牙周炎对肥胖大鼠脂肪组织炎症状态的影响及其促胰岛素抵抗机制初探
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摘要
研究背景
肥胖作为一种亚健康状态,与机体一些重要的生理指标密切相关,如血脂,血压,胰岛素敏感性等,它是促进机体发生某些慢性疾病如2型糖尿病(T2DM)和动脉粥样硬化的重要病理特征。肥胖状态下,机体最早发生的,也是最重要的病理改变就是脂肪组织的增生肥大,尤其是内脏脂肪。目前已知,脂肪组织不仅仅是一个能量储存器官,而且是具有活跃分泌功能的内分泌器官。脂肪组织可以分泌大量的细胞因子,这些因子统称为脂肪细胞因子,它们作为炎症介质和信号分子参与机体的能量代谢,免疫调控和炎症介导
在众多脂肪细胞因子中,肿瘤坏死因子α (tumor necrosis factor α, TNF-α)与白介素6(Interleukin6, IL-6)最早受到关注,并且被证实与胰岛素抵抗和T2DM的病理机制有关。研究证实,在健康个体以及患有T2DM的个体体内,脂肪组织中TNF-α的浓度均与空腹血糖,胰岛素,甘油三酯的浓度以及胰岛素抵抗有关。IL-6的表达在肥胖个体的脂肪组织中也明显升高,并且是健康个体的10倍。脂肪组织是肥胖状态下血清IL-6浓度的一个主要来源,30%的血清IL-6来自于脂肪组织。TNF-α和IL-6的联合作用,增加了脂肪细胞中脂质的溶解和脂肪酸的氧化,对脂肪组织的这种局部调控作用,最终促进了外周组织的胰岛素抵抗[7-10]。近年来,更多新的脂肪细胞因子被发现并进行研究,其中瘦素和脂联素受到的关注最多。瘦素经脂肪细胞分泌,进入血循环,主要作用于下丘脑,但在胰岛素敏感的外周靶器官如肝脏和骨骼肌中发现有瘦素受体的表达,说明瘦素参与了外周胰岛素敏感性的调控。瘦素可通过自分泌和神经内分泌途径两种不同方式调控胰岛素信号[12]。相反,脂联素是所有脂肪细胞因子中唯一一个具有抗炎,保护血管内皮功能和胰岛素增敏作用[13-18]的细胞因子。脂肪细胞因子因此成为了联系脂肪组织与全身病变,尤其是胰岛素抵抗的关键纽带。
牙周炎是发生在口腔内最常见的炎症性疾病,主要表现为牙周局部软组织的破坏和骨组织的吸收[19]。大量流行病学研究证实牙周炎与肥胖有关,纵向临床研究也表明牙周炎的发展与体重指数(body mass index, BMI)的增加有关,并且使糖尿病患者糖化血红蛋白的控制不良[20-241。在牙周炎,肥胖和T2DM之间,慢性炎症状态成为了其共同的病理机制。
然而,牙周炎的局部炎症反应是否能造成机体胰岛素抵抗的发生发展,或者在机体本身已存在病变如肥胖的情况下,牙周炎是否能加重机体的全身性的炎症状态,牙周炎参与胰岛素抵抗的调控机制是否与脂肪细胞因子有关,这些问题都尚未得到解决,而且人体学研究也无法完成这些研究。因此,我们采用建立疾病动物模型的方法,研究牙周炎对机体炎症状态的影响,以及这种影响足否与机体的胰岛素有关,从而揭示牙周炎与T2DM病变发生发展的关系,为临床治疗和预防提供理论依据。
第一章牙周炎合并肥胖大鼠模型的建立
目的
通过谷氨酸钠(MSG)诱导建立肥胖大鼠模型,牙周局部结扎联合涂菌建立牙周炎模型；检测肥胖大鼠的Lee's指数,血压和血糖水平,评估大鼠的肥胖状态；Micro-CT扫描大鼠颌骨样本并进行三维重建,计算牙周结扎造成的线性和体积骨丧失量；HE染色评估牙周组织的病理改变。
方法
(1)80只新出生的雄性Sprague-Dawley (SD)大鼠随机分为4组(n=20)：牙周炎联合肥胖组(OB+CP),肥胖组(OB),牙周炎组(CP)和正常对照组(C);
(2) OB+CP组与OB组的新生大鼠在出生后的第2,4,6,8,10天分别皮下注射MSG诱导肥胖,于12周龄时检测体重,体长和血压变化,取尾静脉血测定空腹血糖浓度。判定肥胖模型成功建立的大鼠进入牙周炎模型的建立；
(3) OB+CP组与CP组的大鼠在局麻下结扎双侧上颌第一,第二磨牙结扎处涂布四种牙周致病菌的混悬液,诱导实验性牙周炎8周；
(4)20周龄时,处死所有实验大鼠,采集血清和组织样本,颌骨样本采用Micro-CT扫描,EDTA脱钙和HE染色。
结果
本实验通过MSG药物诱导新生大鼠,使其在成年期后表现出向心性肥胖,血压升高,血糖异常等症状和体征,成功建立肥胖大鼠模型。采用丝线结扎双侧上颌磨牙并涂布牙周致病菌,经8周的诱导,成功建立慢性牙周炎模型。两种疾病模型在SD大鼠体内成功复合。Micro-CT的三维重建显示,结扎的牙周组织表现出明显的线性和体积骨丧失量；HE染色切片显示牙周组织附着丧失明显,胶原纤维破坏,牙周袋底炎症细胞浸润,肉芽组织形成,牙槽骨吸收明显。
结论
采用MSG诱导新生大鼠,可成功建立成年期肥胖大鼠模型,且该模型鼠表现出某些前糖尿病状态的体征,该方法简单,易行,重复性好,模型稳定。丝线结扎联合涂菌可诱导大鼠典型的牙周组织破坏,病理改变明显,慢性炎症状态维持稳定。
第二章牙周炎对大鼠内脏脂肪组织炎症状态的影响
目的
研究牙周炎对肥胖大鼠和非肥胖大鼠诱导内脏脂肪组织的形态学改变以及炎症状态变化的影响,探讨牙周炎和肥胖对脂肪组织炎症的联合作用,为牙周炎参与调控肥胖相关性疾病的病理机制提供理论基础。
方法
四组大鼠的内脏脂肪组织经福尔马林固定后,制成6um的切片,分别用于HE染色,甲苯胺蓝染色和CD68/CD14的免疫组化染色。软件分析四组大鼠脂肪细胞个数和直径的变化,血管基质成分的增生以及CD68/CD14标记的巨噬细胞的浸润状态。
结果
四组大鼠脂肪组织的HE染色结果显示,和CP组与C组相比,OB+CP组与OB组大鼠的脂肪细胞数目增加,有显著性差异(P<0.05),但OB+CP组与OB组之间(t=0.503,P=0.616)以及CP组与C组之间(t=0.215,P=0.830)的差别无统计学意义。OB组与OB+CP组的脂肪细胞直径明显大于C组和CP组,差别有统计学意义(P<0.05)；但C组与CP组(t=0.480,P=0.632),OB组与OB+CP组之间无统计学差异(t=0.773,P=0.441)。甲苯胺蓝染色显示OB+CP组大鼠脂肪组织中的血管基质部分出现明显的增生扩张和炎症细胞浸润,而CP组的变化不明显。比较四组大鼠脂肪组织中巨噬细胞的浸润,OB+CP组的CD68蛋白表达明显优于OB组(t=3.944,P=0.000),且CP组的蛋白表达量也高于C组,差别有统计学意义(t=8.332,P=0.000)。脂肪组织中CD14蛋白的表达分析显示,OB+CP组的平均光密度值高于OB组,有显著统计学差异(t=5.051,P=0.000)；CP组与C组的脂肪组织内偶见阳性染色细胞,CP组的平均光密度值高于C组,差别有统计学差异(t=3.382,P=0.000)。肥胖和牙周炎对大鼠脂肪组织中CD14蛋白的表达存在交互作用(F=7.481,P=0.007)。
结论
肥胖可造成大鼠脂肪细胞的个数增加,体积增大,血管基质扩张,炎症细胞浸润。而单纯牙周感染,并不能引起脂肪细胞发生明显的增生和肥大,但能使脂肪组织表现出轻度的炎症状态,表现为脂肪组织中的血管基质部分轻度增生,巨噬细胞浸润增加。在联合了肥胖后,牙周炎可使脂肪细胞发生更明显的增生和肥大,并且血管基质部分增生扩张更明显,巨噬细胞浸润更严重。基于以上证据,我们认为,慢性牙周炎症可引起脂肪组织的轻度炎症性改变,但不发生明显的形态学变化,但在机体本身存在炎症的前提下,牙周炎对脂肪组织炎症的加重具有促进作用。
第三章牙周炎对脂肪细胞因子表达的影响及其与胰岛素抵抗的关系
目的
