| Objective: Type 2 diabetes mellitus(T2DM) is a kind of metabolic disease caused by genetic and environmental factors. Epidemiological survey shows that genetic factors play an important role in the pathogenesis of T2 DM and T2 DM with macroangiopathy. Searching for predisposing genes of T2 DM is the focal point of the study for genetic mechanism of T2 DM. Sever lesions and high mortality is feature of T2 DM with macroangiopathy. Its typical pathological change is atherosclerosis(AS). Recent studies have shown that NADPH oxidase is the principal source of is reactive oxide species(ROS), which is closely associated with the development of T2 DM and AS. P47 phox is a part of NADPH oxidase, which plays an important role in producing ROS. As a new tool of studying gene polymorphism, single nucleotide polymorphisms(SNP) have been widely used in identifying susceptibility genes for common diseases. At present, there is no report on the study of relationship between T2 DM and SNP of NADPH oxidase p47 phox gene. 8-hydroxy-2-deoxyguanosine(8-OHd G) has a constant steady state and high sensitivity in body, and has thought to be the ideal indicator to measure oxidative stress level. Therefore, the aims of our research are following: firstly, acquiring a further understanding of the relationship between SNP of NADPH oxidase p47 phox gene and T2 DM with macroangiopathy, and elucidating the molecular mechanism of T2 DM and T2 DM with macroangiopathy by measuring the genotypes in T2 DM patient; secondly, determining the phycological fuctions of NADPH oxidase by measuring serum 8-OHd G level in T2 DM subjects.Methods: According to the T2 DM diagnosis and classification standard proposed from the WHO in 1999, a total of unrelated 300 T2 DM patients was selected and further divided into macroangiopathy group and non macroangiopathy group. T2 DM without macroangiopathy group had 156 cases, including 90 male and 66 female, with age 51.6±10.0 years,course 4.2±1.3 years. T2 DM with macroangiopathy group had 144 cases, including 84 male and 60 female, with age 55.0±11.3 years,course 10.6±2.3 years. The 140 healthy individuals in control group were collected from the medical center of the third hospital of Hebei Medical University during the same period, including 85 male and 55 female, with age 53.0±9.1 years. After overnight fasting, a 4ml vein blood sample was collected and used to extract genomic DNA. Another was collected and used to measure the biochemical indicators and 8-OHd G. Serum and blood samples were frozen and stored at-20℃ until use.1 The measurement of clinical indicators: height, weight, waist circumfer- ence, hip circumference, systolic blood pressure(SBP), diastolic blood pressure(DBP), ankle blood pressure, body mass index(BMI), waist-to-hip ratio(WHR) and Ankle brachial pressure index(ABI) were measured and calculated. Circulating fasting blood-glucose(FBG), triglycerides(TG), total cholesterol(TC), low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol(HDL-C) were measured by automatic biochemical analyzer. Hb A1 c were measured in all subjects.2 The measurement of 8-OHd G: serum level was measured by enzyme linked immunosorbent assay(ELISA).3 Detection of G338 A polymorphism: genomic DNA extraction: a blood genomic DNA extraction kit extracted DNA from whole blood in EDTA, then the spectrophotometer determined the concentration and purity of DNA. Polymerase chain reaction method to detect p47 phox gene G338 A loci polymorphism: The forward primer 5’-TCCCAAGTGGTTTGACGG-3’ and reverse primer 5’-CCTCCTCTTTCTGGCTGTG-3’. The amplification was performed in a 25μl volume containing 2μl of DNA, 1μl of upstream primers, 1μl of downstream primers, 12.5 μl of PCR Premix, 8.5 ml of sterilized distilled water. Samples were initially denatured at 94℃ for 3min, denatured at 94℃ for 30 s, annealed at 54℃ for 30 s, extended at 72℃ for 40 s, and these four steps were repeated for 35 times. At a final step extended at 72℃ for 3min. Then the products were digested at 4℃ for 10 min.4 Statistical methods: using software SPSS19.0 version analyzed all data. Measurement data with normal distribution were expressed as mean ± standard deviation(?