDL increased, the difference was statistically significant; acute myocardial infarction group and the unstable angina group, systolic blood pressure, diastolic blood pressure than normal were significantly higher, the difference was statistically significant; triglycerides acute myocardial infarction group levels were significantly higher than the unstable group and stable group, the LDL levels of the acute group was significantly higher than the stable group (P<0.05). The remaining groups of data, the difference is not statistically significant.2.1mmunofluorescence staining:under the laser scanning confocal microscope, we observed the expression of NONO protein within the monocytes nucleus of the normal group and the disease groups.3. Quantitative Real-Time PCR: Compared with the normal group, the expression of NONO protein mRNA level in the disease group increased significantly. Followed by the AMI group, the expression of NONO protein at mRNA level in the UAP group increased most significantly (P<0.01). The difference among the SAP group, the UAP group was statistically significant (P<0.05). Yet the difference between the UAP group and the AMI group was not statistically significant.(fig3:one-way ANOVA, F3.167, df3,75, P<0.05)4. Western-Blot:Compared with the normal group, the expression of NONO protein in the UA group was significantly high (P<0.05), However, the difference among other groups was not statistically significant.(fig3:one-way ANOVA, F3.154, df3,40, P<0.05)Conclusions1. The expression of NONO protein in the UA group was significantly increased, which suggests that the NONO protein can be used as an early warning indicator of myocardial infarction.2. The expression of NONO protein is higher in UAP and AMI patients than that of SAP patients, which indicates that NONO protein is associated closely with the plaque vulnerability. |
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