检测肥胖和非肥胖状态下,牙周炎对瘦素和脂联素这两个关键的脂肪细胞因子在脂肪组织中表达的影响和血清浓度变化的影响,并分析这两个脂肪细胞因子的局部和全身水平与机体胰岛素抵抗的关系。
方法
采用免疫荧光的方法标记四组大鼠脂肪组织中瘦素和脂联素蛋白表达的变化,进行半定量分析；ELISA法检测大鼠血清中瘦素,脂联素和胰岛素的浓度；葡萄糖氧化酶法检测血清中空腹血糖的浓度。根据空腹胰岛素(mIU/L)和空腹血糖(mmol/L)浓度计算稳态模型下的胰岛素抵抗指数(homeostasis model assessment-insulin resistance, HOMA-IR),并采用Pearson相关分析评估瘦素和脂联素在脂肪组织和血清中的表达水平是否与机体的胰岛素抵抗有关。
结果
瘦素蛋白在OB+CP组大鼠脂肪组织中的表达量最高,显著高于OB组(t=2.046,P=0.044)；CP组大鼠脂肪组织中的瘦素蛋白表达高于C组,差别有统计学意义(t=5.237,P=0.000)；血清中的瘦素水平与脂肪组织中的表达水平一致,OB+CP组高于OB组(t=11.197,P=0.004),CP组高于C组(t=7.156,P=0.000),差别有显著的统计学差异。脂联素蛋白在C组大鼠脂肪组织中的表达量最高,而OB+CP组中最低,CP组的脂联素蛋白表达量显著低于C组(t=-3.661,P=0.000),OB+CP组低于OB组(t=-2.932,P=0.004),有显著统计学差异；但OB+CP组的血清脂联素浓度最高,高于OB组(t=3.624,P=0.003),而CP组的血清脂联素浓度略低于C组,但无显著性统计学差异(t=-1.323,P=0.207)。肥胖和牙周炎对脂肪组织中瘦素蛋白的表达(F=14.859,P=0.001)以及血清中瘦素(F=54.254,P=0.000)和脂联素水平(F=5.191,P=0.024)存在交互效应。
OB+CP组大鼠的空腹血糖浓度高于OB组,差别有显著统计学差异(t=7.694,P=0.000),CP组的血糖稍高于C组,差别有统计学意义(t=3.369,P=0.005)。胰岛素的血清浓度在OB+CP组比OB组稍高,但无统计学差异(t=0.658,P=0.521)；CP组血清中胰岛素浓度高于C组,有统计学差异(t=6.462,P=0.000)。肥胖和牙周炎对空腹血糖存在交互作用(F=17.699,P=0.000),但对胰岛素水平不存在交互作用(F=1.606,P=0.215)。
HOMA-IR的评分显示,从高到低依次为：OB+CP组>OB组>CP组>C组。其中,OB+CP组的HOMA-IR得分高于OB组,差别有统计学意义(t=5.662,P=0.000)；CP组的HOMA-IR得分高于C组,差别有统计学差异(t=9.992,P=0.000)。肥胖和牙周炎对大鼠HOMA-IR得分存在交互效应(F=12.340,P=0.002)。大鼠脂肪组织中瘦素的表达水平与HOMA-IR呈正相关(r=0.844,P=0.000),随着瘦素在局部脂肪组织中表达量的增多,大鼠机体的胰岛素抵抗水平增高；脂肪组织中脂联素蛋白表达水平与HOMA-IR呈负相关(r=-0.886,P=0.000),随着脂肪组织中脂联素蛋白表达的降低,胰岛素抵抗水平升高；大鼠血清中的瘦素(r=0.875,P=0.000)和脂联素(r=0.735,P=0.000)水平与HOMA-IR也呈正相关,随着瘦素和脂联素血清水平的不断升高,大鼠机体的胰岛素抵抗也加重。
结论
慢性牙周炎可诱导大鼠脂肪组织中瘦素和脂联素蛋白的表达失调,这种影响在合并了肥胖以后更为明显。瘦素和脂联素的血清浓度也受牙周炎的调控,并且牙周炎以及牙周炎联合肥胖所造成的瘦素和脂联素在局部和血清中水平的变化,与机体的胰岛素抵抗密切相关,说明牙周炎可能通过调控脂肪组织表达瘦素和脂联素,参与了机体胰岛素抵抗的发生发展。
第四章牙周炎对骨骼肌中脂肪细胞因子受体表达的影响
目的
研究各实验组大鼠骨骼肌中瘦素受体和脂联素受体1蛋白的表达,探讨牙周炎对胰岛素靶组织中脂肪细胞因子受体表达的调控以及在骨骼肌胰岛素抵抗中的作用。
方法
采用免疫组化的方法标记四组大鼠骨骼肌组织中瘦素受体和脂联素受体1蛋白的表达,并进行半定量分析。
结果
瘦素受体在C组和CP组的骨骼肌中广泛表达,染色呈强阳性,而在OB组和OB+CP组骨骼肌组织中表达量减少,表达强度减弱,尤其是OB+CP组中表达量最少。半定量分析显示CP组与C组,蛋白表达量无显著统计学差异(t=0.272,P=0.786)；但OB组中,瘦素受体的表达量显著高于OB+CP组,差别有统计学意义(t=-3.490,P=0.001)。肥胖和牙周炎对骨骼肌中瘦素受体的表达存在交互效应(F=4.341,P=0.039)。
OB+CP组大鼠骨骼肌中脂联素受体1蛋白的表达最弱,而且表达量明显低于OB组,差别有显著的统计学意义(t=-3.470,P=0.001)；CP组和C组骨骼肌切片中可见肌细胞的胞膜广泛表达脂联素受体1,但两组之间的蛋白表达量未见显著的统计学差异(t=0.543,P=0.598)。肥胖和牙周炎对大鼠骨骼肌中脂联素受体1的表达存在交互效应(F=6.364,P=0.013)。
结论
牙周炎的局部感染不能下调大鼠骨骼肌组织中瘦素受体和脂联素受体1的表达,但在肥胖状态下,牙周炎可促进大鼠骨骼肌中瘦素受体和脂联素受体1的表达明显下调,这可能造成骨骼肌组织中瘦素与瘦素受体,脂联素与脂联素受体1之间的结合障碍,从而影响骨骼肌组织中胰岛素的敏感性,有促进骨骼肌发生胰岛素抵抗的趋势。
Background:
Obesity, a subclinical state with multiple of lipid stored in adipose tissue, is closely correlated with important physiological parameters such as lipid profile, blood pressure and systemic insulin sensitivity. Obesity is a major contributor to systemic chronic disease, including type2diabetes militus (T2DM) and atherosclerosis. The first and the important pathological changes in obese state is the adipocyte hypertrophy (increased size) and hyperplasia (increased number). It is well established that the role of adipose tissue in obesity is not only a passive energy storage site, but it is understood that adipose tissue plays a much more active role as an endocrine organ. Adipose tissue can secrete a plenty of cytokines, so-called "adipokines". The adipokines can act as mediators and signal factors, and interact with energy metabolism, immune and inflammatory regulation.