χ± s). Hardy-Weinberg equilibrium tested each gene frequency of representative group. Measurement data among groups was compared by ANOVA. Enumeration data among groups was compared by Chi-square test. The relevance analysis of 8-OHd G concentration and other factors was estimated by Spearman or Pearson correlation analysis. The contribution of risk factors for type 2 diabetic macroangiopathy was measured by logistic stepwise regression analysis.Results:1 There was no significant difference in constituent ratios of the age, gender and BMI between T2 DM group and control group. Smoking, WHR, SBP, DBP, TC, TG, LDL-C and FBG in T2 DM groups were significantly higher than that in control group(t=-1.563,-1.793,-2.474,-2.296,-1.952,-1.504,-2.261,-7.191 and-1.784,-2.410,-2.774,-2.576,-1.569,-1.752,-2.710,-8.675, all P<0.05), and HDL-C was lower than that in control group(t=2.183 and 4.224, both P<0.05). The patients with macroangiopathy had higher levels of course, moking, WHR, SBP, DBP, TG, LDL-C, FBP and Hb A1 c were higher than that in T2 DM without complications group(t=-2.681,-2.716,-3.328,-3.002,-2.863,-3.619,-2.299,-1.979 and-2.037, all P<0.05), and had lower levels of ABI and HDL-C than that in T2 DM without complications group(t=1.913 and 1.863, both P<0.05). In T2 DM without complications group, the serum 8-OHd G was 6.41±2.04ng/ml, which was higher than 4.16±2.11ng/ml in control group(t=-4.662, P < 0.01). And the 8-OHd G level of patients with diabetic macroangiopathy was 7.88±2.80ng/ml, which was higher than that in T2 DM without complications and control group(t=-3.215 and-6.354, both P<0.05).2 8-OHd G was positively correlated with course, smoking, TC, FBG and Hb A1c(r=0.324, 0.337, 0.172, 0.213 and 0.328, all P<0.05), and was negatively correlated with ABI and HDL-C(r=-0.383 and-0.173, both P<0.05). There was no significant correlation between serum 8-OHd G and other factors.3 In T2 DM macroangiopathy group and T2 DM without complications group, the frequencies of genotype GA+AA and allele A were 29.17%, 21.15% and 15.97%, 11.54%, which were significantly higher than 7.14% and 3.57% in control group(X2=23.020, 11.665 and 24.566, 13.070, all P<0.05). There was no significant difference in the distribution of allele A frequency and genotype GA+AA frequency between T2 DM with complications and T2 DM without complications.4 Blood pressure, BMI, SBP, DBP, FBG, Hb A1 c and 8-OHd G in GA+AA genotype group were higher than that in GG genotype group(t=-2.163,-2.090,-2.624,-3.011,-2.870,-1.989 and-2.037, all P<0.05) and ABI was lower(t=2.184, P<0.05). The morbidity of macroangiopathy group in GA+AA genotype group(49.4%) was higher than 28.7% of GG genotype(X2 = 13.32, P < 0.01).5 Logistic analysis showed that course, SBP, ABI, LDL-C and circulating 8-OHd G level were the independent risk factors of T2 DM macroangiopathy(β=0.227, 0.484,-1.162, 1.756 and 0.224, all P<0.05). Course, ABI, LDL-C, 8-OHd G and allele A were the independent risk factors of T2 DM macroangiopathy with blood pressure(β=0.534,-0.765, 1.467, 0.526 and 0.509, all P<0.05).Conclusions:1 Serum 8-OHd G of T2 DM patients was higher and T2 DM with macroangiopathy was highest. Serum 8-OHd G was an independent risk factor for T2 DM macroangiopathy. Therefore, oxidative stress may play an important role in T2 DM macroangiopathy.2 Serum 8-OHd G level of T2 DM patients may be closely related to the NADPH oxidase p47 phox gene G338 A polymorphism, and serum 8-OHd G level was high in T2 DM patients with allele A.3 P47 phox gene G338 A polymorphism may have no correlation with T2 DM macroangiopathy, but it was an independent risk factor for T2 DM macroangiopathy with blood pressure. |
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