| Part I The changes and clinical significance of levels of serum TNF-a, hs-CRP,Adiponectin,HMGBl in patients of Type 2 Diabetes Mellitus with Coronary Heart DiseaseObjective:To observe the changes of levels of tumor necrosis factor (TNF-α), high sensitivity C-reactive protein(hs-CRP),adiponectin(APN),high mobility group box chromosomal protein 1(HMGB1) in patients of Type 2 Diabetes Mellitus with Coronary Heart Disease,and to investigate the atherosclerotic pathogenesis of type 2 diabetes mellitus.Methods:324 cases were enrolled, consisted of A group (pure diabetic patients group, 50 cases), B group(pure coronary heart disease group,64 cases),C group (diabetes mellitus with coronary heart disease group,90 cases), and control group (normal persons,120 cases).The clinical characteristics were collected for each patient. Serum fasting plasma glucose(FPG),glycation hemoglobin Alc(GHbAlc),lipid profiles levels were measured.Levels of serum hs-CRP,TNF-a,APN and HMGB1 were detected by enzyme-linked immunosorbent assay (ELISA).Results:Levels of serum total cholesterol(TC), triglyceride (TG),low density lipoprotein cholesterol (LDL-C) were significantly higher in A group,B group and C group than that of in normal control group(all P<0.05orP<0.01).Levels of serum triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) were significantly higher in C group than that of in A group (all P<0.05).There were no significant differences in levels of serum HDL-C among four groups(all P>0.05).Levels of FPQGHbAlc,hs-CRP;TNF-α,HMGB1 were significantly higher in A group and C group than that of in normal control group (all P <0.01), serum adiponectin levels in A group and C group were markedly lower than that of in control group (allP<0.01).Levels of hs-CRP,TNF-α,HMGB1 were significantly higher in C group than that of in A group and B group (all P<0.01), serum adiponectin levels in C group were markedly lower than that of in A group and B group (all P< 0.01).Levels of hs-CRP,TNF-α,HMGB1 were significantly higher in B group than that of in A group (all P <0.01), serum adiponectin levels in B group were markedly lower than that of in A group (P<0.01).In C group, serum TNF-α, hs-CRP and HMGB1 levels gradually increased along with the elevation of the number of the coronary artery pathological change, while serum APN levels gradually decreased. In comparison, there was no significant difference in levels of hs-CRP between single and double branch lesions group(P>0.05),but there were significant differences in levels of the remainder indexs (such as TNF-α, HMGB1 and Gensini’s degree integral) in C group between the random two subgroups (P<0.01).In C group,there were positive correlation between FPG and hs-CRP and TNF-α (separately,r=0.909,0.979,P<0.01),between GHbAlc and hs-CRP and TNF-α (separately,r=0.884,0.973,P<0.01), between HMGB1 and TNF-α and hs-CRP (separately,r=0.971,0.970,P<0.01),there were negative correlation between adiponecin and TNF-a and hs-CRP (separately,r=-0.979,-0.908,alLP<0.01).Pearson correlation analysis showed that Gensini score was positively correlated to HMGB1 and TNF-α and hs-CRP (separately,r=0.869,0.697,0.598,allP<0.01)Conclusions:The simultaneous expression levels of TNF-a,hs-CRP,adiponectin and HMGB1 could participate in the mechanism of atherosclerotic immune pathological in type 2 diabetes mellitus complicated with CHD, may have a guiding significance to the monitoring of pathogenetic condition and treatment.Part Ⅱ:Distribution of NURRl in peripheral blood mononuclear cells from patients with Type 2 DiabetesObjective:To investigate the expression levels of nuclear receptor subfamily 4 group A member 2(NURR1) in peripheral blood mononuclear cells (PBMCs), and explore its correlation with inflammatory cytokine productionsMethods:40 healthy subjects and 40 patients with newly diagnosed Type 2 Diabetes (T2DM) were