There are a couple of adipokines that are of particular interest to researchers and TNF-α and IL-6are the ones of those. Previous reports shows that TNF-α and IL-6are closed related with pathological mechanism of insulin resistance and T2DM. Adipose tissue level of TNF-α is correlated with fasting plasma glucose, insulin, triacylglycerol concentrations and insulin resistance in patients with and without T2DM.The expression for IL-6is also increased in obese adipose tissue and is10-fold that in adipose tissue from lean individuals. In obese condition, adipose tissue is a major source of circulating IL-6concentrations, and contributes as much as30%of total body production. The combination of TNF-a and IL-6increases lipolysis and fat oxidation, which induce adipose tissue inflammation and followed by the development of systemic insulin resistance. Recently, leptin and adiponectin has been paid much attention as new adipokines and take part in the regulation of metabolism and inflammation. Previous experimental data indicated that leptin modulates glucose homeostasis. Leptin is secreted by adipocytes into circulation. Leptin acts mainly in the hypothalamus and has an impact on appetite, whereas expression of the leptin receptor has been observed in peripheral insulin-target tissues, including fat, liver and muscle, suggesting potential effects of leptin on peripheral insulin actions. In adipocytes, leptin inhibits insulin signaling by two different ways:as an autocrine signal and through neuroendocrine pathways. In contrast, adiponectin possesses anti-inflammatory or insulin-sensitizing properties. Thus, these adipokines can act as regulating mediators between adipose tissue and systemic disorders including insulin resistance.
Chronic periodontitis is one of the most widespread inflammatory diseases, characterized by inflammation and destruction of connective tissue attachment and alveolar bone. Published cross-sectional epidemiologic studies have suggested that periodontal disease, particularly periodontitis is associated with obesity. Finding from a longitudinal study has demonstrated a dose-response relationship between body mass index (BMI) and the development of periodontal disease. Periodontitis has been reported to adversely affect glycemic control in patients with diabetes mellitus and contribute to the development of diabetic complications. It is well established that inflammatory processes as common pathways involve in the pathogenesis of obesity, periodontitis and T2DM.
However, the mechanistic relationship between IR and periodontitis in obesity remains unclear. Whether chronic periodontitis can regulate the expression of key adipokines and result in the inflammation of adipose tissue and whether the combination of chronic periodontitis and obesity can exacerbate the inflammatory condition of adipose tissue and systemic insulin resistance. The establishment of experimental animal models is ideal to investigate the potential mechanism between periodontitis and T2DM and thus provide some useful theoretical evidence.
Part one
Establishment Experimental Periodontitis in Obese Rat Model
Objective
To establish obese rat model by monosodium glutamate (MSG) inducement and experimental periodontitis by ligature inducement with periodontal pathogens infection; To evaluate obese condition by investigating Lee's index, blood pressure, and fasting glucose; To evaluate periodontal infection by Micro-CT scanning and histological sections.
Methods
All animal's procedures were performed in accordance with the guidelines of the Southern Medical University Animal Care and Use Committee.
(1) Eighty natal SD male rats were randomly divided into four groups of20rats each, respectively:obese rats with periodontitis (OB+CP group), obese rats without periodontitis (OB group), normal rats with periodontitis (CP group) and normal rats without periodontitis (C group).
(2) In the OB group and OB+CP group, rats received subcutaneous injection of MSG (3mg/g of body weight) on alternate days for the first10postnatal days. The animals were weaned on the21st postnatal day and fed normally. At12weeks of age, body weight, body length, blood pressure, fasting glucose were investigated.
(3) At3months of age,3/0silk ligatures soaked with periodontal pathogens, were placed in subgingival position of the bilateral maxillary first and second molars for8weeks in the CP group and C group, whereas the control group were free of MSG-injection and ligation.
(4) At5months of age, rats were sacrificed under general anesthesia. Samples of the bilateral maxillary molar regions were resected from each rat and immediately fixed in10%neutral formalin for2days, and stored in70%ethanol for scanning by micro-CT. Maxillary samples were further decalcified with10%tetrasodium-EDTA equeous solution (PH7.0) for4weeks at4℃after scaning. The tissue blocks of periodontal samples were embedded in paraffin and sections (thickness:5um) were stained with hematoxylin and eosin (HE). Serum samples were also collected and stored in-80℃.
Results
In this study, we employed postnatal administration of MSG to rats and induced central obesity in adulthood. Ligatures soaked with periodontal pathogen were used to induce chronic periodontitis for8weeks. Rats in the OB+CP group and CP group showed apparent inflammatory cellular infiltration, collagenous fibers destruction and alveolar bone resorption, which were similar to features of human chronic periodontitis. This suggests that an ideal model of obesity and chronic periodontitis was established.
Conclusions
Administration of MSG can induce obese rat model. Besides, MSG-treated rats also have metabolic state of prediabetes with hypertention and hyperglycemia. This method is simple, effective and can be well repeated. In this obese state, ligature-induced periodontitis can result in typical periodontal destruction and sustain a low-grade chronic inflammation. The combination of periodontitis and obesity were successfully established in rat model.
Part two
The Effect of periodontitis on the Inflammation of Adipose Tissue in Rat Model
Objective
To investigate the effect of experimental periodontitis on the morphological changes and inflammation of adipose tissue, and to explore the addictive effect of periodontitis and obesity with the aim of providing theoretical basis on the interrelationships between periodontitis and systemic inflammatory disorders.
Methods
At the time of sacrifice, visceral adipose tissues were fixed in10%neutral formalin. After dehydration with increasing concentrations of ethanol, samples were incubated in xylenes and embedded in paraffin at60℃. Six-micron sections were cut and stained with HE, toluidine blue O or CD68/CD14immunohistochemistry. Quantification of adipocytes was done on HE-stained sections using Image-ProPlus software. Stromal-vascular fractions were observed on toluidine blue O-stained sections. Macrophage infiltration was detected on CD68/CD14immunohistochemical stained sections.
Results
Adipocyte size and amount were measured on HE-stained sections. The numbers of adipocytes in the OB+CP and OB groups were significantly higher than that in the CP and C groups (P<0.05). However, there were no significant differences in the number of adipocytes between the CP group and the C group (t=0.215, P=0.830) and that between the OB+CP group and the OB group (t=0.503, P=0.616). The cell diameter changes in the OB and OB+CP groups were striking. Enlarged cells were prevalent and mean diameters of adipocytes were167.21um and170.02um, respectively, which were higher than that in the CP and C groups (P<0.05). However, adipocyte diameters between CP and C groups were similar (t=0.480, P=0.632), and there was also no difference between OB+CP group and OB group (t=0.773, P=0.441).
