| IntroductionAccording to World Health Organization, obese and overweight together with cardiovascular and cerebrovascular diseases, diabetes mellitus, and carcinoma, whose definite relationship with obesity and overweight have been proved, had caused nearly2.8million of the world’s adults’death, which enough attention is desperately in needed.Periodontitis, which has a dozen of complicated causes, characterized as localized chronic inflammation of the periodontium which can damage the dental supportive tissue and ultimately cause tooth loss. Chronic periodontits has the highest mobidity in Chinese adults and serves as the leading cause of tooth loss. Many literatures have indicated that systematic diseases such as metabolic syndrome, diabetes mellitus, cardiovascular disease can give rise to more severe and more rapidly destruction of periodontal support tissue. Meanwhile, uncontrolled periodontitis, can also play a negative role on systemic disease, which we dentists, physician together with the patients should attach more emphasis on.The majority of adipose tissue is consists of adipocyte and thin layer of connective tissue. Adipose tissue not only serves as an energy storage, but also an active endocrine organ. In the past, majority of researchers had an eye only on adipocytes, while now, more and more literatures concentrate on vascular stromal cells, neurons, macrophages, cause of their great importance during inflammation and cytokines secretion.Since1993, Hotamisligil noted inflammatory factor TNF-a increased in adipose tissue of obese individual which unprecedentedly put an linkage between obesity and inflammation. Later, variety of study have been made to certified that adipose tissue could secrete cytokines which could affect the whole body. This has conformity with the pathophysiology mechanism of diabetes mellitus, insulin resistance, atherosclerosis and metabolic syndrome which put cytokines to a key situation during pathogenesis and development progress of systematic diseases. Insulin resistance serves as one of the most important characteristics of T2DM, which means weaken cellular efficiency of insulin-inducing cell. Insulin β cell has to secrete more insulin to maintain its function towards peripheral tissue such as liver, muscle and adipose tissue. TNF-a and IL-1β are important inflammatory factors which could be potential contributor towards relevant disease. TNF-a and IL-1β could lead to insulin resistance and diabetes mellitus by damaging insulin β cells. Receptor antagonists and antibody of inflammatory factors can be effective in treatment of T2DM. TNF-a and IL-1β also can’t be ignore in the progress of peridontium damage during periodontitis. Concentration detection of TNF-a and IL-1β in gingival crevicular fluid in inflammatory gingiva shows that that of which increased compared with health site. After treatment, concentration of the inflammatory factors can be reduced. The severity of periodontitis has a positive correlationship with the local concentration of cytokines. Periodontitis has a correlationship with obesity, and uncontrolled periodontitis has a positive correlationship with increaing body mass index, uncontrolled glycated hemoglobin, and high level of fasting blood glucose, and plays a negative effect of periodontal therapy.This experiment is going to compare localized and serum inflammatory TNF-α and IL-1β level and compare its correlationship with insulin resistance hoping to explore the proper mechanism of chronic periodontitis towards the inflammatory degree of the entire body and insulin resistance.Chapter I Establishment of Periodontitis Composite Obese Rat ModelsObjectivesAll of the32experimental rats were equally allocated into four groups randomly, namely control group (C), periodontitis group (CP), obese group (OB) and periodontitis composite obese group(CP+OB).MethodsAll of the32experimental models were equally allocation into four groups randomly, namely control group (C), periodontitis group (CP), obese group (OB) and periodontitis composite obese group (CP+OB).(1) Model Construction1) C group:control group, imposing no treatment factors;2) CP Group:Chronic periodontitis group.12weeks after birth, anesthesia with10%chloral hydrate solution(0.3ml/100g·bw), intraperitoneal injection. Ligature first and second molar of both sides of the maxillary, place the ligature into the gingival sulcus, injected with periodontal pathogens:Porphyromonas Gingivalis, Pg ATCC33277; Hctinobacillus Actinomycetemcomitans, Ha ATCC29523; Prevotella Intermedia, Pi ATCC25611; Fusobacterium Nucleatum, Fn ATCC25586,109CFU/mL. Feed with normal diet; daily recorded rats normal condition; observe the silk thread, if slipped, replace it as soon as possible. It takes8weeks to fulfilled model construction. 