recruited from Wuhan University Zhongnan Hospital. Physical examination were given, including height, weight and blood pressure. We calculated body mass index (BMI) and lab tests, including fasting glucose (FBG), fasting insulin (Fins), total cholesterol(TC), triglyceride(TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), C-reactive protein (CRP) and homeostasis model assessment (HOMA-IR) were given. Blood plasma was collected for detection of tumor necrosis factor α (TNF-α), interleukin (IL-6). Density gradient centrifugation method was used for collecting PBMCs. NURR1 expression levers were detected by western-blot and real-time PCR respectively in PBMCs from healthy subjects and T2DM patients. The association between NURR1 mRNA lever and HOMA-IR, TNF-α, IL-6 were disclosed. Gsk-3β phosphorylation expression levels in PBMCs from healthy subjects and T2DM patients were detected through western-blot.Results:There was no significant difference in age, sex, ALT, BUN, Cr, BMI, TC, LDL-C between the patients and healthy controls. The levels of plasma FBG、Fins、HOMA-IR、 TG、、CRP、TNF-α and IL-6 were higher absolutely compared to controls, and lower lever of blood HDL-C lever, Gsk-3β phosphorylation and NURR1 mRNA expression levels in PBMCs were relatively lower in T2DM patients. Pearson’s correlation analysis showed that NURR1 mRNA expression level positively correlated with HDL-c (r=0.45, p=0.005), and negatively correlated with IL-6 (r=-0.353, p=0.030), TNF-α (r=-0.426, p=0.008), FINS (r=-0.485, p=0.002) and HOMA-IR (r=-0.424, p=0.008). HOMA-IR which was used to evaluate the insulin resistance was positively correlated with TNF-α mRNA (r= 0.384, p=0.017), and IL-6 protein (r=0.36, p=0.027).Conclusions:The expression of NURR1 and Gsk-3β phosphorylation were decreased in PBMCs from newly diagnosed T2DM patients. The expression of NURR1 was negatively correlated with HOMA-IR, TNF-A and IL-6.Part Ⅲ:Impact of high lever of fat or glucose on NURR1 expression in peripheral blood mononuclear cells from healthy populationObjective:To investigate the impact of high level of fat or glucose stimulation on NURR1 expression in PBMCs from healthy population.Methods:Healthy subjects were recruited from Wuhan University Zhongnan Hospital. PBMCs from healthy subjects were collected through Density gradient centrifugation method and cultured with 250 or 500 μM palmitic acid or 16.7 or 33.3 mM glucose for 45 min and 24h. cells were collected and proteins were extracted. NURR1 expression was detected using western blot. PBMCs from healthy people, cultured with 250 or 500 μM palmitic acid for 24h. TNF-α and IL-6 were detected by western blot.Results:The levels of TNF-α and IL-6 increased in 250μM induced PBMCs compared to controls (0.85 ± 0.14vs 0.56 ± 0.09 p<0.05,0.90 ± 0.09 vs 0.49 ± 0.14 p=0.01), even more in 500μM P induced PBMCs(1.14±0.11 vs 0.56±0.09 p<0.01、1.08±0.11 vs0.49± 0.14 p<0.01). The expression of Nurrl in PBMCs treated with 16.7mM glucose for 45min was not significantly different from that of the control group (0.85 ± 0.23vs 1.17 ± 0.33, p>0.05); compared with the control group, the expression of Nurrl could be inhibited in PBMCs treated with 33.3mM glucose for 45 minutes (1.17 ± 0.33 vs0.54 ± 0.17 p=0.04), and the expression of Nurrl was inhibited by 16.7mM or 33.3mM glucose for 24 hours (1.21 ± 0.23 vs0.52 ± 0.22, p<0.05; 1.21 ± 0.23 vs0.38 ± 0.15, p<0.01).A similar pattern of reduced levels of Nurrl expression was observed in PBMCs following treatment with different concentrations of palmitic acid for varying periods in vitro.ConcIusions:High-level fat and glucose on human PBMCs induced TNF-α and IL-6 expression, simultaneously, inhibited NURR1 expression. High glucose or palmitic acid inhibited the Nurrl expression in PBMCs in a dose- and time-dependent manner. |
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