Toluidine blue O-stained sections described the presence of SVF and nucleated stromal cells. The stromal multinucleated cells were greatly increased in the OB and OB+CP groups when compared to the CP and C groups. The presence of nucleated stromal cells in the CP group was similar to those in the C group.
In the visceral fat tissue, immunohistochemical analysis showed that the protein expression for CD68in the OB+CP was significantly higher than that in the OB group (t=3.944, P=0.000); and the expression in the CP group was also significantly higher than that in the C group (t=8.332, P=0.000). Furthermore, the protein expression for CD14in the OB+CP group was significantly higher than that in the OB group (t=5.051, P=0.000).And the difference between the CP group and the C group was statistically significant (t=3.382, P=0.000). A synergistic interaction between obesity and periodontitis was evident to the expression for CD14in the adipose tissue.
Conclusions
Obesity can induce adipocyte hypertrophy (increased size) and hyperplasia (increased number), enlarged stromal vascular fraction and macrophage infiltration in rat model. In contrast, experimental periodontitis can barely induce similar morphological changes in adipose tissue. However, periodontitis can result in slightly inflammatory disorder of adipose tissue, with characteristic of early enlarged stromal vascular fraction and several macrophage infiltrations. When combined with obesity, periodontitis can induce further hypertrophy and hyperplasia of adipocytes; furthermore, significant enlargement of stromal vascular fraction and macrophage infiltration can be observed in the OB+CP group. Based on the data, we accept that experimental periodontitis has a little impact on the morphological changes of adipose tissue but has remarkable effects on the inflammatory condition of adipose tissue. Periodontitis can exacerbate this inflammatory condition with the presence of obesity.
Part three
The Effect of Periodontitis on Protein Expression of Adipokines and the Relationships with Insulin Resistance
Objective
To investigate the impact of experimental periodontitis on the protein expression for leptin and adiponectin protein in adipose tissues and the concentrations in circulation; and further to explore the relationships between the expression of leptin and adiponectin locally and systemically and the systemic insulin resistance.
Methods
Immunofluorescence analysis of leptin and adiponectin expression of adipose tissue in rat model was employed and semi-quantitative method was used. Concentrations of serum leptin, adiponectin and insulin were determined using enzyme-linked immunosorbent assay (ELISA) kit; glucose levels were measured by glucose oxidase method. IR was calculated using the homeostasis model assessment (HOMA-IR), where IR=In[(Fasting glucose[mmol/l]×fasting insulin[mU/l])/22.5]. Pearson's correlation coefficient was used to determine the correlation between the levels of leptin and adiponectin and IR.
Results
In the visceral fat tissue, immunofluorescence analysis showed that the protein expression for leptin in the OB+CP, CP and OB groups were significantly higher than in the C group (P<0.05). Furthermore, the expression for leptin in the OB+CP group was significantly higher than that in the OB group (t=2.046, P=0.044). And the difference between the CP group and the C group was statistically significant (t=5.237,P=0.000). In contrast, the protein expressions for adiponectin in the OB+CP, OB and CP groups were decreased. Weak protein expression was detected in the OB+CP group and was significantly lower than that in the OB group (t=-2.932, P=0.004). In the CP group, the adiponectin expression level was decreased relative to C group (t=-3.661,P=0.000)
The mean levels of serum leptin in the OB+CP, OB and CP groups were significantly greater than that of the C group (P<0.01). Serum leptin in the OB+CP group was significantly higher than that in the OB (t=11.197, P=0.004). And serum leptin in the CP group was higher than in the C group (t=7.156, P=0.000). In adipose tissue, the expression for adiponectin was lower in the OB+CP group than that in the OB group (t=-2.932, P=0.004); and adiponectin in the CP group was also lower than that in the C group (t=-3.661, P=0.000). The circulating level of adiponectin in the OB+CP group was significantly higher than each of the three groups (P<0.01). In the OB+CP group, the levels of adiponectin were significantly higher than that in the OB group(t=3.624, P=0.003).However, adiponectin level was declined in the CP group, but there was no significant difference between CP group and C group (t=-1.323,P=0.207). A synergistic interaction between obesity and periodontitis was evident to the expression for leptin in the adipose tissue and the circulating levels of leptin and adiponectin.
Fasting glucose level in the OB+CP group was significantly higher than that in the OB group (t=7.694,P=0.000),and glucose level in the CP group was also higher than that in C groups (t=3.369,.P=0.005). In contrast, there was no difference between OB+CP and OB groups in the insulin levels (t=0.658, P=0.521) and the insulin level in the CP group was higher than that in the C group (t=6.462, P=0.000). A synergistic interaction between obesity and periodontitis was evident to the fasting glucose level (F=17.669, P=0.000).
HOMA-IR analysis indicated that MSG-induced obesity was associated with an increase in IR in the OB and OB+CP groups compared to non-obesity rats. Further, HOMA-IR was significantly greater in OB+CP group compared to the OB group (t=5.662,P=0.000). In contrast, there was an increase in IR in the CP group, and the difference between CP group and C group was statistically significant (t=9.992, P=0.000). Significant positive relationships were observed between IR and leptin expression levels in adipose tissue (r=0.844, P=0.000), serum leptin (r=0.875, P=0.000) and adiponectin level (r=0.735, P=0.000). And negative relationship was observed between IR and adiponectin level in the adipose tissue (r=-0.886, P=0.000)
Conclusions
Experimental periodontitis can affect expression for leptin and adiponectin protein directly in adipose tissues:upregulation of leptin and downregulation of adiponectin. When combined with systemic pathological changes such as obesity, periodontitis can exacerbate dysregulation of leptin and adiponectin in adipose tissue. Expremental periodontitis can also regulate circulating levels of leptin and adiponectin. When combined with obesity, periodontitis aggravates hyperleptinemia and hyperadiponectinemia and contribute to the development of IR.
Part four
The Effect of Periodontitis on Receptors Expression of Adipokines in Skeletal Muscle
Objective
To invesitgate the protein expression for leptin receptor and adiponectin receptor1protein in skeletal muscle, with the aim of exploring the effect of experimental periodontitis on adipokines receptors in insulin-targeting tissues and underlying mechanisms of skeletal muscle insulin resistance.
Methods
Immunohistochemical analysis of leptin receptor and adiponectin receptor1expression in skeletal muscle was employed and semi-quantitative method was used.
Results
In the skeletal muscle, the protein expressions for leptin receptor in the OB+CP, OB and CP groups were decreased, but in the C group, the protein expression were popular and strong positive. Fewer protein expression was detected in the OB+CP group when compared with OB group (t=-3.490, P-0.001). In the CP group, the protein expression level were decreased relative to C group but not significant difference (t=0.272, P=0.786). In contrast, the expression for adiponectin receptor1was greatly lower than that in the OB group (t=-3.470, P=0.001); and the expression in the CP group was similar to the C group (t=0.543, P=0.598). A synergistic interaction between obesity and periodontitis was evident to the expression for the leptin receptor and adiponectin receptor1.
Conclusions
Chronic low-grade inflammation induced by experimental periodontitis can barely down-regulate the protein expression for leptin receptor and adiponectin receptor1in the skeletal muscle. However, periodontitis can significantly down-regulate the protein expressions with the presence of obesity. This may contribute to the binding disruption of these two adipokines and their receptors in skeletal muscle samples, and may further decrease the insulin sensitivity, which accelerate the onset and development of insulin resistance in the skeletal muscle.
