| BACKGROUND Atherosclerosis is the main cause of death in western developed countries. With development of living standards and change in diet of Chinese people, it also has become one of main fatal diseases in China. Atherosclerosis is a disease with chronic inflammatory lesion of blood vessel walls, of which the pathogenesis has not been fully elucidated, but the cytokines theory is one of the main pathogenesis of atherosclerotic. Cytokines play an important role in atherosclerosis due to widely involvement in inflammation development. The study on new cytokines and their interaction with atherosclerosis will play an important role in early diagnosis, risk assessment of vascμlar disease and targeting therapy. Research indicated that patients with type 2 diabetes were subject to artery lesions and the risk of atherosclerosis was significantly higher. Therefore, through detection NLRP3 inflammasome mRNA expression of mononuclear cells and inflammatory cytokines such as interleukin 1 beta and interleukin 18 in peripheral blood, this research analyzed the correlation between these factors and type 2 diabetes patients complicated with atherosclerosis.NLRP3, as the typical representative of NLRPs protein family, can not only identify the ligand, cell wall acyl dipeptide (MDP), by its leucine rich repeat sequence (LRR) but also activate NLRP3 inflammasome signaling pathways to cause inflammation by binding uric acid, adenosine triphosphate (ATP), endotoxin and cellμlar lysate. Domestic research on correlation between NLRP3 and atherosclerosis is still less. In the abroad study, NLRP3 inflammasome was considered as a new type of medium between cholesterol metabolism and inflammation, which was very important in the process of atherosclerosis, but other studies found no correlation between inflammatory factor and the development of atherosclerosis in mouse model of ApoE gene defect, so the role of NLRP3 in atherosclerosis is unclear.Inflammatory factors are associated with the development of diabetes, so the the study on relationship between inflammatory factors and diabetes with vascμlar disease has become a hot spot and also imperative.Objective In order to understand the correlation in expression of NLRP3 inflammasome mRNA and level of IL-18, IL-1βand type 2 diabetes mellitus (T2DM) patients complicated with carotid atherosclerosis (CAS).Methods Recruited 107 patients with T2DM,35 cases of T2DM patients with CAS and 35 cases of healthy physical examination were used in this study.107 T2DM patients were enrolled and divided into T2DM group (n=26),T2DM small plaque group (n=32),T2DM moderate plaque group (n=38), T2DM serious plaque group (n=11) according to atherosclerosis plaque μltrasonic classification constituted by Washington university. Non T2DM CAS patients were enrolled as CAS group (n=35) and healthy persons as normal control group (n=35). To establish and evaluate real-time fluorescence quantitative polymerase chain reaction (RT-FQ-PCR) for NLRP3 inflammasome mRNA detection and Through the probe stability, detection sensitivity, specificity and repeatability four aspects carries on the appraisal. It was used to detect expression of NLRP3 inflammasome mRNA in peripheral blood mononuclear cells (PBMCs). The NLRP3 inflammasome mRNA expression levels of PBMCs in each group was detected by real-time fluorescent quantitative polymerase chain reaction (RT-FQ-PCR) technology. Glucose oxidase peroxidase coupling method was used for determination of fasting blood glucose. Enzymatic method was used to detect serum total cholesterol and serum triglycerides. Double reagent for direct determination of homogeneous method was used to detect high-density lipoprotein cholesterol and low density lipoprotein cholesterol. Enzyme-linked immunosorbent assay method was used to detect IL concentration. Patients with SPSS 18.0 software is used to statistical analysis of each group differences and in PBMCs NLRP3 inflammatory mRNA expression and IL-18, IL-1 beta concentration and correlation between the severity of the CAS plaques.Resμlts1. The results of evaluation of