The Relationship Of Inflammatory Mediators And Periodontal Parameters In Type2 Diabetes Patients With Chronic Periodontitis

Background:The relationship between chronic periodontits and type 2 diabetes is most likely bidrectional. And the inflammatory mediators play an important role in mechanism of the interaction. Periodontal inflamed burden may contribute to the systemic inflammatory condition through inflammatory mediators, which may have an effect on the systemic inflammatory condition and immunity reactive function. The genetic factors could contribute to the variation of the inflammatory mediator concentrations. The genetic variation in cytokine production may explain some of the differences among individuals in the initiate and the progression of the complex diseases.CRP may act as a classical member of the innate immune system. CRP is induced by proinflammatory cytokines like IL-6 in the liver and is able to activate the complement and play an important role in inflammatory process. A persistently raised CRP level was associated with advanced periodontal disease. Furthermore, it is reported that elevated levels of CRP can serve as a risk factor for the development of type 2 diabetes.The aims of this cross-sectional study are to evaluate the effect of severity of the local periodontal inflammation on systematic inflammatory mediators and metabolic levels in individuals with periododntitis and type 2 diabetes, and the influence of the IL-6 and CRP polymorphism on baseline CRP and glucose metabolic levels.The contents of this study include:①The relationship between periodontal inflammatory burden and systemic inflammatory markers in patients with periododntitis and type 2 diabetes;②The association between periodontal inflammatory burden and metabolic levels in patients with periododntitis and type 2 diabetes;③The influence of the IL-6-572(rs 1800796) and CRP1846(rsl205) polymorphism on baseline CRP;④The influence of the IL-6-572 and CRP 1846 polymorphism on HbAlc levels.Materials and Methods1、The relationship among the local periodontal inflammatory burden, systemic inflammatory mediators and glycemic metabolic levels in individuals with periododntitis and type 2 diabetes(cross-sectional study on clinical parameters)(1) Study design and samplingA total of 142 subjects, aged 32-84 years old,76 males and 66 females, with type 2 diabetes and periodontitis were collected at five diabetes centers in Guangzhou City (China) between September 2008 and June 2010. Patients recruited were diagnosed with diabetes for more than 1 year and receive periodontal treatment in the past 12 months. Exclusion criteria were:presence of systemic disease which will bring the threat to periodontal conditions, like atherosclerosis and retinopathy; had antibiotics treatment within the last 4 weeks; presence of acute infection other than periodontitis and CRP>10mg/L; women with pregnancy, lactation or planning to have a baby; using any medication known to affect the secrum inflammatory markers or immune cells; someone refuse to join the study. All of participants were asked to complete a questionnaire full-mouth periodontal assessment and blood sample analysis. The questionnaire gathered their demographic information, diabetes treatment and family history. Periodontal examinations, including visible plaque index, probing depth, bleeding on probing, gingival recession and clinical attachment level, were recorded. Blood analysis included glycated hemoglobin(HbAlc), fasting glucose, high-sensitivity C-reaction protein(hsCRP) and lipid profiles.(2) The relationship between periodontal inflamed surface area and systemic biomarkers in type 2 diabetics patients with chronic periodontitisAccording to the method reported by Nesse, periodontal inflamed surface area(PISA) was calculated from probing depth(PD), bleeding on probing(BOP), gingival recession(GR) and clinical attachment level(CAL). Subjects were divided into two groups according to medians of PISA. Multiple linear regression analyses were used to calculate the dose-response association with PISA to hsCRP levels, after controlling for the confounding factors. Multivariate logistic regression analysis was further used to assess the potential risk factor by odd ratios (ORs), using hsCRP (>50th percentile) as dependent variables.The results show there were also no significant difference between groups in age, BMI, duration of type 2 diabetes mellitus, sex, diabetic family history, alcohol consumption, regular physical exercise, smoke states and stress states(P>0.05). Subjects having greater degree of PISA had a significant higher level of hsCRP, HbAlc, TC, HDL-C and LDL-C, but not the levels of TNF and TG The multiple linear regression anslysis show that PISA (P<0.0001,β=0.002,95%CI=0.001-0.002, r2=6.3%) was a significant factor has the dose-response relationship with hsCRP base levels, adjusting for HbAlc, TC, HDL-C and LDL-C. An increase of PISA with 500mm2 was associated with a 1.0mg/L increase in plasma hsCRP, independent of the influence of other factors. In multiple logistic regression models using hsCRP (50%) as the dependant variable, PISA, BMI, stress states, FPG and duration of diabetes became significant predictors for serum hsCRP, in which PISA (P=0.020, OR =1.002), BMI(P=0.009, OR=1.215), stress states(P=0.026, OR=3.791) and FPG(P=0.011, OR=1.318) are the risk factors for elevated hsCRP levels, after adjusting for possible confounders.