肥胖作为一种亚健康状态,与机体一些重要的生理指标密切相关,如血脂,血压,胰岛素敏感性等,它是促进机体发生某些慢性疾病如2型糖尿病(T2DM)和动脉粥样硬化的重要病理特征。肥胖状态下,机体最早发生的,也是最重要的病理改变就是脂肪组织的增生肥大,尤其是内脏脂肪。目前已知,脂肪组织不仅仅是一个能量储存器官,而且是具有活跃分泌功能的内分泌器官。脂肪组织可以分泌大量的细胞因子,这些因子统称为脂肪细胞因子,它们作为炎症介质和信号分子参与机体的能量代谢,免疫调控和炎症介导
在众多脂肪细胞因子中,肿瘤坏死因子α (tumor necrosis factor α, TNF-α)与白介素6(Interleukin6, IL-6)最早受到关注,并且被证实与胰岛素抵抗和T2DM的病理机制有关。研究证实,在健康个体以及患有T2DM的个体体内,脂肪组织中TNF-α的浓度均与空腹血糖,胰岛素,甘油三酯的浓度以及胰岛素抵抗有关。IL-6的表达在肥胖个体的脂肪组织中也明显升高,并且是健康个体的10倍。脂肪组织是肥胖状态下血清IL-6浓度的一个主要来源,30%的血清IL-6来自于脂肪组织。TNF-α和IL-6的联合作用,增加了脂肪细胞中脂质的溶解和脂肪酸的氧化,对脂肪组织的这种局部调控作用,最终促进了外周组织的胰岛素抵抗[7-10]。近年来,更多新的脂肪细胞因子被发现并进行研究,其中瘦素和脂联素受到的关注最多。瘦素经脂肪细胞分泌,进入血循环,主要作用于下丘脑,但在胰岛素敏感的外周靶器官如肝脏和骨骼肌中发现有瘦素受体的表达,说明瘦素参与了外周胰岛素敏感性的调控。瘦素可通过自分泌和神经内分泌途径两种不同方式调控胰岛素信号[12]。相反,脂联素是所有脂肪细胞因子中唯一一个具有抗炎,保护血管内皮功能和胰岛素增敏作用[13-18]的细胞因子。脂肪细胞因子因此成为了联系脂肪组织与全身病变,尤其是胰岛素抵抗的关键纽带。
牙周炎是发生在口腔内最常见的炎症性疾病,主要表现为牙周局部软组织的破坏和骨组织的吸收[19]。大量流行病学研究证实牙周炎与肥胖有关,纵向临床研究也表明牙周炎的发展与体重指数(body mass index, BMI)的增加有关,并且使糖尿病患者糖化血红蛋白的控制不良[20-241。在牙周炎,肥胖和T2DM之间,慢性炎症状态成为了其共同的病理机制。
然而,牙周炎的局部炎症反应是否能造成机体胰岛素抵抗的发生发展,或者在机体本身已存在病变如肥胖的情况下,牙周炎是否能加重机体的全身性的炎症状态,牙周炎参与胰岛素抵抗的调控机制是否与脂肪细胞因子有关,这些问题都尚未得到解决,而且人体学研究也无法完成这些研究。因此,我们采用建立疾病动物模型的方法,研究牙周炎对机体炎症状态的影响,以及这种影响足否与机体的胰岛素有关,从而揭示牙周炎与T2DM病变发生发展的关系,为临床治疗和预防提供理论依据。
第一章牙周炎合并肥胖大鼠模型的建立
目的
通过谷氨酸钠(MSG)诱导建立肥胖大鼠模型,牙周局部结扎联合涂菌建立牙周炎模型；检测肥胖大鼠的Lee's指数,血压和血糖水平,评估大鼠的肥胖状态；Micro-CT扫描大鼠颌骨样本并进行三维重建,计算牙周结扎造成的线性和体积骨丧失量；HE染色评估牙周组织的病理改变。
方法
(1)80只新出生的雄性Sprague-Dawley (SD)大鼠随机分为4组(n=20)：牙周炎联合肥胖组(OB+CP),肥胖组(OB),牙周炎组(CP)和正常对照组(C);
(2) OB+CP组与OB组的新生大鼠在出生后的第2,4,6,8,10天分别皮下注射MSG诱导肥胖,于12周龄时检测体重,体长和血压变化,取尾静脉血测定空腹血糖浓度。判定肥胖模型成功建立的大鼠进入牙周炎模型的建立；
(3) OB+CP组与CP组的大鼠在局麻下结扎双侧上颌第一,第二磨牙结扎处涂布四种牙周致病菌的混悬液,诱导实验性牙周炎8周；
(4)20周龄时,处死所有实验大鼠,采集血清和组织样本,颌骨样本采用Micro-CT扫描,EDTA脱钙和HE染色。
结果
本实验通过MSG药物诱导新生大鼠,使其在成年期后表现出向心性肥胖,血压升高,血糖异常等症状和体征,成功建立肥胖大鼠模型。采用丝线结扎双侧上颌磨牙并涂布牙周致病菌,经8周的诱导,成功建立慢性牙周炎模型。两种疾病模型在SD大鼠体内成功复合。Micro-CT的三维重建显示,结扎的牙周组织表现出明显的线性和体积骨丧失量；HE染色切片显示牙周组织附着丧失明显,胶原纤维破坏,牙周袋底炎症细胞浸润,肉芽组织形成,牙槽骨吸收明显。
结论
采用MSG诱导新生大鼠,可成功建立成年期肥胖大鼠模型,且该模型鼠表现出某些前糖尿病状态的体征,该方法简单,易行,重复性好,模型稳定。丝线结扎联合涂菌可诱导大鼠典型的牙周组织破坏,病理改变明显,慢性炎症状态维持稳定。
第二章牙周炎对大鼠内脏脂肪组织炎症状态的影响
目的
研究牙周炎对肥胖大鼠和非肥胖大鼠诱导内脏脂肪组织的形态学改变以及炎症状态变化的影响,探讨牙周炎和肥胖对脂肪组织炎症的联合作用,为牙周炎参与调控肥胖相关性疾病的病理机制提供理论基础。
方法
四组大鼠的内脏脂肪组织经福尔马林固定后,制成6um的切片,分别用于HE染色,甲苯胺蓝染色和CD68/CD14的免疫组化染色。软件分析四组大鼠脂肪细胞个数和直径的变化,血管基质成分的增生以及CD68/CD14标记的巨噬细胞的浸润状态。
结果
四组大鼠脂肪组织的HE染色结果显示,和CP组与C组相比,OB+CP组与OB组大鼠的脂肪细胞数目增加,有显著性差异(P<0.05),但OB+CP组与OB组之间(t=0.503,P=0.616)以及CP组与C组之间(t=0.215,P=0.830)的差别无统计学意义。OB组与OB+CP组的脂肪细胞直径明显大于C组和CP组,差别有统计学意义(P<0.05)；但C组与CP组(t=0.480,P=0.632),OB组与OB+CP组之间无统计学差异(t=0.773,P=0.441)。甲苯胺蓝染色显示OB+CP组大鼠脂肪组织中的血管基质部分出现明显的增生扩张和炎症细胞浸润,而CP组的变化不明显。比较四组大鼠脂肪组织中巨噬细胞的浸润,OB+CP组的CD68蛋白表达明显优于OB组(t=3.944,P=0.000),且CP组的蛋白表达量也高于C组,差别有统计学意义(t=8.332,P=0.000)。脂肪组织中CD14蛋白的表达分析显示,OB+CP组的平均光密度值高于OB组,有显著统计学差异(t=5.051,P=0.000)；CP组与C组的脂肪组织内偶见阳性染色细胞,CP组的平均光密度值高于C组,差别有统计学差异(t=3.382,P=0.000)。肥胖和牙周炎对大鼠脂肪组织中CD14蛋白的表达存在交互作用(F=7.481,P=0.007)。
结论
肥胖可造成大鼠脂肪细胞的个数增加,体积增大,血管基质扩张,炎症细胞浸润。而单纯牙周感染,并不能引起脂肪细胞发生明显的增生和肥大,但能使脂肪组织表现出轻度的炎症状态,表现为脂肪组织中的血管基质部分轻度增生,巨噬细胞浸润增加。在联合了肥胖后,牙周炎可使脂肪细胞发生更明显的增生和肥大,并且血管基质部分增生扩张更明显,巨噬细胞浸润更严重。基于以上证据,我们认为,慢性牙周炎症可引起脂肪组织的轻度炎症性改变,但不发生明显的形态学变化,但在机体本身存在炎症的前提下,牙周炎对脂肪组织炎症的加重具有促进作用。
第三章牙周炎对脂肪细胞因子表达的影响及其与胰岛素抵抗的关系
目的
检测肥胖和非肥胖状态下,牙周炎对瘦素和脂联素这两个关键的脂肪细胞因子在脂肪组织中表达的影响和血清浓度变化的影响,并分析这两个脂肪细胞因子的局部和全身水平与机体胰岛素抵抗的关系。