3) OB group:obese group. Rat models subcutaneously injected with MSG3mg/g.bw,2nd,4th,6th,8th,10th day postnatal, in order to construct obese rat models;4) CP+OB group:constructed obese model same as mentioned above until12weeks then conducted methods inducing periodontitis as aforementioned until20weeks to fulfilled the periodontitis composite obesity rat models;(2)20week, execute, collected serum samples and periodontium tissue examples1) Index of periodontitis model:clinical manifestation of localized gingival inflammation; HE staining of tissue section and observation using microscope; micro-CT to examine and evaluate alveolar bone loss;2) Index of obese model:measure body mass, body length, calculate Lee’s index and examine fasting blood glucose, fasting insulin and homeostasis model assessment of insulin resistance(HOMA-IR);3) Index of chronic periodontitis composite obese model:composite index of chronic periodontits and obese models to decide whether the models are constructed successfully.ResultsMSG injected group exhibits central obesity, weight gain and high Lee’s index, high fasting blood-glucose, high fasting insulin and high HOMA-IR which altogether indicated successfully construction of obese rat models. Ligatured with localized injected with periodontal pathogens group shows clinical manifestation of localized inflammation, histological examination manifests inflammatory cells infiltration and alveolar bone resorption together with micro-CT result indicates bone height and bone mass reduction, demonstrated periodontitis models build accomplishment. And the CP+OB group express both of the two treatments’characteristics.Conclusion Subcutaneous injection of MSG toward neonatal rats could be a method to construct obese rat model, and the models can be constructed in12weeks. Ligatured molars and injected with periodontal pathogens to construct periodontitis models, and the expanding time is8weeks. MSG injection neonatally in combination with ligatured and injected periodontal pathogens on the12week could build periodontitis composite obese rat model, and the altogether requiring time is20weeks.Chapter II Expression of TNF-a and IL-1β in Serum and Correlationship with Insulin ResistanceObjectiveDetect the expression of inflammatory factors TNF-a and IL-1β in C group, OB group, CP group and CP+OB group seperately in serum in order to analysis mechanisms of localized periodontal cytokines towards entirety inflammatory response of the whole body and the correlationship of inflammatory factors with insulin resistance.MethodsExecuted, obtained serum, then used ELISA to detect TNF-a and IL-1β expression in serum. Statistical analysis of the concentration and calculate insulin resistance index. The experimental data are indicated as mean(x)±standard deviation(s). Statistical analysis was documented using SPSS(?) v13.0Softwear (SPSS Inc., Chicago, USA). Factorial design variance analysis was also used to analysis the main effects and interaction of the factors. The correlation analyses were using Pearson method.ResultsELISA detection indicates that, compared with control group, serum TNF-a increasing significantly in OB (t=5.680, P=0.000) and CP group (t=2.449, P=0.028); serum IL-1β also increasing significantly in OB (t=2.790, P=0.000) and CP group (t=3.417, P=0.028). Compared with OB group, TNF-a (t=10.049, P=0.000) and IL-1β(t=11.577, P=0.000) in CP+OB group increasing significantly; Also, compared with CP, TNF-a (t=9.873, P=0.000) and IL-1β(t=10.142, P=0.000) level also increasing significantly in CP+OB group. Compared with OB and CP group, that of OB+CP group increasing significantly. Periodontitis and obese treatment measures could interact towards inflammatory factors raising(F=28.061, P=0.000). Results of HOMA-IR are as followed, OB+CP group>OB group>CP group>C group, serum TNF-a (r=0.854) and IL-1β(r=0.730) has a positive correlation with HOMA-IR.ConclusionsObese and periodontitis individually can contribute to the serum inflammatory factors increasement, and obese and periodontits treatment factors could interaction towards TNF-a and IL-1β raising. Increasing TNF-a and IL-1β have a positive correlation with insulin resistance.Chapter III Expression of TNF-a and IL-1β in Periodontal TissueObjectsDetect expression of inflammatory factors TNF-a and IL-1β in C, OB, CP and CP+OB group in periodontal tissue in order to analysis mechanisms of pathological obese individual’s respond towards localized periodontal inflammation stimuli.MethodsExecuted, using immunohistochemical staining technique to detect TNF-a and IL-1β expression in periodontal tissue of C, OB, CP, CP+OB group. Image Pro Plus6.0system was used to capture photos and then calculate the optical density. Statistical analysis of the experimental data are indicated as mean(x)±standard deviation(s). Statistical analysis was documented using SPSS(?) v13.0Softwear (SPSS Inc., Chicago, USA).Factorial design variance analysis was also used to analysis the main effects and interaction of the factors.ResultsImmunohistochemical staining results shows that TNF-a and IL-1β could present in fibroblasts, epithelial fibrosis and only a few numbers of epithelial in CP group and CP+OB group which under microscope brown particles as positive expression. CP group shows significant high average optical density of TNF-a (P=0.000)and IL-1β(P=0.002), while TNF-a (P=0.078) and IL-1β(P=0.467) of OB group compared with C group, just the opposite, with no statistical difference. CP+OB group indicates a significant low density of TNF-a (P=0.000) and IL-1β (P=0.000) than CP group.ConclusionsLocal periodontal inflammation could increased the TNF-a and IL-1β expression, while in obese individual, localized inflammation decreased may due to innate immune system damage caused by obesity. |
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