(3) The relationship between periodontal inflamed surface area and glycaemic metabolic level in type 2 diabetics patients with chronic periodontitisMultiple linear regression analyses were used to calculate the dose-response association with PISA to HbAlc levels, after controlling for the confounding factors. Multivariate logistic regression analysis was further used to assess the potential risk factor by odd ratios (ORs), using HbAl c (>7.0%) as dependent variables.Duration of type 2 diabetes mellitus(P=0.032,β=0.041), HDL-C (P=0.009,β=-0.621) and PISA(P=0.026,β=0.001) have the dose-response relationship with HbAlc (r2=9.0%). In the model of HbAlc (>7.0%), FPG was a significant high risk factor for HbAlc levels P<0.0001, OR=1.864. PISA with OR=1.003, P<0.05, and duration of type 2 diabetes mellitus (P=0.026, OR=1.098) is also an significant variables in the regression model.2、The influence of the IL-6-572 and CRP1846 polymorphism on baseline CRP and glucose metabolic levels in type 2 diabetes patients with chronic periodontitis(1)Genotype detection of IL-6-572G/C(rs 1800796)and CRP 1846C/T(rs1205)The buccal swab samples were collected. Genomic DNA was extracted from buccal swabs by Chelex-100 or extracted from blood by QIAamp DNA Blood Kits. The DNA concentration was determined by ultraviolet (UV) spectrophotometry. Genotyping of IL-6-572G/C and CRP1846C/T was performed by the polymerase chain reaction restriction fragment length polymorphism (PCR-RELP) method. To confirm the results of the PCR-RELP, the PCR products of two or more samples were randomly selected to sequence directly with two-blind method.There are 142 samples tested and 129 were successful, of which 13 samples failed. The genotypes at two loci were in Hardy-Weinberg equilibrium (P>0.05). The locus IL-6-572:the GG genotype frequency was 5.4%, GG was 35.4%,CC was 59.2%. The C allele frequency was 23.1%. The detection of CC/CT/TT genotype of CRP1846 locus was 22.7%/45.5%/31.8%. The C allele frequency was 45.5%.(2) Influence of IL-6-572C/G polymorphism on CRP baseline levels in type 2 diabetes patients with chronic periodontitisThe study subjects were divided into two groups by hsCRP median. The comparisons of IL-6-572G/C and CRP1846C/T alleles and genotypes distributions were carried out by chi-square test. The effect of genotypes were further analyzed through logistic regression models with the adjustment for stress status, BMI, duration of type 2 diabetes and PISA. A level of P>0.05 was accepted as being statistically significant. Statistics were calculated by SPSS 13.0.There was no significant effect on CRP levels of IL-6-572 locus. The G allele frequency was significantly higher in CRP high level group (P=0.046, OR=1.816, 95%CI 1.006-3.278). And both the distribution of CRP 1846 genotype and allele frequency show no significant difference between two group. In the logistic regression model, the results showed IL-6-572 was a risk factor for elevated hsCRP levels (P=0.011, OR=3.075,95%CI:1.300-7.276), after controlling for BMI, duration of type 2 diabetes mellitus, PISA and stress. IL-6-572 G allele may be associated with the high level of hsCRP in patients with periodontitis and type 2 diabetes. Meanwhile, BMI,stress status and PISA may be the risk factors for high level of hsCRP (P<0.05,OR>1). And there are no significant P-value for CRP1846 locus. (3) Influence of IL-6-572C/G polymorphism on glucose metabolic levels in type 2 diabetes patients with chronic periodontitisThe study subjects were divided into two groups according to the level of HbAlc with the threshold of 7.0%. The comparisons of IL-6-572G/C and CRP1846C/T alleles and genotypes distributions were carried out by chi-square test. The effect of genotypes were further analyzed through logistic regression models with the adjustment for age, FPG, HDL-C, duration of type 2 diabetes and PISA. A level of p>0.05 was accepted as being statistically significant. Statistics were calculated by SPSS 13.0.Both the distribution of IL-6-572 genotype and allele frequency show no significant