方法
采用免疫荧光的方法标记四组大鼠脂肪组织中瘦素和脂联素蛋白表达的变化,进行半定量分析；ELISA法检测大鼠血清中瘦素,脂联素和胰岛素的浓度；葡萄糖氧化酶法检测血清中空腹血糖的浓度。根据空腹胰岛素(mIU/L)和空腹血糖(mmol/L)浓度计算稳态模型下的胰岛素抵抗指数(homeostasis model assessment-insulin resistance, HOMA-IR),并采用Pearson相关分析评估瘦素和脂联素在脂肪组织和血清中的表达水平是否与机体的胰岛素抵抗有关。
结果
瘦素蛋白在OB+CP组大鼠脂肪组织中的表达量最高,显著高于OB组(t=2.046,P=0.044)；CP组大鼠脂肪组织中的瘦素蛋白表达高于C组,差别有统计学意义(t=5.237,P=0.000)；血清中的瘦素水平与脂肪组织中的表达水平一致,OB+CP组高于OB组(t=11.197,P=0.004),CP组高于C组(t=7.156,P=0.000),差别有显著的统计学差异。脂联素蛋白在C组大鼠脂肪组织中的表达量最高,而OB+CP组中最低,CP组的脂联素蛋白表达量显著低于C组(t=-3.661,P=0.000),OB+CP组低于OB组(t=-2.932,P=0.004),有显著统计学差异；但OB+CP组的血清脂联素浓度最高,高于OB组(t=3.624,P=0.003),而CP组的血清脂联素浓度略低于C组,但无显著性统计学差异(t=-1.323,P=0.207)。肥胖和牙周炎对脂肪组织中瘦素蛋白的表达(F=14.859,P=0.001)以及血清中瘦素(F=54.254,P=0.000)和脂联素水平(F=5.191,P=0.024)存在交互效应。
OB+CP组大鼠的空腹血糖浓度高于OB组,差别有显著统计学差异(t=7.694,P=0.000),CP组的血糖稍高于C组,差别有统计学意义(t=3.369,P=0.005)。胰岛素的血清浓度在OB+CP组比OB组稍高,但无统计学差异(t=0.658,P=0.521)；CP组血清中胰岛素浓度高于C组,有统计学差异(t=6.462,P=0.000)。肥胖和牙周炎对空腹血糖存在交互作用(F=17.699,P=0.000),但对胰岛素水平不存在交互作用(F=1.606,P=0.215)。
HOMA-IR的评分显示,从高到低依次为：OB+CP组>OB组>CP组>C组。其中,OB+CP组的HOMA-IR得分高于OB组,差别有统计学意义(t=5.662,P=0.000)；CP组的HOMA-IR得分高于C组,差别有统计学差异(t=9.992,P=0.000)。肥胖和牙周炎对大鼠HOMA-IR得分存在交互效应(F=12.340,P=0.002)。大鼠脂肪组织中瘦素的表达水平与HOMA-IR呈正相关(r=0.844,P=0.000),随着瘦素在局部脂肪组织中表达量的增多,大鼠机体的胰岛素抵抗水平增高；脂肪组织中脂联素蛋白表达水平与HOMA-IR呈负相关(r=-0.886,P=0.000),随着脂肪组织中脂联素蛋白表达的降低,胰岛素抵抗水平升高；大鼠血清中的瘦素(r=0.875,P=0.000)和脂联素(r=0.735,P=0.000)水平与HOMA-IR也呈正相关,随着瘦素和脂联素血清水平的不断升高,大鼠机体的胰岛素抵抗也加重。
结论
慢性牙周炎可诱导大鼠脂肪组织中瘦素和脂联素蛋白的表达失调,这种影响在合并了肥胖以后更为明显。瘦素和脂联素的血清浓度也受牙周炎的调控,并且牙周炎以及牙周炎联合肥胖所造成的瘦素和脂联素在局部和血清中水平的变化,与机体的胰岛素抵抗密切相关,说明牙周炎可能通过调控脂肪组织表达瘦素和脂联素,参与了机体胰岛素抵抗的发生发展。
第四章牙周炎对骨骼肌中脂肪细胞因子受体表达的影响
目的
研究各实验组大鼠骨骼肌中瘦素受体和脂联素受体1蛋白的表达,探讨牙周炎对胰岛素靶组织中脂肪细胞因子受体表达的调控以及在骨骼肌胰岛素抵抗中的作用。
方法
采用免疫组化的方法标记四组大鼠骨骼肌组织中瘦素受体和脂联素受体1蛋白的表达,并进行半定量分析。
结果
瘦素受体在C组和CP组的骨骼肌中广泛表达,染色呈强阳性,而在OB组和OB+CP组骨骼肌组织中表达量减少,表达强度减弱,尤其是OB+CP组中表达量最少。半定量分析显示CP组与C组,蛋白表达量无显著统计学差异(t=0.272,P=0.786)；但OB组中,瘦素受体的表达量显著高于OB+CP组,差别有统计学意义(t=-3.490,P=0.001)。肥胖和牙周炎对骨骼肌中瘦素受体的表达存在交互效应(F=4.341,P=0.039)。
OB+CP组大鼠骨骼肌中脂联素受体1蛋白的表达最弱,而且表达量明显低于OB组,差别有显著的统计学意义(t=-3.470,P=0.001)；CP组和C组骨骼肌切片中可见肌细胞的胞膜广泛表达脂联素受体1,但两组之间的蛋白表达量未见显著的统计学差异(t=0.543,P=0.598)。肥胖和牙周炎对大鼠骨骼肌中脂联素受体1的表达存在交互效应(F=6.364,P=0.013)。
结论
牙周炎的局部感染不能下调大鼠骨骼肌组织中瘦素受体和脂联素受体1的表达,但在肥胖状态下,牙周炎可促进大鼠骨骼肌中瘦素受体和脂联素受体1的表达明显下调,这可能造成骨骼肌组织中瘦素与瘦素受体,脂联素与脂联素受体1之间的结合障碍,从而影响骨骼肌组织中胰岛素的敏感性,有促进骨骼肌发生胰岛素抵抗的趋势。
Background:
Obesity, a subclinical state with multiple of lipid stored in adipose tissue, is closely correlated with important physiological parameters such as lipid profile, blood pressure and systemic insulin sensitivity. Obesity is a major contributor to systemic chronic disease, including type2diabetes militus (T2DM) and atherosclerosis. The first and the important pathological changes in obese state is the adipocyte hypertrophy (increased size) and hyperplasia (increased number). It is well established that the role of adipose tissue in obesity is not only a passive energy storage site, but it is understood that adipose tissue plays a much more active role as an endocrine organ. Adipose tissue can secrete a plenty of cytokines, so-called "adipokines". The adipokines can act as mediators and signal factors, and interact with energy metabolism, immune and inflammatory regulation.