methodology was as follows:1.1 Stability of the probe:No fluorescent signal emerged in RT-FQ-PCR testing system without primers after 30 amplification cycles, which ensured the stability and no abnormal fracture of the probes.1.2 The detection sensitivity:NLRP3 inflammatory mRNA and beta actin mRNA amplification, according to the results of CT value in the range of 15-25, reaction in good condition.1.3 Detection of specificity:By 2.0% agarose gel electrophoresis of the PCR amplification product, a single strip appeared between 50-100 bp fragments of DNA marker, in accordance with the purposed fragments of 84 bp,85 bp,76 bp and 80 bp.1.4 Detection of repetitive:Samples from the normal control group and T2DM complicated with CAS group samples were determined repeatedly for 10 times. The intraassay coefficients of variation were 3.21%、3.68%、3.24% and 3.55% respectively. These samples were determined in 10 consecutive days and the interassay coefficients of variation were 3.26%、3.75%、3.53% and 3.90% respectively.2. In patients with type 2 diabetes, the correlation between NLRP3 inflammasome mRNA expression in mononuclear cells and inflammatory factor in peripheral blood and atherosclerosis was as follows:2.1 Level of NLRP3 inflammasome mRNA was significantly higher in the pathological group than the normal control group (P<0.05). Level of NLRP3 inflammasome mRNA was significantly higher in the T2DM complicated with CAS group than the T2DM and CAS groups (P<0.01). Level of ASC mRNA was significantly higher in the T2DM combined with CAS group than the CAS group (P<0.01).2.2 Levels of NLRP3 mRNA, ASC mRNA and Caspase-1 mRNA were higher in the T2DM plaque groups and were positively correlated with one another (r= 0.61,P< 0.01 between NLRP3 mRNA and ASC mRNA, r= 0.52,P= 0.03 between NLRP3 mRNA and Caspase-1 mRNA, r= 0.70,P<0.01 between ASC mRNA and Caspase-1 mRNA).2.3 In the T2DM mild, moderate and severe plaque groups, NLRP3 mRNA level showed a gradually decreasing trend one by one, which meant level of NLRP3 mRNA and plaques were significantly negatively correlated (r=-0.70,P<0.01). ASC mRNA level in the small plaque group was greater than in the moderate plaque group and the latter was great than severe plaque group. ASC mRNA level was negatively correlated with in CAS plaque degree of T2DM(r=-0.43,P=0.02). Caspase-1 mRNA expression in the small plaque group was greater than the moderate plaque group and the latter was greater than severe plaque group, showing a negative correlation (r=-0.47,P< 0.01),but Differences of Caspase-1 mRNA level among the three groups had no statistical significance (P> 0.05).3. The concentrations of IL-1β and IL-18 were higher in the plaque groups than the normal control group and positively correlated with level of NLRP3 inflammasome mRNA, which meant the concentrations of IL-1βand IL-18 were positively correlated with NLRP3 mRNA (r=0.70, P<0.01 and r=0.76, P<0.01), ASC mRNA (r=0.69, P<0.01 and r=0.77, P<0.01), and Caspase 1mRNA (r=0.70, P <0.01 and r=0.74, P<0.01), but had no correlation with T2DM plaques (r=0.07, P=0.80 and r=0.26, P=0.29).Conclusion1. The conclusion of establishment and evaluation of methodology was as follows:The established RT-FQ-PCR assay had the characteristics of rapidness and accuracy. Its detection for NLRP3 inflammasome mRNA was stable, specific, repeatable and reliable.2.The NLRP3 inflammasome mRNA level was higher in patients with T2DM complicated with CAS and was highest at the beginning of the CAS plaque formation, while along with gradually mature of the CAS plaque the expression turned to be silent. Expression higher of NLRP3 inflammasome mRNA were correlated with occurrence and development of T2DM patients complicated with CAS.3. Concentrations of inflammatory cytokines IL-1β and IL-18 increased in blood of T2DM patients complicated with CAS were correlated with NLRP3 inflammasome mRNA. Concentration increases of inflammatory cytokines IL-18 and IL-1β were correlated with occurrence and development of T2DM patients complicated with CAS. |
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