difference between two groups with different HbAlc, after controlling for BMI, gender, smoke status, alcohol consumption and regular physical exercise(P >0.05). The distribution of CRP1846 genotype also showed no significant difference between two groups. In the other side, CRP1846 T allele frequency of low HbAlc group was significant higher than the other (P=0.041, OR=0.598,95%CI:0.365-0.979). In the logistic regression model, using the HbAlc(>7.0%) as the dependent variable, the results showed neither of these two locus were significant variables in the model (P=0.162, OR=2.033,95%CI:0.751-5.500和P=0.688). after controlling for age, FPG, HDL-C, PISA and duration of type 2 diabetes mellitus. Meanwhile, FPG and PISA may be risk factors of elevated HbAlc levels.(4) The influence of CRP1846 genotype on baseline CRP and glucose metabolic levels in patients with type 2 diabetes patients with chronic periodontitisAccording to the genotype of rs 1205, the subjects were divided into three groups. Multivariate logistic regression analysis was performed using hsCRP (>50th percentile) and HbAlc (>7.0%) as dependent variables, respectively. In the regression model of hsCRP (>50th percentile), the independent variables include BMI, stress status, duration of type 2 diabetes. mellitus, PISA, HbAlc and mean CAL. In the model of HbAlc (>7.0%), the independent variables include age, BMI, duration of type 2 diabetes mellitus, PISA, hsCRP, FPG, HDL-C and mean CAL. A level of p>0.05 was accepted as being statistically significant. Statistics were calculated by SPSS 13.0.The subjects were divided by the genotype of CRP1846. There was no significant difference among the groups of all the study variables (including continuous variables, they are, age, BMI, FPG, HbAlc, TC, HDL-C, LDL-C, TG, duration of type 2 diabetes mellitus, TNF-a, hsCRP, PISA, mean PD,mean CAL; ordinal variables, they are, gender, smoke status, stress status, alcohol consumption, regular physical exercise and diabetic family history), except for BMI. That is to say, the basic conditions of these three group are almost balanced.In the three different logistic regression models appealling to people with different CRP1846 genotypes, with hsCRP(>50th percentile) as the dependent variables, we can see the predicting factor of hsCRP level was different along with the genotypes. For CRP1846 CC genotype, stress status may be the risk factor for the high level of baseline hsCRP (P=0.043, OR=55.862). For CRP1846 CT genotype, both the stress status and PISA may be the risk factors, with P=0.036, OR=6.669 and P=0.019, OR=1.004,respectively. For TT genotype, there find no significant variables in our group.In the three different logistic regression models appealling to people with different CRP 1846 genotypes, with HbAlc (>7.0%) as the dependent variables, we can see the predicting factor of HbAlc level was different along with the genotypes. For CRP1846 CC genotype, there find no significant variables in our group. For CRP 1846 CT genotype, both FPG and duration of type 2 diabetes mellitus may be the risk factors, P=0.002, OR=1.784 and P=0.034, OR=1.166, respectively. For TT genotype, FPG may be the only risk factor. Conclusion1、An positive dose-response relationship exists between PISA and hsCRP levels. An increase of PISA with 500mm2 was in relation of a 1.0 mg/L increase of hsCRP, adjusting for the influence of the other factors.2、Peridontal inflamed surface area may be not an risk factor that influence the baseline hsCRP and HbA1c level directly. There may be something that serve as the mediator between the local periodontal inflammation, systemic inflammatory mediators and metabolic levels.3、Stress, BMI and FPG may be risk factors for the elevated baseline hsCRP.4、There is a dose-response relationship between HDL-C, PISA and duration of type 2 diabetes mellitus and HbAlc levels in patients with periodontitis and type 2 diabetes.5、IL-6-572 G allele may be risk factors for the elevated baseline hsCRP in patients with type 2 diabetes and periodontitis. Further studies were needed to confirm this point.6、Both the CRP 1846 genotype and allele frequency show no significant with elevated baseline hsCRP and HbA1c levels.7、The predicting factor of hsCRP and HbA1c levels was different with CRP 1846 genotype.8、For CRP 1846 CC genotype, stress status may be the risk factor for the high level of baseline hsCRP. For CT genotype, both the stress status and PISA may be the risk factors. For TT genotype, there find no significant variables in our group.9、For CRP 1846 CC genotype, there find no significant variables in our group. For CT genotype, both FPG and duration of type 2 diabetes mellitus may be the risk factors. For TT genotype, FPG may be the only risk factor.