There are a couple of adipokines that are of particular interest to researchers and TNF-α and IL-6are the ones of those. Previous reports shows that TNF-α and IL-6are closed related with pathological mechanism of insulin resistance and T2DM. Adipose tissue level of TNF-α is correlated with fasting plasma glucose, insulin, triacylglycerol concentrations and insulin resistance in patients with and without T2DM.The expression for IL-6is also increased in obese adipose tissue and is10-fold that in adipose tissue from lean individuals. In obese condition, adipose tissue is a major source of circulating IL-6concentrations, and contributes as much as30%of total body production. The combination of TNF-a and IL-6increases lipolysis and fat oxidation, which induce adipose tissue inflammation and followed by the development of systemic insulin resistance. Recently, leptin and adiponectin has been paid much attention as new adipokines and take part in the regulation of metabolism and inflammation. Previous experimental data indicated that leptin modulates glucose homeostasis. Leptin is secreted by adipocytes into circulation. Leptin acts mainly in the hypothalamus and has an impact on appetite, whereas expression of the leptin receptor has been observed in peripheral insulin-target tissues, including fat, liver and muscle, suggesting potential effects of leptin on peripheral insulin actions. In adipocytes, leptin inhibits insulin signaling by two different ways:as an autocrine signal and through neuroendocrine pathways. In contrast, adiponectin possesses anti-inflammatory or insulin-sensitizing properties. Thus, these adipokines can act as regulating mediators between adipose tissue and systemic disorders including insulin resistance.
Chronic periodontitis is one of the most widespread inflammatory diseases, characterized by inflammation and destruction of connective tissue attachment and alveolar bone. Published cross-sectional epidemiologic studies have suggested that periodontal disease, particularly periodontitis is associated with obesity. Finding from a longitudinal study has demonstrated a dose-response relationship between body mass index (BMI) and the development of periodontal disease. Periodontitis has been reported to adversely affect glycemic control in patients with diabetes mellitus and contribute to the development of diabetic complications. It is well established that inflammatory processes as common pathways involve in the pathogenesis of obesity, periodontitis and T2DM.
However, the mechanistic relationship between IR and periodontitis in obesity remains unclear. Whether chronic periodontitis can regulate the expression of key adipokines and result in the inflammation of adipose tissue and whether the combination of chronic periodontitis and obesity can exacerbate the inflammatory condition of adipose tissue and systemic insulin resistance. The establishment of experimental animal models is ideal to investigate the potential mechanism between periodontitis and T2DM and thus provide some useful theoretical evidence.
Part one
Establishment Experimental Periodontitis in Obese Rat Model
Objective
To establish obese rat model by monosodium glutamate (MSG) inducement and experimental periodontitis by ligature inducement with periodontal pathogens infection; To evaluate obese condition by investigating Lee's index, blood pressure, and fasting glucose; To evaluate periodontal infection by Micro-CT scanning and histological sections.
Methods
All animal's procedures were performed in accordance with the guidelines of the Southern Medical University Animal Care and Use Committee.
(1) Eighty natal SD male rats were randomly divided into four groups of20rats each, respectively:obese rats with periodontitis (OB+CP group), obese rats without periodontitis (OB group), normal rats with periodontitis (CP group) and normal rats without periodontitis (C group).
(2) In the OB group and OB+CP group, rats received subcutaneous injection of MSG (3mg/g of body weight) on alternate days for the first10postnatal days. The animals were weaned on the21st postnatal day and fed normally. At12weeks of age, body weight, body length, blood pressure, fasting glucose were investigated.
(3) At3months of age,3/0silk ligatures soaked with periodontal pathogens, were placed in subgingival position of the bilateral maxillary first and second molars for8weeks in the CP group and C group, whereas the control group were free of MSG-injection and ligation.
(4) At5months of age, rats were sacrificed under general anesthesia. Samples of the bilateral maxillary molar regions were resected from each rat and immediately fixed in10%neutral formalin for2days, and stored in70%ethanol for scanning by micro-CT. Maxillary samples were further decalcified with10%tetrasodium-EDTA equeous solution (PH7.0) for4weeks at4℃after scaning. The tissue blocks of periodontal samples were embedded in paraffin and sections (thickness:5um) were stained with hematoxylin and eosin (HE). Serum samples were also collected and stored in-80℃.
Results
In this study, we employed postnatal administration of MSG to rats and induced central obesity in adulthood. Ligatures soaked with periodontal pathogen were used to induce chronic periodontitis for8weeks. Rats in the OB+CP group and CP group showed apparent inflammatory cellular infiltration, collagenous fibers destruction and alveolar bone resorption, which were similar to features of human chronic periodontitis. This suggests that an ideal model of obesity and chronic periodontitis was established.
Conclusions
Administration of MSG can induce obese rat model. Besides, MSG-treated rats also have metabolic state of prediabetes with hypertention and hyperglycemia. This method is simple, effective and can be well repeated. In this obese state, ligature-induced periodontitis can result in typical periodontal destruction and sustain a low-grade chronic inflammation. The combination of periodontitis and obesity were successfully established in rat model.
Part two
The Effect of periodontitis on the Inflammation of Adipose Tissue in Rat Model
Objective
To investigate the effect of experimental periodontitis on the morphological changes and inflammation of adipose tissue, and to explore the addictive effect of periodontitis and obesity with the aim of providing theoretical basis on the interrelationships between periodontitis and systemic inflammatory disorders.
Methods
At the time of sacrifice, visceral adipose tissues were fixed in10%neutral formalin. After dehydration with increasing concentrations of ethanol, samples were incubated in xylenes and embedded in paraffin at60℃. Six-micron sections were cut and stained with HE, toluidine blue O or CD68/CD14immunohistochemistry. Quantification of adipocytes was done on HE-stained sections using Image-ProPlus software. Stromal-vascular fractions were observed on toluidine blue O-stained sections. Macrophage infiltration was detected on CD68/CD14immunohistochemical stained sections.
Results
Adipocyte size and amount were measured on HE-stained sections. The numbers of adipocytes in the OB+CP and OB groups were significantly higher than that in the CP and C groups (P<0.05). However, there were no significant differences in the number of adipocytes between the CP group and the C group (t=0.215, P=0.830) and that between the OB+CP group and the OB group (t=0.503, P=0.616). The cell diameter changes in the OB and OB+CP groups were striking. Enlarged cells were prevalent and mean diameters of adipocytes were167.21um and170.02um, respectively, which were higher than that in the CP and C groups (P<0.05). However, adipocyte diameters between CP and C groups were similar (t=0.480, P=0.632), and there was also no difference between OB+CP group and OB group (t=0.773, P=0.441).
Toluidine blue O-stained sections described the presence of SVF and nucleated stromal cells. The stromal multinucleated cells were greatly increased in the OB and OB+CP groups when compared to the CP and C groups. The presence of nucleated stromal cells in the CP group was similar to those in the C group.
In the visceral fat tissue, immunohistochemical analysis showed that the protein expression for CD68in the OB+CP was significantly higher than that in the OB group (t=3.944, P=0.000); and the expression in the CP group was also significantly higher than that in the C group (t=8.332, P=0.000). Furthermore, the protein expression for CD14in the OB+CP group was significantly higher than that in the OB group (t=5.051, P=0.000).And the difference between the CP group and the C group was statistically significant (t=3.382, P=0.000). A synergistic interaction between obesity and periodontitis was evident to the expression for CD14in the adipose tissue.
Conclusions
Obesity can induce adipocyte hypertrophy (increased size) and hyperplasia (increased number), enlarged stromal vascular fraction and macrophage infiltration in rat model. In contrast, experimental periodontitis can barely induce similar morphological changes in adipose tissue. However, periodontitis can result in slightly inflammatory disorder of adipose tissue, with characteristic of early enlarged stromal vascular fraction and several macrophage infiltrations. When combined with obesity, periodontitis can induce further hypertrophy and hyperplasia of adipocytes; furthermore, significant enlargement of stromal vascular fraction and macrophage infiltration can be observed in the OB+CP group. Based on the data, we accept that experimental periodontitis has a little impact on the morphological changes of adipose tissue but has remarkable effects on the inflammatory condition of adipose tissue. Periodontitis can exacerbate this inflammatory condition with the presence of obesity.
Part three
The Effect of Periodontitis on Protein Expression of Adipokines and the Relationships with Insulin Resistance
Objective
To investigate the impact of experimental periodontitis on the protein expression for leptin and adiponectin protein in adipose tissues and the concentrations in circulation; and further to explore the relationships between the expression of leptin and adiponectin locally and systemically and the systemic insulin resistance.
Methods
Immunofluorescence analysis of leptin and adiponectin expression of adipose tissue in rat model was employed and semi-quantitative method was used. Concentrations of serum leptin, adiponectin and insulin were determined using enzyme-linked immunosorbent assay (ELISA) kit; glucose levels were measured by glucose oxidase method. IR was calculated using the homeostasis model assessment (HOMA-IR), where IR=In[(Fasting glucose[mmol/l]×fasting insulin[mU/l])/22.5]. Pearson's correlation coefficient was used to determine the correlation between the levels of leptin and adiponectin and IR.
Results
In the visceral fat tissue, immunofluorescence analysis showed that the protein expression for leptin in the OB+CP, CP and OB groups were significantly higher than in the C group (P<0.05). Furthermore, the expression for leptin in the OB+CP group was significantly higher than that in the OB group (t=2.046, P=0.044). And the difference between the CP group and the C group was statistically significant (t=5.237,P=0.000). In contrast, the protein expressions for adiponectin in the OB+CP, OB and CP groups were decreased. Weak protein expression was detected in the OB+CP group and was significantly lower than that in the OB group (t=-2.932, P=0.004). In the CP group, the adiponectin expression level was decreased relative to C group (t=-3.661,P=0.000)
The mean levels of serum leptin in the OB+CP, OB and CP groups were significantly greater than that of the C group (P<0.01). Serum leptin in the OB+CP group was significantly higher than that in the OB (t=11.197, P=0.004). And serum leptin in the CP group was higher than in the C group (t=7.156, P=0.000). In adipose tissue, the expression for adiponectin was lower in the OB+CP group than that in the OB group (t=-2.932, P=0.004); and adiponectin in the CP group was also lower than that in the C group (t=-3.661, P=0.000). The circulating level of adiponectin in the OB+CP group was significantly higher than each of the three groups (P<0.01). In the OB+CP group, the levels of adiponectin were significantly higher than that in the OB group(t=3.624, P=0.003).However, adiponectin level was declined in the CP group, but there was no significant difference between CP group and C group (t=-1.323,P=0.207). A synergistic interaction between obesity and periodontitis was evident to the expression for leptin in the adipose tissue and the circulating levels of leptin and adiponectin.
Fasting glucose level in the OB+CP group was significantly higher than that in the OB group (t=7.694,P=0.000),and glucose level in the CP group was also higher than that in C groups (t=3.369,.P=0.005). In contrast, there was no difference between OB+CP and OB groups in the insulin levels (t=0.658, P=0.521) and the insulin level in the CP group was higher than that in the C group (t=6.462, P=0.000). A synergistic interaction between obesity and periodontitis was evident to the fasting glucose level (F=17.669, P=0.000).
HOMA-IR analysis indicated that MSG-induced obesity was associated with an increase in IR in the OB and OB+CP groups compared to non-obesity rats. Further, HOMA-IR was significantly greater in OB+CP group compared to the OB group (t=5.662,P=0.000). In contrast, there was an increase in IR in the CP group, and the difference between CP group and C group was statistically significant (t=9.992, P=0.000). Significant positive relationships were observed between IR and leptin expression levels in adipose tissue (r=0.844, P=0.000), serum leptin (r=0.875, P=0.000) and adiponectin level (r=0.735, P=0.000). And negative relationship was observed between IR and adiponectin level in the adipose tissue (r=-0.886, P=0.000)
Conclusions
Experimental periodontitis can affect expression for leptin and adiponectin protein directly in adipose tissues:upregulation of leptin and downregulation of adiponectin. When combined with systemic pathological changes such as obesity, periodontitis can exacerbate dysregulation of leptin and adiponectin in adipose tissue. Expremental periodontitis can also regulate circulating levels of leptin and adiponectin. When combined with obesity, periodontitis aggravates hyperleptinemia and hyperadiponectinemia and contribute to the development of IR.
Part four
The Effect of Periodontitis on Receptors Expression of Adipokines in Skeletal Muscle
Objective
To invesitgate the protein expression for leptin receptor and adiponectin receptor1protein in skeletal muscle, with the aim of exploring the effect of experimental periodontitis on adipokines receptors in insulin-targeting tissues and underlying mechanisms of skeletal muscle insulin resistance.
Methods
Immunohistochemical analysis of leptin receptor and adiponectin receptor1expression in skeletal muscle was employed and semi-quantitative method was used.
Results
In the skeletal muscle, the protein expressions for leptin receptor in the OB+CP, OB and CP groups were decreased, but in the C group, the protein expression were popular and strong positive. Fewer protein expression was detected in the OB+CP group when compared with OB group (t=-3.490, P-0.001). In the CP group, the protein expression level were decreased relative to C group but not significant difference (t=0.272, P=0.786). In contrast, the expression for adiponectin receptor1was greatly lower than that in the OB group (t=-3.470, P=0.001); and the expression in the CP group was similar to the C group (t=0.543, P=0.598). A synergistic interaction between obesity and periodontitis was evident to the expression for the leptin receptor and adiponectin receptor1.
Conclusions
Chronic low-grade inflammation induced by experimental periodontitis can barely down-regulate the protein expression for leptin receptor and adiponectin receptor1in the skeletal muscle. However, periodontitis can significantly down-regulate the protein expressions with the presence of obesity. This may contribute to the binding disruption of these two adipokines and their receptors in skeletal muscle samples, and may further decrease the insulin sensitivity, which accelerate the onset and development of insulin resistance in the skeletal muscle.
引文
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